Search results for: cell invasion

Authors: Tanushree Jaitly, Shailendra Gupta, Leila Taher, Gerold Schuler, Julio Vera

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Genomics-based personalized medicine is a promising approach to fight aggressive tumors based on patient's specific tumor mutation and expression profiles. A remarkable case is, dendritic cell-based immunotherapy, in which tumor epitopes targeting patient's specific mutations are used to design a vaccine that helps in stimulating cytotoxic T cell mediated anticancer immunity. Here we present a computational pipeline for epitope-based personalized cancer vaccines using patient-specific haplotype and cancer mutation profiles. In the workflow proposed, we analyze Whole Exome Sequencing and RNA Sequencing patient data to detect patient-specific mutations and their expression level. Epitopes including the tumor mutations are computationally predicted using patient's haplotype and filtered based on their expression level, binding affinity, and immunogenicity. We calculate binding energy for each filtered major histocompatibility complex (MHC)-peptide complex using docking studies, and use this feature to select good epitope candidates further.

Keywords: cancer immunotherapy, epitope prediction, NGS data, personalized medicine

Procedia PDF Downloads 238

Authors: Marek Halenár, Eva Tvrdá, Simona Baldovská, Ľubomír Ondruška, Peter Massányi, Adriana Kolesárová

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This study aimed to assess the in vitro effects of different concentrations of the Viscum album extract on the motility, viability, and reactive oxygen species (ROS) production by rabbit spermatozoa during different time periods (0, 2, and 8h). Spermatozoa motility was assessed by using the CASA (Computer aided sperm analysis) system. Cell viability was evaluated by using the metabolic activity MTT assay, and the luminol-based luminometry was applied to quantify the ROS formation. The CASA analysis revealed that low Viscum concentrations were able to prevent a rapid decline of spermatozoa motility, especially in the case of concentrations ranging between 1 and 5 µg/mL (P<0.05 with respect to time 8h). At the same time, concentrations ranging between 1 and 100 µg/mL of the extract led to a significant preservation of the cell viability (P<0.05 in case of 5, 50 and 100 µg/mL; P<0.01 with respect to 1 and 10 µg/mL, time 8h). 1 and 5 µg/mL of the extract exhibited antioxidant characteristics, translated into a significant reduction of the ROS production, particularly notable at time 8h (P<0.01). The results indicate that the Viscum extract is capable of delaying the damage inflicted to the spermatozoon by the in vitro environment.

Keywords: CASA, mistletoe, mitochondrial activity, motility, reactive oxygen species, rabbits, spermatozoa, Viscum album

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Authors: Amir Peyman Soleymani, Jasna Jankovic

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The augmented demand of the clean and renewable energy has necessitated the fuel cell and battery industries to produce more efficient devices at the lower prices, which can be achieved through the improvement of the electrode. Microstructural characterization, as one of the main materials development tools, plays a pivotal role in the production of better clean energy devices. In this study, methods for characterization and studying of the defects and components distribution were performed on the polymer electrolyte membrane fuel cell (PEMFC) and Li-ion battery (LIB) electrodes in 2D and 3D. The particles distribution, porosity, mechanical defects, and component distribution were studied by Scanning Electron Microscope (SEM), SEM-Focused Ion Beam (SEM-FIB), and Scanning Transmission Electron Microscope equipped with Energy Dispersive Spectroscopy (STEM-EDS). The 3D results obtained from X-ray Computed Tomography (XCT) revealed the pathways for electron and ion conductivity and defects progression maps. Computer-aided methods (Avizo) were employed to simulate the properties and performance of the microstructure in the electrodes. The suggestions were provided to improve the performance of PEMFCs and LIBs by adjusting the microstructure and the distribution of the components in the electrodes.

Keywords: PEM fuel cells, Li-ion batteries, 2D and 3D imaging, materials characterizations

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Authors: Arunas Stirke, Neringa Bakute, Gatis Mozolevskis

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A technology of microfluidics is an emerging tool in the field of biology, medicine and chemistry. Microfluidic device is also known as ‘lab-on-a-chip’ technology [1]. In moving from macro- to microscale, there is unprecedented control over spatial and temporal gradients and patterns that cannot be captured in conventional Petri dishes and well plates [2]. However, there is not a single standard microfluidic chip designated for all purposes – every different field of studies needs a specific microchip with certain geometries, inlet/outlet, channel depth and other parameters to precisely regulate the required function. Since our group is studying an effect of pulsed electric field (PEF) to the cells, we have manufactured a microfluidic chip designated for high-throughput electroporation of cells. In our microchip, a cell culture chamber is divided into two parallel channels by a membrane, meanwhile electrodes for electroporation are attached to the wall of the channels. Both microchannels have their own inlet and outlet, enabling injection of transfection material separately. Our perspective is to perform electroporation of mammalian cells in two different ways: (1) plasmid and cells are injected in the same microchannel and (2) injected into separate microchannels. Moreover, oxygen and pH sensors are integrated on order to analyse cell viability parameters after PEF treatment.

Keywords: microfluidics, chip, fabrication, electroporation

Procedia PDF Downloads 63

Authors: S. K. Thind, Aparjot Kaur

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Heat stress is one of the major environmental factors drastically reducing wheat production. Crop heat tolerance can be enhanced by preconditioning of plants by exogenous application of osmoprotectants. Presently, the effect of trehalose pretreatment (at 1 mM, and 1.5 nM) under heat stress of 35±2˚C (moderate) and 40±2˚ (severe) for four and eight hour was conducted in wheat (Tricticum aestivum L.) genotypes viz. HD2967, PBW 175, PBW 343, PBW 621, and PBW 590. Heat stress affects wide spectrum of physiological processes within plants that are irreversibly damaged by stress. Membrane thermal stability (MTS) and cell viability was significantly decreased under heat stress for eight hours. Pretreatment with trehalose improved MTS and cell viability under stress and this effect was more promotory with higher concentration. Thermal stability of photosynthetic apparatus differed markedly between genotypes and Hill reaction activity was recorded more in PBW621 followed by C306 as compared with others. In all genotypes photolysis of water showed decline with increase in temperature stress. Trehalose pretreatment helped in sustaining Hill reaction activity probably by stabilizing the photosynthetic apparatus against heat-induced photo inhibition. Both plant growth and development were affected by temperature in both shoot and root under heat stress. The reduction was compensated partially by trehalose (1.5 mM) application. Adaption to heat stress is associated with the metabolic adjustment which led to accumulation of soluble sugars including non-reducing and reducing for their role in adaptive mechanism. Higher acid invertase activity in shoot of tolerant genotypes appeared to be a characteristic for stress tolerance. As sucrose synthase play central role in sink strength and in studied wheat genotype was positively related to dry matter accumulation. The duration of heat stress for eight hours had more severe effect on these parameters and trehalose application at 1.5 mM ameliorated it to certain extent.

