Search results for: cancer cell lines

Authors: Jin Young Kim, Kwon Kyoo Kang

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The SGR1 (STAYGREEN1) protein is a critical regulator of plant leaves in chlorophyll degradation and senescence. The functions and mechanisms of tomato SGR1 action are poorly understood and worthy of further investigation. To investigate the function of the SGR1 gene, we generated a SGR1-knockout (KO) null line via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing and conducted RNA sequencing and gas chromatography tandem mass spectrometry (GC-MS/MS) analysis to identify the differentially expressed genes. The SlSGR1 (Solanum lycopersicum SGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid content compared to wild-type (WT) fruit. Differential gene expression analysis revealed 728 differentially expressed genes (DEGs) between WT and sgr1 #1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, for which fold change was >2, and the adjusted p-value was <0.05. Most of the DEGs were related to photosynthesis and chloroplast function. In addition, the pigment, carotenoid changes in sgr1 #1-6 line was accumulated of key primary metabolites such as sucrose and its derivatives (fructose, galactinol, raffinose), glycolytic intermediates (glucose, G6P, Fru6P) and tricarboxylic acid cycle (TCA) intermediates (malate and fumarate). Taken together, the transcriptome and metabolite profiles of SGR1-KO lines presented here provide evidence for the mechanisms underlying the effects of SGR1 and molecular pathways involved in chlorophyll degradation and carotenoid biosynthesis.

Keywords: tomato, CRISPR/Cas9, null line, RNA-sequencing, metabolite profiling

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Authors: S. Najafi, E. Bertini, M. Pezzotti, G.B. Tornielli, S. Zenoni

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Grapevine is an important agricultural fruit crop plant consumed worldwide and with a key role in the global economy. Grapevine is strongly affected by both biotic and abiotic stresses, which impact grape growth at different stages, such as during plant and berry development and pre- and post-harvest, consequently causing significant economic losses. Recently global warming has propelled the anticipation of the onset of berry ripening, determining the reduction of a grape color and increased volatilization of aroma compounds. Climate change could negatively alter the physiological characteristics of the grape and affect the berry and wine quality. Modern plant breeding can provide tools such as genome editing for improving grape resilience traits while maintaining intact the viticultural and oenological quality characteristics of the genotype. This study aims at developing a platform for genome editing application in grapevine plants with the final goal to improve berry quality, biotic, and abiotic resilience traits. We chose to directly deliver ribonucleoproteins (RNP, preassembled Cas protein and guide RNA) into plant protoplasts, and, from these cell structures, regenerate grapevine plants edited in specific selected genes controlling traits of interest. Edited plants regenerated by somatic embryogenesis from protoplasts will then be sequenced and molecularly characterized. Embryogenic calli of Sultana and Shiraz cultivars were initiated from unopened leaves of in-vitro shoot tip cultures and from stamens, respectively. Leaves were placed on NB2 medium while stamens on callus initiation medium (PIV) medium and incubated in the dark at 28 °C for three months. Viable protoplasts, tested by FDA staining, isolated from embryogenic calli were cultured by disc method at 1*105 protoplasts/ml. Mature well-shaped somatic embryos developed directly in the protoplast culture medium two months later and were transferred in the light into to shooting medium for further growth. Regenerated plants were then transferred to the greenhouse; no phenotypic alterations were observed when compared to non in-vitro cultured plants. The performed experiments allowed to established an efficient protocol of embryogenic calli production, protoplast isolation, and regeneration of the whole plant through somatic embryogenesis in both Sultana and Shiraz. Regenerated plants, through direct somatic embryogenesis deriving from a single cell, avoid the risk of chimerism during the regeneration process, therefore improving the genome editing process. As pre-requisite of genome editing, an efficient method for transfection of protoplast by yellow fluorescent protein (YFP) marker genes was also established and experiments of direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) in protoplasts to achieve efficient DNA-free targeted mutations are in progress.

Keywords: CRISPR-cas9, plant regeneration, protoplast isolation, Vitis vinifera

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Authors: Yo-Sheng Lin, Jen-How Lee, Chien-Chin Wang

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A 60-GHz receiver front-end using standard 90-nm CMOS technology is reported. The receiver front-end comprises a wideband low-noise amplifier (LNA), and a double-balanced Gilbert cell mixer with a current-reused RF single-to-differential (STD) converter, an LO Marchand balun and a baseband amplifier. The receiver front-end consumes 34.4 mW and achieves LO-RF isolation of 60.7 dB, LO-IF isolation of 45.3 dB and RF-IF isolation of 41.9 dB at RF of 60 GHz and LO of 59.9 GHz. At IF of 0.1 GHz, the receiver front-end achieves maximum conversion gain (CG) of 26.1 dB at RF of 64 GHz and CG of 25.2 dB at RF of 60 GHz. The corresponding 3-dB bandwidth of RF is 7.3 GHz (58.4 GHz to 65.7 GHz). The measured minimum noise figure was 5.6 dB at 64 GHz, one of the best results ever reported for a 60 GHz CMOS receiver front-end. In addition, the measured input 1-dB compression point and input third-order inter-modulation point are -33.1 dBm and -23.3 dBm, respectively, at 60 GHz. These results demonstrate the proposed receiver front-end architecture is very promising for 60 GHz direct-conversion transceiver applications.

Keywords: CMOS, 60 GHz, direct-conversion transceiver, LNA, down-conversion mixer, marchand balun, current-reused

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Authors: Michael Anthony Doherty

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Modern Methods of Construction (MMC) is a significant topic in the construction industry; while reviewing (MMC) over different fields that are significant in the modern construction world, the following areas were assessed where (MMC) is developing, supply chain management, automation, digital technology, and new construction technologies. Different methods were considered as an approach to research and exploring areas highlighted within the construction industry that are making advancements using Modern Methods of Construction Methods and Technologies (MCMTs). The research was conducted using the following methodologies, literature review of academic sources, primary and secondary data sources, questionaries, and interviews. The paper is composed of two parts, firstly a literature review and secondly a questionnaire used as the basis for interviews were utilised to achieve the following key objectives: to identify (MCMTs) being implemented in the construction industry, research and compile information with regards to these methods, determine their purpose and their application in the industry, establishing what (MCMTs) are being used in the industry while also determining the success of the methods. The research considers the evolution and development of these methods in projects and within the industry itself. Major findings were as follows; automation technologies such as robotics, offsite fabrication utilising automated production lines are increasingly part of project execution, digital technologies such as AR and VR are increasingly utilised in project co-ordination, (MMCTs) are proving to be a solution to the construction industry problems such as a lack of skilled workforce, hazardous work tasks, and situations, new construction technologies are available and finding their place in mainstream construction, (SCM) and (GSCM) are evolving to new levels using new systems and technologies such as block chain technology as well as Company Size and Project size influence the use of (MMCTs) and the adoption of (MMCTS). In summary the paper endeavours to identify and detail how areas of (MMCTs) are developing and are gaining traction within mainstream construction.

