Search results for: extracellular vesicles

Authors: Ainoa Guinart, Maria Korpidou, Daniel Doellerer, Cornelia Palivan, Ben L. Feringa

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Stimuli responsive systems arise from the need to meet unsolved needs of current molecular drugs. Our study presents the design of a delivery system with high spatiotemporal control and tuneable release profiles. We study the incorporation of a hydrophobic synthetic molecular motor into PDMS-b-PMOXA block copolymer vesicles to create a self-assembled system. We prove their successful incorporation and selective activation by low powered visible light (λ 430 nm, 6.9 mW). We trigger the release of a fluorescent dye with high release efficiencies over sequential cycles (up to 75%) with the ability to turn on and off the release behaviour on demand by light irradiation. Low concentrations of photo-responsive units are proven to trigger release down to 1 mol% of molecular motor. Finally, we test our system in relevant physiological conditions using a lung cancer cell line and the encapsulation of an approved drug. Similar levels of cell viability are observed compared to the free-given drugshowing the potential of our platform to deliver functional drugs on demand with the same efficiency and lower toxicity.

Keywords: molecular motor, polymer, drug delivery, light-responsive, cancer, selfassembly

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Authors: Yuanyuan Zhang, Yujuan Zheng, Giuseppina Barutello, Sumako Kameishi, Kungchun Chiu, Katharina Hennig, Martial Balland, Federica Cavallo, Lars Holmgren

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Angiogenesis describes that new blood vessels migrate from pre-existing ones to form 3D lumenized structure and remodeling. During directional migration toward the gradient of pro-angiogenic factors, the endothelial cells, especially the tip cells need filopodia to sense the environment and exert the pulling force. Of particular interest are the integrin proteins, which play an essential role in focal adhesion in the connection between migrating cells and extracellular matrix (ECM). Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving angiogenesis. We have previously identified Angiomotin (Amot), a member of Amot scaffold protein family, as a promoter for endothelial cell migration in vitro and zebrafish models. Hence, we established inducible endothelial-specific Amot knock-out mice to study normal retinal angiogenesis as well as tumor angiogenesis. We found that the migration ratio of the blood vessel network to the edge was significantly decreased in Amotec- retinas at postnatal day 6 (P6). While almost all the Amot defect tip cells lost migration advantages at P7. In consistence with the dramatic morphology defect of tip cells, there was a non-autonomous defect in astrocytes, as well as the disorganized fibronectin expression pattern correspondingly in migration front. Furthermore, the growth of transplanted LLC tumor was inhibited in Amot knockout mice due to fewer vasculature involved. By using MMTV-PyMT transgenic mouse model, there was a significantly longer period before tumors arised when Amot was specifically knocked out in blood vessels. In vitro evidence showed that Amot binded to beta-actin, Integrin beta 1 (ITGB1), Fibronectin, FAK, Vinculin, major focal adhesion molecules, and ITGB1 and stress fibers were distinctly induced by Amot transfection. Via traction force microscopy, the total energy (force indicater) was found significantly decreased in Amot knockdown cells. Taken together, we propose that Amot is a novel partner of the ITGB1/Fibronectin protein complex at focal adhesion and required for exerting force transition between endothelial cell and extracellular matrix.

Keywords: angiogenesis, angiomotin, endothelial cell migration, focal adhesion, integrin beta 1

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Authors: Er-Yuan Chuang

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Bio-mimetic matters have biological functionalities. This might be valuable in the development of versatile biomaterials. At biological fields, protein-based materials might be components to form a 3D network of extracellular biomolecules, containing growth factors. Also, the protein-based biomaterial provides biochemical and structural assistance of adjacent cells. In this study, we try to prepare protein based biomaterial, which was harvested from living animal. We analyzed it’s chemical, physical and biological property in vitro. Besides, in vivo bio-interaction of the prepared biomimetic matrix was tested in an animal model. The protein-based biomaterial has degradability and biocompatibility. This development could be used for tissue regenerations and be served as platform technologies.

Keywords: protein based, in vitro study, in vivo study, biomaterials

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Authors: Ghoul Rachid Brahim, Trad Khodja Rafik

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Vertebral localization of the hydatid cyst is a severe form of bone hydatidosis, is a parasitic infection caused by the larval forms of the tapeworms Echinococcus granulosus, The disease is slowly remaining silent (a long incubation period) which may explain why this pathology is often discovered at the stage of neurological complications. The objective of this study is to recall the clinical and radiological aspects of this condition and the importance of early diagnosis and appropriate management. We report a study of 5 patients with vertebral hydatidosis, four men and one woman, four (04) patients operated in the emergency setting for spinal cord compression (decompression by wide laminectomy with evacuation of intra and extra canal vesicles).Albendazole-based medical treatment is instituted in all patients. Results: The evolution was favorable for three patients, the other two patients reoperated for a local recurrence. Conclusion: Vertebral hydatidosis is a rare condition with a poor prognosis due to the risk of neurological damage, the infiltrating nature of bone lesions, the frequency of relapses and therapeutic difficulties. The only curative method remains surgery, which must aim for complete and large excision of the lesions as if it were a “malignant tumour”.