Keywords: heat stress, Triticum aestivum, trehalose, membrane thermal stability, triphenyl tetrazolium chloride, reduction test, growth, sugar metabolism

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Authors: Eitan Yefenof

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Glucocorticoid (GC) hormones, e.g. Dexamethasone and Prednisone, are widely used in the therapy of leukemia and lymphoma owing to their apoptogenic effect on lymphoid cells. However, the emergence of GC resistant cells during therapy is a major cause for treatment failure, urging the need for novel strategies that maintain leukemia sensitivity to the pro-apoptotic activity of GCs. GCs act by binding to the GC receptor (GR), which, in its inactive state, is sequestered in the cytosol by a multi-subunit complex of heat shock proteins. Upon ligand binding, the complex dissociates, allowing GR activation and translocation to the nucleus, where it regulates transcription of multiple genes. We demonstrated that in addition to gene expression, GR also regulates microRNA (miR) expression. Deep-sequencing analysis revealed 14 miRs that are regulated in GC-sensitive but resistant leukemias upon treatment with GC. GC up-regulates miR-103, miR-15~16 and miR-30e/d, while down-regulates miR-17, mir-18a, miR-19a, miR-19b, miR-20a and miR-92a (members of the miR-17∼92a multi-cistron). Upon transfection, miR-103 confers GC apoptotic sensitivity to otherwise GC-resistant cell. Furthermore, knocking down miR-103 expression reduces the GC apoptotic response of sensitive cells. miR-103 abrogates c-Myc expression, an oncogenic transcription factor which is deregulated in many cancers. In addition, miR-103 up-regulates Bim, a pro-apoptotic protein crucial for GC-induced death. Activated glycogen synthase kinase 3 (GSK3) is also crucial for GC-induced apoptosis. GSK3 is active in GC-sensitive but not in GC-resistant cells. We found that GSK3 associates with the GR multi-subunit complex. Upon GC exposure, it dissociates from the GR and interacts with Bim to enable activation of the mitochondrial apoptosis pathway. miR-103 mediated c-Myc ablation is followed by down-regulation of the multi-cistron miR-17~92a, in particular miR-18a and miR-20a. miR-18a targets GR for degradation whereas miR-20a targets Bim degradation. Hence, miR-103 acts, in concert with Bim and GR, as a "tumor suppressor" that leads to reduced proliferation, cell-cycle arrest and cell death. We suggest that miR-103 can provide a diagnostic tool that predicts the sensitivity of leukemia to GC based therapy. Furthermore, exosomal delivery of miR-103 or up-regulation of the endogenous miR-103 could confer apoptotic sensitivity to resistant cells at the outset, thus becoming a useful therapeutic tool combined with GCs.

Keywords: apoptosis, leukemia, micro-RNA, steroids

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Authors: Aoxue Yang, Weili Tian, Yonghe Wu, Haikun Liu

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Brain tumor stem cells (BTSCs) are resistant to therapy and give rise to recurrent tumors. These rare and elusive cells are likely to disseminate during cancer progression, and some may enter dormancy, remaining viable but not increasing. The identification of dormant BTSCs is thus necessary to design effective therapies for glioblastoma (GBM) patients. Little progress has been made in therapeutic treatment of glioblastoma in the last decade despite rapid progress in molecular understanding of brain tumors1. Here we show that the stress hormone glucocorticoid is essential for the maintenance of brain tumor stem cells (BTSCs), which are resistant to conventional therapy. The glucocorticoid receptor (GR) regulates metabolic plasticity and chemoresistance of the dormant BTSC via controlling expression of GPD1 (glycerol-3-phosphate dehydrogenase 1), which is an essential regulator of lipid metabolism in BTSCs. Genomic, lipidomic and cellular analysis confirm that GR/GPD1 regulation is essential for BTSCs metabolic plasticity and survival. We further demonstrate that the GR agonist dexamethasone (DEXA), which is commonly used to control edema in glioblastoma, abolishes the effect of chemotherapy drug temozolomide (TMZ) by upregulating GPD1 and thus promoting tumor cell dormancy in vivo, this provides a mechanistic explanation and thus settle the long-standing debate of usage of steroid in brain tumor patient edema control. Pharmacological inhibition of GR/GPD1 pathway disrupts metabolic plasticity of BTSCs and prolong animal survival, which is superior to standard chemotherapy. Patient case study shows that GR antagonist mifepristone blocks tumor progression and leads to symptomatic improvement. This study identifies an important mechanism regulating cancer stem cell dormancy and provides a new opportunity for glioblastoma treatment.

Keywords: cancer stem cell, dormancy, glioblastoma, glycerol-3-phosphate dehydrogenase 1, glucocorticoid receptor, dexamethasone, RNA-sequencing, phosphoglycerides.

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Authors: Eva Tvrdá, Hana Greifová, Peter Ivanič, Norbert Lukáč

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The aim of this study was to evaluate the dose- and time-dependent in vitro effects of berberine (BER), a natural alkaloid with numerous biological properties on bovine spermatozoa during three time periods (0 h, 2 h, 24 h). Bovine semen samples were diluted and cultivated in physiological saline solution containing 0.5% DMSO together with 200, 100, 50, 10, 5, and 1 μmol/L BER. Spermatozoa motility was assessed using the computer assisted semen analyzer. The viability of spermatozoa was assessed by the metabolic (MTT) assay, production of superoxide radicals was quantified using the nitroblue tetrazolium (NBT) test, and chemiluminescence was used to evaluate the generation of reactive oxygen species (ROS). Cell lysates were prepared and the extent of lipid peroxidation (LPO) was evaluated using the TBARS assay. The results of the movement activity showed a significant increase in the motility during long term cultivation in case of concentrations ranging between 1 and 10 μmol/L BER (P < 0.01; P < 0.001; 24 h). At the same time, supplementation of 1, 5 and 10 μmol/L BER led to a significant preservation of the cell viability (P < 0.001; 24 h). BER addition at a range of 1-50 μmol/L also provided a significantly higher protection against superoxide (P < 0.05) and ROS (P < 0.001; P < 0.01) overgeneration as well as LPO (P < 0.01; P<0.05) after a 24 h cultivation. We may suggest that supplementation of BER to bovine spermatozoa, particularly at concentrations ranging between 1 and 50 μmol/L, may offer protection to the motility, viability and oxidative status of the spermatozoa, particularly notable at 24 h.

