Search results for: esophagus squamous cell carcinoma

Authors: Joseph M. Rich, Vinay A. Duddalwar, Assad A. Oberai

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Prostate adenocarcinoma is the most common cancer in males, with osseous metastases as the commonest site of metastatic prostate carcinoma (mPC). Treatment monitoring is based on the evaluation and characterization of lesions on multiple imaging studies, including Computed Tomography (CT). Monitoring of the osseous disease burden, including follow-up of lesions and identification and characterization of new lesions, is a laborious task for radiologists. Deep learning algorithms are increasingly used to perform tasks such as identification and segmentation for osseous metastatic disease and provide accurate information regarding metastatic burden. Here, nnUNet was used to produce a model which can segment CT scan images of prostate adenocarcinoma vertebral bone metastatic lesions. nnUNet is an open-source Python package that adds optimizations to deep learning-based UNet architecture but has not been extensively combined with transfer learning techniques due to the absence of a readily available functionality of this method. The IRB-approved study data set includes imaging studies from patients with mPC who were enrolled in clinical trials at the University of Southern California (USC) Health Science Campus and Los Angeles County (LAC)/USC medical center. Manual segmentation of metastatic lesions was completed by an expert radiologist Dr. Vinay Duddalwar (20+ years in radiology and oncologic imaging), to serve as ground truths for the automated segmentation. Despite nnUNet’s success on some medical segmentation tasks, it only produced an average Dice Similarity Coefficient (DSC) of 0.31 on the USC dataset. DSC results fell in a bimodal distribution, with most scores falling either over 0.66 (reasonably accurate) or at 0 (no lesion detected). Applying more aggressive data augmentation techniques dropped the DSC to 0.15, and reducing the number of epochs reduced the DSC to below 0.1. Datasets have been identified for transfer learning, which involve balancing between size and similarity of the dataset. Identified datasets include the Pancreas data from the Medical Segmentation Decathlon, Pelvic Reference Data, and CT volumes with multiple organ segmentations (CT-ORG). Some of the challenges of producing an accurate model from the USC dataset include small dataset size (115 images), 2D data (as nnUNet generally performs better on 3D data), and the limited amount of public data capturing annotated CT images of bone lesions. Optimizations and improvements will be made by applying transfer learning and generative methods, including incorporating generative adversarial networks and diffusion models in order to augment the dataset. Performance with different libraries, including MONAI and custom architectures with Pytorch, will be compared. In the future, molecular correlations will be tracked with radiologic features for the purpose of multimodal composite biomarker identification. Once validated, these models will be incorporated into evaluation workflows to optimize radiologist evaluation. Our work demonstrates the challenges of applying automated image segmentation to small medical datasets and lays a foundation for techniques to improve performance. As machine learning models become increasingly incorporated into the workflow of radiologists, these findings will help improve the speed and accuracy of vertebral metastatic lesions detection.

Keywords: deep learning, image segmentation, medicine, nnUNet, prostate carcinoma, radiomics

Procedia PDF Downloads 97

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

Procedia PDF Downloads 112

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)

Procedia PDF Downloads 341

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

Procedia PDF Downloads 39

Authors: Eddy K. Mukadi

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This work is a documentary and descriptive study devoted to the epidemiological, clinical, histopathological and therapeutic profile of breast cancer deals with the department of gynecology and obstetrics of the university clinics of Kinshasa during the period from 1 January 2014 to 31 December 2014. We have identified 56 cases of breast cancer. These cancers accounted for 45.2% of gynecological mammary cancers. The youngest in our series was 18 years old while the oldest was 74 years old; And the mean age of these patients was 43.4 years and mostly multiparous (35.7%). Brides (60.7%) and bachelors (26.8%) were the most affected by breast cancer. The reasons for consultation were dominated by nodules in the breast (48.2%) followed by pain (35.7%) and nipple discharge (14.3%). In 89.2% of the cases, it was the advanced clinical stage (stage 3 and 4) and the infiltrating ductal carcinoma was the most frequent histological type (75%) The malignant tumor was mainly in the left breast (55.3%), and chemotherapy with hormone therapy and patey was the most convenient treatment (42.8%), while patey mastectomy was performed in 12.5% of patients. Because of the high incidence of breast cancer identified in our study, some preventive measures must be taken into account to address this public health problem, including breast autopalpation once a month, Early detection system development of a national breast cancer policy and the implementation of a national breast cancer control program.

