Search results for: osteoblastic MG-63 cells

Authors: Elif Gencturk, Ekin Yurdakul, Ahmet Y. Celik, Senol Mutlu, Kutlu O. Ulgen

Abstract:

Yeast cells are generally used as a model system of eukaryotes due to their complex genetic structure, rapid growth ability in optimum conditions, easy replication and well-defined genetic system properties. Thus, yeast cells increased the knowledge of the principal pathways in humans. During fermentation, carbohydrates (hexoses and pentoses) degrade into some toxic by-products such as 5-hydroxymethylfurfural (5-HMF or HMF) and furfural. HMF influences the ethanol yield, and ethanol productivity; it interferes with microbial growth and is considered as a potent inhibitor of bioethanol production. In this study, yeast single cell behavior under HMF application was monitored by using a continuous flow single phase microfluidic platform. Microfluidic device in operation is fabricated by hot embossing and thermo-compression techniques from cyclo-olefin polymer (COP). COP is biocompatible, transparent and rigid material and it is suitable for observing fluorescence of cells considering its low auto-fluorescence characteristic. The response of yeast cells was recorded through Red Fluorescent Protein (RFP) tagged Nop56 gene product, which is an essential evolutionary-conserved nucleolar protein, and also a member of the box C/D snoRNP complexes. With the application of HMF, yeast cell proliferation continued but HMF slowed down the cell growth, and after HMF treatment the cell proliferation stopped. By the addition of fresh nutrient medium, the yeast cells recovered after 6 hours of HMF exposure. Thus, HMF application suppresses normal functioning of cell cycle but it does not cause cells to die. The monitoring of Nop56 expression phases of the individual cells shed light on the protein and ribosome synthesis cycles along with their link to growth. Further computational study revealed that the mechanisms underlying the inhibitory or inductive effects of HMF on growth are enriched in functional categories of protein degradation, protein processing, DNA repair and multidrug resistance. The present microfluidic device can successfully be used for studying the effects of inhibitory agents on growth by single cell tracking, thus capturing cell to cell variations. By metabolic engineering techniques, engineered strains can be developed, and the metabolic network of the microorganism can thus be manipulated such that chemical overproduction of target metabolite is achieved along with the maximum growth/biomass yield.  

Keywords: COP, HMF, ribosome biogenesis, thermoplastic microbioreactor, yeast

Procedia PDF Downloads 171

Authors: A. Vijayendra Chary, R. Hemalatha

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Background: Vitamin D is a potent modulator of the immune system and is involved in regulating cell proliferation and differentiation. Vitamin D deficiency has been linked to increased severity of asthma in children. Asthma has dramatically increased in past decades, particular in developing countries and affects up to 20% of the population. IgE and its receptors, CD23 (FcεRII) and CD 21, play an essential role in all allergic conditions. Methods: A case control study was conducted on asthma and age and sex matched control children. 25 hydroxyvitamin D3 was quantified by HPLC; CD23; and CD21 expression on B cells were performed by flow cytometry. Total Histamine, total IGE and IL-5 and IFN-γ cytokines were determined by ELISA in blood samples of bronchial asthma (n=45) and control children (n=45). Results: The mean ± SE of vitamin D was significantly (p<0.05) low in asthma children (13.6±0.54 ng/mL) than in controls (17.4 ± 0.37 ng/mL). The mean (%) ± SE of CD23 and CD21 expression on B cells were significantly (p<0.01) high in asthma (1.02±0.09; 1.67± 0.13), when compared to controls (0.24±0.01; 0.94±0.03) respectively. The mean± SE of Serum IgE and blood histamine levels in asthma children (354.52 ± 17.33 IU/mL; 53.27 ± 2.54 nM/mL) were increased (P<0.05) when compared to controls (183.12±17.62 IU/mL 39.34±4.16 nM/mL) respectively and IFN-γ (Th1 cytokine) was lower (P<0.01) (16.37±1.27 pg/mL) than in controls (43.34±6.21 pg/mL). Conclusion: Our study provides evidence that low vitamin D levels are associated with increased IgE receptors CD23 and CD21 on B cells. In addition, there was preferential activation of Th2 (IL-5) and suppression of Th1 (IFN-γ) cytokines in children with asthma.

Keywords: bronchial asthma, CD23, IgE, vitamin D

Procedia PDF Downloads 476

Authors: Gupta Renu, T. S. Roy

Abstract:

Introduction: For the prospect of successful replacement therapies in treatment of Diabetes mallitus it is necessary to know events occurring during normal human pancreas development. Literature of human pancreas development are few in number as well as mainly related to first trimester because of ethical and technical difficulties. So the study was conducted on 12 fetuses from 12 gestational weeks (GW) to 5 months of infant to know normal development of exocrine and endocrine part of human pancreas. Material and Methods: Human fetalpancreases were screened by haematoxyline and eosin staining and done electron microscopy for suitable specimens to know ultrastructural detail of fetal pancreas. Results:It was observed arborized tubules, the cells budding out from these tubules differentiated into primitive acini and islets in 12thGW. At 14 weeks scanty granules were observed in the endocrine cells which coincided with the capillary invasion of the islets. The ducts and acini were surrounded by well-organized connective tissue. The acinihad elongated cells, small amount of cytoplasm and large open face euchromatic nuclei with single nucleolus. The mature form of islets of Langerhans was observed close to the acini and duct in 20 GW fetus. Connective tissue around the duct was well organized.No significant developmental change was observed early postnatal, infant. Conclusion: The development of both component exocrine as well as endocrine part of human fetal pancreas was studied by light and electron microscopy. Observations suggested that the fetal pancreas contained mainly ducts, few acini, many centroacinar cells, and large undifferentiated tissue.