Keywords: berberine, bulls, motility, oxidative profile, spermatozoa, viability

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Authors: Jayarama Reddy, Anand S., H., Sundaram, Jeldi Hemachandran

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Forty two strains of Trichoderma sp. were isolated from cultivated lands around Bangalore and analyzed for their antagonistic potential against Macrophomina phaseolina. The potential of biocontrol agents ultimately lies in their capacity to control pathogens in vivo. Bioefficacy studies were hence conducted using chickpea (Cicer arientum c.v. Annigeri) as an experimental plant by the roll paper towel method. Overall the isolates T6, T35, T30, and T25 showed better antagonistic potential in addition to enhancing plant growth. The production of chitinases to break down the mycelial cell walls of fungal plant pathogens has been implicated as a major cause of biocontrol activity. In order to study the mechanism of biocontrol against Macrophomina phaseolina, ten better performing strains were plated on media, amended with colloidal chitin and Sclerotium rolfsii cell wall extract. All the isolates showed chitinolytic activity on day three as well as day five. Production of endochitinase and exochitinase were assayed in liquid media using colloidal chitin amended broth. Strains T35 and T6 displayed maximum endochitinase and exochitinase activity. Although all strains exhibited cellulase activity, the quantum of enzyme produced was higher in T35 and T6. The results also indicate a positive correlation between enzyme production and bioefficacy.

Keywords: biocontrol, bioefficacy, cellulase, chitinase

Procedia PDF Downloads 358

Authors: Xinyong Li

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A new coupling system was constructed by combining photo-electrochemical cell with eletro-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 photo-induced 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

Procedia PDF Downloads 127

Authors: Peerawat Khongkliang, Prawit Kongjan, Tsuyoshi Imai, Poonsuk Prasertsan, Sompong O-Thong

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A two-stage thermophilic fermentation and electrohydrogenesis process was used to convert cassava starch processing wastewater into hydrogen gas. Maximum hydrogen yield from fermentation stage by Thermoanaerobacterium thermosaccharolyticum PSU-2 was 248 mL H2/g-COD at optimal pH of 6.5. Optimum hydrogen production rate of 820 mL/L/d and yield of 200 mL/g COD was obtained at HRT of 2 days in fermentation stage. Cassava starch processing wastewater fermentation effluent consisted of acetic acid, butyric acid and propionic acid. The effluent from fermentation stage was used as feedstock to generate hydrogen production by microbial electrolysis cell (MECs) at an applied voltage of 0.6 V in second stage with additional 657 mL H2/g-COD was produced. Energy efficiencies based on electricity needed for the MEC were 330 % with COD removals of 95 %. The overall hydrogen yield was 800-900 mL H2/g-COD. Microbial community analysis of electrohydrogenesis by DGGE shows that exoelectrogens belong to Acidiphilium sp., Geobacter sulfurreducens and Thermincola sp. were dominated at anode. These results show two-stage thermophilic fermentation, and electrohydrogenesis process improved hydrogen production performance with high hydrogen yields, high gas production rates and high COD removal efficiency.

Keywords: cassava starch processing wastewater, biohydrogen, thermophilic fermentation, microbial electrolysis cell

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Authors: Indu Lata Kanwar, Preeti K. Suresh

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The aim of this study is to fabricate hydroxyapatite based nanocomposites for local drug delivery in periodontal pockets. Hydroxyapatite is chemically similar to the mineral component of bones and hard tissues in mammals. Synthetic biocompatibility and bioactivity with human teeth and bone, making it very attractive for biomedical applications. Nanocomposite is a multiphase solid material where one of the phases has one, two or three dimensions of less than 100 nanometres (nm), or structures having nano­scale repeat distances between the different phases that make up the material. Nanostructured calcium phosphate materials play an important role in the formation of hard tissues in nature. It is reported that calcium phosphates materials in nano-size can mimic the dimensions of constituent components of calcified tissues. Nano-sized materials offer improved performances compared with conventional materials due to their large surface-to-volume ratios. The specific biological properties of the nanocomposites, as well as their interaction with cells, include the use of bioactive molecules. The approach of periodontal tissue engineering is considered promising to restore bone defect through the use of engineered materials with the aim that they will prohibit the invasion of fibrous connective tissue and help repair the function during bone regeneration.

Keywords: bioactive, hydroxyapatite, nanocomposities, periondontal

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Authors: Sanat K. Dash, Rama S. Verma, Sarit K. Das

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A logarithmic microfluidic shear device was designed and fabricated for cellular mechanotransduction studies. The device contains four cell culture chambers in which flow was modulated to achieve a logarithmic increment. Resistance values were optimized to make the device compact. The network of resistances was developed according to a unique combination of series and parallel resistances as found via optimization. Simulation results done in Ansys 16.1 matched the analytical calculations and showed the shear stress distribution at different inlet flow rates. Fabrication of the device was carried out using conventional photolithography and PDMS soft lithography. Flow profile was validated taking DI water as working fluid and measuring the volume collected at all four outlets. Volumes collected at the outlets were in accordance with the simulation results at inlet flow rates ranging from 1 ml/min to 0.1 ml/min. The device can exert fluid shear stresses ranging four orders of magnitude on the culture chamber walls which will cover shear stress values from interstitial flow to blood flow. This will allow studying cell behavior in the long physiological range of shear stress in a single run reducing number of experiments.

