Search results for: aggregated stem cells
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 3835

Search results for: aggregated stem cells

2845 SUMOylation Enhances Nurr1/1a Mediated Transactivation in a Neuronal Cell Type

Authors: Jade Edey, Andrew Bennett, Gareth Hathway

Abstract:

Nuclear receptor-related 1 protein (also known as Nurr1 or NR4A2) is an orphan nuclear receptor which plays a vital role in the development, survival and maintenance of dopaminergic (DA) neurons particularly in the substantia nigra (SN). Increasing research has investigated Nurr1’s additional role within microglia and astrocytes where it has been suggested to act as a negative regulator of inflammation; potentially offering neuroprotection. Considering both DA neurodegeneration and neuroinflammation are commonly accepted constituents of Parkinson’s Disease (PD), understanding the mechanisms by which Nurr1 regulates inflammatory processes could provide an attractive therapeutic target. Nurr1 regulates inflammation via a transrepressive mechanism possibly dependent upon SUMOylation. In addition, Nurr1 can transactivate numerous genes involved in DA synthesis, such as Tyrosine Hydroxylase (TH). A C-terminal splice variant of Nurr1, Nurr-1a, has been reported in both neuronal and glial cells. However, research into its transcriptional activity is minimal. We employed in vitro methods such as SUMO-Pulldown experiments alongside Luciferase reporter assays to investigate the SUMOylation status and transactivation capabilities of Nurr1 and Nurr-1a respectively. The SUMO-Pulldown assay demonstrated Nurr-1a undergoes significantly more SUMO modification than its full-length variant. Consequently, despite having less transcriptional activation than Nurr1, Nurr1a may play a more prominent role in repression of microglial inflammation. Contrary to published literature we also identified that SUMOylation enhances transcriptional activation by Nurr1 and Nurr1a. SUMOylation-dependent increases in Nurr1 and Nurr1a transcriptional activation were only evident in neuronal SHSY5Y cells but not in HEK293 cells. This research provides novel insight into the regulation of Nurr-1a and indicates differential effects of SUMOylation dependent regulation in neuronal and inflammatory cells.

Keywords: nuclear receptors, Parkinson’s disease, inflammation, transcriptional regulation

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2844 Antiplasmodial Activity of Drimane Sesquiterpene Isolated from Warburgia salutaris

Authors: Mthokozisi Simelane

Abstract:

Background: Malaria remains a life-threatening disease in tropical regions despite the advances in the treatment of this disease, it still remains a significant burden as some parasites have become resistant to the currently available drugs. This has created a necessity for the development of alternative, more efficient antimalarial drugs. Warburgia salutaris is a traditional medicinal plant used in malaria treatment by Zulu traditional healers. Materials and methods: The W. salutaris stem-bark was extracted with dichloromethane and the compound was isolated through column chromatography. The compound was identified and characterized by spectroscopic analysis (1H NMR, 13C NMR, IR and MS) and the structure was also confirmed by x-ray crystallography. The anti-plasmodial activity (in vitro) was studied on NF54 Plasmodium falciparum strain (CQS). Cytotoxicity was measured using the MTT assay on HEK239 and HEPG2 cell lines. Docking of Mukaadial acetate was conducted in AutoDock Vina. Structural modifications were conducted in UCSF Chimera and molecular interactions examined in LigPlot. Results: The compound, Mukaadial Acetate showed appreciable inhibition (IC50 0.44±0.10 µg/ml) of the parasite growth and cytotoxicity activity of 0.124±0.109 and 0.199±0.083 (µg/ml) on HEK293 and HEPG2 cells respectively. Molecular docking revealed that Mukaadial Acetate binds to the purine, pyrophosphate and ribose binding sites of the PfHGXPRT with an optimum binding conformation and forms hydrogen bond, steric and hydrophobic interactions with the residues inhabiting the respective binding sites. Conclusion: It is apparent that W. salutaris contains components (including Mukaadial Acetate) that exhibit antimalarial activity. This study scientifically validates the use of this plant in folk medicine.

Keywords: plasmodium falciparum, molecular docking, antimalarial activity, PfHGXPRT, Warburgia salutaris, mukaadial acetate

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2843 Virtual Engineers on Wheels: Transitioning from Mobile to Online Outreach

Authors: Kauser Jahan, Jason Halvorsen, Kara Banks, Kara Natoli, Elizabeth McWeeney, Brittany LeMasney, Nicole Caramanna, Justin Hillman, Christopher Hauske, Meghan Sparks

Abstract:

The Virtual Engineers on Wheels (ViEW) is a revised version of our established mobile K-12 outreach program Engineers on Wheels in order to address the pandemic. The Virtual Engineers on Wheels' (VIEW) goal has stayed the same as in prior years: to provide K-12 students and educators with the necessary resources to peak interest in the expanding fields of engineering. With these trying times, the Virtual Engineers on Wheels outreach has adapted its medium of instruction to be more seamless with the online approach to teaching and outreach. In the midst of COVID-19, providing a safe transfer of information has become a constraint for research. The focus has become how to uphold a level of quality instruction without diminishing the safety of those involved by promoting proper health practices and giving hope to students as well as their families. Furthermore, ViEW has created resources on effective strategies that minimize risk factors of COVID-19 and inform families that there is still a promising future ahead. To obtain these goals while still maintaining true to the hands-on learning that is so crucial to young minds, the approach is online video lectures followed by experiments within different engineering disciplines. ViEW has created a comprehensive website that students can leverage to explore the different fields of study. One of the experiments entails teaching about drone usage and how it might play a factor in the future of unmanned deliveries. Some of the other experiments focus on the differences in mask materials and their effectiveness, as well as their environmental outlook. Having students perform from home enables them a safe environment to learn at their own pace while still providing quality instruction that would normally be achieved in the classroom. Contact information is readily available on the website to provide interested parties with a means to ask their inquiries. As it currently stands, the interest in engineering/STEM-related fields is underrepresented from women and certain minority groups. So alongside the desire to grow interest, helping balance the scales is one of the main priorities of VIEW. In previous years, VIEW surveyed students before and after instruction to see if their perception of engineering has changed. In general, it is the understanding that being exposed to engineering/STEM at a young age increases the chances that it will be pursued later in life.

