Search results for: lymphocyte cell line

Authors: Fui Ping Lim, Wen Choong Chua, Toan Thang Phan

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Aim: This study investigates the roles of Cord Lining Stem Cells (CLSCs) as potential therapeutic agents for diabetic wounds. Method: 20 genetically diabetic db/db mice were randomly assigned to two arms; (i) control group received placebo treatment (sham media or cells delivery material), and (ii) active comparator received CLSCs. Two full-thickness wounds, each sized 10mm X 10mm were created, one on each side of the midline on the back of the mice. Digital pictures were taken on day 1, 3, 7, 10, 14, 17, 21, 24, 28. Wound areas were analyzed with ImageJ TM software and calculated as percentage of the original wound. Time to closure was defined as the day the wound bed was completely epithelized and filled with new tissues. Results: The CLSCs-treated wounds, showed a significant increase in the percentage of wound closure and achieved 100% closure of the wound sooner than the control group by an average of 3.7 days. The mice treated with CLSCs have a shorter wound closure time (mean closure day: 19.8 days) as compared to the control group (mean closure day: 23.5 days). Conclusion: Our preliminary findings inferred that CLSCs treated wound achieved higher percentage of wound closure within a shorter duration of time.

Keywords: cord lining stem cell, diabetic wound, stem cell, wound

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Authors: Jer Ping Ooi, Shah Rizal Bin Kasim, Nor Aini Saidin

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The objective of this study was to synthesize nano-sized β-tricalcium phosphate (β-TCP) powder and assess its cytotoxic effects on human osteoblast (hFOB1.19) by using four cytotoxicity assays, namely, lactose dehydrogenase (LDHe), tetrazolium hydroxide (XTT), neutral red (NR), and sulforhodamine B (SRB) assays. β-tricalcium phosphate (β-TCP) is a calcium phosphate compound commonly used as an implant material. To date, bulk-sized β-TCP is reported to be readily tolerated by the osteogenic cells and body based on in vitro, in vivo experiments and clinical studies. However, to what extent of nano-sized β-TCP will react in models as compared to bulk β-TCP is yet to be investigated. Thus, in this project, the cells were treated with nano β-TCP powder within a range of concentrations from 0 to 1000 μg/mL for 24, 48, and 72 h. The cytotoxicity tests showed that loss of cell viability ( > 50%) was high for hFOB1.19 cells in all assays. Cell cycle and apoptosis analysis of hFOB1.19 cells revealed that 50 μg/mL of the compound led to 30.5% of cells being apoptotic after 72 h of incubation, and the percentage was increased to 58.6% when the concentration was increased to 200 μg/mL. When the incubation time was increased from 24 to 72 h, the percentage of apoptotic cells increased from 17.3% to 58.6% when the hFOB1.19 were exposed with 200 μg/mL of nano β-TCP powder. Thus, both concentration and exposure duration affected the cytotoxicity effects of the nano β-TCP powder on hFOB1.19. We hypothesize that these cytotoxic effects on hFOB1.19 are related to the nano-scale size of the β-TCP.

Keywords: β-tricalcium phosphate, hFOB1.19, adipose-derived mesenchymal stem cells, cytotoxicity

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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

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Authors: Onur Kaya, Ilknur Tukenmez

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In this study, we consider vehicle routing problems on networks with alternative direct links between nodes, and we analyze a multi-objective problem considering the financial, environmental and social objectives in this context. In real life, there might exist several alternative direct roads between two nodes, and these roads might have differences in terms of their lengths and durations. For example, a road might be shorter than another but might require longer time due to traffic and speed limits. Similarly, some toll roads might be shorter or faster but require additional payment, leading to higher costs. We consider such alternative links in our problem and develop a mixed integer linear programming model that determines which alternative link to use between two nodes, in addition to determining the optimal routes for different vehicles, depending on the model objectives and constraints. We consider the minimum cost routing as the financial objective for the company, minimizing the CO2 emissions and gas usage as the environmental objectives, and optimizing the driver working conditions/working hours, and minimizing the risks of accidents as the social objectives. With these objective functions, we aim to determine which routes, and which alternative links should be used in addition to the speed choices on each link. We discuss the results of the developed vehicle routing models and compare their results depending on the system parameters.

Keywords: vehicle routing, alternative links between nodes, mixed integer linear programming, triple bottom line accounting

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Authors: Bhavini Patel, Aslihan Ugun-Klusek, Ellen Billet

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The two main contributing factors to familial and idiopathic form of Parkinson’s disease (PD) are oxidative stress and altered proteolysis. Monoamine oxidase-A (MAO-A) plays a significant role in redox homeostasis by producing reactive oxygen species (ROS) via deamination of for example, dopamine. The ROS generated induces chemical modification of proteins resulting in altered biological function. The ubiquitin-proteasome system, which consists of three different types or proteolytic activity, namely “chymotrypsin-like” activity (CLA), “trypsin-like” activity (TLA) and “post acidic-like” activity (PLA), is responsible for the degradation of ubiquitinated proteins. Defects in UPS are known to be strongly correlated to PD. Herein, the effect of ROS generated by MAO-A on proteasome activity and the effects of proteasome inhibition on MAO-A protein levels in WT, mock and MAO-A overexpressed (MAO-A+) SHSY5Y neuroblastoma cell lines were investigated. The data in this study report increased proteolytic activity when MAO-A protein levels are significantly increased, in particular CLA and PLA. Additionally, 20S proteasome inhibition induced a decrease in MAO-A levels in WT and mock cells in comparison to MAO-A+ cells in which 20S proteasome inhibition induced increased MAO-A levels to be further increased at 48 hours of inhibition. This study supports the fact that MAO-A could be a potential pharmaceutical target for neuronal protection as data suggests that endogenous MAO-A levels may be essential for modulating cell death and survival.