Keywords: microfluidics, mechanotransduction, fluid shear stress, physiological shear

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Authors: Grigoria Athanasaki, Veerarajan Vimala, A. M. Kannan, Louis Cindrella

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Energy usage has been increased throughout the years, leading to severe environmental impacts. Since the majority of the energy is currently produced from fossil fuels, there is a global need for clean energy solutions. Proton Exchange Membrane Fuel Cells (PEMFCs) offer a very promising solution for transportation applications because of their solid configuration and low temperature operations, which allows them to start quickly. One of the main components of PEMFCs is the Gas Diffusion Layer (GDL), which manages water and gas transport and shows direct influence on the fuel cell performance. In this work, a novel dual-layer GDL with gradient porosity was prepared, using polyethylene glycol (PEG) as pore former, to improve the gas diffusion and water management in the system. The microporous layer (MPL) of the fabricated GDL consists of carbon powder PUREBLACK, sodium dodecyl sulfate as a surfactant, 34% wt. PTFE and the gradient porosity was created by applying one layer using 30% wt. PEG on the carbon substrate, followed by a second layer without using any pore former. The total carbon loading of the microporous layer is ~ 3 mg.cm-2. For the assembly of the catalyst layer, Nafion membrane (Ion Power, Nafion Membrane NR211) and Pt/C electrocatalyst (46.1% wt.) were used. The catalyst ink was deposited on the membrane via microspraying technique. The Pt loading is ~ 0.4 mg.cm-2, and the active area is 5 cm2. The sample was ex-situ characterized via wetting angle measurement, Scanning Electron Microscopy (SEM), and Pore Size Distribution (PSD) to evaluate its characteristics. Furthermore, for the performance evaluation in-situ characterization via Fuel Cell Testing using H2/O2 and H2/air as reactants, under 50, 60, 80, and 100% relative humidity (RH), took place. The results were compared to a single layer GDL, fabricated with the same carbon powder and loading as the dual layer GDL, and a commercially available GDL with MPL (AvCarb2120). The findings reveal high hydrophobic properties of the microporous layer of the GDL for both PUREBLACK based samples, while the commercial GDL demonstrates hydrophilic behavior. The dual layer GDL shows high and stable fuel cell performance under all the RH conditions, whereas the single layer manifests a drop in performance at high RH in both oxygen and air, caused by catalyst flooding. The commercial GDL shows very low and unstable performance, possibly because of its hydrophilic character and thinner microporous layer. In conclusion, the dual layer GDL with PEG appears to have improved gas diffusion and water management in the fuel cell system. Due to its increasing porosity from the catalyst layer to the carbon substrate, it allows easier access of the reactant gases from the flow channels to the catalyst layer, and more efficient water removal from the catalyst layer, leading to higher performance and stability.

Keywords: gas diffusion layer, microporous layer, proton exchange membrane fuel cells, relative humidity

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Authors: T. Ferreira, A. Kulkarni, C. Bretscher, K. Richter, M. Ehrlich, A. Marchini

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H-1 protoparvovirus (H-1PV) is a virus with inherent oncolytic and oncosuppressive activities while remaining non-pathogenic in humans. H-1PV was the first oncolytic parvovirus to undergo clinical testing. Results from trials in patients with glioblastoma or pancreatic carcinoma showed an excellent safety profile and first signs of efficacy. H-1PV infection is vastly dependent on cellular factors, from cell attachment and entry to viral replication and egress. Hence, we believe that the characterisation of the parvovirus life cycle would ultimately help further improve H-1PV clinical outcome. In the present study, we explored the entry pathway of H-1PV in cervical HeLa and glioma NCH125 cancer cell lines. Electron and confocal microscopy showed viral particles associated with clathrin-coated pits and vesicles, providing the first evidence that H-1PV cell entry occurs through clathrin-mediated endocytosis. Accordingly, we observed that by blocking clathrin-mediated endocytosis with hypertonic sucrose, chlorpromazine, or pitstop 2, H-1PV transduction was markedly decreased. Accordingly, siRNA-mediated knockdown of AP2M1, which retains a crucial role in clathrin-mediated endocytosis, verified the reliance of H-1PV on this route to enter HeLa and NCH125 cancer cells. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. Indeed, pre-treatment of cells with nystatin or methyl-β-cyclodextrin, both inhibitors of caveolae-mediated endocytosis, did not affect viral transduction levels. Unexpectedly, siRNA-mediated knockdown of caveolin-1, the main driver of caveolae-mediated endocytosis, increased H-1PV transduction, suggesting caveolin-1 is a negative modulator of H-1PV infection. We also show that H-1PV entry is dependent on dynamin, a protein responsible for mediating the scission of vesicle neck and promoting further internalisation. Furthermore, since dynamin inhibition almost completely abolished H-1PV infection, makes it unlikely that H-1PV uses macropinocytosis as an alternative pathway to enter cells. After viral internalisation, H-1PV passes through early to late endosomes as observed by confocal microscopy. Inside these endocytic compartments, the acidic environment proved to be crucial for a productive infection. Inhibition of acidification of pH dramatically reduced H-1PV transduction. Besides, a fraction of H-1PV particles was observed inside LAMP1-positive lysosomes, most likely following a non-infectious route. To the author's best knowledge, this is the first study to characterise the cell entry pathways of H-1PV. Along these lines, this work will further contribute to understand H-1PV oncolytic properties as well as to improve its clinical potential in cancer virotherapy.

Keywords: clathrin-mediated endocytosis, H-1 parvovirus, oncolytic virus, virus entry

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Authors: B. Gonzalez-Yebra, H. Barajas, P. Palomares, M. Hernandez, O. Torres, M. Ayala, A. L. González, G. Vazquez-Ortiz, M. L. Guzman

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Leukemia arises by molecular alterations of the normal hematopoietic stem cell (HSC) transforming it into a leukemic stem cell (LSC) with high cell proliferation, self-renewal, and cell differentiation. Chronic myeloid leukemia (CML) originates from an LSC-leading to elevated proliferation of myeloid cells and acute lymphoblastic leukemia (ALL) originates from an LSC development leading to elevated proliferation of lymphoid cells. In both cases, LSC can be identified by multicolor flow cytometry using several antibodies. However, to date, LSC levels in peripheral blood (PB) are not established well enough in ALL and CML patients. On the other hand, the detection of the minimal residue disease (MRD) in leukemia is mainly based on the identification of the mRNA bcr-abl gene in CML patients and some other genes in ALL patients. There is no a properly biomarker to detect MDR in both types of leukemia. The objective of this study was to determine mRNA bcr-abl and the percentage of LSC in peripheral blood of patients with CML and ALL and identify a possible association between the amount of LSC in PB and clinical data. We included in this study 19 patients with Leukemia. A PB sample was collected per patient and leukocytes were obtained by Ficoll gradient. The immunophenotype for LSC CD34+ was done by flow cytometry analysis with CD33, CD2, CD14, CD16, CD64, HLA-DR, CD13, CD15, CD19, CD10, CD20, CD34, CD38, CD71, CD90, CD117, CD123 monoclonal antibodies. In addition, to identify the presence of the mRNA bcr-abl by RT-PCR, the RNA was isolated using TRIZOL reagent. Molecular (presence of mRNA bcr-abl and LSC CD34+) and clinical results were analyzed with descriptive statistics and a multiple regression analysis was performed to determine statistically significant association. In total, 19 patients (8 patients with ALL and 11 patients with CML) were analyzed, 9 patients with de novo leukemia (ALL = 6 and CML = 3) and 10 under treatment (ALL = 5 and CML = 5). The overall frequency of mRNA bcr-abl was 31% (6/19), and it was negative in ALL patients and positive in 80% in CML patients. On the other hand, LSC was determined in 16/19 leukemia patients (%LSC= 0.02-17.3). The Novo patients had higher percentage of LSC (0.26 to 17.3%) than patients under treatment (0 to 5.93%). The amount of LSC was significantly associated with the amount of LSC were: absence of treatment, the absence of splenomegaly, and a lower number of leukocytes, negative association for the clinical variables age, sex, blasts, and mRNA bcr-abl. In conclusion, patients with de novo leukemia had a higher percentage of circulating LSC than patients under treatment, and it was associated with clinical parameters as lack of treatment, absence of splenomegaly and a lower number of leukocytes. The mRNA bcr-abl detection was only possible in the series of patients with CML, and molecular detection of LSC could be identified in the peripheral blood of all leukemia patients, we believe the identification of circulating LSC may be used as biomarker for the detection of the MRD in leukemia patients.