Keywords: automation, digital technology, new construction technologies, supply chain management

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Authors: Salman Naveed, Nitant Gandhi, Grant Billings, Zachary Jones, B. Todd Campbell, Michael Jones, Sachin Rustgi

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Cotton (Gossypium spp.) is the primary source of natural fiber in the U.S. and a major crop in the Southeastern U.S. Despite constant efforts to increase the cotton fiber yield, the yield gain has stagnated. Therefore, we undertook a novel approach to improve the cotton fiber yield by altering its growth habit from perennial to annual. In this effort, we identified genotypes with high-expression alleles of five floral induction and meristem identity genes (FT, SOC1, FUL, LFY, and AP1) from an upland cotton mini-core collection and crossed them in various combinations to develop cotton lines with annual growth habit, optimal flowering time and enhanced productivity. To facilitate the characterization of genotypes with the desired combinations of stacked alleles, we identified markers associated with the gene expression traits via genome-wide association analysis using a 63K SNP Array (Hulse-Kemp et al. 2015 G3 5:1187). Over 14,500 SNPs showed polymorphism and were used for association analysis. A total of 396 markers showed association with expression traits. Out of these 396 markers, 159 mapped to genes, 50 to untranslated regions, and 187 to random genomic regions. Biased genomic distribution of associated markers was observed where more trait-associated markers mapped to the cotton D sub-genome. Many quantitative trait loci coincided at specific genomic regions. This observation has implications as these traits could be bred together. The analysis also allowed the identification of candidate regulators of the expression patterns of these floral induction and meristem identity genes whose functions will be validated via virus-induced gene silencing.

Keywords: cotton, GWAS, QTL, expression traits

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Authors: Eunseop Yeom

Abstract:

Many cardiovascular diseases, such as thrombosis and atherosclerosis, can change biochemical molecules in plasma and red blood cell. These alterations lead to excessive increase of blood viscosity contributing to peripheral vascular diseases. In this study, a simple microfluidic-based method is used to measure blood viscosity. Microfluidic device is composed of two parallel side channels and a bridge channel. To estimate blood viscosity, blood samples and reference fluid are separately delivered into each inlet of two parallel side channels using pumps. An interfacial line between blood samples and reference fluid occurs by blocking the outlet of one side-channel. Since width for this interfacial line is determined by pressure ratio between blood and reference flows, blood viscosity can be estimated by measuring width for this interfacial line. This microfluidic-based method can be used for evaluating variations in the viscosity of animal models with cardiovascular diseases under flow conditions.

Keywords: blood viscosity, microfluidic chip, pressure, shear rate

Procedia PDF Downloads 372

Authors: Parul Kulshreshtha, Subrata Sinha, Rakesh Bhatnagar

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This study was conducted by taking B. anthracis as a model pathogen. On infecting a host, B. anthracis secretes three proteins, namely, protective antigen (PA, 83kDa), edema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). These three proteins are the components of two anthrax toxins. PA binds to the cell surface receptors, namely, tumor endothelial marker (TEM) 8 and capillary morphogenesis protein (CMG) 2. TEM8 and CMG2 interact with LDL-receptor related protein (LRP) 6 for endocytosis of EF and LF. On entering the cell, EF acts as a calmodulin-dependent adenylate cyclase that causes a prolonged increase of cytosolic cyclic adenosine monophosphate (cAMP). LF is a metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MAPKK/MEK) close to their N-terminus. By secreting these two toxins, B.anthracis ascertains death of the host. Once the systemic levels of the toxins rise, antibiotics alone cannot save the host. Therefore, toxin-specific inhibitors have to be developed. In this wake, monoclonal antibodies have been developed for the neutralization of toxic effects of anthrax toxins. We created hybridomas by using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor of B. anthracis) to obtain anti-toxin antibodies. Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immunized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies from all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H8 and H10) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). The protective efficacy of H7, H8, H10 and H11 was investigated. H7, H8 and H10 were found to be protective. H11 was found to have disease enhancing characteristics in-vitro and in mouse model of challenge with B. anthracis. In this study the disease enhancing character of H11 monoclonal antibody and anti-rLFn polyclonal sera was investigated. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature both in-vitro and in-vivo. But combination of H11 with LETscFv (an scFv with VH and VL identical to H10 but lacking Fc region) could not abrogate the disease-enhancing character of H11 mAb. Therefore it was concluded that for suppression of disease enhancement, Fc portion was absolutely essential for interaction of H10 with H11. Our study indicates that the protective potential of an antibody depends equally on its idiotype/ antigen specificity and its isotype. A number of monoclonal and engineered antibodies are being explored as immunotherapeutics but it is absolutely essential to characterize each one for their individual and combined protective potential. Although new in the sphere of toxin-based diseases, it is extremely important to characterize the disease-enhancing nature of polyclonal as well as monoclonal antibodies. This is because several anti-viral therapeutics and vaccines have failed in the face of this phenomenon. The passive –immunotherapy thus needs to be well formulated to avoid any contraindications.