Keywords: gestational weeks (GW), acini, islets of Langerhans, ducts

Procedia PDF Downloads 263

Authors: Prajna Paramita Naik, Sujit Kumar Bhutia

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Background: Regardless of the development multimodal treatment strategies, oral squamous cell carcinoma (OSCC) is often associated with a high rate of recurrence, metastasis and chemo- and radio- resistance. The present study inspected the relevance of CD44, ABCB1 and ADAM17 expression as a putative stem cell compartment in oral squamous cell carcinoma (OSCC) and deciphered the role of autophagy in regulating the expression of aforementioned proteins, stemness and chemoresistance. Methods: A retrospective analysis of CD44, ABCB1 and ADAM17 expression with respect to the various clinicopathological factors of sixty OSCC patients were determined via immunohistochemistry. The correlation among CD44, ABCB1 and ADAM17 expression was established. Sphere formation assay, flow cytometry and fluorescence microscopy were conducted to elucidate the stemness and chemoresistance nature of established cisplatin-resistant oral cancer cells (FaDu). The pattern of expression of CD44, ABCB1 and ADAM17 in parental (FaDu-P) and resistant FaDu cells (FaDu-CDDP-R) were investigated through fluorescence microscopy. Western blot analysis of autophagy marker proteins was performed to compare the status of autophagy in parental and resistant FaDu cell. To investigate the role of autophagy in chemoresistance and stemness, sphere formation assay, immunofluorescence and Western blot analysis was performed post transfection with siATG14 and the level of expression of autophagic proteins, mitochondrial protein and stemness-associated proteins were analyzed. The statistical analysis was performed by GraphPad Prism 4.0 software. p-value was defined as follows: not significant (n.s.): p > 0.05;*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001 were considered statistically significant. Results: In OSCC, high CD44, ABCB1 and ADAM17 expression were significantly correlated with higher tumor grades and poor differentiation. However, the expression of these proteins was not related to the age and sex of OSCC patients. Moreover, the expression of CD44, ABCB1 and ADAM17 were positively correlated with each other. In vitro and OSCC tissue double labeling experiment data showed that CD44+ cells were highly associated with ABCB1 and ADAM17 expression. Further, FaDu-CDDP-R cells showed higher sphere forming capacity along with increased fraction of the CD44+ population and β-catenin expression FaDu-CDDP-R cells also showed accelerated expression of CD44, ABCB1 and ADAM17. A comparatively higher autophagic flux was observed in FaDu-CDDP-R against FaDu-P cells. The expression of mitochondrial proteins was noticeably reduced in resistant cells as compared to parental cells indicating the occurrence of autophagy-mediated mitochondrial degradation in oral cancer. Moreover, inhibition of autophagy was coupled with the decreased formation of orospheres suggesting autophagy-mediated stemness in oral cancer. Blockade of autophagy was also found to induce the restoration of mitochondrial proteins in FaDu-CDDP-R cells indicating the involvement of mitophagy in chemoresistance. Furthermore, a reduced expression of CD44, ABCB1 and ADAM17 was also observed in ATG14 deficient cells FaDu-P and FaDu-CDDP-R cells. Conclusion: The CD44+ ⁄ABCB1+ ⁄ADAM17+ expression in OSCC might be associated with chemoresistance and a putative CSC compartment. Further, the present study highlights the contribution of mitophagy in chemoresistance and confirms the potential involvement of autophagic regulation in acquisition of stem-like characteristics in OSCC.

Keywords: ABCB1, ADAM17, autophagy, CD44, chemoresistance, mitophagy, OSCC, stemness

Procedia PDF Downloads 195

Authors: Abdulrahman M. Alajlan, Saichao Dang, Qiaoqiang Gan

Abstract:

Photovoltaic (PV) cells, while pivotal for solar energy harnessing, confront a challenge due to the presence of persistent residual heat. This thermal energy poses significant obstacles to the performance and longevity of PV cells. Mitigating this thermal issue is imperative, particularly in tropical regions where solar abundance coexists with elevated ambient temperatures. In response, a sustainable and economically viable solution has been devised, incorporating water-passive cooling within a Photovoltaic-Thermoelectric (PV-TEG) hybrid system to address PV cell overheating. The implemented system has significantly reduced the operating temperatures of PV cells, achieving a notable reduction of up to 15 °C below the temperature observed in standalone PV systems. In addition, a thermoelectric generator (TEG) integrated into the system significantly enhances power generation, particularly during nighttime operation. The developed hybrid system demonstrates its capability to generate power at a density of 0.5 Wm⁻² during nighttime, which is sufficient to concurrently power multiple light-emitting diodes, demonstrating practical applications for nighttime power generation. Key findings from this research include a consistent temperature reduction exceeding 10 °C for PV cells, translating to a 5% average enhancement in PV output power compared to standalone PV systems. Experimental demonstrations underscore nighttime power generation of 0.5 Wm⁻², with the potential to achieve 0.8 Wm⁻² through simple geometric optimizations. The optimal cooling of PV cells is determined by the volume of water in the heat storage unit, exhibiting an inverse relationship with the optimal performance for nighttime power generation. Furthermore, the TEG output effectively powers a lighting system with up to 5 LEDs during the night. This research not only proposes a practical solution for maximizing solar radiation utilization but also charts a course for future advancements in energy harvesting technologies.

Keywords: photovoltaic-thermoelectric systems, nighttime power generation, PV thermal management, PV cooling

Procedia PDF Downloads 87

Authors: Roberto Coppo, Francesca Orso, Daniela Dettori, Elena Quaglino, Lei Nie, Mehran M. Sadeghi, Daniela Taverna