Keywords: STEM, engineering outreach, teaching pedagogy, pandemic

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2842 Survey and Identification of Coinfecting Botryosphaeriales Causing Stem Canker Diseases of Eucalyptus camaldulensis in Ethiopia

Authors: Wendu Admasu, Assefa Sintayehu, Alemu Gezahgne, Zewdu Terefework

Abstract:

Eucalyptus is the most widely planted forest tree species in the world. In Ethiopia, pathogenic fungi pose an increasing threat to Eucalyptus species. Due to limited research, there is insufficient information on the associated diseases and pathogens. This study investigated Eucalyptus diseases, the extent of their damage, and the causal fungal pathogens. A Eucalyptus disease survey was conducted in the Eucalyptus forestry areas of Ethiopia during the growth years 2019/20 and 2020/21. Disease assessment and sampling were carried out in eighteen plantations at nine locations. E. camaldulensis was the most dominant species planted in the surveyed areas. The field study shows a high incidence and severity of canker diseases. Diseased stem and branch samples were collected, cultured on malt extract agar media and studied. The results of morphological and ITS sequence analysis confirmed that the fungal species Neofusicoccum parvum, Lasiodiplodia theobromae, and Aplosporella hesperidica caused the observed canker symptoms. This is the first report of Lasiodiplodia theobromae and Aplosporella hesperidica causing diseases in Eucalyptus plants in Ethiopia. Changes in global climate and environmental factors, such as altitude, are believed to have a strong impact on the susceptibility of Eucalyptus plants to diseases. Strict quarantine practices and continuous monitoring of pathogenic and endophytic fungal species associated with Eucalyptus trees are issued to be prioritized to effectively control and manage the disease.

Keywords: Neofusicoccum, Lasiodiplodia, Aplosporella, pathogenicity, phylogeny, severity

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2841 Destruction of Colon Cells by Nanocontainers of Ferromagnetic

Authors: Lukasz Szymanski, Zbigniew Kolacinski, Grzegorz Raniszewski, Slawomir Wiak, Lukasz Pietrzak, Dariusz Koza, Karolina Przybylowska-Sygut, Ireneusz Majsterek, Zbigniew Kaminski, Justyna Fraczyk, Malgorzata Walczak, Beata Kolasinska, Adam Bednarek, Joanna Konka

Abstract:

The aim of this work is to investigate the influence of electromagnetic field from the range of radio frequencies on the desired nanoparticles for cancer therapy. In the article, the development and demonstration of the method and the model device for hyperthermic selective destruction of cancer cells are presented. This method was based on the synthesis and functionalization of carbon nanotubes serving as ferromagnetic material nanocontainers. The methodology of the production carbon - ferromagnetic nanocontainers (FNCs) includes: The synthesis of carbon nanotubes, chemical, and physical characterization, increasing the content of a ferromagnetic material and biochemical functionalization involving the attachment of the key addresses. The ferromagnetic nanocontainers were synthesised in CVD and microwave plasma system. Biochemical functionalization of ferromagnetic nanocontainers is necessary in order to increase the binding selectively with receptors presented on the surface of tumour cells. Multi-step modification procedure was finally used to attach folic acid on the surface of ferromagnetic nanocontainers. Pristine ferromagnetic carbon nanotubes are not suitable for application in medicine and biotechnology. Appropriate functionalization of ferromagnetic carbon nanotubes allows to receiving materials useful in medicine. Finally, a product contains folic acids on the surface of FNCs. The folic acid is a ligand of folate receptors – α which is overexpressed on the surface of epithelial tumours cells. It is expected that folic acids will be recognized and selectively bound by receptors presented on the surface of tumour cells. In our research, FNCs were covalently functionalized in a multi-step procedure. Ferromagnetic carbon nanotubes were oxidated using different oxidative agents. For this purpose, strong acids such as HNO3, or mixture HNO3 and H2SO4 were used. Reactive carbonyl and carboxyl groups were formed on the open sides and at the defects on the sidewalls of FNCs. These groups allow further modification of FNCs as a reaction of amidation, reaction of introduction appropriate linkers which separate solid surface of FNCs and ligand (folic acid). In our studies, amino acid and peptide have been applied as ligands. The last step of chemical modification was reaction-condensation with folic acid. In all reaction as coupling reagents were used derivatives of 1,3,5-triazine. The first trials in the device for hyperthermal RF generator have been done. The frequency of RF generator was in the ranges from 10 to 14Mhz and from 265 to 621kHz. Obtained functionalized nanoparticles enabled to reach the temperature of denaturation tumor cells in given frequencies.

Keywords: cancer colon cells, carbon nanotubes, hyperthermia, ligands

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2840 Modified 'Perturb and Observe' with 'Incremental Conductance' Algorithm for Maximum Power Point Tracking

Authors: H. Fuad Usman, M. Rafay Khan Sial, Shahzaib Hamid

Abstract:

The trend of renewable energy resources has been amplified due to global warming and other environmental related complications in the 21st century. Recent research has very much emphasized on the generation of electrical power through renewable resources like solar, wind, hydro, geothermal, etc. The use of the photovoltaic cell has become very public as it is very useful for the domestic and commercial purpose overall the world. Although a single cell gives the low voltage output but connecting a number of cells in a series formed a complete module of the photovoltaic cells, it is becoming a financial investment as the use of it fetching popular. This also reduced the prices of the photovoltaic cell which gives the customers a confident of using this source for their electrical use. Photovoltaic cell gives the MPPT at single specific point of operation at a given temperature and level of solar intensity received at a given surface whereas the focal point changes over a large range depending upon the manufacturing factor, temperature conditions, intensity for insolation, instantaneous conditions for shading and aging factor for the photovoltaic cells. Two improved algorithms have been proposed in this article for the MPPT. The widely used algorithms are the ‘Incremental Conductance’ and ‘Perturb and Observe’ algorithms. To extract the maximum power from the source to the load, the duty cycle of the convertor will be effectively controlled. After assessing the previous techniques, this paper presents the improved and reformed idea of harvesting maximum power point from the photovoltaic cells. A thoroughly go through of the previous ideas has been observed before constructing the improvement in the traditional technique of MPP. Each technique has its own importance and boundaries at various weather conditions. An improved technique of implementing the use of both ‘Perturb and Observe’ and ‘Incremental Conductance’ is introduced.