Keywords: breast cancer, histopathological profile, epidemiological profile, Kinshasa

Procedia PDF Downloads 217

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

Procedia PDF Downloads 95

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

Procedia PDF Downloads 267

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

Procedia PDF Downloads 302

Authors: Nkechi V. Enwuru, Bullum Nkeki, Elizabeth A. Adekoya, Olumide A. Adebesin, Rebecca F. Peters, Victoria A. Aikhomu, Mendie E. U.

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Background: Probiotics are live microbial feed supplement beneficial for host. Probiotics and their postbiotic products have been used to prevent or treat various health conditions. However, the products cell viability is often low due to harsh conditions subjected during processing, handling, storage, and gastrointestinal transit. These strongly influence probiotics’ benefits; thus, viability is essential for probiotics to produce health benefits for the host. Microencapsulation is a promising technique with considerable effects on probiotic survival. The study is aimed to formulate a microencapsulated probiotic and evaluate its viability, antimicrobial efficacy, and cytotoxic activity of its postbiotic on the MCF-7 breast cancer cell line. Method: Human and animal raw milk were sampled for lactic acid bacteria. The isolated bacteria were identified using conventional and VITEK 2 systems. The identified lactic acid bacterium was encapsulated using spray-dried and extrusion methods. The free, encapsulated, and chitosan-coated encapsulated probiotics were tested for viability in simulated-gastric intestinal (SGI) fluid and different storage conditions at refrigerated (4oC) and room (25oC) temperatures. The disintegration time and weight uniformity of the spray-dried hard gelatin capsules were tested. The antimicrobial property of free and encapsulated probiotics was tested against enteric pathogenic isolates from antiretroviral therapy (ART) treated HIV-positive patients. The postbiotic of the free cells was extracted, and its cytotoxic effect on the MCF-7 breast cancer cell line was tested through an MTT assay. Result: The Lactobacillus plantarum was isolated from animal raw milk. Zero-size hard gelatin L. plantarum capsules with granules within a size range of 0.71–1.00 mm diameter was formulated. The disintegration time ranges from 2.14±0.045 to 2.91±0.293 minutes, while the average weight is 502.1mg. Simulated gastric solution significantly affected viability of both free and microcapsules. However, the encapsulated cells were more protected and viable due to impermeability in the microcapsules. Furthermore, the viability of free cells stored at 4oC and 25oC were less than 4 log CFU/g and 6 log CFU/g respectively after 12 weeks. However, the microcapsules stored at 4oC achieved the highest viability among the free and microcapsules stored at 25oC and the free cells stored at 4oC. Encapsulated cells were released in the simulated gastric fluid, viable and effective against the enteric pathogens tested. However, chitosan-coated calcium alginate encapsulated probiotics significantly inhibited Shigella flexneri, Candida albicans, and Escherichia coli. The Postbiotic Metabolites (PM) of L. plantarum produced a cytotoxic effect on the MCF-7 breast cancer cell line. The postbiotic showed significant cytotoxic activity similar to 5FU, a standard antineoplastic agent. The inhibition concentration of 50% growth (IC50) of postbiotic metabolite K3 is low and consistent with the IC50 of the positive control (Cisplatin). Conclusions: Lactobacillus plantarum postbiotic exhibited a cytotoxic effect on the MCF-7 breast cancer cell line and could be used as combined adjuvant therapy in breast cancer management. The microencapsulation technique protects the probiotics, improving their viability and delivery to the gastrointestinal tract. Chitosan enhances antibacterial efficacy; thus, chitosan-coated microencapsulated L. plantarum probiotics could be more effective and used as a combined therapy in HIV management of opportunistic enteric infection.