Keywords: immunotherapy, polyclonal, monoclonal, antibody-dependent disease enhancement

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Authors: M. Khalfa, H. Khachai, F. Chiker, K. Bougherara, R. Khenata, G. Murtaza, M. Harmel

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A theoretical study of structural, elastic, electronic and thermodynamic properties of Fe2VX, (with X = Al and Ga), were studied by means of the full-relativistic version of the full-potential augmented plane wave plus local orbitals method. For exchange and correlation potential we used both generalized-gradient approximation (GGA) and local-density approximation (LDA). Our calculated ground state properties like as lattice constants, bulk modulus and elastic constants appear more accurate when we employed the GGA rather than the LDA approximation, and these results agree very well with the available experimental and theoretical data. Further, prediction of the thermal effects on some macroscopic properties of Fe2VAl and Fe2VGa are given in this paper using the quasi-harmonic Debye model in which the lattice vibrations are taken into account. We have obtained successfully the variations of the primitive cell volume, volume expansion coefficient, heat capacities and Debye temperature with pressure and temperature in the ranges of 0–40 GPa and 0–1500 K.

Keywords: full Heusler, FP-LAPW, electronic properties, thermal properties

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Authors: Jing Zhang, Yvonne Reinwald, Nick Poulson, Alicia El Haj, Chung See, Mike Somekh, Melissa Mather

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Biomedical imaging of native tissue using light offers the potential to obtain excellent structural and functional information in a non-invasive manner with good temporal resolution. Image contrast can be derived from intrinsic absorption, fluorescence, or scatter, or through the use of extrinsic contrast. A major challenge in applying optical microscopy to in vivo tissue imaging is the effects of light attenuation which limits light penetration depth and achievable imaging resolution. Recently Selective Plane Illumination Microscopy (SPIM) has been used to map the 3D distribution of fluorophores dispersed in biological structures. In this approach, a focused sheet of light is used to illuminate the sample from the side to excite fluorophores within the sample of interest. Images are formed based on detection of fluorescence emission orthogonal to the illumination axis. By scanning the sample along the detection axis and acquiring a stack of images, 3D volumes can be obtained. The combination of rapid image acquisition speeds with the low photon dose to samples optical sectioning provides SPIM is an attractive approach for imaging biological samples in 3D. To date all implementations of SPIM rely on the use of fluorescence reporters be that endogenous or exogenous. This approach has the disadvantage that in the case of exogenous probes the specimens are altered from their native stage rendering them unsuitable for in vivo studies and in general fluorescence emission is weak and transient. Here we present for the first time to our knowledge a label-free implementation of SPIM that has downstream applications in the clinical setting. The experimental set up used in this work incorporates both label-free and fluorescent illumination arms in addition to a high specification camera that can be partitioned for simultaneous imaging of both fluorescent emission and scattered light from intrinsic sources of optical contrast in the sample being studied. This work first involved calibration of the imaging system and validation of the label-free method with well characterised fluorescent microbeads embedded in agarose gel. 3D constructs of mammalian cells cultured in agarose gel with varying cell concentrations were then imaged. A time course study to track cell proliferation in the 3D construct was also carried out and finally a native tissue sample was imaged. For each sample multiple images were obtained by scanning the sample along the axis of detection and 3D maps reconstructed. The results obtained validated label-free SPIM as a viable approach for imaging cells in a 3D gel construct and native tissue. This technique has the potential use in a near-patient environment that can provide results quickly and be implemented in an easy to use manner to provide more information with improved spatial resolution and depth penetration than current approaches.

Keywords: bioimaging, optics, selective plane illumination microscopy, tissue imaging

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Authors: Eleonora Di Salvo, Antonio Panebianco, Graziella Ziino

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Background: Vibrio parahaemolyticus is a human pathogen that is widely distributed in marine environments. It is frequently isolated from raw seafood, particularly shellfish. Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus may lead to acute gastroenteritis. Vibrio spp. has excellent resistance to low temperatures so it can be found in frozen products for a long time. Recently, the viable but non-culturable state (VBNC) of bacteria has attracted great attention, and more than 85 species of bacteria have been demonstrated to be capable of entering this state. VBNC cells cannot grow in conventional culture medium but are viable and maintain metabolic activity, which may constitute an unrecognized source of food contamination and infection. Also V. parahaemolyticus could exist in VBNC state under nutrient starvation or low-temperature conditions. Aim: The aim of the present study was to optimize methods and investigate V. parahaemolyticus VBNC cells and their presence in frozen bivalve molluscs, regularly marketed. Materials and Methods: propidium monoazide (PMA) was integrated with real-time polymerase chain reaction (qPCR) targeting the tl gene to detect and quantify V. parahaemolyticus in the VBNC state. PMA-qPCR resulted highly specific to V. parahaemolyticus with a limit of detection (LOD) of 10-1 log CFU/mL in pure bacterial culture. A standard curve for V. parahaemolyticus cell concentrations was established with the correlation coefficient of 0.9999 at the linear range of 1.0 to 8.0 log CFU/mL. A total of 77 samples of frozen bivalve molluscs (35 mussels; 42 clams) were subsequently subjected to the qualitative (on alkaline phosphate buffer solution) and quantitative research of V. parahaemolyticus on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (DIFCO) NaCl 2.5%, and incubation at 30°C for 24-48 hours. Real-time PCR was conducted on homogenate samples, in duplicate, with and without propidium monoazide (PMA) dye, and exposed for 45 min under halogen lights (650 W). Total DNA was extracted from cell suspension in homogenate samples according to bolliture protocol. The Real-time PCR was conducted with species-specific primers for V. parahaemolitycus. The RT-PCR was performed in a final volume of 20 µL, containing 10 µL of SYBR Green Mixture (Applied Biosystems), 2 µL of template DNA, 2 µL of each primer (final concentration 0.6 mM), and H2O 4 µL. The qPCR was carried out on CFX96 TouchTM (Bio-Rad, USA). Results: All samples were negative both to the quantitative and qualitative detection of V. parahaemolyticus by the classical culturing technique. The PMA-qPCR let us individuating VBNC V. parahaemolyticus in the 20,78% of the samples evaluated with a value between the Log 10-1 and Log 10-3 CFU/g. Only clams samples were positive for PMA-qPCR detection. Conclusion: The present research is the first evaluating PMA-qPCR assay for detection of VBNC V. parahaemolyticus in bivalve molluscs samples, and the used method was applicable to the rapid control of marketed bivalve molluscs. We strongly recommend to use of PMA-qPCR in order to identify VBNC forms, undetectable by the classic microbiological methods. A precise knowledge of the V.parahaemolyticus in a VBNC form is fundamental for the correct risk assessment not only in bivalve molluscs but also in other seafood.