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Malignant melanoma is currently the fifth most common cancer in the white population and it is fatal in its metastatic stage. Several research studies in recent years have provided evidence that cancer initiation and progression are driven by genetic alterations of the tumor and paracrine interactions between tumor and microenvironment. Scattered data show that the Endothelial and Smooth muscle cell-Derived Neuropilin-like molecule (ESDN) controls cell proliferation and movement of stroma and tumor cells. To investigate the role of ESDN in the tumor microenvironment during melanoma progression, murine melanoma cells (B16 or B16-F10) were injected in ESDN knockout mice in order to evaluate how the absence of ESDN in stromal cells could influence melanoma progression. While no effect was found on primary tumor growth, increased cell extravasation and lung metastasis formation was observed in ESDN knockout mice compared to wild type controls. In order to understand how cancer cells cross the endothelial barrier during metastatic dissemination in an ESDN-null microenvironment, structure, and permeability of lung blood vessels were analyzed. Interestingly, ESDN knockout mice showed structurally altered and more permeable vessels compared to wild type animals. Since cell surface molecules mediate the process of tumor cell extravasation, the expression of a panel of extravasation-related ligands and receptors was analyzed. Importantly, modulations of N-cadherin, E-selectin, ICAM-1 and VAP-1 were observed in ESDN knockout endothelial cells, suggesting the presence of a favorable tumor microenvironment which facilitates melanoma cell extravasation and metastasis formation in the absence of ESDN. Furthermore, a potential contribution of immune cells in tumor dissemination was investigated. An increased recruitment of macrophages in the lungs of ESDN knockout mice carrying subcutaneous B16-F10 tumors was found. In conclusion, our data suggest a functional role of ESDN in the tumor microenvironment during melanoma progression and the identification of the mechanisms that regulate tumor cell extravasation could lead to the development of new therapies to reduce metastasis formation.

Keywords: melanoma, tumor microenvironment, extravasation, cell surface molecules

Procedia PDF Downloads 335

Authors: Mahfuzur Rahman

Abstract:

Back-contact solar cells improve optical properties by moving all electrically conducting parts to the back of the cell. The cell's structure allows silicon solar cells to surpass the 25% efficiency barrier and interdigitated solar cells are now the most efficient. In this work, the fabrication of a light, efficient and temperature resistant interdigitated back contact (IBC) solar cell is investigated. This form of solar cell differs from a conventional solar cell in that the electrodes are located at the back of the cell, eliminating the need for grids on the top, allowing the full surface area of the cell to receive sunlight, resulting in increased efficiency. In this project, we will use SILVACO TCAD, an optoelectronic device simulator, to construct a very thin solar cell with dimensions of 100x250um in 2D Luminous. The influence of sunlight intensity and atmospheric temperature on solar cell output power is highly essential and it has been explored in this work. The cell's optimum performance with 150um bulk thickness provides 28.81% efficiency with an 87.68% fill factor rate making it very thin, flexible and resilient, providing diverse operational capabilities.

Keywords: interdigitated, shading, recombination loss, incident-plane, drift-diffusion, luminous, SILVACO

Procedia PDF Downloads 146

Authors: Leander Van Neste, Kirk Wojno

Abstract:

The innate immune system reacts to foreign insult in several unique ways, one of which is phagocytosis of perceived threats such as cancer, bacteria, and viruses. The goal of this study was to look for evidence of phagocytosed RNA from tumor cells in circulating monocytes. While all monocytes possess phagocytic capabilities, the non-classical CD14+/FCGR3A+ monocytes and the intermediate CD14++/FCGR3A+ monocytes most actively remove threatening ‘external’ cellular materials. Purified CD14-positive monocyte samples from fourteen patients recently diagnosed with clinically localized prostate cancer (PCa) were investigated by single-cell RNA sequencing using the 10X Genomics protocol followed by paired-end sequencing on Illumina’s NovaSeq. Similarly, samples were processed and used as controls, i.e., one patient underwent biopsy but was found not to harbor prostate cancer (benign), three young, healthy men, and three men previously diagnosed with prostate cancer that recently underwent (curative) radical prostatectomy (post-RP). Sequencing data were mapped using 10X Genomics’ CellRanger software and viable cells were subsequently identified using CellBender, removing technical artifacts such as doublets and non-cellular RNA. Next, data analysis was performed in R, using the Seurat package. Because the main goal was to identify differences between PCa patients and ‘control’ patients, rather than exploring differences between individual subjects, the individual Seurat objects of all 21 patients were merged into one Seurat object per Seurat’s recommendation. Finally, the single-cell dataset was normalized as a whole prior to further analysis. Cell identity was assessed using the SingleR and cell dex packages. The Monaco Immune Data was selected as the reference dataset, consisting of bulk RNA-seq data of sorted human immune cells. The Monaco classification was supplemented with normalized PCa data obtained from The Cancer Genome Atlas (TCGA), which consists of bulk RNA sequencing data from 499 prostate tumor tissues (including 1 metastatic) and 52 (adjacent) normal prostate tissues. SingleR was subsequently run on the combined immune cell and PCa datasets. As expected, the vast majority of cells were labeled as having a monocytic origin (~90%), with the most noticeable difference being the larger number of intermediate monocytes in the PCa patients (13.6% versus 7.1%; p<.001). In men harboring PCa, 0.60% of all purified monocytes were classified as harboring PCa signals when the TCGA data were included. This was 3-fold, 7.5-fold, and 4-fold higher compared to post-RP, benign, and young men, respectively (all p<.001). In addition, with 7.91%, the number of unclassified cells, i.e., cells with pruned labels due to high uncertainty of the assigned label, was also highest in men with PCa, compared to 3.51%, 2.67%, and 5.51% of cells in post-RP, benign, and young men, respectively (all p<.001). It can be postulated that actively phagocytosing cells are hardest to classify due to their dual immune cell and foreign cell nature. Hence, the higher number of unclassified cells and intermediate monocytes in PCa patients might reflect higher phagocytic activity due to tumor burden. This also illustrates that small numbers (~1%) of circulating peripheral blood monocytes that have interacted with tumor cells might still possess detectable phagocytosed tumor RNA.

Keywords: circulating monocytes, phagocytic cells, prostate cancer, tumor immune response

Procedia PDF Downloads 162

Authors: Caroline Mendes, Mary McNamara, Orla Howe

Abstract:

Drug delivery systems are proposed for use in cancer treatment to specifically target cancer cells and deliver a therapeutic dose without affecting normal cells. For that purpose, the use of folate receptors (FR) can be considered a key strategy, since they are commonly over-expressed in cancer cells. In this study, cyclodextrins (CD) have being used as vehicles to target FR and deliver the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within their cavities. Here, β-CD has been modified using folic acid so as to specifically target the FR. Thus, this drug delivery system consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 15.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 16.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 10.5 for A549 and 132.6 µM ± 16.1 and 288.1 µM ± 26.3 for BEAS-2B. These results demonstrate that free MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug. The use of cell imaging by confocal microscopy has allowed visualisation of FR targeting in cancer cells, as well as the identification of the interlisation pathway of the drug. Hence, the cellular uptake and internalisation process of this drug delivery system is being addressed.