Keywords: stem cells, leukemia, biomarkers, flow cytometry

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Authors: Mehdi Ghatus

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Two simple apparatus based on the hammer and anvil principle have been constructed and used to study the microstructure and micro-hardness characteristics of some AL-base alloys. Foils with thicknesses arranging from 20 µm up to 600 µm have been obtained. The cooling rate was estimated to be in the range 10^4 - 10^5 K/sec. Microstructure study of rapidly quenched Al-30% Si foils indicated that with decreasing the foil thickness the size of primary Si crystallites decreases in the whole investigated range (0.64-0.15 mm). However, the volume fraction of the primary Si crystals in the structure remained constant down to thickness the primary Si volume fraction started to decrease. Rapid quenching of Al- 14-16% Cu showed single phase cell structure. In foils up to 0.55 mm with decreasing the foil thickness the cell size decreases and micro-hardness increases particularly in foils below 0.3 mm in thickness. Isochronal annealing of theses foils show that the highly supersaturated Al-14-16% Cu solid solution decomposes readily at relatively low temperature and short time intervals. The maximum hardness is obtained after annealing at 100 °C for 30 minutes. However with decreasing the Cu content of the foils the precipitation process is largely delayed. Eight hours of annealing at 100 °C was not enough to achieve the maximum hardness in Al-4% Cu thin foils. The achieved hardness value was more than twice of the maximum hardness obtained in articles of similar composition but conventionally aged.

Keywords: aluminum, hardness, alloys, quenched aluminum

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Authors: Kenichi Yamahara, Makiko Ohshima, Shunsuke Ohnishi, Hidetoshi Tsuda, Akihiko Taguchi, Toshihiro Soma, Hiroyasu Ogawa, Jun Yoshimatsu, Tomoaki Ikeda

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We have previously reported the therapeutic potential of rat fetal membrane(FM)-derived mesenchymal stem cells (MSCs) using various rat models including hindlimb ischemia, autoimmune myocarditis, glomerulonephritis, renal ischemia-reperfusion injury, and myocardial infarction. In this study, 1) we isolated and characterized MSCs from human amnion and chorion; 2) we examined their differences in the expression profile of growth factors and cytokines; and 3) we investigated the therapeutic potential and difference of these MSCs using murine hindlimb ischemia and acute graft-versus-host disease (GVHD) models. Isolated MSCs from both amnion and chorion layers of FM showed similar morphological appearance, multipotency, and cell-surface antigen expression. Conditioned media obtained from amnion- and chorion-derived MSCs inhibited cell death caused by serum starvation or hypoxia in endothelial cells and cardiomyocytes. Amnion and chorion MSCs secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Transplantation of human amnion or chorion MSCs significantly increased blood flow and capillary density in a murine hindlimb ischemia model. In addition, compared to human chorion MSCs, human amnion MSCs markedly reduced T-lymphocyte proliferation with the enhanced secretion of PGE2, and improved the pathological situation of a mouse model of GVHD disease. Our results highlight that human amnionand chorion-derived MSCs, which showed differences in their soluble factor secretion and angiogenic/immuno-suppressive function, could be ideal cell sources for regenerative medicine.

Keywords: amnion, chorion, fetal membrane, mesenchymal stem cells

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Authors: Gehan Hussein, Hala Agha, Rasha Abdelraof, Marina George, Antoine Fakhri

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Background: β-Thalassemia major (TM) and sickle cell disease (SCD) are inherited hemoglobin disorders resulting in chronic hemolytic anemia. Cardiovascular involvement is an important cause of morbidity and mortality in these groups of patients. The narrow border is between overt myocardial dysfunction and clinically silent left ventricular (LV) and / or right ventricular (RV) dysfunction in those patients. 3 D Speckle tracking echocardiography (3D STE) is a novel method for the detection of subclinical myocardial involvement. We aimed to study myocardial affection in SCD and TM using 3D STE, comparing it with conventional echocardiography, correlate it with serum ferritin level and lactate dehydrogenase (LDH). Methodology: Thirty SCD and thirty β TM patients, age range 4-18 years, were compared to 30 healthy age and sex matched control group. Cases were subjected to clinical examination, laboratory measurement of hemoglobin level, serum ferritin, and LDH. Transthoracic color Doppler echocardiography, 3D STE, tissue Doppler echocardiography, and aortic speckle tracking were performed. Results: significant reduction in global longitudinal strain (GLS), global circumferential strain (GCS), and global area strain (GAS) in SCD and TM than control (P value <0.001) there was significantly lower aortic speckle tracking in patients with TM and SCD than control (P value< 0.001). LDH was significantly higher in SCD than both TM and control and it correlated significantly positive mitral inflow E, (p value:0.022 and 0.072. r: 0.416 and -0.333 respectively) lateral E/E’ (p value.<0.001and 0.818. r. 0.618 and -0. 044.respectively) and septal E/E’ (p value 0.007 and 0.753& r value 0.485 and -0.060 respectively) in SCD but not TM and significant negative correlation between LDH and aortic root speckle tracking (value 0.681& r. -0.078.). The potential diagnostic accuracy of LDH in predicting vascular dysfunction as represented by aortic root GCS with a sensitivity 74% and aortic root GCS was predictive of LV dysfunction in SCD patients with sensitivity 100% Conclusion: 3D STE LV and RV systolic dysfunction in spite of their normal values by conventional echocardiography. SCD showed significantly lower right ventricular dysfunction and aortic root GCS than TM and control. LDH can be used to screen patients for cardiac dysfunction in SCD, not in TM