Keywords: duty cycle, MPPT (Maximum Power Point Tracking), perturb and observe (P&O), photovoltaic module

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2839 Dissimilarity-Based Coloring for Symbolic and Multivariate Data Visualization

Authors: K. Umbleja, M. Ichino, H. Yaguchi

Abstract:

In this paper, we propose a coloring method for multivariate data visualization by using parallel coordinates based on dissimilarity and tree structure information gathered during hierarchical clustering. The proposed method is an extension for proximity-based coloring that suffers from a few undesired side effects if hierarchical tree structure is not balanced tree. We describe the algorithm by assigning colors based on dissimilarity information, show the application of proposed method on three commonly used datasets, and compare the results with proximity-based coloring. We found our proposed method to be especially beneficial for symbolic data visualization where many individual objects have already been aggregated into a single symbolic object.

Keywords: data visualization, dissimilarity-based coloring, proximity-based coloring, symbolic data

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2838 Software Quality Measurement System for Telecommunication Industry in Malaysia

Authors: Nor Fazlina Iryani Abdul Hamid, Mohamad Khatim Hasan

Abstract:

Evolution of software quality measurement has been started since McCall introduced his quality model in year 1977. Starting from there, several software quality models and software quality measurement methods had emerged but none of them focused on telecommunication industry. In this paper, the implementation of software quality measurement system for telecommunication industry was compulsory to accommodate the rapid growth of telecommunication industry. The quality value of the telecommunication related software could be calculated using this system by entering the required parameters. The system would calculate the quality value of the measured system based on predefined quality metrics and aggregated by referring to the quality model. It would classify the quality level of the software based on Net Satisfaction Index (NSI). Thus, software quality measurement system was important to both developers and users in order to produce high quality software product for telecommunication industry.

Keywords: software quality, quality measurement, quality model, quality metric, net satisfaction index

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2837 Understanding the Role of Nitric Oxide Synthase 1 in Low-Density Lipoprotein Uptake by Macrophages and Implication in Atherosclerosis Progression

Authors: Anjali Roy, Mirza S. Baig

Abstract:

Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid rich plaque enriched with necrotic core, modified lipid accumulation, smooth muscle cells, endothelial cells, leucocytes and macrophages. Macrophage foam cells play a critical role in the occurrence and development of inflammatory atherosclerotic plaque. Foam cells are the fat-laden macrophages in the initial stage atherosclerotic lesion formation. Foam cells are an indication of plaque build-up, or atherosclerosis, which is commonly associated with increased risk of heart attack and stroke as a result of arterial narrowing and hardening. The mechanisms that drive atherosclerotic plaque progression remain largely unknown. Dissecting the molecular mechanism involved in process of macrophage foam cell formation will help to develop therapeutic interventions for atherosclerosis. To investigate the mechanism, we studied the role of nitric oxide synthase 1(NOS1)-mediated nitric oxide (NO) on low-density lipoprotein (LDL) uptake by bone marrow derived macrophages (BMDM). Using confocal microscopy, we found that incubation of macrophages with NOS1 inhibitor, TRIM (1-(2-Trifluoromethylphenyl) imidazole) or L-NAME (N omega-nitro-L-arginine methyl ester) prior to LDL treatment significantly reduces the LDL uptake by BMDM. Further, addition of NO donor (DEA NONOate) in NOS1 inhibitor treated macrophages recovers the LDL uptake. Our data strongly suggest that NOS1 derived NO regulates LDL uptake by macrophages and foam cell formation. Moreover, we also checked proinflammatory cytokine mRNA expression through real time PCR in BMDM treated with LDL and copper oxidized LDL (OxLDL) in presences and absences of inhibitor. Normal LDL does not evoke cytokine expression whereas OxLDL induced proinflammatory cytokine expression which significantly reduced in presences of NOS1 inhibitor. Rapid NOS-1-derived NO and its stable derivative formation act as signaling agents for inducible NOS-2 expression in endothelial cells, leading to endothelial vascular wall lining disruption and dysfunctioning. This study highlights the role of NOS1 as critical players of foam cell formation and would reveal much about the key molecular proteins involved in atherosclerosis. Thus, targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages at early stage of disease and hence dampening the atherosclerosis progression.

Keywords: atherosclerosis, NOS1, inflammation, oxidized LDL

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2836 Forecasting Amman Stock Market Data Using a Hybrid Method

Authors: Ahmad Awajan, Sadam Al Wadi

Abstract:

In this study, a hybrid method based on Empirical Mode Decomposition and Holt-Winter (EMD-HW) is used to forecast Amman stock market data. First, the data are decomposed by EMD method into Intrinsic Mode Functions (IMFs) and residual components. Then, all components are forecasted by HW technique. Finally, forecasting values are aggregated together to get the forecasting value of stock market data. Empirical results showed that the EMD- HW outperform individual forecasting models. The strength of this EMD-HW lies in its ability to forecast non-stationary and non- linear time series without a need to use any transformation method. Moreover, EMD-HW has a relatively high accuracy comparing with eight existing forecasting methods based on the five forecast error measures.

Keywords: Holt-Winter method, empirical mode decomposition, forecasting, time series

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2835 Successes on in vitro Isolated Microspores Embryogenesis

Authors: Zelikha Labbani

Abstract:

The In Vitro isolated micro spore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a micro spore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the micro spore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of micro spore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, micro spore became a strategy to achieve various objectives particularly in genetic engineering. In this study we would show the most recent advances in the producing haploid embryos via In Vitro isolated micro spore culture.

Keywords: haploid cells, In Vitro isolated microspore culture, success

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2834 Metal Nanoparticles Caused Death of Metastatic MDA-MB-231 Cells

Authors: O. S. Adeyemi, C. G. Whiteley

Abstract:

The present study determined the toxic potential of metal nanoparticles in cell culture system. Silver and gold nanoparticles were synthesized and characterized following established "green" protocols. The synthesized nanoparticles, in varying concentrations ranging from 0.1–100 µM were evaluated for toxicity in metastatic MDA-MB-231 cells. The nanoparticles promoted a generation of reactive oxygen species and reduced cell viability to less than 50% in the demonstration of cellular toxicity. The nanoparticles; gold and the silver-gold mixture had IC50 values of 56.65 and 18.44 µM respectively. The IC50 concentration for silver nanoparticles could not be determined. Furthermore, the probe of the cell death using flow cytometry and confocal microscopy revealed the partial involvement of apoptosis as well as necrosis. Our results revealed cellular toxicity caused by the nanoparticles but the mechanism remains yet undefined.