Keywords: cancer treatment, cyclodextrins, drug delivery, folate receptors, reduced folate carriers

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Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

Abstract:

PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provides better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

Procedia PDF Downloads 69

Authors: Nancy M. El-Baz, Laila Ziko, Rania Siam, Wael Mamdouh

Abstract:

Cancer is one of the most devastating diseases, and has over than 10 million new cases annually worldwide. Despite the effectiveness of chemotherapeutic agents, their systemic toxicity and non-selective anticancer actions represent the main obstacles facing cancer curability. Due to the effective enhanced permeability and retention (EPR) effect of nanomaterials, nanoparticles (NPs) have been used as drug nanocarriers providing targeted cancer drug delivery systems. In addition, several inorganic nanoparticles such as silver (AgNPs) nanoparticles demonstrated a potent anticancer activity against different cancers. The present study aimed at formulating core-shell inorganic NPs-based combinatorial therapy based on combining the anticancer activity of AgNPs along with doxorubicin (DOX) and evaluating their cytotoxicity on MCF-7 breast cancer cells. These inorganic NPs-based combinatorial therapies were designed to (i) Target and kill cancer cells with high selectivity, (ii) Have an improved efficacy/toxicity balance, and (iii) Have an enhanced therapeutic index when compared to the original non-modified DOX with much lower dosage The in-vitro cytotoxicity studies demonstrated that the NPs-based combinatorial therapy achieved the same efficacy of non-modified DOX on breast cancer cell line, but with 96% reduced dose. Such reduction in DOX dose revealed that the combination between DOX and NPs possess a synergic anticancer activity against breast cancer. We believe that this is the first report on a synergic anticancer effect at very low dose of DOX against MCF-7 cells. Future studies on NPs-based combinatorial therapy may aid in formulating novel and significantly more effective cancer therapeutics.

Keywords: nanoparticles-based combinatorial therapy, silver nanoparticles, doxorubicin, breast cancer

Procedia PDF Downloads 437

Authors: Mohamed A. Baba, Gazy Rodowan, Brigita Abakevičienė, Sigitas Tamulevičius, Bartlomiej Lemieszek, Sebastian Molin, Tomas Tamulevičius

Abstract:

Solid oxide fuel cells (SOFC) efficiently convert hydrogen to energy without producing any disturbances or contaminants. The core of the cell is electrolyte. For improving the performance of electrolyte-supported cells, it is desirable to extend the available exchange surface area by micro-structuring of the electrolyte with laser-based micromachining. This study investigated the electrochemical performance of cells micro machined using a femtosecond laser. Commercial ceramic SOFC (Elcogen, AS) with a total thickness of 400 μm was structured by 1030 nm wavelength Yb: KGW fs-laser Pharos (Light Conversion) using 100 kHz repetition frequency and 290 fs pulse length light by scanning with the galvanometer scanner (ScanLab) and focused with a f-Theta telecentric lens (SillOptics). The sample height was positioned using a motorized z-stage. The microstructures were formed using a laser spiral trepanning in Ni/YSZ anode supported membrane at the central part of the ceramic piece of 5.5 mm diameter at active area of the cell. All surface was drilled with 275 µm diameter holes spaced by 275 µm. The machining processes were carried out under ambient conditions. The microstructural effects of the femtosecond laser treatment on the electrolyte surface were investigated prior to the electrochemical characterisation using a scanning electron microscope (SEM) Quanta 200 FEG (FEI). The Novo control Alpha-A was used for electrochemical impedance spectroscopy on a symmetrical cell configuration with an excitation amplitude of 25 mV and a frequency range of 1 MHz to 0.1 Hz. The fuel cell characterization of the cell was examined on open flanges test setup by Fiaxell. Using nickel mesh on the anode side and au mesh on the cathode side, the cell was electrically linked. The cell was placed in a Kittec furnace with a Process IDentifier temperature controller. The wires were connected to a Solartron 1260/1287 frequency analyzer for the impedance and current-voltage characterization. In order to determine the impact of the anode's microstructure on the performance of the commercial cells, the acquired results were compared to cells with unstructured anode. Geometrical studies verified that the depth of the -holes increased linearly according to laser energy and scanning times. On the other hand, it reduced as the scanning speed increased. The electrochemical analysis demonstrates that the open circuit voltage OCV values of the two cells are equal. Further, the modified cell's initial slope reduces to 0.209 from 0.253 of the unmodified cell, revealing that the surface modification considerably decreases energy loss. Plus, the maximum power density for the cell with the microstructure and the reference cell respectively, are 1.45 and 1.16 Wcm⁻².

Keywords: electrochemical performance, electrolyte-supported cells, laser micro-structuring, solid oxide fuel cells

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Authors: Fethi Benyettou, Abdelkader Aissat, M. A. Benammar

Abstract:

In this work, we use Silvaco TCAD software for modeling and simulations of standard GaAs solar cell, InAs/GaAs and GaSb/GaAs p-i-n quantum dot solar cell. When comparing 20-layer InAs/GaAs, GaSb/GaAs quantum dots solar cells with standard GaAs solar cell, the conversion efficiency in simulation results increased from 16.48 % to 22.6% and 16.48% to 22.42% respectively. Also, the absorption range edge of photons with low energies extended from 900 nm to 1200 nm.