Keywords: monoamine oxidase, neurodegeneration, Parkinson's disease, proteasome

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Authors: Ferreira RC, Simons HZ, Thompson WS, Cutler AJ, Dopico XC, Smyth DJ, Mashar M, Schuilenburg H, Walker NM, Dunger DB, Wallace C, Todd JA, Wicker LS, Pekalski ML

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Type 1 diabetes is caused by autoimmune destruction of insulin-secreting beta cells in the pancreas. T cells are known to play an important role in this immune-mediated destruction; however, there is no general consensus regarding alterations in cytokine production or T cell subsets in peripheral blood of patients with type 1 diabetes. Using polychromatic flow cytometry of peripheral blood mononuclear cells (PBMCs), we assessed production of the proinflammatory cytokines IL-21, IFN-γ and IL-17 by memory CD4 T effector (Teff) cells in 69 patients with type 1 diabetes and 61 healthy donors. We found a 21.9% (95% CI 5.8, 40.2; p = 3.9 × 10(-3)) higher frequency of IL-21(+) CD45RA(-) memory CD4(+) Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). In a separate cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. Consistent with the increased production of IL-21, we also found a 14.9% increase in circulating Tfh cells in the patients with type 1 diabetes (95% CI 2.9, 26.9; p = 0.016). Analysis of IL-21 production by PBMCs from a subset of 46 of the 62 donors immunophenotyped for Tfh showed that frequency of Tfh cells was associated with the frequency of IL-21+ cells (r2 = 0.174, p = 0.004). These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.

Keywords: T follicular helper cell, IL-21, IL-17, type 1 diabetes

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Authors: Ashit Syngle, Inderjit Verma, Pawan Krishan

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Aim: Endothelial progenitor cells (EPCs) are unique populations which have reparative potential in overcoming the endothelial damage and reducing cardiovascular risk. Patients with ankylosing spondylitis (AS) have increased risk of cardiovascular morbidity and mortality. The aim of this study was to investigate the endothelial progenitor cell population in AS patients and its potential relationships with disease variables. Methods: Endothelial progenitor cells were measured in peripheral blood samples from 20 AS and 20 healthy controls by flow cytometry on the basis of CD34 and CD133 expression. Disease activity was evaluated by using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Functional ability was monitored by using Bath Ankylosing Spondylitis Functional Index (BASFI). Results: EPCs were depleted in AS patients as compared to the healthy controls (CD34+/CD133+: 0.027 ± 0.010 % vs. 0.044 ± 0.011 %, p<0.001). EPCs depletion were significantly associated with disease duration (r=-0.52, p=0.01) and BASDAI (r=-0.45, p=0.04). Conclusion: This is the first study to demonstrate endothelial progenitor cells depletion in AS patients. EPCs depletion inversely correlates with disease duration and disease activity, suggesting the pivotal role of inflammation in depletion of EPCs. EPC would possibly also serve as a therapeutic target for preventing cardiovascular disease in AS.

Keywords: ankylosing spondylitis, endothelial progenitor cells, inflammation, vascular damage

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Authors: P. N. Salini, B. Anish Kini, R. Ashalatha

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At signalized intersections with heterogeneous traffic, the percentage share of different vehicle categories have a bearing on the inter-vehicle space utilization, which eventually impacts the saturation flow. This paper analyzed the impact of the percentage share of various vehicle categories in the traffic stream on the saturation flow at signalized intersections by video graphing major intersections with varying geometry in Kerala, India. It was found that as the percentage share of two-wheelers increases, the saturation flow at signalized intersections increases and vice-versa for the percentage share of cars. The effect of bus blockage and parking maneuvers on the saturation flow were also studied. As the distance of bus blockage increases from the stop line, the effect on the saturation flow decreases, while with more buses stopping at the same bus stop, the saturation flow reduces further. The study revealed that with higher kerbside parking maneuvers on the upstream, the saturation flow reduces, and with an increase in the distance of the parking maneuver from the stop line, the effect on the saturation flow decreases. The adjustment factors for bus blockage due to bus stops within 75m downstream and parking maneuvers within 75m upstream of the intersection have been established for mixed traffic conditions. These adjustment factors could empower the urban planners, enforcement personnel and decision-makers to estimate the reduction in the capacity of signalized intersections for suggesting improvements in the form of parking restrictions/ bus stop relocation for existing intersections or make design changes for planned intersections.

Keywords: signalized intersection, saturation flow, adjustment factors, capacity

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Authors: Michael P. Mannino, Gerald W. Hart

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The majority of glucose metabolism proceeds through glycolytic pathways such as glycolysis or pentose phosphate pathway, however, about 5% is shunted through the hexosamine biosynthetic pathway, producing uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). This precursor can then be incorporated into complex oligosaccharides decorating the cell surface or remain as an intracellular post-translational-modification (PTM) of serine/threonine residues (O-GlcNAcylation, OGN), which has been identified on over 4,000 cytosolic or nuclear proteins. Intracellular OGN has major implications on cellularprocesses, typically by modulating protein localization, protein-protein interactions, protein degradation, and gene expression. Additionally, OGN is known to have an extensive cross-talk with phosphorylation, be in a competitive or cooperative manner. Unlike other PTMs there are only two cycling enzymes that are capable of adding or removing the GlcNAc moiety, O-linked N-aceytl glucosamine Transferase (OGT) and O-linked N-acetyl glucoamidase (OGA), respectively. The activity of OGT has been shown to be sensitive to cellular UDP-GlcNAc levels, even changing substrate affinity. Owing to this and that the concentration of UDP-GlcNAc is related to the metabolisms of glucose, amino acid, fatty acid, and nucleotides, O-GlcNAc is often referred to as a nutrient sensing rheostat. Indeed OGN is known to regulate several signaling pathways as a result of nutrient levels, such as insulin signaling. Dysregulation of OGN is associated with several disease states such as cancer, diabetes, and neurodegeneration. Improvements in glycomics over the past 10-15 years has significantly increased the OGT substrate pool, suggesting O-GlcNAc’s involvement in a wide variety of signaling pathways. However, O-GlcNAc’s role at the receptor level has only been identified in a case-by-case basis of known pathways. Examining the OGN of the plasma membrane (PM) may better focus our understanding of O-GlcNAc-effected signaling pathways. In this current study, PM fractions were isolated from several cell types via ultracentrifugation, followed by purification and MS/MS analysis in several cell lines. This process was repeated with or without OGT/OGA inhibitors or with increased/decreased glucose levels in media to ascertain the importance of OGN. Various pathways are followed up on in more detailed studies employing methods to localize OGN at the PM specifically.