Keywords: probiotics, encapsulation, gastrointestinal conditions, antimicrobial effect, postbiotic, cytotoxicity effect

Procedia PDF Downloads 125

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

Procedia PDF Downloads 179

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

Procedia PDF Downloads 361

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

Procedia PDF Downloads 527

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: 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

Procedia PDF Downloads 185

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

Procedia PDF Downloads 129

Authors: Roland Benedict Reyes, Marc Edsel Ayes, Regina Berba, Cybele Lara Abad

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Background: Three AIDS-defining malignancies have been associated with the human immunodeficiency virus (HIV): Kaposi’s sarcoma, non-Hodgkin’s lymphoma, and cervical carcinoma. However, new cases of non-AIDS defining malignancies also have been increasingly associated with HIV. One of these is a rare intracranial malignancy, meningeal hemangiopericyotma. Case Description: A 32-year old HIV-positive male, not on highly active antiretroviral therapy, was admitted to our hospital due to generalized weakness and sudden onset hearing loss. Cranial MRI was done, which revealed a temporal nodule with the following considerations: granuloma, meningioma or metastases. A craniotomy was performed and the mass excised. Results from the biopsy showed meningeal hemangiopericytoma. The patient was then started on antiretroviral therapy (Lamivudine, Tenofovir, and Efavirenz) and was discharged for radiation therapy and metastatic work-up as an outpatient. On follow-up seven months later, metastatic work up revealed multiple hepatic foci not previously documented suggestive of metastasis short of biopsy sampling. Conclusions: This case of an intracranial hemangiopericytoma in an HIV-positive patient is the second case thus far presented, based on our systematic and extensive search of the literature.

Keywords: Hemangiopericytoma, Human Immunodeficiency Virus, Meningeal hemangiopericytoma, Neoplasm

Procedia PDF Downloads 463

Authors: Ayda Youssef, Tarek Rafaat, Iman zaky

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Purpose: Langerhans’ cell histiocytosis (LCH) is not uncommon pathology that implies aberrant proliferation of a specific dendritic (Langerhans) cell. These atypical but mature cells of monoclonal origin can infiltrate many sites of the body and may occur as localized lesions or as widespread systemic disease. Liver is one of the uncommon sites of affection. The twofold objective of this study is to illustrate the radiological presentation of this disease, and to compare these results with previously reported series. Methods and Materials: Between 2007 and 2012, 150 patients with biopsy-proven LCH were treated in our hospital, a paediatric cancer tertiary care center. A retrospective review of radiographic images and reports was performed. There were 33 patients with liver affection are stratified. All patients underwent imaging studies, mostly US and CT. A chart review was performed to obtain demographic, clinical and radiological data. They were analyzed and compared to other published series. Results: Retrospective assessment of 150 patients with LCH was performed, among them 33 patients were identified who had liver involvement. All these patients developed multisystemic disease; They were 12 females and 21 males with (n= 32), seven of them had marked hepatomegaly. Diffuse hypodense liver parenchyma was encountered in five cases, the periportal location has a certain predilection in cases of focal affection where three cases has a hypodense periportal soft tissue sheets, one of them associated with dilated biliary radicals, only one case has multiple focal lesions unrelated to portal tracts. On follow up of the patients, two cases show abnormal morphology of liver with bossy outline. Conclusion: LCH is a not infrequent disease. A high-index suspicion should be raised in the context of diagnosis of liver affection. A biopsy is recommended in the presence of radiological suspicion. Chemotherapy is the preferred therapeutic modality. Liver histiocytosis are not disease specific features but should be interpreted in conjunction with the clinical history and the results of biopsy. Clinical Relevance/Application: Radiologist should be aware of different patterns of hepatobiliary histiocytosis, Thus early diagnosis and proper management of patient can be conducted.

Keywords: langerhans’ cell histiocytosis, liver, medical and health sciences, radiology