Keywords: thalassemia major, sickle cell disease, 3d speckle tracking echocardiography, LDH

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Authors: Arifa Batool, Ghulam Hussain Bhatti, Syed Mujtaba Shah

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Inorganic n-type (TiO2, CdO) and p-type (NiO, CuO) metal oxide nanoparticles were synthesized by a facile wet chemical method at room temperature. The morphological, compositional, structural and optical properties were investigated by scanning electron microscopy, energy dispersive X-ray spectroscopy, FT-IR, XRD analysis, UV/Visible and fluorescence spectroscopy. All semiconducting nanoparticles were photosensitized with Ru (II) based Z907 dye in ethanol solvent by grafting. Grafting of dye on the surface of nanoparticles was confirmed by UV/Visible and FT-IR spectroscopy. The synthesized photo-active nanohybrid was thoroughly blended with P3HT, a solid electrolyte and I-V measurements under solar stimulated radiations 1000 W/m2 (AM 1.5) were recorded. Maximum incident photon to current conversion efficiency (IPCE) of 0.9% was achieved with dye functionalized Z907-TiO2 hybrid, IPCE of 0.72% was achieved with bulk-heterojunction of TiO2-Z907-CuO and IPCE of 0.68% was attained with nanocomposite of TiO2-CdO. TiO2 based Solar cells have maximum Jscvalue i.e.4.63 mA/cm2. Dye-functionalized TiO2-based photovoltaic devices were found more efficient than the reference device but the morphology of the device was a major check in progress.

Keywords: solar cell, bulk heterojunction, nanocomposites, photosensitization, dye sensitized solar cell

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Authors: Peramachi Sathiyamoorthy, Jacop Gopas, Avi Golan Goldhirsh

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Corchorus olitorius is a leafy vegetable and an industrial crop. The herb has antioxidant, anti inflammatory, and anti-cancer properties. To assay the pharmaceutical properties, aqueous extracts of leaves and seeds from C. olitorius were tested against drug resistant melanoma cell line. The test showed LC50 of the extract was 0.08µg/ml. Aqueous seed extract exhibited higher melanoma inhibiting activity than leaf extract. Dialysis of seed extract showed that the active compound is less than 12 KDa. The compound with <3 KDa MW separated by microconcentration of seed extract showed 70.5 % inhibition of melanoma cell growth. Among the two fractions obtained by Gel filtration with G10 column, the first fraction at 1:2000 dilutions exhibited 100% inhibition of melanoma growth. The compound with Rf value 0.86 (MA4) isolated by TLC separation showed about 98% cytotoxicity against melanoma at 1: 1000 dilutions. Furthermore, HPLC separation of MA4 compound with Superdex 75 column resulted in 4 compounds. Out of 4, one compound showed melanoma inhibition. The active compound is identified by reagent methods as Strophanthidin. Further toxicological and clinical studies will lead to the development of a potential drug to treat drug resistant melanoma.

Keywords: corchorus olitorius, melanoma, drug development, strophanthidin

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Authors: A. Alao Olumuyiwa

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Health hazards reported to be associated with exposure to electromagnetic radiations which include brain tumors, genotoxic effects, neurological effects, immune system deregulation, allergic responses and some cardiovascular effects are discussed under a closed tabular model in this study. This review however showed that there is strong and robust evidence that chronic exposures to electromagnetic frequency across the spectrum, through strength, consistency, biological plausibility and many dose-response relationships, may result in brain cancer and other carcinogenic disease symptoms. There is therefore no safe threshold because of the genotoxic nature of the mechanism that may however be involved. The discussed study explains that the cell phone has induced effects upon the blood –brain barrier permeability and the cerebellum exposure to continuous long hours RF radiation may result in significant increase in albumin extravasations. A physical Biomodeling approach is however employed to review this health effects using Specific Absorption Rate (SAR) of different GSM machines to critically examine the symptoms such as a decreased loco motor activity, increased grooming and reduced memory functions in a variety of animal spices in classified grouped and sub grouped models.

Keywords: brain cancer, electromagnetic radiations, physical biomodeling, specific absorption rate (SAR)

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Authors: Daniel Phifer, Anna Prokhodtseva

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DualBeam (FIB-SEM) has long been the technology of choice to sub-sample and characterize materials at site-specific locations which are difficult or impossible to extract by conventional embedding/polishing methods. Whereas Ga based FIB provides excellent resolution and enables precise material removal, the current is usually limited and only allows the extraction of small material biopsies typically ranging from 5-70um wide. Xe Plasma FIB, by contrast, has around 38x more current and can remove more material at the same time to extract significant sized chunks (100-1000um) of materials for further analysis. This increased volume has enabled time-prohibitive investigations like large grain 3D serial sectioning and EBSD and micro-machining for micro-mechanical testing. Investigation of the pore spaces with 3D modeling can determine the relative characteristics of the materials to help design or select properties for best function. Pore spaces can be described with a tortuosity number which is calculated by modules in the 3D analysis software. Xe Plasma FIB technology provides a workflow with sufficient volume to characterize porosity when both large-volume 3D materials characterization and nanometer resolution is required to understand the system.

Keywords: dual-beam, FIB-SEM, porosity, SOFC, solid oxide fuel cell

Procedia PDF Downloads 191

Authors: M. Toghyani, A. Ghasemi, S. A. Tabeidian

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The present study was carried out to evaluate the effect of different levels of dietary seed and extract of Harmal (Peganum harmala L.) on immunity of broiler chicks. A total of 350 one-day old broiler chicks (Ross 308) were randomly allocated to five dietary treatments with four replicates pen of 14 birds each. Dietary treatments consisted of control, 1 and 2 g/kg Harmal seed in diet, 100 and 200 mg/L Harmal seed extract in water. Broilers received dietary treatments from 1 to 42 d. Two birds from each pen were randomly weighed and sacrificed at 42 d of age, the relative weight of lymphoid organs (bursa of Fabercius and spleen) to live weight were calculated. Antibody titers against Newcastle and influenza viruses and sheep red blood cell were measured at 30 d of age. Results showed that the relative weights of lymphoid organs were not affected by dietary treatments. Furthermore, antibody titer against Newcastle and influenza viruses as well as sheep red blood cell antigen were significantly (P<0.05) enhanced by feeding Harmal seed and extract. In conclusion, the results indicated that dietary inclusion of Harmal seed and extract enhanced immunological responses in broiler chicks.