Keywords: food safety, frozen bivalve molluscs, PMA dye, Real-time PCR, VBNC state, Vibrio parahaemolyticus

Procedia PDF Downloads 139

Authors: Shu Hwang Ang, Choo Yee Yu, Geik Yong Ang, Yean Yean Chan, Yatimah Binti Alias, And Sook Mei Khor

Abstract:

With the high prevalence of Type 2 diabetes mellitus across the world, the morbidities and mortalities associated with Type 2 diabetes have significant impact on the production line for a nation. With routine scheduled clinical visits to manage Type 2 diabetes, diabetic patients with hectic lifestyles can have low clinical compliance. Hence, it often decreases the effectiveness of diabetic management personalized for each diabetic patient. Here, we report a useful developed point-of-care (POC) device that detect glycated hemoglobin (HbA1c, biomarker for long-term Type 2 diabetic management). In fact, the established POC devices certified to be used in clinical setting are not only expensive ($ 8 to $10 per test), they also require skillful practitioners to perform sampling and interpretation. As a paper-based biosensor, the developed HbA1c biosensor utilized lateral flow principle to offer an alternative for cost-effective (approximately $2 per test) and end-user friendly device for household testing. Requiring as little as 2 L of finger-picked blood, the test can be performed at the household with just simple dilution and washings. With visual interpretation of numbers of test lines shown on the developed biosensor, it can be interpreted as easy as a urine pregnancy test, aided with scale of intensity provided. In summary, the developed HbA1c immunosensor has been tested to have high selectivity towards HbA1c, and is stable with reasonably good performance in clinical testing. Therefore, our developed HbA1c immunosensor has high potential to be an effective diabetic management tool to increase patient compliance and thus contain the progression of the diabetes.

Keywords: blood, glycated hemoglobin (HbA1c), lateral flow, type 2 diabetes mellitus

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Authors: Armenio Lopes, Carlos Ferreira

Abstract:

In the specific context of the Iberian Union between 1580 and 1640, characteristics emerged in Portuguese architecture that stood out from the main architectural production of the period. Recognised and identified aspects that had begun making their appearance decades before (1521) became significantly more marked during the Hapsburg-Spanish occupation. Distinctive even from the imperialist language of Spain, this trend would endure even after the restoration of independence (1706), continuing through to the start of the age of absolutism. Or perhaps not. This trend, recognised as Plain Style (Kubler), associated with a certain scarcity of resources, involved a certain formal and decorative simplification, as well as a particular set of conventions that would subsequently mark the landscape. This expression could also be seen as a means of asserting a certain spirit of independence as the Iberian Union breathed its last. The image of a simple, bare-bones architecture with purer design lines is associated by various authors –most notably Kubler– with the narratives of modernism, to whose principles it is similar, in a context-specific to the period. There is a contrast with some of the exuberance of the baroque or its expression in the Manueline period, in a similar fashion to modernism's responses to nineteenth-century eclecticism. This assertion and practice of simple architecture, drafted from the interpretation of the treaties, and highlighting a certain classical inspiration, was to become a benchmark in the theory of architecture, spanning the Baroque and Mannerism, until achieving contemporary recognition within certain originality and modernity. At a time when the baroque and its scenography became generally very widespread, it is important also to recognise the role played by plain style architecture in the construction of a rather complex and contradictory waterfront landscape, featuring promises of exuberance and more discrete practices.

Keywords: Carlos Mardel, Lisbon's waterfront, plain style, urban image on the waterfront

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Authors: Weiying Wang, Qi Li, Huiwen Deng, Weirong Chen

Abstract:

The abrupt light change and uneven illumination will make the PV system get rid of constant output power, which will affect the efficiency of the grid connected inverter as well as the stability of the system. To solve this problem, this paper presents a strategy to control the stability of photovoltaic power system under the condition of partial shading of PV array, leading to constant power output, improving the capacity of resisting interferences. Firstly, a photovoltaic cell model considering the partial shading is established, and the backtracking search algorithm is used as the maximum power point to track algorithm under complex illumination. Then, the energy storage system based on the constant power control strategy is used to achieve constant power output. Finally, the effectiveness and correctness of the proposed control method are verified by the joint simulation of MATLAB/Simulink and RTLAB simulation platform.

Keywords: backtracking search algorithm, constant power control, hybrid system, partial shading, stability

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Authors: Dandan Shen, Hui-zhu Wang, Xi Yu, LiLi Gui, Xiao Wei, Xiao-yan Jiang, Da-wei Wang, Min Hong

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Yu-ping-feng-san (YPFS) is a clinical medicine for asthma and other allergic diseases, but the mechanism of YPFS on relapse of allergy is unclear. Currently, people come to realize the epithelial cells(EC) play a key role in stimulating and regulating local immune response. The study of thymic stromal lymphopoietin（TSLP derived from EC provides an important evidence that the EC can regulate immune response to stimulate allergic response. In this study, we observed the effect of YPFS on TSLP in vivo and in vitro. We established a method by using bronchial epithelial cells (16HBE) for screening potential bioactive components by HPLC-MS in YPFS and then analyzed the components in serum containing YPFS by UPLC-MS. The results showed that YPFS could decrease TSLP protein level in OVA-sensitized mice and 16HBE cells. Five components combing with the 16HBE cells were both detected in the serum.

Keywords: TSLP, bronchial epithelial cells, cell-binding, drug-containing serum

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Authors: P. Moskal, T. Bednarski, P. Bialas, E. Czerwinski, A. Gajos, A. Gruntowski, D. Kaminska, L. Kaplon, G. Korcyl, P. Kowalski, T. Kozik, W. Krzemien, E. Kubicz, Sz. Niedzwiecki, M. Palka, L. Raczynski, Z. Rudy, P. Salabura, N. G. Sharma, M. Silarski, A. Slomski, J. Smyrski, A. Strzelecki, A. Wieczorek, W. Wislicki, M. Zielinski, N. Zon

Abstract:

A new concept and results of the performance tests of the TOF-PET detection system developed at the Jagiellonian University will be presented. The novelty of the concept lies in employing long strips of polymer scintillators instead of crystals as detectors of annihilation quanta, and in using predominantly the timing of signals instead of their amplitudes for the reconstruction of Lines-of-Response. The diagnostic chamber consists of plastic scintillator strips readout by pairs of photo multipliers arranged axially around a cylindrical surface. To take advantage of the superior timing properties of plastic scintillators the signals are probed in the voltage domain with the accuracy of 20 ps by a newly developed electronics, and the data are collected by the novel trigger-less and reconfigurable data acquisition system. The hit-position and hit-time are reconstructed by the dedicated reconstruction methods based on the compressing sensing theory and the library of synchronized model signals. The solutions are subject to twelve patent applications. So far a time-of-flight resolution of ~120 ps (sigma) was achieved for a double-strip prototype with 30 cm field-of-view (FOV). It is by more than a factor of two better than TOF resolution achievable in current TOF-PET modalities and at the same time the FOV of 30 cm long prototype is significantly larger with respect to typical commercial PET devices. The Jagiellonian PET (J-PET) detector with plastic scintillators arranged axially possesses also another advantage. Its diagnostic chamber is free of any electronic devices and magnetic materials thus giving unique possibilities of combining J-PET with CT and J-PET with MRI for scanning the same part of a patient at the same time with both methods.

Keywords: PET-CT, PET-MRI, TOF-PET, scintillator

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Authors: Amna B. Medani, M. A. Elbadwi Samia, Hassan A. Khalid

Abstract:

In our study, nine Nubian goat kids were obtained, allotted into three groups, and healthily adapted in pens within the premises of the Veterinary Teaching Hospital, University of Khartoum to be given the oral doses of the dried herb shoots at daily doses of 1 and 5 gm/kg/day with drinking water, while the kids of the control group were left undosed. All goats were slaughtered,if not died, after 35 days. S. argel at the given doses caused signs of arched posture, ruffled hair, shivering and paralysis of limbs. On post mortem, lesions were seen to be hepatic fatty changes, renal necrosis, congested lungs and inflamed intestines. Serum chemistry investigations revealed significant increase (P< 0.05-0.01) in the activities of ALP(alkaline phosphates) and AST( aspartate-aminotransferase) in goats dosed with 5 gm /kg/ day. Also observed were significant increases in inorganic phosphorus and urea concentrations (P < 0.05-0.01) in both dosed goat groups. .Other investigations including the activity of GGT( gamma glutamyltransferase), creatinine, calcium, total protein and albumin illustrated no significant difference from that of the undosed controls. On haematological evaluation , the goat kids dosed with 5 gm/kg/dayshowed a decrease in haemoglobin concentration and red blood cells count of (P < 0.05-0.01).Both groups of dosed goats showed a higher packed cell volume values of (P < 0.05) when compared to the control goats .Mean corpuscular haemoglobin values were not different from those of the control kids. S. argel at the given doses caused signs of arched posture, ruffled hair, shivering and paralysis of limbs. On post mortem, lesions were seen to be hepatic fatty changes, renal necrosis, congested lungs and inflamed intestines. Serum chemistry investigations revealed significant increase (P < 0.05-0.01) in the activities of ALP(alkaline phosphates) and AST( aspartate-aminotransferase) in goats dosed with 5 gm /kg/ day. Also observed were significant increases in inorganic phosphorus and urea concentrations (P < 0.05-0.01) in both dosed goat groups. .Other investigations including the activity of GGT( gamma-glutamyltransferase), creatinine, calcium, total protein and albumin illustrated no significant difference from that of the undosed controls. calcium, total protein and albumin illustrated no significant difference from that of the undosed controls. On haematological evaluation , the goat kids dosed with 5 gm/kg/dayshowed a decrease in haemoglobin concentration and red blood cells count of (P < 0.05-0.01).Both groups of dosed goats showed a higher packed cell volume values of (P < 0.05) when compared to the control goats .Mean corpuscular haemoglobin values were not different from those of the control kids. Data obtained were then discussed to find S. argel irritable to intestines , toxic to the kidney and liver and a haematological mild toxin.Suggestions for future were forwarded.

Keywords: hargal, nubian goats, solenstemma argel, toxicity

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Authors: Chih-Hung Liang, Jung-Jung Chen, Shen-Shih Chiang

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Obesity is defined as abnormal or excessive fat accumulation that presents a risk to health, which are major risk factors for a number of chronic diseases, including diabetes, cardiovascular diseases and cancer. Cordyceps militaris (CM) is a well-known traditional medicine in Asian countries and a rich source of biologically active components. Black rice (Oryza sativa L.) is a special cultivar of rice that contains rich anthocyanins and regarded as a health-promoting food in China and other Eastern. The aim of this study was to investigate the anti-obesity effect of Cordyceps militaris fermented black rice (CB) on HFD-induced BALB/c mice model. The results indicated that administration of low and high dosage of CB powder significantly reduced the body weights (7.38% and 7.78%), body fat ratio (2.37% and 2.78%), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels compared to the HF group (p<0.05). Histopathological analysis showed that the score of fatty liver in HF group (5.0) was significantly higher than CB groups (2.1 and 3.6) (p<0.05). In conclusion, Cordyceps militaris fermented black rice can reduce the body weight via inhibition of the fat accumulation in liver and body and possess the anti-obesity potency.

Keywords: Cordyceps militaris, black rice, obesity, HFD-induced mice

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Authors: Bayan Almofty, Yuto Yamaki, Tadamasa Terai, Sadahito Uto

Abstract:

Culture techniques of skeletal muscle cells are advanced in the field of regenerative medicine and applied research of cultured muscle. As applied research of cultured muscle, myopathy (muscles disease) treatment is expected and development bio of actuator is also expected in biomedical engineering. Grayanotoxins (GTXs) is known as neurotoxins that enhance the permeability of cell membrane for Na ions. Grayanotoxins are extracted from a famous Pieris japonica and Ericaceae as well as a phytotoxin. In this study, we investigated the effect of GTXs on muscle cells (C2C12) contraction and membrane potential. Contraction of myotubes is induced by applied external electrical stimulation. Contraction and membrane potential change of skeletal muscle cells are induced by injection of current. We, therefore, concluded that effect of Grayanotoxins on contraction and membrane potential of C2C12 relate to acute toxicity of GTXs.