Procedia PDF Downloads 284

Authors: Anna Cameron, Chunxia Zhao, Haofei Wang, Yun Liu, Guang Ze Yang

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Since the first publication in 1775, cancer research has built a comprehensive understanding of how cellular components of the tumor niche promote disease development. However, only within the last decade has research begun to establish the impact of non-cellular components of the niche, particularly the extracellular matrix (ECM). The ECM, a three-dimensional scaffold that sustains the tumor microenvironment, plays a crucial role in disease progression. Cancer cells actively deregulate and remodel the ECM to establish a tumor-promoting environment. Recent work has highlighted the need to further our understanding of the complexity of this cancer-ECM relationship. In vitro models use hydrogels to mimic the ECM, as hydrogel matrices offer biological compatibility and stability needed for long term cell culture. However, natural hydrogels are being used in these models verbatim, without tuning their biophysical characteristics to achieve pathophysiological relevance, thus limiting their broad use within cancer research. The biophysical attributes of these gels dictate cancer cell proliferation, invasion, metastasis, and therapeutic response. Evaluating the three most widely used natural hydrogels, Matrigel, collagen, and agarose gel, the permeability, stiffness, and pore-size of each gel were measured and compared to the in vivo environment. The pore size of all three gels fell between 0.5-6 µm, which coincides with the 0.1-5 µm in vivo pore size found in the literature. However, the stiffness for hydrogels able to support cell culture ranged between 0.05 and 0.3 kPa, which falls outside the range of 0.3-20,000 kPa reported in the literature for an in vivo ECM. Permeability was ~100x greater than in vivo measurements, due in large part to the lack of cellular components which impede permeation. Though, these measurements prove important when assessing therapeutic particle delivery, as the ECM permeability decreased with increasing particle size, with 100 nm particles exhibiting a fifth of the permeability of 10 nm particles. This work explores ways of adjusting the biophysical characteristics of hydrogels by changing protein concentration and the trade-off, which occurs due to the interdependence of these factors. The global aim of this work is to produce a more pathophysiologically relevant model for each tumor type.

Keywords: cancer, extracellular matrix, hydrogel, microfluidic

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Authors: Harish K. Chandrawanshi, Mithun S. Rajput, Neelima Choure, Purnima Dey Sarkar, Shailesh Jain

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The present research study aimed to fabricate and evaluate biodegradable nanoparticles of aromatase inhibitor letrozole, intended for breast cancer therapy. Letrozole loaded poly(D,L-lactide-co-glycolide acid) nanoparticles were prepared by solvent evaporation method using dichlorometane as solvent (oil phase) and polyvinyl alcohol (PVA) as aqueous phase. Prepared nanoparticles were characterized by particle size, infrared spectra, drug loading efficiency, drug entrapment efficiency and in vitro release and also evaluated for in vivo anticancer activity. The high speed homogenizer was used to produce stable nanoparticles of mean size range 198.35 ± 0.04 nm with high entrapment efficiency (69.86 ± 2.78%). Percentage of drug and homogenization speed significantly influenced the particle size, entrapment efficiency and release (p<0.05). The nanoparticles show significant in vivo anticancer activity against Ehrlich ascites carcinoma in mice. The significant system sustained the release of letrozole drug effectively and further investigation could exhibit its potential usefulness in breast cancer therapy.

Keywords: breast cancer/therapy, letrozole, nanoparticles, PLGA

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

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Authors: Mengfei Peng, Serajus Salaheen, Debabrata Biswas

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Lactobacillus casei found in the human intestine and mouth is commonly applied for dairy production. Recently, it was found that some byproducts produced by Lactobacillus exhibited antimicrobial activities against multiple bacteria. Meanwhile, introduction of prebiotic-like foods (e.g. cocoa) or probiotics or both of them as food supplements in human diets as well as in farm animal feeds is believed to be an effective ways in control/reduce the colonization of foodborne bacterial pathogens infection in the gut environment. We hypothesized that cocoa may stimulate the production antimicrobial components of Lactobacillus casei and may potentially inhibit/reduce the colonization and infection of foodborne bacterial pathogens in the gut. Mixed culture of L. casei (LC) with enterohemorrhagic E. coli EDL933 (EHEC), Salmonella Typhimurium LT2 (ST), or Listeria monocytogenes LM2 (LM) showed that LC could competitively exclude (100%) them within 72 h. Further, investigation of cell-free culture supernatant (CFCS) revealed that the antimicrobial effects of LC came from CFCS. CFCS of LC eliminated (100%) EHEC, ST, and LM within 72 h, and 2 h CFCS treatment increased the hydrophobicity of EHEC (5.10 folds), ST (8.48 folds), and LM (2.03 folds). In addition, LC cells exhibited more inhibitive effects than CFCS on cell adhesive and invasive activities of EHEC (52.14% & 90.45%), ST (66.89% & 93.83%), and LM (61.10% & 83.40%). Two clusters of poly-peptides in CFCS were identified by SDS-PAGE, the molecular weights of which are ≈5 KD and 40-45 KD. LC CFCS with overnight growth in the presence of 3% strengthened all of the antimicrobial activities (growth inhibition, outer membrane disruption, and cell infective ability reduction). Liquid chromatography/Mass spectrometry analysis detected 5 unique components in class of flavonoids in LC CFCS with overnight 3% cocoa supplement. Furthermore, qPCR results showed that CFCSs up-regulated the expression level of genes responsible for flagellin synthesis and motility, but down-regulated genes for specific binding and invasion-associated proteins synthesis. The stimulatory effects of cocoa in producing bioactive components of probiotics may aid prevention of foodborne illness caused by major foodborne enteric bacterial pathogens.