Keywords: broiler chicks, Harmal, immunity, Peganum harmala

Procedia PDF Downloads 529

Authors: S. V. Ukwungwu, A. J. Abbas, G. G. Nasr

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The increasing high price of natural gas and oil with attendant increase in energy demand on world markets in recent years has stimulated interest in recovering residual oil saturation across the globe. In order to meet the energy security, efforts have been made in developing new technologies of enhancing the recovery of oil and gas, utilizing techniques like CO2 flooding, water injection, hydraulic fracturing, surfactant flooding etc. Surfactant flooding however optimizes production but poses risk to the environment due to their toxic nature. Amongst proven records that have utilized other type of bacterial in producing biosurfactants for enhancing oil recovery, this research uses a technique to combine biosurfactants that will achieve a scale of EOR through lowering interfacial tension/contact angle. In this study, three biosurfactants were produced from three Bacillus species from freeze dried cultures using sucrose 3 % (w/v) as their carbon source. Two of these produced biosurfactants were screened with the TEMCO Pendant Drop Image Analysis for reduction in IFT and contact angle. Interfacial tension was greatly reduced from 56.95 mN.m-1 to 1.41 mN.m-1 when biosurfactants in cell-free culture (Bacillus licheniformis) were used compared to 4. 83mN.m-1 cell-free culture of Bacillus subtilis. As a result, cell-free culture of (Bacillus licheniformis) changes the wettability of the biosurfactant treatment for contact angle measurement to more water-wet as the angle decreased from 130.75o to 65.17o. The influence of microbial treatment on crushed rock samples was also observed by qualitative wettability experiments. Treated samples with biosurfactants remained in the aqueous phase, indicating a water-wet system. These results could prove that biosurfactants can effectively change the chemistry of the wetting conditions against diverse surfaces, providing a desirable condition for efficient oil transport in this way serving as a mechanism for EOR. The environmental friendly effect of biosurfactants applications for industrial purposes play important advantages over chemically synthesized surfactants, with various possible structures, low toxicity, eco-friendly and biodegradability.

Keywords: bacillus, biosurfactant, enhanced oil recovery, residual oil, wettability

Procedia PDF Downloads 269

Authors: S. Khosravi, X. Chelius, A. Unger, D. Rieger, J. Frickel, T. Sachsenheimer, C. Luechtenborg, R. Schieweck, B. Bruegger, B. Westermann, T. Klecker, W. Neupert, M. E. Harner

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The use of Saccharomyces cerevisiae as a model organism to study eukaryotic cell functions has been used successfully for decades. Like virtually all eukaryotic cells, they contain mitochondria as essential organelles performing various functions, including participation in lipid metabolism. They are separated from the cytosol by a double membrane system consisting of the mitochondrial inner membrane (MIM) and the mitochondrial outer membrane (MOM). This physical separation of the mitochondria requires an exchange of metabolites, proteins, and lipids. Proteinaceous contact sites are thought to be important for this communication. Recently, it was found that Cqd1, in cooperation with Cqd2, controls the distribution of Coenzyme Q within the cell. In this study, a contact site is described, formed by the MOM protein complex Por1-Om14 and the UbiB protein kinase-like MIM protein Cqd1. The present results suggest the additional involvement of Cqd1 in the homeostasis of phospholipids. Moreover, we show that overexpression of the UbiB family proteins also causes tethering of the mitochondria to the endoplasmatic reticulum. Due to the conservation of the subunits of this contact site to higher eukaryotes, its identification in S. cerevisiae might provide promising avenues for further research in other organisms.

Keywords: contact sites, mitochondrial architecture, mitochondrial proteins, yeast mitochondria

Procedia PDF Downloads 77

Authors: G. Cunningham, E. Cantor, X. Wu, F. Shen, G. Jiang, S. Philips, C. Bales, Y. Xiao, T. R. Cummins, J. C. Fehrenbacher, B. P. Schneider

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Background: Taxane-induced peripheral neuropathy (TIPN) is the most devastating survivorship issue for patients receiving therapy. Dose reductions due to TIPN in the curative setting lead to inferior outcomes for African American patients, as prior research has shown that this group is more susceptible to developing severe neuropathy. The mechanistic underpinnings of TIPN, however, have not been entirely elucidated. While it would be appealing to use primary tissue to study the development of TIPN, procuring nerves from patients is not realistically feasible, as nerve biopsies are painful and may result in permanent damage. Therefore, our laboratory has investigated paclitaxel-induced neuronal morphological and molecular changes using an ex vivo model of human-induced pluripotent stem cell (iPSC)-derived neurons. Methods: iPSCs are undifferentiated and endlessly dividing cells that can be generated from a patient’s somatic cells, such as peripheral blood mononuclear cells (PBMCs). We successfully reprogrammed PBMCs into iPSCs using the Erythroid Progenitor Reprograming Kit (STEMCell Technologiesᵀᴹ); pluripotency was verified by flow cytometry analysis. iPSCs were then induced into neurons using a differentiation protocol that bypasses the neural progenitor stage and uses selected small-molecule modulators of key signaling pathways (SMAD, Notch, FGFR1 inhibition, and Wnt activation). Results: Flow cytometry analysis revealed expression of core pluripotency transcription factors Nanog, Oct3/4 and Sox2 in iPSCs overlaps with commercially purchased pluripotent cell line UCSD064i-20-2. Trilineage differentiation of iPSCs was confirmed with immunofluorescent imaging with germ-layer-specific markers; Sox17 and ExoA2 for ectoderm, Nestin, and Pax6 for mesoderm, and Ncam and Brachyury for endoderm. Sensory neuron markers, β-III tubulin, and Peripherin were applied to stain the cells for the maturity of iPSC-derived neurons. Patch-clamp electrophysiology and calcitonin gene-related peptide (CGRP) release data supported the functionality of the induced neurons and provided insight into the timing for which downstream assays could be performed (week 4 post-induction). We have also performed a cell viability assay and fluorescence-activated cell sorting (FACS) using four cell-surface markers (CD184, CD44, CD15, and CD24) to select a neuronal population. At least 70% of the cells were viable in the isolated neuron population. Conclusion: We have found that these iPSC-derived neurons recapitulate mature neuronal phenotypes and demonstrate functionality. Thus, this represents a patient-derived ex vivo neuronal model to investigate the molecular mechanisms of clinical TIPN.