Keywords: skeletal muscle cells C2C12, grayanotoxins, contraction, membrane potential, acute toxicity, pytotoxin, motubes

Procedia PDF Downloads 505

Authors: Rajkumar Ghosh, Bhabani Prasad Mukhopadhay, Ananya Mukhopadhyay, Harendra Nath Bhattacharya

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This study investigates the global issue of electronic waste (e-waste), focusing on its prevalence in India and other regions. E-waste has emerged as a significant worldwide problem, with India contributing a substantial share of annual e-waste generation. The primary sources of e-waste in India are computer equipment and mobile phones. Many developed nations utilize India as a dumping ground for their e-waste, with major contributions from the United States, China, Europe, Taiwan, South Korea, and Japan. The study identifies Maharashtra, Tamil Nadu, Mumbai, and Delhi as prominent contributors to India's e-waste crisis. This issue is contextualized within the broader framework of the United Nations' 2030 Agenda for Sustainable Development, which encompasses 17 Sustainable Development Goals (SDGs) and 169 associated targets to address poverty, environmental preservation, and universal prosperity. The study underscores the interconnectedness of e-waste management with several SDGs, including health, clean water, economic growth, sustainable cities, responsible consumption, and ocean conservation. Central Pollution Control Board (CPCB) data reveals that e-waste generation surpasses that of plastic waste, increasing annually at a rate of 31%. However, only 20% of electronic waste is recycled through organized and regulated methods in underdeveloped nations. In Europe, efficient e-waste management stands at just 35%. E-waste pollution poses serious threats to soil, groundwater, and public health due to toxic components such as mercury, lead, bromine, and arsenic. Long-term exposure to these toxins, notably arsenic in microchips, has been linked to severe health issues, including cancer, neurological damage, and skin disorders. Lead exposure, particularly concerning for children, can result in brain damage, kidney problems, and blood disorders. The study highlights the problematic transboundary movement of e-waste, with approximately 352,474 metric tonnes of electronic waste illegally shipped from Europe to developing nations annually, mainly to Africa, including Nigeria, Ghana, and Tanzania. Effective e-waste management, underpinned by appropriate infrastructure, regulations, and policies, offers opportunities for job creation and aligns with the objectives of the 2030 Agenda for SDGs, especially in the realms of decent work, economic growth, and responsible production and consumption. E-waste represents hazardous pollutants and valuable secondary resources, making it a focal point for anthropogenic resource exploitation. The United Nations estimates that e-waste holds potential secondary raw materials worth around 55 billion Euros. The study also identifies numerous challenges in e-waste management, encompassing the sheer volume of e-waste, child labor, inadequate legislation, insufficient infrastructure, health concerns, lack of incentive schemes, limited awareness, e-waste imports, high costs associated with recycling plant establishment, and more. To mitigate these issues, the study offers several solutions, such as providing tax incentives for scrap dealers, implementing reward and reprimand systems for e-waste management compliance, offering training on e-waste handling, promoting responsible e-waste disposal, advancing recycling technologies, regulating e-waste imports, and ensuring the safe disposal of domestic e-waste. A mechanism, Buy-Back programs, will compensate customers in cash when they deposit unwanted digital products. This E-waste could contain any portable electronic device, such as cell phones, computers, tablets, etc. Addressing the e-waste predicament necessitates a multi-faceted approach involving government regulations, industry initiatives, public awareness campaigns, and international cooperation to minimize environmental and health repercussions while harnessing the economic potential of recycling and responsible management.

Keywords: e-waste management, sustainable development goal, e-waste disposal, recycling technology, buy-back policy

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Authors: Maurizio E. Picarella, Ludovica Fumelli, Francesca Siligato, Andrea Mazzucato

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Studies on reproductive dynamics in crops subjected to heat stress are crucial to breed more tolerant cultivars. In tomato, cultivars, breeding lines, and wild species have been thoroughly evaluated for the response to heat stress in several studies. Here, we address the reaction to temperature stress in a panel of selected landraces representing genotypes cultivated before the advent of professional varieties that usually show high adaptation to local environments. We adopted an experimental design with two open field trials, where transplanting was spaced by one month. In the second field, plants were thus subjected to mild stress with natural temperature fluctuations. The genotypes showed wide variation for both vegetative (plant height) and reproductive (stigma exsertion, pollen viability, number of flowers per inflorescence, and fruit set) traits. On average, all traits were affected by heat conditions; except for the number of flowers per inflorescence, the “G*E” interaction was always significant. In agreement with studies based on different materials, estimated broad sense heritability was high for plant height, stigma exsertion, and pollen viability and low for the number of flowers per inflorescence and fruit set. Despite the interaction, traits recorded in control and in heat conditions were positively correlated. The first two principal components estimated by multivariate analysis explained more than 50% of the total variability. The study indicated that landraces present a wide variability for the response of reproductive traits to temperature stress and that such variability could be very informative to dissect the traits with higher heritability and identify new QTL useful for breeding more resilient varieties.

Keywords: fruit set, heat stress, solanum lycopersicum L., style exsertion, tomato

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Authors: Amin Abbasi, Mahdi Asghari Ozma

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Background and Objective:Enterococcus faecium is a normal flora of the human gastrointestinal tract that causes infection in the host body under conditions such as biofilm formation, in which the use of antibiotics causes changes in these pathogenic mechanisms. In this study, we aimed to evaluate comprehensively the changes in E.faecium when exposed to sub-MIC of the gentamicin,especiallythe biofilm formation rate. Materials and Methods: For this study, the keywords "Enterococcus faecium ", "Biofilm", and "Gentamicin" in the databases PubMed, Google Scholar, Sid, and MagIran between 2015 and 2021 were searched, and 14 articles were chosen, studied, and analyzed. Results: Gentamicin significantly had increased biofilm formation in most of the isolates in the studies. Increased expression of the genes (efaA and esp) and proteins involved in biofilm formation and decreased expression of the genes (gelE and cylA) involved in spreading and proteins involved in metabolism and cell division in E.faecium were the most significant cause of the biofilm formation, which were increased in sub-MIC gentamicin-treated situation. Conclusion: Inadequate use of gentamicin intensify biofilm formation of E.faecium, which can make the treatment of infections caused by this bacterium difficult.