Keywords: foodborne pathogens, probiotics, prebiotics, pathogen exclusion

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Authors: Xin Chen, Xinyong Li, Qidong Zhao, Dong Wang

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A new coupling system was constructed by combining photo-electrochemical cell with electro-fenton cell (PEC-EF). The electrode material in this system was derived from MnyFe₁₋yCo Prussian-Blue-Analog (PBA). Mn₀.₄Fe₀.₆Co₀.₆₇-N@C spin-coated on carbon paper behaved as the gas diffusion cathode and Mn₀.₄Fe₀.₆Co₀.₆₇O₂.₂ spin-coated on fluorine-tin oxide glass (FTO) as anode. The two separated cells could degrade Sulfamethoxazole (SMX) simultaneously and some coupling mechanisms by PEC and EF enhancing the degradation efficiency were investigated. The continuous on-site generation of H₂O₂ at cathode through an oxygen reduction reaction (ORR) was realized over rotating ring-disk electrode (RRDE). The electron transfer number (n) of the ORR with Mn₀.₄Fe₀.₆Co₀.₆₇-N@C was 2.5 in the selected potential and pH range. The photo-electrochemical properties of Mn₀.₄Fe₀.₆Co₀.₆₇O₂.₂ were systematically studied, which displayed good response towards visible light. The photoinduced electrons at anode can transfer to cathode for further use. Efficient photo-electro-catalytic performance was observed in degrading SMX. Almost 100% SMX removal was achieved in 120 min. This work not only provided a highly effective technique for antibiotic treatment but also revealed the synergic effect between PEC and EF.

Keywords: electro-fenton, photo-electrochemical, synergic effect, sulfamethoxazole

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Authors: Santosh Tummidi, Pragati Sathe, Kanchan Kothari, Prachi Gholap, Mona Agnihotri, Gwendolyn Fernandes, Leena Naik, Rachana Chaturvedi

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Introduction: EUS-Guided Fine Needle Aspiration/Brush (EUS-FNA/Brush) has become increasingly popular for the diagnosis and staging of gastrointestinal and peri-gastrointestinal lesions. Objective: To evaluate the diagnostic accuracy and spectrum of lesions in gastrointestinal EUS-FNA. Material and Methods: A total of 124 EUS-FNA during the period from Aug 2015-Nov 2016 were studied. Results: Age ranged from 13-80 years with a slight female predominance. CBD was the most common site with 47 cases amongst which were 9 adenocarcinoma, and 7 cases were suspicious for malignancy. Pancreatic EUS-FNA showed 5 adenocarcinoma, 2 SPEN, 1 case each of neuroendocrine tumor, anaplastic carcinoma and NHL. Amongst oesophageal lesions, 3 cases were suspicious for malignancy, and 4 were inflammatory, 4 showed SCC, 1case each adenocarcinoma and leiomyoma. Stomach- 1 case each of adenocarcinoma, granulomatous inflammation, and GIST. Periportal lymph nodes were the commonest nodes, and there were 11 necrotising granulomatous inflammations, 3 metastatic adenocarcinoma, 2 cases of atypical cells and 1 case of NHL. 17 cases were unsatisfactory, 41 cases had histopathology follow up with 85% cases being concordant. Conclusion: EUS-FNA is reliable, sensitive and specific. It can be utilized for better management of intra-abdominal lesions.