Keywords: SILVACO TCAD, the quantum dot, simulation, materials engineering

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Authors: Hyunjung Lim, Jeonghun Nam, Hyuk Choi

Abstract:

High-throughput separation is an essential technique for cancer research and diagnosis. Here, we propose a viscoelastic microfluidic device for sheathless, high-throughput isolation of circulating tumor cells (CTCs) from white blood cells. Here, we demonstrate a viscoelastic method for separation and concentration of CTCs using closed-loop microfluidics. Our device is a rectangular straight channel with a low aspect ratio. Also, to achieve high-efficiency, high-throughput processing, we used a polymer solution with low viscosity. At the inlet, CTCs and white blood cells (WBCs) were randomly injected into the microchannel. Due to the viscoelasticity-induced lateral migration to the equilibrium positions, large CTCs could be collected from the side outlet while small WBCs were removed at the center outlet. By recirculating the collected CTCs from the side outlet back to the sample reservoir, continuous separation and concentration of CTCs could be achieved with high separation efficiency (~ 99%). We believe that our device has the potential to be applied in resource-limited clinical settings.

Keywords: circulating tumor cell, closed-loop microfluidics, concentration, separation, viscoelastic fluid

Procedia PDF Downloads 153

Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

Abstract:

PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provide better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

Procedia PDF Downloads 71

Authors: Maryam Radan, Fereshteh Nejad Dehbashi, Vahid Bayati, Mahin Dianat, Seyyed Ali Mard, Zahra Mansouri

Abstract:

Pulmonary emphysema is a pathological respiratory condition identified by alveolar destruction which leads to limitation of airflow and diminished lung function. A substantial body of evidence suggests that mesenchymal stem cells (MSCs) have the ability to induce tissue repair primarily through a paracrine effect. In this study, we aimed to determine the efficacy of Intratracheal adipose-derived mesenchymal stem cells (ADSCs) therapy in comparison to this approach with that of Intravenous (Systemic) therapy. Fifty adult male Sprague–Dawley rats weighing between 180 and 200 g were used in this experiment. The animals were randomized to Control groups (Intratracheal or Intravenous vehicle), Elastase group (intratracheal administration of porcine pancreatic elastase; 25 U/kg on day 0 and day 10th), Elastase+Intratracheal ADSCs therapy (1x107 Cells, on day 28) and Elastase+Systemic ADSCs therapy (1x107 Cells, on day 28). The rats which not subjected to any treatment, considered as the control. All rats were sacrificed 3 weeks later. Morphometric findings in lung tissues (Mean linear intercept) confirmed the establishment of the emphysema model via alveolar disruption. Contrarily, ADSCs administration partially restored alveolar architecture. These results were associated with improving arterial oxygenation, reducing lung edema, and decreasing lung inflammation with higher significant effects in the Intratracheal therapy route. These results documented that the efficacy of intratracheal ADSCs was comparable with intravenous ADSCs therapy. Accordingly, the obtained data suggested that intratracheal delivery of ADSCs would enhance lung repair in pulmonary emphysema. Moreover, this method provides benefits over a systemic administration, such as the reduction of cell number and the low risk to engraft other organs.

Keywords: mesenchymal stem cell, emphysema, Intratracheal, systemic

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Authors: Francisco Dos Santos, Diogo Fonseca-Pereira, Sílvia Arroz-Madeira, Henrique Veiga-Fernandes

Abstract:

Haematopoiesis is a developmental process that generates all blood cell lineages in health and disease. This relies on quiescent haematopoietic stem cells (HSCs) that are able to differentiate, self renew and expand upon physiological demand. HSCs have great interest in regenerative medicine, including haematological malignancies, immunodeficiencies and metabolic disorders. However, the limited yield from existing HSC sources drives the global need for reliable techniques to expand harvested HSCs at high quality and sufficient quantities. With the extensive use of cord blood progenitors for clinical applications, there is a demand for a safe and efficient expansion protocol that is able to overcome the limitations of the cord blood as a source of HSC. StemCell2MAXTM developed a technology that enhances the survival, proliferation and transplantation efficiency of HSC, leading the way to a more widespread use of HSC for research and clinical purposes. StemCell2MAXTM MIX is a solution that improves HSC expansion up to 20x, while preserving stemness, when compared to state-of-the-art. In a recent study by a leading cord blood bank, StemCell2MAX MIX was shown to support a selective 100-fold expansion of CD34+ Hematopoietic Stem and Progenitor Cells (when compared to a 10-fold expansion of Total Nucleated Cells), while maintaining their multipotent differentiative potential as assessed by CFU assays. The technology developed by StemCell2MAXTM opens new horizons for the usage of expanded hematopoietic progenitors for both research purposes (including quality and functional assays in Cord Blood Banks) and clinical applications.

Keywords: cord blood, expansion, hematopoietic stem cell, transplantation

Procedia PDF Downloads 268

Authors: Malgorzata Rudnicka, Waldemar Karcz

Abstract:

Introduction: Naphthoquinones are widely occurring organic compounds produced by bacteria, fungi, and plants. They can act as the functional components of biochemical systems (plastoquinone) as well as biologically active substances, which have a negative impact on environmental processes. Naphthoquinones seem to act through two mechanisms: a covalent modification of biological molecules at their nucleophilic sites or by generation of reactive oxygen species (ROS) connected with redox cycling. Investigating the effect of naphthoquinones (1,4-naphthoquinone, lawsone and naphthazarin) on the elongation growth, membrane potential and the level of oxidative stress of maize cells seems to be important due to the possibility of using these substances as herbicides. Methods: All experiments were performed on etiolated maize coleoptile segments. Simultaneous measurements of elongation growth and pH of the incubation medium were carried out using an angular position transducer, allowing a precise record of the growth kinetics. To compare the oxidative stress level induced by all tested naphthoquinones, the changes in malondialdehyde content, as well as superoxide dismutase and catalase activities were measured. In order to measure the membrane potential of parenchymal cells the standard electrophysiology technique was used. Results: Naphthoquinones such as: 1,4-naphthoquinone, lawsone and naphthazarin were studied. It was found that all of the naphthoquinones diminished the growth of the maize coleoptile cells depending on the type of naphthoquinones and their concentration. Interestingly, naphthazarin at the intermediate concentration was less toxic compared to the others. In addition, the effect of naphthoquinones on the oxidative stress was dependent on their concentration as well. Superoxide dismutase and catalase activities were changed in the presence of higher concentrations of naphthoquinones. Similar interrelations were observed for membrane potential changes. Conclusion: It can be concluded that naphthoquinones tested differ in their toxic effect on the growth of maize coleoptile cells. Furthermore, naphthoquinones can be distinguish considering the oxidative stress level and membrane potential changes. The results presented here give new insight into the possible opportunities of practical usage of naphthoquinones for herbicides improvement.