Keywords: biofilm, enterococcus faecium, gentamicin, proteome

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Authors: Kwang-Ho Kim, Kwan-Hong Min, Pyungwoo Jang, Chisup Jung, Kyu Seomoon

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By restricting the dimensions of silicon to less than Bohr radius of bulk crystalline silicon (∼5 nm), quantum confinement causes its effective bandgap to increase. Therefore, silicon quantum wells (QWs) using these quantum phenomena could be a good candidate to achieve high performance silicon solar cells. The Al2O3/Si QW structures were fabricated by using the successive deposition technique, as a quantum confinement device to increase the effective energy bandgap and passivation effect in Si surface for the 3rd generation solar cell applications. In Si/Al2O3 QWs, the thicknesses of Si layers and Al2O3 layers were varied between 1 to 5 nm, respectively. The roughness of deposited Si on Al2O3 was less than 4 Å in the thickness of 2 nm. By using the Al2O3/Si QW structures on Si surfaces, the lifetime measured by u-PCD technique increased as a result of passivated surface effects. The discussion about the other properties such as electrical and optical properties of the QWs structures as well as the fabricated solar cells will be presented in this paper.

Keywords: Al2O3/Si quantum well, quantum confinement, solar cells, third generation, successive deposition technique

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Authors: Promothes Saha

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Recently, transportation engineering industry members at the study university showed concern that students lacked the skills needed to solve real-world engineering problems using spreadsheet data analysis. In response to the concerns shown by industry members, this study investigated how to engage students in a better way by incorporating spreadsheet analysis during class - also, help them learn the course topics. Helping students link theoretical knowledge to real-world problems can be a challenge. In this effort, in-class activities and worksheets were redesigned to integrate with Excel to solve example problems using built-in tools including cell referencing, equations, data analysis tool pack, solver tool, conditional formatting, charts, etc. The effectiveness of this technique was investigated using students’ evaluations of the course, enrollment data, and students’ comments. Based on the data of those criteria, it is evident that the spreadsheet activities may increase student learning.

Keywords: civil, engineering, active learning, transportation

Procedia PDF Downloads 138

Authors: P. S. Surya Meghana, K. Lingeshwaran, C. Kannan, V. Raghavendran, C. Priya

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One of the most basic and powerful tools in all of science and medicine is the light microscope, the fundamental device for laboratory as well as research purposes. With the improving technology, the need for portable, economic and user-friendly instruments is in high demand. The conventional microscope fails to live up to the emerging trend. Also, adequate access to healthcare is not widely available, especially in developing countries. The most basic step towards the curing of a malady is the diagnosis of the disease itself. The main aim of this paper is to diagnose Malaria with the most common device, cell phones, which prove to be the immediate solution for most of the modern day needs with the development of wireless infrastructure allowing to compute and communicate on the move. This opened up the opportunity to develop novel imaging, sensing, and diagnostics platforms using mobile phones as an underlying platform to address the global demand for accurate, sensitive, cost-effective, and field-portable measurement devices for use in remote and resource-limited settings around the world.

Keywords: cellular, hand-held, health care, image processing, malarial parasites, microscope

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Authors: Bo Ram Choi, Ji Su Kim, Juyeon Cho, Hyukjin Lee

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Food contaminated by bacteria (E.coli), causes food poisoning, which occurs to many patients worldwide annually. We have introduced an application of rolling circle amplification (RCA) as a versatile biosensor and developed a diagnostic platform composed of capillary tube and microbeads for rapid and easy detection of Escherichia coli (E. coli). When specific mRNA of E.coli is extracted from cell lysis, rolling circle amplification (RCA) of DNA template can be achieved and can be visualized by beads aggregation in capillary tube. In contrast, if there is no bacterial pathogen in sample, no beads aggregation can be seen. This assay is possible to detect visually target gene without specific equipment. It is likely to the development of a genetic kit for point of care testing (POCT) that can detect target gene using microbeads.

Keywords: rolling circle amplification (RCA), Escherichia coli (E. coli), point of care testing (POCT), beads aggregation, capillary tube

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Authors: Choo Fatt Foo

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The Five Aggregates is core to Buddhism teaching. According to Buddhism, human beings and all sentient beings are made up of nothing but the Five Aggregates. If that is the case, the Five Aggregates must be found in all natural sciences. So far, there has not been any systematic connection between the Five Aggregates and natural sciences. This study aims at identifying traces of the Five Aggregates in various levels of natural sciences and pointing possible directions for future research. The following areas are briefly explored to identify the connection with the Five Aggregates: physics, chemistry, organic chemistry, DNA, cell, and human body and brain. Traces of the Five Aggregates should be found in each level of this hierarchy of natural sciences for human and sentient beings to be said to be made up of the Five Aggregates. This study proposes a hierarchical structure of nature cutting every level with the Five Aggregates and the Four Great Elements as its basis. The structure proposed by this study would revolutionize how we look at nature. Hopefully, better understanding of sciences in this manner will steer the application of scientific methods and technology towards a brighter future with compassion and tolerance.

Keywords: the five aggregates, Buddhism, four great elements, physics, calabi-yau manifold

Procedia PDF Downloads 190

Authors: David A. Harness

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Natural evolution of ATP cognitive systems is to meet AI peer review standards. ATP process of axiom selection from Mizar to prove a conjecture would be further refined, as in all human and machine learning, by solving the real world problem of the proposed AI peer review challenge: Determine which conjecture forms the higher confidence level constructive proof between Standard Model of Physics SU(n) lattice gauge group operation vs. present non-standard 4D GEM EOS SU(n) lattice gauge group spatially extended operation in which the photon and electron are the first two trace angular momentum invariants of a gravitoelectromagnetic (GEM) energy momentum density tensor wavetrain integration spin-stress pressure-volume equation of state (EOS), initiated via 32 lines of Mathematica code. Resulting gravitoelectromagnetic spectrum ranges from compressive through rarefactive of the central cosmological constant vacuum energy density in units of pascals. Said self-adjoint group operation exclusively operates on the stress energy momentum tensor of the Einstein field equations, introducing quantization directly on the 4D spacetime level, essentially reformulating the Yang-Mills virtual superpositioned particle compounded lattice gauge groups quantization of the vacuum—into a single hyper-complex multi-valued GEM U(1) × SU(1,3) lattice gauge group Planck spacetime mesh quantization of the vacuum. Thus the Mizar corpus already contains all of the axioms required for relevant DeepMath premise selection and unambiguous formal natural language parsing in context deep learning.