Keywords: EUS-FNA, brush, cytology, histopathology

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Authors: Ali Mirtaghavi, Andy Baldwin, Rajendarn Muthuraj, Jack Luo

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Recent advances in biomaterials have led to utilizing biopolymers to develop 3D scaffolds in tissue regeneration. One of the major challenges of designing biomaterials for 3D scaffolds is to mimic the building blocks similar to the extracellular matrix (ECM) of the native tissues. Biopolymer based aerogels obtained by freeze-drying have shown to provide structural similarities to the ECM owing to their 3D format and a highly porous structure with interconnected pores, similar to the ECM. Gelatin (GEL) is known to be a promising biomaterial with inherent regenerative characteristics owing to its chemical similarities to the ECM in native tissue, biocompatibility abundance, cost-effectiveness and accessible functional groups, which makes it facile for chemical modifications with other biomaterials to form biocomposites. Despite such advantages, gelatin offers poor mechanical properties, sensitive enzymatic degradation and high viscosity at room temperature which limits its application and encourages its use to develop biocomposites. Hydrophilic biomass-based cellulose nanofibrous (CNF) has been explored to use as suspension for biocomposite aerogels for the development of 3D porous structures with excellent mechanical properties, biocompatibility and slow enzymatic degradation. In this work, CNF biocomposite aerogels with various ratios of CNF:GEL) (90:10, 70:30 and 50:50) were prepared by freeze-drying technique, and their properties were investigated in terms of physicochemical, mechanical and biological characteristics. Epichlorohydrin (EPH) was used to investigate the effect of chemical crosslinking on the molecular interaction of CNF: GEL, and its effects on physicochemical, mechanical and biological properties of the biocomposite aerogels. Ultimately, chemical crosslinking helped to improve the mechanical resilience of the resulting aerogels. Amongst all the CNF-GEL composites, the crosslinked CNF: GEL (70:30) biocomposite was found to be favourable for cell attachment and viability. It possessed highly porous structure (porosity of ~93%) with pore sizes ranging from 16-110 µm, adequate mechanical properties (compression modulus of ~47 kPa) and optimal biocompatibility both in-vitro and in-vivo, as well as controlled enzymatic biodegradation, high water penetration, which could be considered a suitable option for wound healing application. In-vivo experiments showed improvement on inflammation and foreign giant body cell reaction for the crosslinked CNF: GEL (70:30) compared to the other samples. This could be due to the superior interaction of CNF with gelatin through chemical crosslinking, resulting in more optimal in-vivo improvement. In-vitro cell culture investigation on human dermal fibroblasts showed satisfactory 3D cell attachment over time. Overall, it has been observed that the developed CNF: GEL aerogel can be considered as a potential scaffold for soft tissue regeneration application.

Keywords: 3D scaffolds, aerogels, Biocomposites , tissue engineering

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Authors: Mrinal Gupta, Mohammad Rumman, Babita Singh Abbas Ali Mahdi, Shivani Pandey

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Introduction: Berberine (BBR), a bioactive compound isolated from Coptidis Rhizoma, possesses diverse pharmacological activities, including anti-bacterial, anti-inflammatory, antitumor, hypolipidemic, and anti-diabetic. However, its role as an anti-diabetic agent in animal models of dexamethasone (Dex)-induced diabetes remains unknown. Studies have shown that natural compounds, including aloe, caper, cinnamon, cocoa, green and black tea, and turmeric, can be used for treating Type 2 diabetes mellitus (DM). Compared to conventional drugs, natural compounds have fewer side effects and are easily available. Herein, we studied the anti-diabetic effects of BBR in a mice model of Dex-induced diabetes. Methods: HepG2 cell line was used for glucose release and glycogen synthesis studies. Cell proliferation was measured by methylthiotetrazole (MTT) assay. For animal studies, mice were treated with Dex (2 mg/kg, i.m.) for 30 days and the effect of BBR at the doses 100, 200, and 500 mg/kg (p.o.) was analyzed. Glucose, insulin, and pyruvate tests were performed to evaluate the development of the diabetic model. An echo MRI was performed to assess the fat mass. Further, to elucidate the mechanism of action of BBR, mRNA expression of genes regulating gluconeogenesis, glucose uptake, and glycolysis were analyzed. Results: In vitro BBR had no impact on cell viability up to a concentration of 50μM. Moreover, BBR suppressed the hepatic glucose release and improved glucose tolerance in HepG2 cells. In vivo, BBR improved glucose homeostasis in diabetic mice, as evidenced by enhanced glucose clearance, increased glycolysis, elevated glucose uptake, and decreased gluconeogenesis. Further, Dex treatment increased the total fat mass in mice, which was ameliorated by BBR treatment. Conclusion: BBR improves glucose tolerance by increasing glucose clearance, inhibiting hepatic glucose release, and decreasing obesity. Thus, BBR may become a potential therapeutic agent for treating glucocorticoid-induced diabetes and obesity in the future.