Keywords: GlcNAc, nutrient sensitive, post-translational-modification, receptor

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Authors: Paua Ketan, Bhagate Rajkumar, Adiga Ganesh, M. Kiran

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Low gear noise is an important sound quality feature in modern passenger cars. Annoying gear noise from the gearbox is influenced by the gear design, gearbox shaft layout, manufacturing deviations in the components, assembly errors and the mounting arrangement of the complete gearbox. Geometrical deviations in the form of profile and lead errors are often present on the flanks of the inspected gears. Ghost frequencies of a gear are very challenging to identify in standard gear measurement and analysis process due to small wavelengths involved. In this paper, gear whine noise occurring at non-integral multiples of gear mesh frequency of passenger car gearbox is investigated and the root cause is identified using the displacement deviation analysis (DDA) method. DDA method is applied to identify ghost frequency excitations on the flanks of gears arising out of generation grinding. Frequency identified through DDA correlated with the frequency of vibration and noise on the end-of-line machine as well as vehicle level measurements. With the application of DDA method along with standard lead profile measurement, gears with ghost frequency geometry deviations were identified on the production line to eliminate defective parts and thereby eliminate ghost frequency noise from a vehicle. Further, displacement deviation analysis can be used in conjunction with the manufacturing process simulation to arrive at suitable countermeasures for arresting the ghost frequency.

Keywords: displacement deviation analysis, gear whine, ghost frequency, sound quality

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Authors: Sirus Mohammadi, Rohala Moghimi

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The main advantages of microgrids are high energy efficiency through the application of Combined Heat and Power (CHP), high quality and reliability of the delivered electric energy and environmental and economic advantages. This study presents an energy management system (EMS) to optimize the operation of the microgrid (MG). In this paper an Adaptive Modified Firefly Algorithm (AMFA) is presented for optimal operation of a typical MG with renewable energy sources (RESs) accompanied by a back-up Micro-Turbine/Fuel Cell/Battery hybrid power source to level the power mismatch or to store the energy surplus when it’s needed. The problem is formulated as a nonlinear constraint problem to minimize the total operating cost. The management of Energy storage system (ESS), economic load dispatch and operation optimization of distributed generation (DG) are simplified into a single-object optimization problem in the EMS. The proposed algorithm is tested on a typical grid-connected MG including WT/PV/Micro Turbine/Fuel Cell and Energy Storage Devices (ESDs) then its superior performance is compared with those from other evolutionary algorithms such as Genetic Algorithm (GA), Particle Swarm Optimization (PSO), Fuzzy Self Adaptive PSO (FSAPSO), Chaotic Particle PSO (CPSO), Adaptive Modified PSO (AMPSO), and Firefly Algorithm (FA).

Keywords: microgrid, operation management, optimization, firefly algorithm (AMFA)

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Authors: Obsi Terfasa, Bhanupriya Das, Mithilish Passawan

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The large-scale application of microbial fuel cell (MFC) technology is significantly hindered by the high cost of the commonly used proton exchange membrane, Nafion. This has led to the recent development of ceramic membranes using various clay minerals. This study evaluates the characteristics and potential use of a new ceramic membrane made from potter’s clay © mixed with different proportions (0, 5, 10 wt%) of fly ash (FA), labeled as CFA0, CFA5, CFA10, for cost-effective and sustainable MFC use. Among these, the CFA10 membrane demonstrated superior quality with a fine pore size distribution (average 0.41 μm), which supports higher water uptake and reduced oxygen diffusion. Its oxygen mass transfer coefficient was 4.13 ± 0.13 × 10⁻⁴ cm/s, about 40% lower than the control. X-ray diffraction analysis revealed that the CFA membrane is rich in quartz, which enhances proton conductance and water retention. Electrochemical kinetics studies, including cyclic voltammetry and electrochemical impedance spectroscopy (EIS), also confirmed the effectiveness of the CFA10 membrane in MFC, showing a peak current output of 15.35 mA and low ohmic resistance (78.2 Ω). The novel CFA10 ceramic membrane, incorporating coal fly ash, a waste material, shows promise for high MFC performance at a significantly reduced cost (96%), making it suitable for sustainable scaling up of the technology.

Keywords: ceramic membrane, Coulombic efficiency, electro-chemical kinetics, fly ash, proton conductivity, microbial fuel cell

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Authors: Ramesh P., Aby Joseph

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Micro Inverters provide Module Embedded Solution for harvesting energy from small-scale solar photovoltaic (PV) panels. In addition to higher modularity & reliability (25 years of life), the MicroInverter has inherent advantages such as avoidance of long DC cables, eliminates module mismatch losses, minimizes partial shading effect, improves safety and flexibility in installations etc. Due to the above-stated benefits, the renewable energy technology with Solar Photovoltaic (PV) Micro Inverter becomes more widespread in Village Nano Grid application ensuring grid independence for rural communities and areas without access to electricity. While the primary objective of this paper is to discuss the problems related to rural electrification, this concept can also be extended to urban installation with grid connectivity. This work presents a comprehensive analysis of the power circuit design, control methodologies and prototyping of 300Wₚ Single Phase PV Micro Inverter. This paper investigates two different topologies for PV Micro Inverters, based on the first hand on Single Stage Flyback/ Forward PV Micro-Inverter configuration and the other hand on the Double stage configuration including DC-DC converter, H bridge DC-AC Inverter. This work covers Power Decoupling techniques to reduce the input filter capacitor size to buffer double line (100 Hz) ripple energy and eliminates the use of electrolytic capacitors. The propagation of the double line oscillation reflected back to PV module will affect the Maximum Power Point Tracking (MPPT) performance. Also, the grid current will be distorted. To mitigate this issue, an independent MPPT control algorithm is developed in this work to reject the propagation of this double line ripple oscillation to PV side to improve the MPPT performance and grid side to improve current quality. Here, the power hardware topology accepts wide input voltage variation and consists of suitably rated MOSFET switches, Galvanically Isolated gate drivers, high-frequency magnetics and Film capacitors with a long lifespan. The digital controller hardware platform inbuilt with the external peripheral interface is developed using floating point microcontroller TMS320F2806x from Texas Instruments. The firmware governing the operation of the PV Micro Inverter is written in C language and was developed using code composer studio Integrated Development Environment (IDE). In this work, the prototype hardware for the Single Phase Photovoltaic Micro Inverter with Double stage configuration was developed and the comparative analysis between the above mentioned configurations with experimental results will be presented.