Keywords: growth rate, membrane potential, naphthoquinones, oxidative stress

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Authors: Abhipsa Mohanty, Harit Jha

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The world's energy demands are continuing to rise, resulting in a worldwide energy crisis and environmental pollution. Because of finite, declining supply and environmental damage, reliance on fossil fuels is unsustainable. As a result, experts are concentrating on alternative, renewable, and carbon-free energy sources. Energy sources that are both environmentally and economically sustainable are required. Microbial fuel cells (MFCs) have recently received a lot of attention due to their low operating temperatures and ability to use a variety of biodegradable substrates as fuel. There are single-chamber MFCs as well as traditional MFCs with anode and cathode compartments. Bioelectricity is produced when microorganisms actively catabolize substrate. MFCs can be used as a power source in small devices like biosensors. Understanding of its components, microbiological processes, limiting variables, and construction designs in MFC systems must be simplified, and large-scale systems must be developed for them to be cost-effective as well as increase electricity production. The purpose of this research was to review current microbiology knowledge in the field of electricity. The manufacturing process, the materials, and procedures utilized to construct the technology, as well as the applications of MFC technology, are all covered.

Keywords: bio-electricity, exoelectrogenic bacteria, microbial fuel cells, soil microorganisms

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Authors: Jehad Dumireih, Malak Dmirieh, Michael Wink

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The present work is aimed to use plant cell suspension cultures of Crataegus monogyna for biosynthesis of valuable natural products by using quercetin as an inexpensive precursor. Suspension cell cultures of C. monogyna were established by using Murashige and Skoog medium (MS) supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid and 1 mg/L kinetin. Cells were harvested from the cultures and extracted by using methanol and ethyl acetate; then the extracts were used for the identification of isoquercetin by HPLC and by mass spectrometry. The incubation of the cells with 0.24 mM quercetin for one week resulted in an 16 fold increase of isoquercetin biosynthesis; the growth rate of the cells increased by 20%. Moreover, the biosynthesis of isoquercetin was enhanced by 40% when we divided the added quercetin into three portions each one with concentration 0.12 mM supplied at 3 days intervals. In addition, we didn’t find any positive effects of adding different concentrations the precursors phenylalanine (0.2 mM) and galactose to the cell cultures. In conclusion, the efficiency of the biotransformation of quercetin into isoquercetin depended on the concentration quercetin, its incubation time and the way of its administration. The results of the present work suggest that the biotechnological methods such as cell suspension cultures could be successfully used to obtain highly valuable natural product starting from inexpensive compound.

Keywords: biosynthesis, biotransformation, Crataegus, isoquercetin

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Authors: Amina Farooq, Nauman Zafar Butt

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This paper is about methods and tools for counting particles of interest, such as cells. A microfluidic system with interconnected electronics on a flexible substrate, inlet-outlet ports and interface schemes, sensitive and selective detection of cells specificity, and processing of cell counting at polymer interfaces in a microscale biosensor for use in the detection of target biological and non-biological cells. The development of fluidic channels, planar fluidic contact ports, integrated metal electrodes on a flexible substrate for impedance measurements, and a surface modification plasma treatment as an intermediate bonding layer are all part of the fabrication process. Magnetron DC sputtering is used to deposit a double metal layer (Ti/Pt) over the polypropylene film. Using a photoresist layer, specified and etched zones are established. Small fluid volumes, a reduced detection region, and electrical impedance measurements over a range of frequencies for cell counts improve detection sensitivity and specificity. The procedure involves continuous flow of fluid samples that contain particles of interest through the microfluidic channels, counting all types of particles in a portion of the sample using the electrical differential counter to generate a bipolar pulse for each passing cell—calculating the total number of particles of interest originally in the fluid sample by using MATLAB program and signal processing. It's indeed potential to develop a robust and economical kit for cell counting in whole-blood samples using these methods and similar devices.

Keywords: impedance, biochip, cell counting, microfluidics

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Authors: H. Djebar, L. Khene, M. Boucenna, M. R. Djebar, M. N. Khebbeb, M. Djekoun

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Currently in toxicology, use of alternative models permits to understand the mechanisms of toxicity at different levels of cells. Objectives of our research concern the determination of NPs ZnO, TiO2, AlO2, and FeO2 effect on ciliate protist freshwater Paramecium sp and Helix aspersa. The result obtained show that NPs increased antioxidative enzyme activity like catalase, glutathione –S-transferase and level GSH. Also, cells treated with high concentrations of NPs showed a high level of MDA. In conclusion, observations from growth and enzymatic parameters suggest on one hand that treatment with NPs provokes an oxidative stress and on the other that snale and paramecium are excellent alternatives models for ecotoxicological studies.

Keywords: NPs, GST, catalase, GSH, MDA, toxicity, snale and paramecium

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Authors: Feng Zhang, Li Chen, Desong Kong, Xiaoping Zhang, Xiaojing Zhu, Yin Lu, Shizhong Zheng

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Sinusoidal pathological angiogenesis is a novel therapeutic target for liver fibrosis. We demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells (LSECs) were not impaired by curcumin. Further investigations showed that curcumin inhibited VEGF expression in hepatic stellate cells (HSCs) by disrupting PDGF-βR/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin via blocking PDGF-βR/FAK/RhoA cascade. Gain- or loss-of-function analyses revealed that activation of PPARγ was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPARγ activation-dependent mechanism. PPARγ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.