Keywords: glucocorticoid, hyperglycemia, berberine, HepG2 cells, insulin resistance, glucose

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Authors: Hawon Lee, Young-Pil Kim

Abstract:

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

Keywords: collagen, ECM, FRET, MMP

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Authors: Ozlem Oral, Emre Taskin, Aysel Yuce, Serap Dokmeci, Devrim Gozuacik

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Autophagy is an evolutionary conserved lysosome-dependent catabolic pathway, responsible for the degradation of long-lived proteins, abnormal aggregates and damaged organelles which cannot be degraded by the ubiquitin-proteasome system. Lysosomes degrade the substrates through the activity of lysosomal hydrolases and lysosomal membrane-bound proteins. Mutations in the coding region of these proteins cause malfunctional lysosomes, which contributes to the pathogenesis of lysosomal storage diseases. Gaucher disease is a lysosomal storage disease resulting from the mutation of a lysosomal membrane-associated glycoprotein called glucocerebrosidase and its cofactor saposin C. The disease leads to intracellular accumulation of glucosylceramide and other glycolipids. Because of the essential role of lysosomes in autophagic degradation, Gaucher disease may directly be linked to this pathway. In this study, we investigated the expression of autophagy and/or lysosome-related genes and proteins in fibroblast cells isolated from patients with different mutations. We carried out confocal microscopy analysis and examined autophagic flux by utilizing the differential pH sensitivities of RFP and GFP in mRFP-GFP-LC3 probe. We also evaluated lysosomal pH by active lysosome staining and lysosomal enzyme activity. Beside lysosomes, we also performed proteasomal activity and cell death analysis in patient samples. Our data showed significant attenuation in the expression of key autophagy-related genes and accumulation of their proteins in mutant cells. We found decreased the ability of autophagosomes to fuse with lysosomes, associated with elevated lysosomal pH and reduced lysosomal enzyme activity. Proteasomal degradation and cell death analysis showed reduced proteolytic activity of the proteasome, which consequently leads to increased susceptibility to cell death. Our data indicate that the major degradation pathways are affected by multifunctional lysosomes in mutant patient cells and may underlie in the mechanism of clinical severity of Gaucher patients. (This project is supported by TUBITAK-3501-National Young Researchers Career Development Program, Project No: 112T130).

Keywords: autophagy, Gaucher's disease, glucocerebrosidase, mutant fibroblasts

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Authors: Manh Tin Ho, Phorl Sophors, Ga Young Seo, Young Mee Kim, Youngho Lim, Moonjae Cho

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UV radiation in sunlight is one of the most potential factor induced skin ageing and photocarcinogenesis. UV may induce the melanin production and wrinkle formation. Recently, the natural secondary compounds have been reported that had the beneficial protective effects from UV light. In this study, we investigated the effects of two different compounds, glycitin and 38, on human dermal fibroblast. We first only treated the 38 on melanocyte cell to test the proliferation inhibition of 38 on this cell line. Then, we induced the combination of glycitin and 38 on human dermal fibroblast in 48h and investigate the proliferation, collagen production and the metalloproteinase family expression. The 38 alone could inhibit the proliferation of melanocyte which indicated the reduction of melanin production. The combination of glycitin and 38 truly increased the fibroblast proliferation and even they could recover the UV-induced and H2O2-induced damaged fibroblast proliferation. The co-treatment also promoted the collagen IV expression significantly and accelerated the total collagen secretion. In addition, metalloproteinase (MMPs) family such as MMP1, MMP2, MMP7 was down-regulated in transcriptional level. In conclusion, the combination of glycitin and 38 has induced the fibroblast proliferation even when it was damaged by UV exposure and H2O2, whereas augmented collagen production and inhibited the MMPs caused the wrinkle formation and decreased the melanocyte proliferation, suggested an potential UV-protective therapy.