Keywords: cell death, nanomedicine, nanotoxicology, toxicity

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2833 Synthesis and Application of an Organic Dye in Nanostructure Solar Cells Device

Authors: M. Hoseinnezhad, K. Gharanjig

Abstract:

Two organic dyes comprising carbazole as the electron donors and cyanoacetic acid moieties as the electron acceptors were synthesized. The organic dye was prepared by standard reaction from carbazole as the starting material. To this end, carbazole was reacted with bromobenzene and further oxidation and reacted with cyanoacetic acid. The obtained organic dye was purified and characterized using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1HNMR), carbon nuclear magnetic resonance (13CNMR) and elemental analysis. The influence of heteroatom on carbazole donors and cyno substitution on the acid acceptor is evidenced by spectral and electrochemical photovoltaic experiments. Finally, light fastness properties for organic dye were investigated.

Keywords: dye-sensitized solar cells, indoline dye, nanostructure, oxidation potential, solar energy

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2832 Influence of Controlled Retting on the Quality of the Hemp Fibres Harvested at the Seed Maturity by Using a Designed Lab-Scale Pilot Unit

Authors: Brahim Mazian, Anne Bergeret, Jean-Charles Benezet, Sandrine Bayle, Luc Malhautier

Abstract:

Hemp fibers are increasingly used as reinforcements in polymer matrix composites due to their competitive performance (low density, mechanical properties and biodegradability) compared to conventional fibres such as glass fibers. However, the huge variation of their biochemical, physical and mechanical properties limits the use of these natural fibres in structural applications when high consistency and homogeneity are required. In the hemp industry, traditional processes termed field retting are commonly used to facilitate the extraction and separation of stem fibers. This retting treatment consists to spread out the stems on the ground for a duration ranging from a few days to several weeks. Microorganisms (fungi and bacteria) grow on the stem surface and produce enzymes that degrade pectinolytic substances in the middle lamellae surrounding the fibers. This operation depends on the weather conditions and is currently carried out very empirically in the fields so that a large variability in the hemp fibers quality (mechanical properties, color, morphology, chemical composition…) is resulting. Nonetheless, if controlled, retting might be favorable for good properties of hemp fibers and then of hemp fibers reinforced composites. Therefore, the present study aims to investigate the influence of controlled retting within a designed environmental chamber (lab-scale pilot unit) on the quality of the hemp fibres harvested at the seed maturity growth stage. Various assessments were applied directly on fibers: color observations, morphological (optical microscope), surface (ESEM), biochemical (gravimetry) analysis, spectrocolorimetric measurements (pectins content), thermogravimetric analysis (TGA) and tensile testing. The results reveal that controlled retting leads to a rapid change of color from yellow to dark grey due to development of microbial communities (fungi and bacteria) at the stem surface. An increase of thermal stability of fibres due to the removal of non-cellulosic components along retting is also observed. A separation of bast fibers to elementary fibers occurred with an evolution of chemical composition (degradation of pectins) and a rapid decrease in tensile properties (380MPa to 170MPa after 3 weeks) due to accelerated retting process. The influence of controlled retting on the biocomposite material (PP / hemp fibers) properties is under investigation.

Keywords: controlled retting, hemp fibre, mechanical properties, thermal stability

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2831 Multicellular Cancer Spheroids as an in Vitro Model for Localized Hyperthermia Study

Authors: Kamila Dus-Szachniewicz, Artur Bednarkiewicz, Katarzyna Gdesz-Birula, Slawomir Drobczynski

Abstract:

In modern oncology hyperthermia (HT) is defined as a controlled tumor heating. HT treatment temperatures range between 40–48 °C and can selectively damage heat-sensitive cancer cells or limit their further growth, usually with minimal injury to healthy tissues. Despite many advantages, conventional whole-body and regional hyperthermia have clinically relevant side effects, including cardiac and vascular disorders. Additionally, the lack of accessibility of deep-seated tumor sites and impaired targeting micrometastases renders HT less effective. It is believed that above disadvantages can significantly overcome by the application of biofunctionalized microparticles, which can specifically target tumor sites and become activated by an external stimulus to provide a sufficient cellular response. In our research, the unique optical tweezers system have enabled capturing the silica microparticles, primary cells and tumor spheroids in highly controllable and reproducible environment to study the impact of localized heat stimulation on normal and pathological cell and within multicellular tumor spheroid. High throughput spheroid model was introduced to better mimic the response to HT treatment on tumors in vivo. Additionally, application of local heating of tumor spheroids was performed in strictly controlled conditions resembling tumor microenvironment (temperature, pH, hypoxia, etc.), in response to localized and nonhomogeneous hyperthermia in the extracellular matrix, which promotes tumor progression and metastatic spread. The lack of precise control over these well- defined parameters in basic research leads to discrepancies in the response of tumor cells to the new treatment strategy in preclinical animal testing. The developed approach enables also sorting out subclasses of cells, which exhibit partial or total resistance to therapy, in order to understand fundamental aspects of the resistance shown by given tumor cells in response to given therapy mode and conditions. This work was funded by the National Science Centre (NCN, Poland) under grant no. UMO-2017/27/B/ST7/01255.

Keywords: cancer spheroids, hyperthermia, microparticles, optical tweezers

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2830 Visualizing Matrix Metalloproteinase-2 Activity Using Extracellular Matrix-Immobilized Fluorescence Resonance Energy Transfer Bioprobe in Cancer Cells

Authors: Hawon Lee, Young-Pil Kim

Abstract:

Visualizing matrix metalloproteinases (MMPs) activity is necessary for understanding cancer metastasis because they are implicated in cell migration and invasion by degrading the extracellular matrix (ECM). While much effort has been made to sense the MMP activity, but extracellularly long-term monitoring of MMP activity still remains challenging. Here, we report a collagen-bound fluorescent bioprobe for the detection of MMP-2 activity in the extracellular environment. This bioprobe consists of ECM-immobilized part (including collagen-bound protein) and MMP-sensing part (including peptide substrate linked with fluorescence resonance energy transfer (FRET) coupler between donor green fluorescent protein (GFP) and acceptor TAMRA dye), which was constructed through intein-mediated self-splicing conjugation. Upon being immobilized on the collagen-coated surface, this bioprobe enabled efficient long-lasting observation of MMP-2 activity in the cultured cells without affecting cell growth and viability. As a result, the FRET ratio (acceptor/donor) decreased as the MMP2 activity increased in cultured cancer cells. Furthermore, unlike wild-type MMP-2, mutated MMP-2 expression (Y580A in the hemopexin region) gave rise to lowering the secretion of MMP-2 in HeLa. Conclusively, our method is anticipated to find applications for tracing and visualizing enzyme activity.