Keywords: angiogenesis, hepatic stellate cell, curcumin, peroxisome proliferator-activated receptor-γ

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Authors: Muhammad Affan, Muhammad Ilyas Raza, Muhammad Harris Hashmi

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This work is attributed to the battery management unit design of student Satellites under Pakistan National Student Satellite Program (PNSSP). The aim has been to design a customized, low-cost, efficient, reliable and less-complex battery management scheme for the Satellite. Nowadays, Lithium Ion (Li-ion) batteries have become the de-facto standard for remote applications, especially for satellites. Li-ion cells are selected for secondary storage. The design also addresses Li-ion safety requirements by monitoring, balancing and protecting cells for safe and prolonged operation. Accurate voltage measurement of individual cells was the main challenge because all the actions triggered were based on the digital voltage measurement. For this purpose, a resistive-divider network is used to maintain simplicity and cost-effectiveness. To cater the problem of insufficient i/o pins on microcontroller, fast multiplexers and de-multiplexers were used. The discrepancy inherited in the given design is the dissipation of heat due to the dissipative resistors. However, it is still considered to be the optimum adoption, considering the simple and cost-effective nature of the passive balancing technique. Furthermore, it is a completely unique solution, customized to meet specific requirements. However, there is still an option for a more advanced and expensive design.

Keywords: satellite, battery module, passive balancing, dissipative

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Authors: Ge Zheng, Jun Peng

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Unbalanced power output from individual cells of Proton Exchange Membrane Fuel Cell (PEMFC) has direct effects on PEMFC stack performance, in particular under transient operation. In the paper, a multi-layer ANN (Artificial Neural Network) model Radial Basis Functions (RBF) has been developed for predicting cells' output profiles by applying gas supply parameters, cooling conditions, temperature measurement of individual cells, etc. The feed-forward ANN model was validated with experimental data. Influence of relevant parameters of RBF on the network accuracy was investigated. After adequate model training, the modelling results show good correspondence between actual measurements and reconstructed output profiles. Finally, after the model was used to optimize the stack output performance under steady-state and transient operating conditions, it suggested that the developed ANN control model can help PEMFC stack to have obvious improvement on power output under fast acceleration process.

Keywords: proton exchange membrane fuel cell, PEMFC, artificial neural network, ANN, cell output profile, transient

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Authors: Qi LI, Xu Lin, Nengming Lin

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Objective: The aim of this study was to investigate the role of desmocollin 2 (DSC2) protein in the proliferation, migration and drug resistance of lung cancer cells. Method: CCK-8 assays and colony formation assays were used to evaluate the effect of dsc2 regulation on cancer cell viability and colony formation. Transwell assays and wound healing assays were also performed. Cell flow double staining was used to detect the apoptosis rate of cells with DSC2, which was added cisplatin. Western blot assay was used to detect cell cycle, PI3k/Akt and apoptosis-related proteins. Results: Our data showed that dsc2 is upregulated in clinical lung cancer tissues compared with pericarcinomatous tissues, and it is differentially expressed in lung cancer cell lines. The down-regulation of dsc2 in A549 and H358 lung cancer cells significantly suppressed the cell proliferation, metastasis, and motility. In contrast, the opposite effects were observed in overexpression of dsc2 both in H23 and PC9 cell lines. In addition to lung adenocarcinoma cell lines, we also examined its expression in lung squamous cell lines, such as H226. Western blotting showed that dsc2 could reduce the level of phosphorylated Akt (Ser 473) and p-mTOR. Thus, it is speculated that dsc2 up-regulation promotes proliferation and invasiveness through activation of the PI3K/AKT pathway. Also, knockdown of dsc2 in A549 and H226 could significantly decreased in the levels of cyclinB and wee1 protein. Additionally, flow cytometry showed that dsc2 knockdown combined with cisplatin could significantly enhance cell apoptosis rate. Conclusion: These data suggest that dsc2 promotes the proliferation and migration of lung cancer cells in vitro. Also, the results suggested that dsc2 could affect the cell cycle and apoptosis of lung cells. Furthermore, knockdown of dsc2 could sensitize cisplatin in both lung adenocarcinoma and lung squamous cell lines. Thus we suggested that dsc2 can be used as a therapeutic target for lung cancer.

Keywords: desmocollin 2, cisplatin, lung cancer, PI3K/AKT, lung squamous cell

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Authors: A. A. Abaker Omer, H. B. Mohamed Balh, W. Liu, A. Abas, J. Yu, S. Li, W. Ma, W. El Kolaly, Y. Y. Ahmed Abuker

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We have discovered an important technical solution that could make new approaches in the processing of wet silicon etching, especially in the production of photovoltaic cells. During its inferior light-trapping and structural properties, the inverted pyramid structure outperforms the conventional pyramid textures and black silicone. The traditional pyramid textures and black silicon can only be accomplished with more advanced lithography, laser processing, etc. Importantly, our data demonstrate the feasibility of an inverted pyramidal structure of silicon via one-step Cu-catalyzed chemical etching (CCCE) in Cu (NO₃)₂/HF/H₂O₂/H₂O solutions. The effects of etching time and reaction temperature on surface geometry and light trapping were systematically investigated. The conclusion shows that the inverted pyramid structure has ultra-low reflectivity of ~4.2% in the wavelength of 300~1000 nm; introduce of Cu particles can significantly accelerate the dissolution of the silicon wafer. The etching and the inverted pyramid structure formation mechanism are discussed. Inverted pyramid structure with outstanding anti-reflectivity includes useful applications throughout the manufacture of semi-conductive industry-compatible solar cells, and can have significant impacts on industry colleagues and populations.