Keywords: double line oscillation, micro inverter, MPPT, nano grid, power decoupling

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Authors: Okyu Kwon, Dongho Kang, Byungsik Kim, Seungkwon Jung

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We propose a technique to measure the impact of loss of traffic function in a particular area to surrounding areas. The proposed method is applied to the city of Seoul, which is the capital of South Korea, with a population of about ten million. Based on the actual road network in Seoul, we construct an abstract road network between 1kmx1km grid cells. The link weight of the abstract road network is re-adjusted considering traffic volume measured at several survey points. On the modified abstract road network, we evaluate the traffic vulnerability by calculating a network measure of betweenness centrality (BC) for every single grid cells. This study analyzes traffic impacts caused by road dysfunction due to heavy rainfall in urban areas. We could see the change of the BC value in all other grid cells by calculating the BC value once again when the specific grid cell lost its traffic function, that is, when the node disappeared on the grid-based road network. The results show that it is appropriate to use the sum of the BC variation of other cells as the influence index of each lattice cell on traffic. This research was supported by a grant (2017-MOIS31-004) from Fundamental Technology Development Program for Extreme Disaster Response funded by Korean Ministry of Interior and Safety (MOIS).

Keywords: vulnerability, road network, beweenness centrality, heavy rainfall, road impact

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Authors: Khobaib Khobaib, Alexander Mikkelsen, Zbigniew Rozynek

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Electric fields have proven useful for inducing droplet deformation and to structure particles adsorbed at droplet interfaces. In this experimental research, direct current electric fields were applied to deform particle-covered droplets made out of silicone oil and immersed in castor oil. The viscosity of the drop and surrounding fluid were changed by external heating. We designed an experimental system in such a way that electric field-induced electrohydrodynamic (EHD) flows were asymmetric and only present on one side of the drop, i.e., the droplet adjoined a washer and adhered to one of the electrodes constituting the sample cell. The study investigated the influence of viscosity on the steady-state deformation magnitude of particle-laden droplets, droplet compression, and relaxation, as well as particle arrangements at drop interfaces. Initially, before the application of an electric field, we changed the viscosity of the fluids by heating the sample cell at different temperatures. The viscosity of the fluids was varied by changing the temperature of the fluids from 25 to 50°C. Under the application of a uniform electric field of strength 290 Vmm⁻¹, electric stress was induced at the drop interface, yielding drop deformation. In our study, we found that by lowering the fluid viscosity, the velocity of the EHD flows was increased, which also increases the deformation of the drop.

Keywords: drop deformation and relaxation, electric field, electrohydrodynamic flow, particle assembly, viscosity

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Authors: Ádám Vass, István Bakos, Irina Borbáth, Zoltán Pászti, István Sajó, András Tompos

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Important requirements for the anode side electrocatalysts of polymer electrolyte membrane (PEM) fuel cells are CO-tolerance, stability and corrosion resistance. Carbon is still the most common material for electrocatalyst supports due to its low cost, high electrical conductivity and high surface area, which can ensure good dispersion of the Pt. However, carbon becomes degraded at higher potentials and it causes problem during application. Therefore it is important to explore alternative materials with improved stability. Molybdenum-oxide can improve the CO-tolerance of the Pt/C catalysts, but it is prone to leach in acidic electrolyte. The Mo was stabilized by isovalent substitution of molybdenum into the rutile phase titanium-dioxide lattice, achieved by a modified multistep sol-gel synthesis method optimized for preparation of Ti0.7Mo.3O2-C composite. High degree of Mo incorporation into the rutile lattice was developed. The conductivity and corrosion resistance across the anticipated potential/pH window was ensured by mixed oxide – activated carbon composite. Platinum loading was carried out using NaBH4 and ethylene glycol; platinum content was 40 wt%. The electrocatalyst was characterized by both material investigating methods (i.e. XRD, TEM, EDS, XPS techniques) and electrochemical methods (cyclic-voltammetry, COads stripping voltammetry, hydrogen oxidation reaction on rotating disc electrode). The electrochemical activity of the sample was compared to commercial 40 wt% Pt/C (Quintech) and PtRu/C (Quintech, Pt= 20 wt%, Ru= 10 wt%) references. Enhanced CO tolerance of the electrocatalyst prepared using the Ti0.7Mo.3O2-C composite material was evidenced by the appearance of a CO-oxidation related 'pre-peak' and by the pronounced shift of the maximum of the main CO oxidation peak towards less positive potential compared to Pt/C. Fuel cell polarization measurements were also carried out using Bio-Logic and Paxitech FCT-150S test device. All details on the design, preparation, characterization and testing by both electrochemical measurements and fuel cell test device of electrocatalyst supported on Ti0.7Mo.3O2-C composite material will be presented and discussed.

Keywords: anode electrocatalyst, composite material, CO-tolerance, TiMoOx

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Authors: Elsadig Naseraddeen Ahmed Mohamed

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In contrast to the uncertainty and complementary principle, it will be shown in the present paper that the probability of the simultaneous occupation event of any definite values of coordinates by any definite values of momentum and energy at any definite instance of time can be described by a binary definite function equivalent to the difference between their numbers of occupation and evacuation epochs up to that time and also equivalent to the number of exchanges between those occupation and evacuation epochs up to that times modulus two, these binary definite quantities can be defined at all point in the time’s real-line so it form a binary signal represent a complete mechanical description of physical reality, the time of these exchanges represent the boundary of occupation and evacuation epochs from which we can calculate these binary signals using the fact that the time of universe events actually extends in the positive and negative of time’s real-line in one direction of extension when these number of exchanges increase, so there exists noninvertible transformation matrix can be defined as the matrix multiplication of invertible rotation matrix and noninvertible scaling matrix change the direction and magnitude of exchange event vector respectively, these noninvertible transformation will be called actual transformation in contrast to information transformations by which we can navigate the universe’s events transformed by actual transformations backward and forward in time’s real-line, so these information transformations will be derived as an elements of a group can be associated to their corresponded actual transformations. The actual and information model of the universe will be derived by assuming the existence of time instance zero before and at which there is no coordinate occupied by any definite values of momentum and energy, and then after that time, the universe begin its expanding in spacetime, this assumption makes the need for the existence of Laplace’s demon who at one moment can measure the positions and momentums of all constituent particle of the universe and then use the law of classical mechanics to predict all future and past of universe’s events, superfluous, we only need for the establishment of our analog to digital converters to sense the binary signals that determine the boundaries of occupation and evacuation epochs of the definite values of coordinates relative to its origin by the definite values of momentum and energy as present events of the universe from them we can predict approximately in high precision it's past and future events.