Keywords: collagen, ECM, FRET, MMP

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2829 Ultrastructural Characterization of Lipid Droplets of Rat Hepatocytes after Whole Body 60-Cobalt Gamma Radiation

Authors: Ivna Mororó, Lise P. Labéjof, Stephanie Ribeiro, Kely Almeida

Abstract:

Lipid droplets (LDs) are normally presented in greater or lesser number in the cytoplasm of almost all eukaryotic and some prokaryotic cells. They are independent organelles composed of a lipid ester core and a surface phospholipid monolayer. As a lipid storage form, they provide an available source of energy for the cell. Recently it was demonstrated that they play an important role in other many cellular processes. Among the many unresolved questions about them, it is not even known how LDs is formed, how lipids are recruited to LDs and how they interact with the other organelles. Excess fat in the organism is pathological and often associated with the development of some genetic, hormonal or behavioral diseases. The formation and accumulation of lipid droplets in the cytoplasm can be increased by exogenous physical or chemical agents. It is well known that ionizing radiation affects lipid metabolism resulting in increased lipogenesis in cells, but the details of this process are unknown. To better understand the mode of formation of LDs in liver cells, we investigate their ultrastructural morphology after irradiation. For that, Wistar rats were exposed to whole body gamma radiation from 60-cobalt at various single doses. Samples of the livers were processed for analysis under a conventional transmission electron microscope. We found that when compared to controls, morphological changes in liver cells were evident at the higher doses of radiation used. It was detected a great number of lipid droplets of different sizes and homogeneous content and some of them merged each other. In some cells, it was observed diffused LDs, not limited by a monolayer of phospholipids. This finding suggests that the phospholipid monolayer of the LDs was disrupted by ionizing radiation exposure that promotes lipid peroxydation of endo membranes. Thus the absence of the phospholipid monolayer may prevent the realization of some cellular activities as follow: - lipid exocytosis which requires the merging of LDs membrane with the plasma membrane; - the interaction of LDs with other membrane-bound organelles such as the endoplasmic reticulum (ER), the golgi and mitochondria and; - lipolysis of lipid esters contained in the LDs which requires the presence of enzymes located in membrane-bound organelles as ER. All these impediments can contribute to lipid accumulation in the cytoplasm and the development of diseases such as liver steatosis, cirrhosis and cancer.

Keywords: radiobiology, hepatocytes, lipid metabolism, transmission electron microscopy

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2828 Prevalence of Anemia and Iron Deficiency in Women of Childbearing Age in the North-West of Libya

Authors: Mustafa Ali Abugila, Basma Nuri Kajruba, Hanan Elhadi, Rehab Ramadan Wali

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Iron deficiency anemia is characterized by a decrease of Hb (hemoglobin), serum iron, ferritin, and RBC (red blood cells) (shape and size). Also, it is characterized by an increase in total iron binding capacity (TIBC). Red blood cells become microctytic and hypochromic due to a decrease in iron content. This study was conducted in the north west of Libya and included 210 women in childbearing age (18-45 years) who were visiting women clinic. After filling the questionnaire, blood samples were taken and analyzed for hematological and biochemical profiles. Biochemical tests included measurement of serum iron, ferritin, and total iron binding capacity (TIBC). Among the total sample (210 women), there were 87 (41.42%) pregnant and 123 (58.57%) non-pregnant women (includes married and single). Pregnant women (87) were classified according to the gestational age into first, second, and third trimesters. The means of biochemical and hematological parameters in the studied samples were: Hb = 10.37± 2.02 g/dl, RBC = 3.78± 1.037 m/m3, serum iron 61.86± 40.28 µg/dl, and TIBC = 386.01 ± 94.91 µg/dl. In this study, we considered that any women have hemoglobin below 11.5 g/dl is anemic. 89.1%, 69.5%, and 47.8% of pregnant women who belong to third trimester had low (below normal value) Hb, serum iron, and ferritin, i.e. iron deficiency anemia was more common in third trimester among the first and the second trimesters. Third trimester pregnant women also had high TIBC more than first and second trimesters.

Keywords: red blood cells, hemoglobin, total iron binding capacity, ferritin

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2827 Sesamol Decreases Melanin Biosynthesis via Melanogenesis-Related Gene Expressions in Melan-a Cells

Authors: Seung-Hwa Baek, In-Jung Nam, Sang-Han Lee

Abstract:

The development of anti-melanogenic agents is important for the prevention of serious esthetic problem like a melasma, freckle, age spots, and chloasma. The aim of this study was to investigate the anti-melanogenic effect of sesamol, an active lignan isolated from sesame seed, by mushroom and cellular tyrosinase assay, melanin content and the analysis of melanogensis-related mRNA expressions in melana cells. Sesamol showed strong inhibitory activity against the mushroom tyrosinase in a dose-dependent manner. Intracellular tyrosinase inhibition activity was also confirmed by zymography. At a concentration of 50 μM, sesamol inhibited melanin production in melan-a cells with no cytoxicity while those of phenylthiourea (PTU) as a positive control were the same condition. Sesamol significantly inhibited the expression of melanogensis-related genes, such as tyrosinase, tyrosinase-related protein-1 (TRP-1), dopachrome tautomerase (Dct), microphthalmia-associated transcription factor (MITF) and melanocortin 1 receptor (MC1R). These findings indicate that sesamol could reduce melanin biosynthesis via the downregulation of tyrosinase activity and melanin production via subsequent gene expression of melanogenesis-related proteins. Together, these results suggest that the sesamol have strong potential in inhibiting melanin biosynthesis, in that the substance may be used as a new skin-whitening agent of cosmetic materials.

Keywords: sesamol, sesame seed, melanin biosynthesis, melanogenesis-related gene, skin-whitening agent

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2826 Biocontrol of Fusarium Crown and Root Rot and Enhancement of Tomato Solanum lycopersicum L. Growth Using Solanum linnaeanum L. Extracts

Authors: Ahlem Nefzi, Rania Aydi Ben Abdallah, Hayfa Jabnoun-Khiareddine, Nawaim Ammar, Sined Medimagh-Saidana, Mejda Daami-Remadi

Abstract:

In the present study, leaf, stem, and fruit aqueous extracts of native wild Solanum linnaeanum L. were screened for their ability to suppress Fusarium Crown and Root Rot disease and to enhance tomato (Solanum lycopersicum L.) growth under greenhouse conditions. Leaf extract used at 30% w/v was the most effective in reducing leaf and root damage index by 92.3% and the extent of vascular discoloration by 97.56% compared to Fusarium oxyxporum f. sp radicis lycopersici -inoculated and untreated control. A significant promotion of growth parameters (root length, shoot height, root and shoot biomass and stem diameter) was recorded on tomato cv. Rio Grande seedlings by 40.3-94.1% as compared to FORL inoculated control and by 9.6-88.8% over pathogen-free control. All S. linnaeanum aqueous extracts tested significantly stimulated the germination by 10.2 to 80.1% relative to the untreated control. FORL mycelial growth, assessed using the poisoned food technique, varied depending on plant organs, extracts, and concentrations used. Butanolic extracts were the most active, leading to 60.81% decrease in FORL mycelial growth. HPLC analysis of butanolic extract revealed the presence of thirteen phenolic compounds. Thus, S. linnaeanum can be explored as a potential natural source of antifungal and biofertilizing compounds.