Keywords: chemotherapy, iPSC-derived neurons, peripheral neuropathy, taxane, paclitaxel

Procedia PDF Downloads 106

Authors: Ning Li, Yaner Yan, Yajun Qiao, Shu-qing An

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Although seed dispersal is considered to be a key process determining the spatial structure and spread of invasive plant populations, few studies have explicitly addressed the link between dispersal vector behaviour, and seedling recruitment to gain insight into the process of exotic species invasion within a urban landscape. The present study tests the effects of native bird species on the dispersal and recruitment of invasive Phytolacca Americana in an urban garden. We found the invasive population of American pokeweed attracted both generalist species and specialist species to forage and disperse its seeds, with generalists Pycnonotus sinensis and Urocissa erythrorhyncha being by far the most important dispersers. Seedling numbers of P. Americana was strongly affected by perching behavior of bird dispersers. Moreover, two main disperser species, P. sinensis and U. erythrorhyncha govern a high quality dispersal service for P. Americana. Our results highlight the ability of invasive P. americana to recruit seed dispersal agents in urban habitats. However, if the newly recruited species could use the seedling safe site for perching shelter, the invasive plants will get a high regenerate rate in the invasive new habitats thus enhancing their invasive ability.

Keywords: frugivorous birds, phytolacca americana, seed dispersal, urban landscape

Procedia PDF Downloads 522

Authors: Parag Katira, Frederika Rentzeperis, Zuzanna Nowicka, Giada Fiandaca, Thomas Veith, Jack Farinhas, Noemi Andor

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Resource limitations shape the outcome of competitions between genetically heterogeneous pre-malignant cells. One example of such heterogeneity is in the ploidy (DNA content) of pre-malignant cells. A whole-genome duplication (WGD) transforms a diploid cell into a tetraploid one and has been detected in 28-56% of human cancers. If a tetraploid subclone expands, it consistently does so early in tumor evolution, when cell density is still low, and competition for nutrients is comparatively weak – an observation confirmed for several tumor types. WGD+ cells need more resources to synthesize increasing amounts of DNA, RNA, and proteins. To quantify resource limitations and how they relate to ploidy, we performed a PAN cancer analysis of WGD, PET/CT, and MRI scans. Segmentation of >20 different organs from >900 PET/CT scans were performed with MOOSE. We observed a strong correlation between organ-wide population-average estimates of Oxygen and the average ploidy of cancers growing in the respective organ (Pearson R = 0.66; P= 0.001). In-vitro experiments using near-diploid and near-tetraploid lineages derived from a breast cancer cell line supported the hypothesis that DNA content influences Glucose- and Oxygen-dependent proliferation-, death- and migration rates. To model how subpopulations with variable DNA content compete in the resource-limited environment of the human brain, we developed a stochastic state-space model of the brain (S3MB). The model discretizes the brain into voxels, whereby the state of each voxel is defined by 8+ variables that are updated over time: stiffness, Oxygen, phosphate, glucose, vasculature, dead cells, migrating cells and proliferating cells of various DNA content, and treat conditions such as radiotherapy and chemotherapy. Well-established Fokker-Planck partial differential equations govern the distribution of resources and cells across voxels. We applied S3MB on sequencing and imaging data obtained from a primary GBM patient. We performed whole genome sequencing (WGS) of four surgical specimens collected during the 1ˢᵗ and 2ⁿᵈ surgeries of the GBM and used HATCHET to quantify its clonal composition and how it changes between the two surgeries. HATCHET identified two aneuploid subpopulations of ploidy 1.98 and 2.29, respectively. The low-ploidy clone was dominant at the time of the first surgery and became even more dominant upon recurrence. MRI images were available before and after each surgery and registered to MNI space. The S3MB domain was initiated from 4mm³ voxels of the MNI space. T1 post and T2 flair scan acquired after the 1ˢᵗ surgery informed tumor cell densities per voxel. Magnetic Resonance Elastography scans and PET/CT scans informed stiffness and Glucose access per voxel. We performed a parameter search to recapitulate the GBM’s tumor cell density and ploidy composition before the 2ⁿᵈ surgery. Results suggest that the high-ploidy subpopulation had a higher Glucose-dependent proliferation rate (0.70 vs. 0.49), but a lower Glucose-dependent death rate (0.47 vs. 1.42). These differences resulted in spatial differences in the distribution of the two subpopulations. Our results contribute to a better understanding of how genomics and microenvironments interact to shape cell fate decisions and could help pave the way to therapeutic strategies that mimic prognostically favorable environments.

Keywords: tumor evolution, intra-tumor heterogeneity, whole-genome doubling, mathematical modeling

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Authors: Surbhi Surbhi, Andrea Erni, Gunter Meister, Harold Cremer, Christophe Beclin

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MicroRNAs (miRNAs) are short 20-23 nucleotide long non-coding RNAs; there are 2605 miRNA in humans and 1936 miRNA in mouse in total (miRBase). The nervous system expresses the most abundant miRNA and most diverse. MiRNAs play a role in many steps during neurogenesis, like cell proliferation, differentiation, neural patterning, axon pathfinding, etc. Moreover, in vitro studies suggested a role in the regulation of local translation at the synapse, thus controlling neuronal plasticity. However, due to the specific structure of miRNA molecules, an in-vivo confirmation of the general role of miRNAs in the control of neuronal plasticity is still pending. For example, their small size and their high level of sequence homology make difficult the analysis of their cellular and sub-cellular localization in-vivo by in-situ hybridization. Moreover, it was found that only 40% of the expressed miRNA molecules in a cell are included in RNA-Induced Silencing Complexes (RISC) and, therefore, involved in inhibitory interactions while the rest is silent. Definitively, the development of new tools is needed to have a better understanding of the cellular function of miRNAs, in particular their role in neuronal plasticity. Here we describe a new technique called in-vivo AGO-APP designed to investigate miRNA expression and function in-vivo. This technique is based on the expression of a small peptide derived from the human RISC-complex protein TNRC6B, called T6B, which binds all known Argonaute (Ago) proteins with high affinity allowing the efficient immunoprecipitation of AGO-bound miRNAs. We have generated two transgenic mouse lines conditionally expressing T6B either ubiquitously in the cell or targeted at the synapse. A comparison of the repertoire of miRNAs immuno-precipitated from mature neurons of both mouse lines will provide us with a list of miRNAs showing a specific activity at the synapse. The physiological role of these miRNAs will be subsequently addressed through gain and loss of function experiments.

Keywords: RNA-induced silencing complexes, TNRC6B, miRNA, argonaute, synapse, neuronal plasticity, neurogenesis

Procedia PDF Downloads 111