Keywords: UV radiation, wrinkle, ageing, glycitin, dermal fibroblast

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Authors: Sang Chul Lee, Gi-Young Park, Dong Rak Kwon

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Objective: The aim of this study was to investigate regenerative effects of ultrasound (US)-guided injection with human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and/or polydeoxyribonucleotide (PDRN) injection in a chronic traumatic full-thickness rotator cuff tendon tear (FTRCTT) in a rabbit model. Material and Methods: Rabbits (n = 32) were allocated into 4 groups. After a 5-mm sized FTRCTT just proximal to the insertion site on the subscapularis tendon was created by excision, the wound was immediately covered by silicone tube to prevent natural healing. After 6 weeks, 4 injections (0.2 mL normal saline, G1; 0.2 mL PDRN, G2; 0.2 mL UCB-MSCs, G3; and 0.2 mL UCB-MSCs with 0.2ml PDRN, G4) were injected into FTRCTT under US guidance. We evaluated gross morphologic changes on all rabbits after sacrifice. Masson’s trichrome, anti-type 1 collagen antibody, bromodeoxyuridine, proliferating cell nuclear antigen, vascular endothelial growth factor and platelet endothelial cell adhesion molecule stain were performed to evaluate histological changes. Motion analysis was also performed. Results: The gross morphologic mean tendon tear size in G3 and 4 was significantly smaller than that of G1 and 2 (p < .05). However, there were no significant differences in tendon tear size between G3 and 4. In G4, newly regenerated collagen type 1 fibers, proliferating cells activity, angiogenesis, walking distance, fast walking time, and mean walking speed were greater than in the other three groups on histological examination and motion analysis. Conclusion: Co-injection of UCB-MSCs and PDRN was more effective than UCB-MSCs injection alone in histological and motion analysis in a rabbit model of chronic traumatic FTRCTT. However, there was no significant difference in gross morphologic change of tendon tear between UCB-MSCs with/without PDRN injection. The results of this study regarding the combination of UCB-MSCs and PDRN are worth additional investigations.

Keywords: mesenchymal stem cell, umbilical cord, polydeoxyribonucleotides, shoulder, rotator cuff, ultrasonography, injections

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Authors: Bremmy Laksono, Nurul Qomarilla, Riksa Parikrama, Dyan K. Nugrahaeni, Willyanti Soewondo, Dadang S. H. Effendi, Eriska Rianti, Arlette S. Setiawan, Ine Sasmita, Risti S. Primanti, Erna Kurnikasari, Yunia Sribudiani

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Down syndrome is a chromosomal abnormality of chromosome 21 which can appear in man or woman. Maternal age and paternal age, history of radiation are the common risk factors. This study was conducted to observe risk factors which related as causes of Down syndrome. In this case control study using purposive sampling technique, 84 respondents were chosen from Cell Culture and Cytogenetics Laboratory patients in Faculty of Medicine, Universitas Padjadjaran, Indonesia. They were used as study samples and divided into 42 Down syndrome cases and 42 control respondents. This study used univariate and bivariate analysis (chi-square). Samples population were West Java residents, the biggest province in Indonesia in number of population. The results showed maternal age, paternal age, history of radiation exposure and family history were not significantly related to Down syndrome baby. Moreover, all of those factors also did not contribute to the risk of having a child with Down syndrome in patients at Cell Culture and Cytogenetics Laboratory, Faculty of Medicine, Universitas Padjadjaran. Therefore, we should investigate other risk factors of Down syndrome in West Java population.

Keywords: down syndrome, family history, maternal age, paternal age, risk factor

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