Keywords: antifungal activity, HPLC-MS analysis, Fusarium oxysporum f. sp. radicis-lycopersici, tomato growth

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2825 Investigation of Cytotoxic Compounds in Ethyl Acetate and Chloroform Extracts of Nigella sativa Seeds by Sulforhodamine-B Assay-Guided Fractionation

Authors: Harshani Uggallage, Kapila D. Dissanayaka

Abstract:

A Sulforhodamine-B assay-guided fractionation on Nigella sativa seeds was conducted to determine the presence of cytotoxic compounds against human hepatoma (HepG2) cells. Initially, a freeze-dried sample of Nigella sativa seeds was sequentially extracted into solvents of increasing polarities. Crude extracts from the sequential extraction of Nigella sativa seeds in chloroform and ethyl acetate showed the highest cytotoxicity. The combined mixture of these two extracts was subjected to bioassay guided fractionation using a modified Kupchan method of partitioning, followed by Sephadex® LH-20 chromatography. This chromatographic separation process resulted in a column fraction with a convincing IC50 (half-maximal inhibitory concentration) value of 13.07µg/ml, which is considerable for developing therapeutic drug leads against human hepatoma. Reversed phase High-Performance Liquid Chromatography (HPLC) was finally conducted for the same column fraction, and the result indicates the presence of one or several main cytotoxic compounds against human HepG2 cells.

Keywords: cytotoxic compounds, half-maximal inhibitory concentration, high-performance liquid chromatography, human HepG2 cells, nigella sativa seeds, Sulforhodamine-B assay

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2824 ORR Electrocatalyst for Batteries and Fuel Cells Development with SIO₂/Carbon Black Based Composite Nanomaterials

Authors: Maryam Kiani

Abstract:

This study focuses on the development of composite nanomaterials based on SiO₂ and carbon black for oxygen reduction reaction (ORR) electrocatalysts in batteries and fuel cells. The aim was to explore the potential of these composite materials as efficient catalysts for ORR, which is a critical process in energy conversion devices. The SiO₂/carbon black composite nanomaterials were synthesized using a facile and scalable method. The morphology, structure, and electrochemical properties of the materials were characterized using various techniques including scanning electron microscopy (SEM), X-ray diffraction (XRD), and electrochemical measurements. The results demonstrated that the incorporation of SiO₂ into the carbon black matrix enhanced the ORR performance of the composite material. The composite nanomaterials exhibited improved electrocatalytic activity, enhanced stability, and increased durability compared to pure carbon black. The presence of SiO₂ facilitated the formation of active sites, improved electron transfer, and increased the surface area available for ORR. This study contributes to the advancement of battery and fuel cell technology by offering a promising approach for the development of high-performance ORR electrocatalysts. The SiO₂/carbon black composite nanomaterials show great potential for improving the efficiency and durability of energy conversion devices, leading to more sustainable and efficient energy solutions.

Keywords: ORR, fuel cells, batteries, electrocatalyst

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2823 Cationic Solid Lipid Nanoparticles Conjugated with Anti-Melantransferrin and Apolipoprotein E for Delivering Doxorubicin to U87MG Cells

Authors: Yung-Chih Kuo, Yung-I Lou

Abstract:

Cationic solid lipid nanoparticles (CSLNs) with anti-melanotransferrin (AMT) and apolipoprotein E (ApoE) were used to carry antimitotic doxorubicin (Dox) across the blood–brain barrier (BBB) for glioblastoma multiforme (GBM) treatment. Dox-loaded CSLNs were prepared in microemulsion, grafted covalently with AMT and ApoE, and applied to human brain microvascular endothelial cells (HBMECs), human astrocytes, and U87MG cells. Experimental results revealed that an increase in the weight percentage of stearyl amine (SA) from 0% to 20% increased the size of AMT-ApoE-Dox-CSLNs. In addition, an increase in the stirring rate from 150 rpm to 450 rpm decreased the size of AMT-ApoE-Dox-CSLNs. An increase in the weight percentage of SA from 0% to 20% enhanced the zeta potential of AMT-ApoE-Dox-CSLNs. Moreover, an increase in the stirring rate from 150 rpm to 450 rpm reduced the zeta potential of AMT-ApoE-Dox-CSLNs. AMT-ApoE-Dox-CSLNs exhibited a spheroid-like geometry, a minor irregular boundary deviating from spheroid, and a somewhat distorted surface with a few zigzags and sharp angles. The encapsulation efficiency of Dox in CSLNs decreased with increasing weight percentage of Dox and the order in the encapsulation efficiency of Dox was 10% SA > 20% SA > 0% SA. However, the reverse order was true for the release rate of Dox, suggesting that AMT-ApoE-Dox-CSLNs containing 10% SA had better-sustained release characteristics. An increase in the concentration of AMT from 2.5 to 7.5 μg/mL slightly decreased the grafting efficiency of AMT and an increase in that from 7.5 to 10 μg/mL significantly decreased the grafting efficiency. Furthermore, an increase in the concentration of ApoE from 2.5 to 5 μg/mL slightly reduced the grafting efficiency of ApoE and an increase in that from 5 to 10 μg/mL significantly reduced the grafting efficiency. Also, AMT-ApoE-Dox-CSLNs at 10 μg/mL of ApoE could slightly reduce the transendothelial electrical resistance (TEER) and increase the permeability of propidium iodide (PI). An incorporation of 10 μg/mL of ApoE could reduce the TEER and increase the permeability of PI. AMT-ApoE-Dox-CSLNs at 10 μg/mL of AMT and 5-10 μg/mL of ApoE could significantly enhance the permeability of Dox across the BBB. AMT-ApoE-Dox-CSLNs did not induce serious cytotoxicity to HBMECs. The viability of HBMECs was in the following order: AMT-ApoE-Dox-CSLNs = AMT-Dox-CSLNs = Dox-CSLNs > Dox. The order in the efficacy of inhibiting U87MG cells was AMT-ApoE-Dox-CSLNs > AMT-Dox-CSLNs > Dox-CSLNs > Dox. A surface modification of AMT and ApoE could promote the delivery of AMT-ApoE-Dox-CSLNs to cross the BBB via melanotransferrin and low density lipoprotein receptor. Thus, AMT-ApoE-Dox-CSLNs have appropriate physicochemical properties and can be a potential colloidal delivery system for brain tumor chemotherapy.