Keywords: hydatidosis, Echinococcosis granulosus, hydatid cyst, spinal cord compression, laminectomy

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Authors: Sanjay Saraf

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The oral cavity can be the site for early manifestations of mucocutaneous disorders (MD) or the only site for occurrence of these disorders. It can also exhibit oral lesions with simultaneous associated skin lesions. The MD involving the oral mucosa commonly presents with signs such as ulcers, vesicles and bullae. The unique environment of the oral cavity may modify these signs of the disease, thereby making the clinical diagnosis an arduous task. In addition to the unique environment of oral cavity, the overlapping of the signs of various mucocutaneous disorders, also makes the clinical diagnosis more intricate. The aim of this review is to present the oral signs of dermatological disorders having common oral involvement and emphasize their importance in early detection of the systemic disorders. The aim is also to highlight the necessity of oral examination by a dermatologist while examining the skin lesions. Prior to the oral examination, it must be imperative for the dermatologists and the dental clinicians to have the knowledge of oral anatomy. It is also important to know the impact of various diseases on oral mucosa, and the characteristic features of various oral mucocutaneous lesions. An initial clinical oral examination is may help in the early diagnosis of the MD. Failure to identify the oral manifestations may reduce the likelihood of early treatment and lead to more serious problems. This paper reviews the oral manifestations of immune mediated dermatological disorders with common oral manifestations.

Keywords: dermatological investigations, genodermatosis, histological features, oral examination

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Authors: Adela Diepoltova, Klara Konecna, Ondrej Jandourek, Petr Nachtigal

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A loss of control over antibiotic-resistant pathogens has become a global issue due to severe and often untreatable infections. This state is reflected in complicated treatment, health costs, and higher mortality. All these factors emphasize the urgent need for the discovery and development of new anti-infectives. One of the most common pathogens mentioned in the phenomenon of antibiotic resistance are bacteria of the genus Staphylococcus. These bacterial agents have developed several mechanisms against the effect of antibiotics. One of them is biofilm formation. In staphylococci, biofilms are associated with infections such as endocarditis, osteomyelitis, catheter-related bloodstream infections, etc. To author's best knowledge, no validated and standardized methodology evaluating candidate compound activity against staphylococcal biofilms exists. However, a variety of protocols for in vitro drug activity testing has been suggested, yet there are often fundamental differences. Based on our experience, a key methodological step that leads to credible results is to form a robust biofilm with appropriate attributes such as firm adherence to the substrate, a complex arrangement in layers, and the presence of extracellular polysaccharide matrix. At first, for the purpose of drug antibiofilm activity evaluation, the focus was put on various conditions (supplementation of cultivation media by human plasma/fetal bovine serum, shaking mode, the density of initial inoculum) that should lead to reproducible and robust in vitro staphylococcal biofilm formation in microtiter plate model. Three model staphylococcal reference strains were included in the study: Staphylococcus aureus (ATCC 29213), methicillin-resistant Staphylococcus aureus (ATCC 43300), and Staphylococcus epidermidis (ATCC 35983). The total biofilm biomass was quantified using the Christensen method with crystal violet, and results obtained from at least three independent experiments were statistically processed. Attention was also paid to the viability of the biofilm-forming staphylococcal cells and the presence of extracellular polysaccharide matrix. The conditions that led to robust biofilm biomass formation with attributes for biofilms mentioned above were then applied by introducing an alternative method analogous to the commercially available test system, the Calgary Biofilm Device. In this test system, biofilms are formed on pegs that are incorporated into the lid of the microtiter plate. This system provides several advantages (in situ detection and quantification of biofilm microbial cells that have retained their viability after drug exposure). Based on our preliminary studies, it was found that the attention to the peg surface and substrate on which the bacterial biofilms are formed should also be paid to. Therefore, further steps leading to the optimization were introduced. The surface of pegs was coated by human plasma, fetal bovine serum, and L-polylysine. Subsequently, the willingness of bacteria to adhere and form biofilm was monitored. In conclusion, suitable conditions were revealed, leading to the formation of reproducible, robust staphylococcal biofilms in vitro for the microtiter model and the system analogous to the Calgary biofilm device, as well. The robustness and typical slime texture could be detected visually. Likewise, an analysis by confocal laser scanning microscopy revealed a complex three-dimensional arrangement of biofilm forming organisms surrounded by an extracellular polysaccharide matrix.

Keywords: anti-biofilm drug activity screening, in vitro biofilm formation, microtiter plate model, the Calgary biofilm device, staphylococcal infections, substrate modification, surface coating

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Authors: Juliana A. Knocikova, Yann Bouret, Médéric Argentina, Laurent Counillon

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Cell volume, together with membrane potential and intracellular hydrogen ion concentration, is an essential biophysical parameter for normal cellular activity. Cell volumes can be altered by osmotically active compounds and extracellular tonicity. In this study, a simple mathematical model of osmotically induced cell swelling and shrinking is presented. Emphasis is given to water diffusion across the membrane. The mathematical description of the cellular behavior consists in a system of coupled ordinary differential equations. We compare experimental data of cell volume alterations driven by differences in osmotic pressure with mathematical simulations under hypotonic and hypertonic conditions. Implications for a future model are also discussed.