Keywords: binary-signal mechanics, actual-information model of the universe, actual-transformation, information-transformation, uncertainty principle, Laplace's demon

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Authors: Taknaz Banihashemi, Jiangang Fei, Peggy Shu-Ling Chen

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Reverse logistics (RL) is a part of the logistics of companies and its aim is to reclaim value from the returned products in an environmentally friendly manner. In recent years, RL has attracted significant attention among both practitioners and academics due to environmental directives and governmental legislation, consumer concerns and social responsibilities for environment, awareness of the limits of natural resources and economic potential. Sustainability development is considered as a critical goal for organisations due to its impact on competitive advantage. With growing environmental concerns and legal regulations related to green and sustainability issues, product disposition through RL can be considered as an environmental, economic and social sound way to achieve sustainable development. When employed properly, RL can help firms to improve their sustainability performance. The aim of this paper is to investigate the sustainability issues in the context of RL in the perspective of the triple-bottom-line approach. Content analysis was used to collect the information. The findings show that there is a research gap to investigate the relationship between RL and sustainability performance. Most of the studies have focused on performance evaluation of RL by considering the factors related to economic and environmental performance. RL can have significant effects on social issues along with economic and environmental issues. The inclusion of the social aspect in the sustainability performance will provide a complete and holistic picture of how RL may impact on the sustainability performance of firms. Generally, there is a lack of research on investigating the relationship between RL and sustainability by integrating the three pillars of triple-bottom-line sustainability performance. This paper provides academics and researchers a broad view of the correlations between RL and sustainability performance.

Keywords: verse Logistics, review, sustainability, sustainability performance

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Authors: Robson da Cunha Santos, Caio Cezar R. Bonifacio, Diego Mureb Quesada, Gerson Gomes Cunha

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The paper is related to the safety of oil wells and environmental preservation on the planet, because they require great attention and commitment from oil companies and people who work with these equipments. This must occur from drilling the well until it is abandoned in order to safeguard the environment and prevent possible damage. The project had as main objective the constitution resulting from comparatives made among books, articles and publications with information gathered in technical visits to operational bases of Petrobras. After the visits, the information from methods of utilization and present managements, which were not available before, became available to the general audience. As a result, it is observed a huge flux of incorrect and out-of-date information that comprehends not only bibliographic archives, but also academic resources and materials. During the gathering of more in-depth information on the manufacturing, assembling, and use aspects of DHSVs, several issues that were previously known as correct, customary issues were discovered to be uncertain and outdated. Information of great importance resulted in affirmations about subjects as the depth of the valve installation that was before installed to 30 meters from the seabed (mud line). Despite this, the installation should vary in conformity to the ideal depth to escape from area with the biggest tendency to hydrates formation according to the temperature and pressure. Regarding to valves with nitrogen chamber, in accordance with books, they have their utilization linked to water line ≥ 700 meters, but in Brazilian exploratory fields, their use occurs from 600 meters of water line. The valves used in Brazilian fields are able to be inserted to the production column and self-equalizing, but the use of screwed valve in the column of production and equalizing is predominant. Although these valves are more expensive to acquire, they are more reliable, efficient, with a bigger shelf life and they do not cause restriction to the fluid flux. It follows that based on researches and theoretical information confronted to usual forms used in fields, the present project is important and relevant. This project will be used as source of actualization and information equalization that connects academic environment and real situations in exploratory situations and also taking into consideration the enrichment of precise and easy to understand information to future researches and academic upgrading.

Keywords: down hole safety valve, security devices, installation, oil-wells

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Authors: Rafael Martins Manzano Silva, Jean-Francois Tremong

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Electrification of railway lines allows to increase speed, power, capacity and energetic efficiency of rolling stocks. However, this process of electrification is complex and costly. An electrification project is not just about design of catenary. It also includes installation of structures around electrification, as substation installation, electrical isolation, signalling, telecommunication and civil engineering structures. France has more than 30,000 km of railways, whose only 53% are electrified. The others 47% of railways use diesel locomotive and represent only 10% of the circulation (tons.km). For this reason, a new type of electrification, less expensive than the usual, is requested to enable the modernization of these railways. One solution could be the use of hybrids trains. This technology opens up new opportunities for less expensive infrastructure development such as the partial electrification of railway lines. In a partially electrified railway, the power supply of theses hybrid trains could be made either by the catenary or by the on-board energy storage system (ESS). Thus, the on-board ESS would feed the energetic needs of the train along the non-electrified zones while in electrified zones, the catenary would feed the train and recharge the on-board ESS. This paper’s objective deals with the technical and economic potential identification of partial electrification of railway lines. This study provides different scenarios of electrification by replacing the most expensive places to electrify using on-board ESS. The target is to reduce the cost of new electrification projects, i.e. reduce the cost of electrification infrastructures while not increasing the cost of rolling stocks. In this study, scenarios are constructed in function of the electrification’s cost of each structure. The electrification’s cost varies considerably because of the installation of catenary support in tunnels, bridges and viaducts is much more expensive than in others zones of the railway. These scenarios will be used to describe the power supply system and to choose between the catenary and the on-board energy storage depending on the position of the train on the railway. To identify the influence of each partial electrification scenario in the sizing of the on-board ESS, a model of the railway line and of the rolling stock is developed for a real case. This real case concerns a railway line located in the south of France. The energy consumption and the power demanded at each point of the line for each power supply (catenary or on-board ESS) are provided at the end of the simulation. Finally, the cost of a partial electrification is obtained by adding the civil engineering costs of the zones to be electrified plus the cost of the on-board ESS. The study of the technical and economic potential ends with the identification of the most economically interesting scenario of electrification.