Keywords: anti-melanotransferrin, apolipoprotein E, cationic catanionic solid lipid nanoparticle, doxorubicin, U87MG cells

Procedia PDF Downloads 278
2822 Study on Surface Morphology and Reflectance of Solar Cells Applied in Pyramid Structures

Authors: Zong-Sheng Chen

Abstract:

With the advancement of technology, human activities have increased greenhouse gas emissions and fossil fuel energy production, leading to increasingly severe global warming. To mitigate global warming, energy conservation and carbon reduction have become global goals. Solar energy, a renewable energy source, not only helps achieve energy conservation and carbon reduction but also serves as an efficient energy generation method. Solar energy, derived from sunlight, is an endless and promising energy source capable of meeting high energy demands sustainably. In recent years, many countries around the world have been developing the solar energy industry, and Taiwan is no exception. Positioned in the subtropical region, Taiwan possesses geographical advantages conducive to solar energy utilization. Furthermore, Taiwan's well-developed semiconductor technology and sophisticated equipment make it highly suitable for the development of high-efficiency solar cells. This study focuses on investigating the anti-reflection properties of solar cells. Through metal-assisted chemical etching, pyramid structures are etched to allow sunlight to pass through, achieving secondary or higher-order reflections on the surface of these structures. This trapping of light within the substrate reduces reflection rates and increases conversion efficiency.

Keywords: solar cell, reflectance, pyramidal structure, potassium hydroxide

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2821 Variation in Wood Anatomical Properties of Acacia seyal var. seyal Tree Species Growing in Different Zones in Sudan

Authors: Hanadi Mohamed Shawgi Gamal, Ashraf Mohamed Ahmed Abdalla

Abstract:

Sudan is endowed by a great diversity of tree species; nevertheless, the utilization of wood resources has traditionally concentrated on a few number of species. With the great variation in the climatic zones of Sudan, great variations are expected in the anatomical properties between and within species. This variation needs to be fully explored in order to suggest the best uses for the species. Modern research on wood has substantiated that the climatic condition where the species grow has significant effect on wood properties. Understanding the extent of variability of wood is important because the uses for each kind of wood are related to its characteristics; furthermore, the suitability or quality of wood for a particular purpose is determined by the variability of one or more of these characteristics. The present study demonstrates the effect of rainfall zones in some anatomical properties of Acacia seyal var. seyal growing in Sudan. For this purpose, twenty healthy trees were collected randomly from two zones (ten trees per zone). One zone with relatively low rainfall (273mm annually) which represented by North Kordofan state and White Nile state and the second with relatively high rainfall (701 mm annually) represented by Blue Nile state and South Kordofan state. From each sampled tree, a stem disc (3 cm thick) was cut at 10% from stem height. One radius was obtained in central stem dices. Two representative samples were taken from each disc, one at 10% distance from pith to bark, the second at 90% in order to represent the juvenile and mature wood. The investigated anatomical properties were fibers length, fibers and vessels diameter, lumen diameter, and wall thickness as well as cell proportions. The result of the current study reveals significant differences between zones in mature wood vessels diameter and wall thickness, as well as juvenile wood vessels, wall thickness. The higher values were detected in the drier zone. Significant differences were also observed in juvenile wood fiber length, diameter as well as wall thickness. Contrary to vessels diameter and wall thickness, the fiber length, diameter as well as wall thickness were decreased in the drier zone. No significant differences have been detected in cell proportions of juvenile and mature wood. The significant differences in some fiber and vessels dimension lead to expect significant differences in wood density. From these results, Acacia seyal var. seyal seems to be well adapted with the change in rainfall and may survive in any rainfall zone.

Keywords: Acacia seyal var. seyal, anatomical properties, rainfall zones, variation

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2820 Development and Evaluation of a Gut-Brain Axis Chip Based on 3D Printing Interconnecting Microchannel Scaffolds

Authors: Zhuohan Li, Jing Yang, Yaoyuan Cui

Abstract:

The gut-brain axis (GBA), a communication network between gut microbiota and the brain, benefits for investigation of brain diseases. Currently, organ chips are considered one of the potential tools for GBA research. However, most of the available GBA chips have limitations in replicating the three-dimensional (3D) growth environment of cells and lack the required cell types for barrier function. In the present study, a microfluidic chip was developed for GBA interaction. Blood-brain barrier (BBB) module was prepared with HBMEC, HBVP, U87 cells and decellularized matrix (dECM). Intestinal epithelial barrier (IEB) was prepared with Caco-2 and vascular endothelial cells and dECM. GBA microfluidic device was integrated with IEB and BBB modules using 3D printing interconnecting microchannel scaffolds. BBB and IEB interaction on this GBA chip were evaluated with lipopolysaccharide (LPS) exposure. The present GBA chip achieved multicellular three-dimensional cultivation. Compared with the co-culture cell model in the transwell, fluorescein was absorbed more slowly by 5.16-fold (IEB module) and 4.69-fold (BBB module) on the GBA chip. Accumulation of Rhodamine 123 and Hoechst33342 was dramatically decreased. The efflux function of transporters on IEB and BBB was significantly increased on the GBA chip. After lipopolysaccharide (LPS) disrupted the IEB, and then BBB dysfunction was further observed, which confirmed the interaction between IEB and BBB modules. These results demonstrated that this GBA chip may offer a promising tool for gut-brain interaction study.

Keywords: decellularized matrix, gut-brain axis, organ-on-chip, three-dimensional printing.