Keywords: automated theorem proving, constructive quantum field theory, information theory, neural networks

Procedia PDF Downloads 179

Authors: D. Ficai, A. Ficai, D. Gudovan, I. A. Gudovan, I. Ardelean, R. Trusca, E. Andronescu, V. Mitran, A. Cimpean

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In the last years, scientists struggled hardly to mimic bone structures to develop implants and biostructures which present higher biocompatibility and reduced rejection rate. One way to obtain this goal is to use similar materials as that of bone, namely collagen/hydroxyapatite composite materials. However, it is very important to tailor both compositions but also the microstructure of the bone that would ensure both the optimal osteointegartion and the mechanical properties required by the application. In this study, new collagen/hydroxyapatites composite materials doped with Cu, Li, Mn, Zn were successfully prepared. The synthesis method is described below: weight the Ca(OH)₂ mass, i.e., 7,3067g, and ZnCl₂ (0.134g), CuSO₄ (0.159g), LiCO₃ (0.133g), MnCl₂.4H₂O (0.1971g), and suspend in 100ml distilled water under magnetic stirring. The solution thus obtained is added a solution of NaH₂PO₄*H2O (8.247g dissolved in 50ml distilled water) under slow dropping of 1 ml/min followed by adjusting the pH to 9.5 with HCl and finally filter and wash until neutral pH. The as-obtained slurry was dried in the oven at 80°C and then calcined at 600°C in order to ensure a proper purification of the final product of organic phases, also inducing a proper sterilization of the mixture before insertion into the collagen matrix. The collagen/hydroxyapatite composite materials are tailored from morphological point of view to optimize their biocompatibility and bio-integration against mechanical properties whereas the addition of the dopants is aimed to improve the biological activity of the samples. The addition of transitional metals can improve the biocompatibility and especially the osteoblasts adhesion (Mn²⁺) or to induce slightly better osteoblast differentiation of the osteoblast, Zn²⁺ being a cofactor for many enzymes including those responsible for cell differentiation. If the amount is too high, the final material can become toxic and lose all of its biocompatibility. In order to achieve a good biocompatibility and not reach the cytotoxic effect, the amount of transitional metals added has to be maintained at low levels (0.5% molar). The amount of transitional metals entering into the elemental cell of HA will be verified using inductively-coupled plasma mass spectrometric system. This highly sensitive technique is necessary, because, at such low levels of transitional metals, the difference between biocompatible and cytotoxic is a very thin line, thus requiring proper and thorough investigation using a precise technique. In order to determine the structure and morphology of the obtained composite materials, IR spectroscopy, X-Ray diffraction (XRD), scanning electron microscopy (SEM), and Energy Dispersive X-Ray Spectrometry (EDS) were used. Acknowledgment: The present work was possible due to the EU-funding grant POSCCE-A2O2.2.1-2013-1, Project No. 638/12.03.2014, code SMIS-CSNR 48652. The financial contribution received from the national project “Biomimetic porous structures obtained by 3D printing developed for bone tissue engineering (BIOGRAFTPRINT), No. 127PED/2017 is also highly acknowledged.

Keywords: collagen, composite materials, hydroxyapatite, bone tissue engineering

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Authors: Saila Tykkyläinen

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The purpose of this study is to pave the way for more focused accumulation of knowledge on social enterprise growth. The body of research touching upon the phenomenon is somewhat fragmented. In order to make an effort to create a solid common ground, this study draws from the theoretical starting points and guidelines developed within small firm growth research. By analyzing their use in social enterprise growth literature, the study offers insights on whether the proven theories and concepts from small firm context could be more systematically applied when investigating growth of social enterprises. Towards this end, the main findings from social enterprise growth research are classified under the three research streams on growth. One of them focuses on factors of growth, another investigates growth as a process and the third is interested in outcomes of growth. During the analysis, special attention is paid on exploring how social mission of the company and the pursuit of augmenting its social impact are dealt within those lines of research. The next step is to scrutinize and discuss some of the central building blocks of growth research, namely the unit of analysis, conceptualization of a firm and operationalizing growth, in relation to social enterprise studies. It appears that the social enterprise growth literature stresses the significance of 'social' both as a main driver and principle outcome of growth. As for the growth process, this emphasis is manifested by special interest in strategies and models tailored to disseminate social impact beyond organizational limits. Consequently, this study promotes more frequent use of business activity as a unit of analysis in the social enterprise context. Most of the times, it is their products, services or programs with which social enterprises and entrepreneurs aim to create the impact. Thus the focus should be placed on activities rather than on organizations. The study also seeks to contribute back to the small firm growth research. Even though the recommendation to think of business activities as an option for unit of analysis stems from there, it is all too rarely used. Social entrepreneurship makes a good case for testing and developing the approach further.

Keywords: conceptual study, growth, scaling, social enterprise

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Authors: Pei-Wen Lin, Ta-I Yang

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Cancer has been one of the leading causes of human death for centuries. Considerable effort has been devoted to developing new treatments to reduce and control cancers. Magnetic particle hyperthermia and near-infrared photothermal therapy are the promising strategies to treat cancers due to its effectiveness with only mild side effects. This study focused on synthesizing magnetic Ag@Fe3O4 nanoparticles applicable for both of magnetic hyperthermia and near-infrared photothermal therapy. The hydrophilic poly(diallyldimethylammonium chloride) polymer was utilized to prepare superparamagnetic Fe3O4 clusters and to promote silver nanoparticles grown on Fe3O4 surfaces, obtaining Ag@Fe3O4 nanoparticles. The morphology (shape and dimension) of Ag nanoparticles was subsequently tailored using commercial LED lights. Therefore, the resulting Ag@Fe3O4 nanoparticles can absorb specific wavelength of light ranging from 400 nm to 800 nm by adjusting the wavelength of LED lights and the free silver ions in reaction solution. Heating performance tests confirmed that the synthesized Ag@Fe3O4 nanoparticles show appreciable heating capability for both of magnetic particle hyperthermia and near-infrared photothermal therapy. The findings in this study could provide new ideas to design functional materials to treat cancers.

Keywords: light-emitting diode assisted synthesis, magnetic particles, photothermal materials, hyperthermia

Procedia PDF Downloads 284