Keywords: electrification, hybrid, railway, storage

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Authors: Ali Riza Perçin, Eser Bingül

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Internet technologies have created opportunities with which people share their ideologies, thoughts and products. This virtual world, named social media has given the chance of gathering individual users and people from the world's remote locations and establishing an interaction between them. However, to an increasingly higher degree terrorist organizations today use the internet and most notably social-network media to create the effects they desire through a series of on-line activities. These activities, designed to support their activities, include information collection (intelligence), target selection, propaganda, fundraising and recruitment to name a few. Meanwhile, these have been used as the most important tool for recruitment especially from the different region of the world, especially disenfranchised youth, in the West in order to mobilize support and recruit “foreign fighters.” The recruits have obtained the statue, which is not accessible in their society and have preferred the style of life that is offered by the terrorist organizations instead of their current life. Like other terrorist groups, for a while now the terrorist organization Islamic State (IS) in Iraq and Syria has employed a social-media strategy in order to advance their strategic objectives. At the moment, however, IS seems to be more successful in their on-line activities than other similar organizations. IS uses social media strategically as part of its armed activities and for the sustainability of their military presence in Syria and Iraq. In this context, “Generation Y”, which could exist at the critical position and undertake active role, has been examined. Additionally, the explained characteristics of “Generation Y” have been put forward and the duties of families and society have been stated as well.

Keywords: social media, "generation Y", terrorist organization, islamic state IS

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Authors: Yanni Chang, Dezhi Dai, Albert Y. Tong

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Piecewise linear interface calculation (PLIC) schemes are widely used in the volume-of-fluid (VOF) method to capture interfaces in numerical simulations of multiphase flows. Dynamic overset meshes can be especially useful in applications involving component motions and complex geometric shapes. In the present study, the VOF value of an acceptor cell is evaluated in a geometric way that transfers the fraction field between the meshes precisely with reconstructed interfaces from the corresponding donor elements. The acceptor cell value is evaluated by using a weighted average of its donors for most of the overset interpolation schemes for continuous flow variables. The weighting factors are obtained by different algebraic methods. Unlike the continuous flow variables, the VOF equation is a step function near the interfaces, which ranges from zero to unity rapidly. A geometric interpolation scheme of the VOF field in overset meshes for the PLIC-VOF method has been proposed in the paper. It has been tested successfully in quadrilateral/hexahedral overset meshes by employing several VOF advection tests with imposed solenoidal velocity fields. The proposed algorithm has been shown to yield higher accuracy in mass conservation and interface reconstruction compared with three other algebraic ones.

Keywords: interpolation scheme, multiphase flows, overset meshes, PLIC-VOF method

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Authors: Fadia Gafri, Kathryn Mckintosh, Louise Young, Alan Harvey, Simon Mackay, Andrew Paul, Robin Plevin

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Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor found originally to play a key role in regulating inflammation. However considerable evidence links this pathway to the suppression of apoptosis, cellular transformation, proliferation and invasion (Aggarwal et al., 2006). Moreover, recent studies have also linked inflammation to cancer progression making NF-κB overall a promising therapeutic target for drug discovery (Dobrovolskaia & Kozlov, 2005). In this study we examined the effect of the natural product canthin-6-one (SU182) as part of a CRUK small molecule drug discovery programme for effects upon the NF-κB pathway. Initial studies demonstrated that SU182 was found to have good potency against the inhibitory kappa B kinases (IKKs) at 30M in vitro. However, at concentrations up to 30M, SU182 had no effect upon TNFα stimulated loss in cellular IκBα or p65 phosphorylation in the keratinocyte cell line NCTC2544. Nevertheless, 30M SU182 reduced TNF-α / PMA-induced NF-κB-linked luciferase reporter activity to (22.9 ± 5%) and (34.6± 3 %, P<0.001) respectively, suggesting an action downstream of IKK signalling. Indeed, SU182 neither decreased NF-κB-DNA binding as assayed by EMSA nor prevented the translocation of p65 (NF-κB) to the nucleus assessed by immunofluorescence and subcellular fractionation. In addition to the inhibition of transcriptional activity of TNFα-induced NF-κB reporter activity SU182 significantly reduced PMA-induced AP-1-linked luciferase reporter activity to about (48± 9% at 30M, P<0.001) . This mode of inhibition was not sufficient to prevent the activation of NF-κB dependent induction of other proteins such as COX-2 and iNOS, or activated MAP kinases (p38, JNK and ERK1/2) in LPS stimulated RAW 264.7 macrophages. Taken together these data indicate the potential for SU182 to interfere with the transcription factors NF-κB and AP-1 at transcriptional level. However, no potential anti-inflammatory effect was indicated, further investigation for other NF-κB dependent proteins linked to survival are also required to identify the exact mechanism of action.

Keywords: Canthin-6-one, NF-κB, AP-1, phosphorylation, Nuclear translocation, DNA-binding activity, inflammatory proteins.

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Authors: Takashi Mitsuishi

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Fuzzy logic has gained acceptance in the recent years in the fields of social sciences and humanities such as psychology and linguistics because it can manage the fuzziness of words and human subjectivity in a logical manner. However, the major field of application of the fuzzy logic is control engineering as it is a part of the set theory and mathematical logic. Mamdani method, which is the most popular technique for approximate reasoning in the field of fuzzy control, is one of the ways to numerically represent the control afforded by human language and sensitivity and has been applied in various practical control plants. Fuzzy logic has been gradually developing as an artificial intelligence in different applications such as neural networks, expert systems, and operations research. The objects of inference vary for different application fields. Some of these include time, angle, color, symptom and medical condition whose fuzzy membership function is a periodic function. In the defuzzification stage, the domain of the membership function should be unique to obtain uniqueness its defuzzified value. However, if the domain of the periodic membership function is determined as unique, an unintuitive defuzzified value may be obtained as the inference result using the center of gravity method. Therefore, the authors propose a method of circular-polar-coordinates transformation and defuzzification of the periodic membership functions in this study. The transformation to circular polar coordinates simplifies the domain of the periodic membership function. Defuzzified value in circular polar coordinates is an argument. Furthermore, it is required that the argument is calculated from a closed plane figure which is a periodic membership function on the circular polar coordinates. If the closed plane figure is continuous with the continuity of the membership function, a significant amount of computation is required. Therefore, to simplify the practice example and significantly reduce the computational complexity, we have discretized the continuous interval and the membership function in this study. In this study, the following three methods are proposed to decide the argument from the discrete polygon which the continuous plane figure is transformed into. The first method provides an argument of a straight line passing through the origin and through the coordinate of the arithmetic mean of each coordinate of the polygon (physical center of gravity). The second one provides an argument of a straight line passing through the origin and the coordinate of the geometric center of gravity of the polygon. The third one provides an argument of a straight line passing through the origin bisecting the perimeter of the polygon (or the closed continuous plane figure).