Procedia PDF Downloads 27
2819 Toward Understanding the Glucocorticoid Receptor Network in Cancer

Authors: Swati Srivastava, Mattia Lauriola, Yuval Gilad, Adi Kimchi, Yosef Yarden

Abstract:

The glucocorticoid receptor (GR) has been proposed to play important, but incompletely understood roles in cancer. Glucocorticoids (GCs) are widely used as co-medication of various carcinomas, due to their ability to reduce the toxicity of chemotherapy. Furthermore, GR antagonism has proven to be a strategy to treat triple negative breast cancer and castration-resistant prostate cancer. These observations suggest differential GR involvement in cancer subtypes. The goal of our study has been to elaborate the current understanding of GR signaling in tumor progression and metastasis. Our study involves two cellular models, non-tumorigenic breast epithelial cells (MCF10A) and Ewing sarcoma cells (CHLA9). In our breast cell model, the results indicated that the GR agonist dexamethasone inhibits EGF-induced mammary cell migration, and this effect was blocked when cells were stimulated with a GR antagonist, namely RU486. Microarray analysis for gene expression revealed that the mechanism underlying inhibition involves dexamenthasone-mediated repression of well-known activators of EGFR signaling, alongside with enhancement of several EGFR’s negative feedback loops. Because GR mainly acts primarily through composite response elements (GREs), or via a tethering mechanism, our next aim has been to find the transcription factors (TFs) which can interact with GR in MCF10A cells.The TF-binding motif overrepresented at the promoter of dexamethasone-regulated genes was predicted by using bioinformatics. To validate the prediction, we performed high-throughput Protein Complementation Assays (PCA). For this, we utilized the Gaussia Luciferase PCA strategy, which enabled analysis of protein-protein interactions between GR and predicted TFs of mammary cells. A library comprising both nuclear receptors (estrogen receptor, mineralocorticoid receptor, GR) and TFs was fused to fragments of GLuc, namely GLuc(1)-X, X-GLuc(1), and X-GLuc(2), where GLuc(1) and GLuc(2) correspond to the N-terminal and C-terminal fragments of the luciferase gene.The resulting library was screened, in human embryonic kidney 293T (HEK293T) cells, for all possible interactions between nuclear receptors and TFs. By screening all of the combinations between TFs and nuclear receptors, we identified several positive interactions, which were strengthened in response to dexamethasone and abolished in response to RU486. Furthermore, the interactions between GR and the candidate TFs were validated by co-immunoprecipitation in MCF10A and in CHLA9 cells. Currently, the roles played by the uncovered interactions are being evaluated in various cellular processes, such as cellular proliferation, migration, and invasion. In conclusion, our assay provides an unbiased network analysis between nuclear receptors and other TFs, which can lead to important insights into transcriptional regulation by nuclear receptors in various diseases, in this case of cancer.

Keywords: epidermal growth factor, glucocorticoid receptor, protein complementation assay, transcription factor

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2818 Investigation of Water Transport Dynamics in Polymer Electrolyte Membrane Fuel Cells Based on a Gas Diffusion Media Layers

Authors: Saad S. Alrwashdeh, Henning Markötter, Handri Ammari, Jan Haußmann, Tobias Arlt, Joachim Scholta, Ingo Manke

Abstract:

In this investigation, synchrotron X-ray imaging is used to study water transport inside polymer electrolyte membrane fuel cells. Two measurement techniques are used, namely in-situ radiography and quasi-in-situ tomography combining together in order to reveal the relationship between the structures of the microporous layers (MPLs) and the gas diffusion layers (GDLs), the operation temperature and the water flow. The developed cell is equipped with a thick GDL and a high back pressure MPL. It is found that these modifications strongly influence the overall water transport in the whole adjacent GDM.

Keywords: polymer electrolyte membrane fuel cell, microporous layer, water transport, radiography, tomography

Procedia PDF Downloads 173
2817 Additive Carbon Dots Nanocrystals for Enhancement of the Efficiency of Dye-Sensitized Solar Cell in Energy Applications Technology

Authors: Getachew Kuma Watiro

Abstract:

The need for solar energy is constantly increasing and it is widely available on the earth’s surface. Photovoltaic technology is one of the most capable of all viable energy technology and is seen as a promising approach to the control era as it is readily available and has zero carbon emissions. Inexpensive and versatile solar cells have achieved the conversion efficiency and long life of dye-sensitized solar cells, improving the conversion efficiency from the sun to electricity. DSSCs have received a lot of attention for Various potential commercial uses, such as mobile devices and portable electronic devices, as well as integrated solar cell modules. The systematic reviews were used to show the critical impact of additive C-dots in the Dye-Sensitized solar cell for energy application technology. This research focuses on the following methods to synthesize nanoparticles such as facile, polyol, calcination, and hydrothermal technique. In addition to these, there are additives C-dots by the Hydrothermal method. This study deals with the progressive development of DSSC in photovoltaic technology. The applications of single and heterojunction structure technology devices were used (ZnO, NiO, SnO2, and NiO/ZnO/N719) and applied some additives C-dots (ZnO/C-dots /N719, NiO/C-dots /N719, SnO2 /C-dots /N719 and NiO/ZnO/C-dots/N719) and the effects of C-dots were reviewed. More than all, the technology of DSSC with C-dots enhances efficiency. Finally, recommendations have been made for future research on the application of DSSC with the use of these additives.

Keywords: dye-sensitized solar cells, heterojunction’s structure, carbon dot, conversion efficiency

Procedia PDF Downloads 115
2816 Expression of Fused Plasmodium falciparum Orotate Phosphoribosyltransferase and Orotidine 5'-Monophosphate Decarboxylase in Escherichia coli

Authors: Waranya Imprasittichai, Patsarawadee Paojinda, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

Abstract:

Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. In this study, we constructed gene fusions of Plasmodium falciparum OMPDC-OPRT (1,836 bp) in pTrcHisA vector and expressed as an 6xHis-tag bifunctional protein in three Escherichia coli strains (BL21, Rosetta, TOP10) at 18 °C, 25 °C and 37 °C. The recombinant bifunctional protein was partially purified by Ni-Nitrilotriacetic acid-affinity chromatography. Specific activities of OPRT and OMPDC domains in the bifunctional enzyme expressed in E. coli TOP10 cells were approximately 3-4-fold higher than those in BL21 cells. There were no enzymatic activities when the construct vector expressed in Rosetta cells. Maximal expression of the fused gene was observed at 18 °C and the bifunctional enzyme had specific activities of OPRT and OMPDC domains in a ratio of 1:2. These results provide greater yields and better catalytic activities of the bifunctional OMPDC-OPRT enzyme for further purification and kinetic study.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

Procedia PDF Downloads 351