Keywords: Cu-catalyzed chemical etching, inverted pyramid nanostructured, reflection, solar cells

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Authors: D. C. Asebiah, D. Saranin, S. Karazhanov, A. R. Tameev, M. Kah

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In this paper, the mesoscopic NiO was used as a hole transport layer in the inverted planar organometallic hybrid perovskite solar cell to study the effect of hysteresis. The devices we fabricated have the structures Fluorine Tin Oxide (FTO)/mesoscopic NiO/perovskite/[6,6]-phenyl C₆₁-butyric acid methyl ester (PC₆₁BM) photovoltaic device. The perovskite solar cell was done by toluene air (TLA) method and horn sonication for the dispersion of the NiO nanoparticles in deionized water. The power conversion efficiency was 12.07% under 1.5 AM illumination. We report hysteresis in the in current-voltage dependence of the solar cells with mesoscopic NiO as a hole transport layer.

Keywords: perovskite, mesoscopic, hysteresis, toluene air

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Authors: Ntokozo Dambuza, Maryna Van De Venter, Trevor Koekemoer

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Oxidative stress contributes to the pathogenesis of many diseases and consequently antioxidant therapy has attracted much attention as a potential therapeutic strategy. Regardless of the quantities ingested, antioxidants need to reach the diseased tissues at concentrations sufficient to combat oxidative stress. Bioavailability is thus a defining criterion for the therapeutic efficacy of antioxidants. In addition, therapeutic antioxidants must possess biologically relevant characteristics which can target the specific molecular mechanisms responsible for disease related oxidative stress. While many chemical antioxidant assays are available to quantify antioxidant capacity, they relate poorly to the biological environment and provide no information as to the bioavailability. The present comparative study thus aims to characterise green and fermented rooibos extracts, well recognized for their exceptional antioxidant capacity, in terms of antioxidant bioavailability and efficacy in a disease relevant cellular setting. Chinese green tea antioxidant activity was also evaluated. Chemical antioxidant assays (FRAP, DPPH and ORAC) confirmed the potent antioxidant capacity of both green and fermented rooibos, with green rooibos possessing antioxidant activity superior to that of fermented rooibos and Chinese green tea. Bioavailability was assessed using the PAMPA assay and the results indicate that green and fermented rooibos have a permeation coefficient of 5.7 x 10-6 and 6.9 x 10-6 cm/s, respectively. Chinese green tea permeability coefficient was 8.5 x 10-6 cm/s. These values were comparable to those of rifampicin, which is known to have a high permeability across intestinal epithelium with a permeability coefficient of 5 x 10 -6 cm/s. To assess the antioxidant efficacy in a cellular context, U937 and red blood cells were pre-treated with rooibos and Chinese green tea extracts in the presence of a dye DCFH-DA and then exposed to oxidative stress. Green rooibos exhibited highest activity with an IC50 value of 29 μg/ml and 70 μg/ml, when U937 and red blood cells were exposed oxidative stress, respectively. Fermented rooibos and Chinese green tea had IC50 values of 61 μg/ml and 57 μg/ml for U937, respectively, and 221 μg/ml and 405 μg/ml for red blood cells, respectively. These results indicate that fermented and green rooibos extracts were able to permeate the U937 cells and red blood cell membrane and inhibited oxidation of DCFH-DA to a fluorescent DCF within the cells.

Keywords: rooibos, antioxidants, permeability, bioavailability

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Authors: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Shohei Koide, Christopher Park, Dimitris Placantonakis

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Background: Glioblastoma (GBM) is the most common and deadly primary brain malignancy in adults. Tumor propagation, brain invasion, and resistance to therapy critically depend on GBM stem-like cells (GSCs); however, the mechanisms that regulate GSC self-renewal are incompletely understood. Given the aggressiveness and poor prognosis of GBM, it is imperative to find biomarkers that could also translate into novel drug targets. Along these lines, we have identified a cell surface antigen, CD97 (ADGRE5), an adhesion G protein-coupled receptor (GPCR), that is expressed on GBM cells but is absent from non-neoplastic brain tissue. CD97 has been shown to promote invasiveness, angiogenesis, and migration in several human cancers, but its frequency of expression and functional role in regulating GBM growth and survival, and its potential as a therapeutic target has not been investigated. Design: We assessed CD97 mRNA and protein expression in patient derived GBM samples and cell lines using publicly available RNA-sequencing datasets and flow cytometry, respectively. To assess CD97 function, we generated shRNA lentiviral constructs that target a sequence in the CD97 extracellular domain (ECD). A scrambled shRNA (scr) with no predicted targets in the genome was used as a control. We evaluated CD97 shRNA lentivirally transduced GBM cells for Ki67, Annexin V, and DAPI. We also tested CD97 KD cells for their ability to self-renew using clonogenic tumorsphere formation assays. Further, we utilized synthetic Abs (sAbs) generated against the ECD of CD97 to test for potential antitumor effects using patient-derived GBM cell lines. Results: CD97 mRNA expression was expressed at high levels in all GBM samples available in the TCGA cohort. We found high levels of surface CD97 protein expression in 6/6 patient-derived GBM cell cultures, but not human neural stem cells. Flow cytometry confirmed downregulation of CD97 in CD97 shRNA lentivirally transduced cells. CD97 KD induced a significant reduction in cell growth in 3 independent GBM cell lines representing mesenchymal and proneural subtypes, which was accompanied by reduced (~20%) Ki67 staining and increased (~30%) apoptosis. Incubation of GBM cells with sAbs (20 ug/ ml) against the ECD of CD97 for 3 days induced GSC differentiation, as determined by the expression of GFAP and Tubulin. Using three unique GBM patient derived cultures, we found that CD97 KD attenuated the ability of GBM cells to initiate sphere formation by over 300 fold, consistent with an impairment in GSC self-renewal. Conclusion: Loss of CD97 expression in patient-derived GBM cells markedly decreases proliferation, induces cell death, and reduces tumorsphere formation. sAbs against the ECD of CD97 reduce tumorsphere formation, recapitulating the phenotype of CD97 KD, suggesting that sAbs that inhibit CD97 function exhibit anti-tumor activity. Collectively, these findings indicate that CD97 is necessary for the proliferation and survival of human GBM cells and identify CD97 as a promising therapeutically targetable vulnerability in GBM.

Keywords: adhesion GPCR, CD97, GBM stem cell, glioblastoma

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