Keywords: defuzzification, fuzzy membership function, periodic function, polar coordinates transformation

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Authors: F. Kashanian, M. M. Masoudi, A. Akbari, A. Shamloo, M. R. Zand, S. S. Salehi

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Nano-sized materials present new opportunities in biology and medicine and they are used as biomedical tools for investigation, separation of molecules and cells. To achieve more effective cancer therapy, it is essential to select cancer cells exactly. This research suggests that using the antibody-functionalized nontoxic Arginine-doped magnetic nanoparticles (A-MNPs), has been prosperous in detection, capture, and magnetic separation of circulating tumor cells (CTCs) in tumor tissue. In this study, A-MNPs were synthesized via a simple precipitation reaction and directly immobilized Ep-CAM EBA-1 antibodies over superparamagnetic A-MNPs for Mucin BCA-225 in breast cancer cell. The samples were characterized by vibrating sample magnetometer (VSM), FT-IR spectroscopy, Tunneling Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). These antibody-functionalized nontoxic A-MNPs were used to capture breast cancer cell. Through employing a strong permanent magnet, the magnetic separation was achieved within a few seconds. Antibody-Conjugated nontoxic Arginine-doped Fe₃O₄ nanoparticles have the potential for the future study to capture CTCs which are released from tumor tissue and for drug delivery, and these results demonstrate that the antibody-conjugated A-MNPs can be used in magnetic hyperthermia techniques for cancer treatment.

Keywords: tumor tissue, antibody, magnetic nanoparticle, CTCs capturing

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Authors: Lynn Lehmann, Dinorah Leyva, Ana Lazic, Stefan Duhr, Philipp Baaske

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Characterization of biomolecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, provides critical insights into cellular processes and is essential for the development of drug diagnostics and therapeutics. Here we present a novel, label-free, and tether-free technology to analyze picomolar to millimolar affinities of biomolecular interactions by Microscale Thermophoresis (MST). The entropy of the hydration shell surrounding molecules determines thermophoretic movement. MST exploits this principle by measuring interactions using optically generated temperature gradients. MST detects changes in the size, charge and hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice, including cell lysates and blood serum. Thus, MST measures interactions under close-to-native conditions, and without laborious sample purification. We demonstrate how MST determines the picomolar affinities of antibody::antigen interactions, and protein::protein interactions measured from directly from cell lysates. MST assays are highly adaptable to fit to the diverse requirements of different and complex biomolecules. NanoTemper´s unique technology is ideal for studies requiring flexibility and sensitivity at the experimental scale, making MST suitable for basic research investigations and pharmaceutical applications.

Keywords: biochemistry, biophysics, molecular interactions, quantitative techniques

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Authors: Salina Yahya Saddick

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Ovarian cancer remains the most lethal gynecological malignancy due to the lack of highly sensitive and specific screening tools for detection of early-stage disease. The OSE provides the progenitor cells for 90% of human ovarian cancers. Recent morphologic, immunohistochemical and molecular genetic studies have led to the development of a new paradigm for the pathogenesis and origin of epithelial ovarian cancer (EOC) based on a ualistic model of carcinogenesis that divides EOC into two broad categories designated Types I and II which are characterized by specific mutations, including KRAS, BRAF, ERBB2, CTNNB1, PTEN PIK3CA, ARID1A, and PPPR1A, which target specific cell signaling pathways. Type 1 tumors rarely harbor TP53. type I tumors are relatively genetically stable and typically display a variety of somatic sequence mutations that include KRAS, BRAF, PTEN, PIK3CA CTNNB1 (the gene encoding beta catenin), ARID1A and PPP2R1A but very rarely TP53 . The cancer stem cell (CSC) hypothesis postulates that the tumorigenic potential of CSCs is confined to a very small subset of tumor cells and is defined by their ability to self-renew and differentiate leading to the formation of a tumor mass. Potential protein biomarker miRNA, are promising biomarkers as they are remarkably stable to allow isolation and analysis from tissues and from blood in which they can be found as free circulating nucleic acids and in mononuclear cells. Recently, genomic anaylsis have identified biomarkers and potential therapeutic targets for ovarian cancer namely, FGF18 which plays an active role in controlling migration, invasion, and tumorigenicity of ovarian cancer cells through NF-κB activation, which increased the production of oncogenic cytokines and chemokines. This review summarizes update information on epithelial ovarian cancers and point out to the most recent ongoing research.

Keywords: epithelial ovarian cancers, somatic sequence mutations, cancer stem cell (CSC), potential protein, biomarker, genomic analysis, FGF18 biomarker

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Authors: Ethan Nikookar, Yalda Boroushaki, Larissa Statsenko, Jorge Ochoa Paniagua

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Given the serious impact that supply chain disruptions can have on a firm's bottom-line performance, both industry and academia are interested in supply chain resilience, a capability of the supply chain that enables it to cope with disruptions. To date, much of the research has focused on the antecedents of supply chain resilience. This line of research has suggested various firm-level capabilities that are associated with greater supply chain resilience. A consensus has emerged among researchers that supply chain flexibility holds the greatest potential to create resilience. Supply chain flexibility achieves resilience by creating readiness to respond to disruptions with little cost and time by means of reconfiguring supply chain resources to mitigate the impacts of the disruption. Decisions related to supply chain disruptions are made by supply chain managers; however, the role played by supply chain managers' reference networks has been overlooked in the supply chain resilience literature. This study aims to understand the impact of supply chain managers on their firms' supply chain resilience. Drawing on social capital theory and social network theory, this paper proposes a conceptual model to explore the role of supply chain managers in developing the resilience of supply chains. Our model posits that higher level of supply chain managers' embeddedness in their reference network is associated with increased resilience of their firms' supply chain. A reference network includes individuals from whom supply chain managers seek advice on supply chain related matters. The relationships between supply chain managers' embeddedness in reference network and supply chain resilience are mediated by supply chain flexibility.

Keywords: supply chain resilience, embeddedness, reference networks, social capitals

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Authors: E. Nakamachi, S. Tanaka, K. Yamamoto, Y. Morita

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In this study, we developed a three-dimensional (3D) direct current electric field (DCEF) stimulation bio-reactor for axonal outgrowth enhancement to generate the neural network of the central nervous system (CNS). By using our newly developed 3D DCEF stimulation bio-reactor, we cultured the rat pheochromocytoma cells (PC12) and investigated the effects on the axonal extension enhancement and network generation. Firstly, we designed and fabricated a 3D bio-reactor, which can load DCEF stimulation on PC12 cells embedded in the collagen gel as extracellular environment. The connection between the electrolyte and the medium using salt bridges for DCEF stimulation was introduced to avoid the cell death by the toxicity of metal ion. The distance between the salt bridges was adopted as the design variable to optimize a structure for uniform DCEF stimulation, where the finite element (FE) analyses results were used. Uniform DCEF strength and electric flux vector direction in the PC12 cells embedded in collagen gel were examined through measurements of the fabricated 3D bio-reactor chamber. Measurement results of DCEF strength in the bio-reactor showed a good agreement with FE results. In addition, the perfusion system was attached to maintain pH 7.2 ~ 7.6 of the medium because pH change was caused by DCEF stimulation loading. Secondly, we disseminated PC12 cells in collagen gel and carried out 3D culture. Finally, we measured the morphology of PC12 cell bodies and neurites by the multiphoton excitation fluorescence microscope (MPM). The effectiveness of DCEF stimulation to enhance the axonal outgrowth and the neural network generation was investigated. We confirmed that both an increase of mean axonal length and axogenesis rate of PC12, which have been exposed 5 mV/mm for 6 hours a day for 4 days in the bioreactor. We found following conclusions in our study. 1) Design and fabrication of DCEF stimulation bio-reactor capable of 3D culture nerve cell were completed. A uniform electric field strength of average value of 17 mV/mm within the 1.2% error range was confirmed by using FE analyses, after the structure determination through the optimization process. In addition, we attached a perfusion system capable of suppressing the pH change of the culture solution due to DCEF stimulation loading. 2) Evaluation of DCEF stimulation effects on PC12 cell activity was executed. The 3D culture of PC 12 was carried out adopting the embedding culture method using collagen gel as a scaffold for four days under the condition of 5.0 mV/mm and 10mV/mm. There was a significant effect on the enhancement of axonal extension, as 11.3% increase in an average length, and the increase of axogenesis rate. On the other hand, no effects on the orientation of axon against the DCEF flux direction was observed. Further, the network generation was enhanced to connect longer distance between the target neighbor cells by DCEF stimulation.

Keywords: PC12, DCEF stimulation, 3D bio-reactor, axonal extension, neural network generation

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Authors: Chang-Hui Shen, Paulina Konarzewska

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Cell homeostasis is regulated by vacuolar activity and it has been shown that lipid composition of the vacuole plays an important role in vacuolar function. The major phosphoinositide species present in the vacuolar membrane include phosphatidylinositol 3,5-biphosphate (PI(3,5)P₂) which is generated from PI(3)P controlled by Fab1p. Deletion of FAB1 gene reduce the synthesis of PI(3,5)P₂ and thus result in enlarged or fragmented vacuoles, with neutral vacuolar pH due to reduced vacuolar H⁺-ATPase activity. These mutants also exhibited poor growth at high extracellular pH and in the presence of CaCl₂. Conversely, VPS34 regulates the synthesis of PI(3)P from phosphatidylinositol (PI), and the lack of Vps34p results in the reduction of vacuolar activity. Although the cellular observations are clear, it is still unknown about the molecular mechanism between the phospholipid biosynthesis pathway and vacuolar activity. Since both VPS34 and FAB1 are important in vacuolar activity, we hypothesize that the molecular mechanism of vacuolar function might be regulated by the transcriptional regulators of phospholipid biosynthesis. In this study, we study the role of the major phospholipid biosynthesis transcription factor, INO2, in the regulation of vacuolar activity. We first performed qRT-PCR to examine the effect of Ino2p on the expression of VPS34 and FAB1. Our results showed that VPS34 was upregulated in the presence of inositol for both WT and ino2Δ cells. However, FAB1 was only upregulated significantly in ino2Δ cells. This indicated that Ino2p might be the negative regulator for FAB1 expression. Next, growth sensitivity experiment showed that WT, vma3Δ, and ino2Δ grew well in growth medium buffered to pH 5.5 containing 10 mM CaCl₂. As cells were switched to growth medium buffered to pH 7 containing CaCl₂ WT, ino2Δ and opi1Δ showed growth reduction, whereas vma3Δ was completely nonviable. As the concentration of CaCl₂ was increased to 60 mM, ino2Δ cells showed moderate growth reduction compared to WT. This result suggests that ino2Δ cells have better vacuolar activity. Microscopic analysis and vacuolar acidification were employed to further elucidate the importance of INO2 in vacuolar homeostasis. Analysis of vacuolar morphology indicated that WT and vma3Δ cells displayed vacuoles that occupied a small area of the cell when grown in media buffered to pH 5.5. Whereas, ino2Δ displayed fragmented vacuoles. On the other hand, all strains grown in media buffered to pH 7, exhibited enlarged vacuoles that occupied most of the cell’s surface. This indicated that the presence of INO2 may play negative effect in vacuolar morphology when cells are grown in media buffered to pH 5.5. Furthermore, vacuolar acidification assay showed that only vma3Δ cells displayed notably less acidic vacuoles as cells were grown in media buffered to pH 5.5 and pH 7. Whereas, ino2Δ cells displayed more acidic pH compared to WT at pH7. Taken together, our results demonstrated the molecular mechanism of the vacuolar activity regulated by the phospholipid biosynthesis transcription factors Ino2p. Ino2p negatively regulates vacuolar activity through the expression of FAB1.

Keywords: vacuole, phospholipid, homeostasis, Ino2p, FAB1

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