Keywords: eukaryotic cell, mathematical modeling, osmosis, volume alterations

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Authors: Shaker Alsharif, Xavier Banquy

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Controlled drug delivery technology represents one of the most rapidly advancing areas of science in which chemists and chemical engineers are contributing to human health care. Such delivery systems provide numerous advantages compared to conventional dosage forms including improved efficacy, and improved patient compliance and convenience. Such systems often use synthetic polymers as carriers for the drugs. As a result, treatments that would not otherwise be possible are now in conventional use. The role of bilayered vesicles as efficient carriers for drugs, vaccines, diagnostic agents and other bioactive agents have led to a rapid advancement in the liposomal drug delivery system. Moreover, the site avoidance and site-specific drug targeting therapy could be achieved by formulating a liposomal product, so as to reduce the cytotoxicity of many potent therapeutic agents. Our project focuses on developing and building hydrogel with nanoinclusion of liposomes loaded with active compounds such as proteins and growth factors able to release them in a controlled fashion. In order to achieve that, we synthesize several liposomes of two different phospholipids concentrations encapsulating model drug. Then, formulating hydrogel with specific mechanical properties embedding the liposomes to manage the release of active compound.

Keywords: controlled release, hydrogel, liposomes, active compounds

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Authors: Neha Singh

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Background: Nowadays, the nanoparticle is gaining unexceptional attention in targeted drug delivery. But before proceeding to this episode of accomplishment, the journey and closure of these nanoparticles inside the cells should be disentangle. Being foreign for the cells, nanoparticles will easily getcleared off without any effective outcome. As the cancer cells withhold these nanoparticles for a longer period of time, more will be the drug’s effect. Chlorpromazine is a cationic amphiphilic drug which is believed to inhibit clathrin-coated pit formation by a reversible translocation of clathrin and its adapter proteins from the plasma membrane to intracellular vesicles. Chlorpromazine has a role in increasing the retention of nanoparticles in cancer cells. The mechanism of action how this small molecule increases the retention of nanoparticles is still uncovered. Method: Polymeric nanoparticle (PLGA) with Cyanine3.5 dye were synthesized by solvent evaporation method and characterized for size and zeta potential. FTIR was also done. Pulse and chase studies with and without inhibitor were done to check the retention of nanoparticle using fluorescence microscopy. Mean fluorescence intensity was measured by ImageJ software. Results: Increased retention of nanoparticle with inhibitor was observed in both pulse and chase studies. Conclusion: Our results demonstrate that by repurposing these small molecule inhibitor, we can increase the retention of nanoparticle at the targeted site.

Keywords: nanoparticle, endocytosis, clathrin inhibitor, cancer cell

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Authors: Rashmi Shukla, Yamini Bhusan Tripathi

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Diabetic nephropathy (DN) is characterized as diabetic kidney disease which involves many pathways e.g. hyperactivated protein kinase c (PKC), polyol pathway, excess production of advanced glycation end product (AGEs) & free radical accumulation etc. All of them results to hypoxia followed by apoptosis of podocytes, glomerulosclerosis, extracellular matrix (ECM) accumulation and fibrosis resulting to irreversible changes in kidney. This is continuously rising worldwide and there are not enough specific drugs, to retard its progress. Due to increasing side effects of allopathic drugs, interest in herbal remedies is growing. Earlier, we have reported that PTY-2 (a phytomedicine, derived from Pueraria tuberosa Linn.) inhibits the accumulation of extracellular matrix (ECM) through activation of MMP-9. Present study exhibited the therapeutic potential of Pueraria tuberosa in the prevention of podocytes apoptosis and modulation of nephrin expression in streptozotocin (STZ) induced DN rats. DN rats were produced by maintaining persistent hyperglycemia for 8 weeks by intra-peritoneal injection of 55 mg/kg streptozotocin (STZ). These rats were randomly divided in 2 groups, i.e. DN control, and DN+ water extract of Pueraria tuberosa (PTW). One group of age-matched normal rats served as non-diabetic control (group-1), The STZ induced DN rats (group-2) and DN+PTW treated rats (group-3). The PTW was orally administered (0.3g/kg) daily to group-2 rats and drug vector (1 ml of 10% tween 20) in control rats. The treatments were continued for 20 days and blood and urine samples were collected. Rats were then sacrificed to investigate the expression Bcl2, Bax and nephroprotective protein i.e. nephrin in kidney glomerulus. The effect of PTW was evaluated, we have found that the PTW significantly(p < .001) reversed the raised serum urea, serum creatinine, urine protein and improved the creatinine clearance in STZ induce diabetic nephropathy in rats and also significantly(p < .001) prevented the rise in urine albumin excretion. The Western blot analysis of kidney tissue homogenate showed increased expression of Bcl2 in PTW treated rats. The RT-PCR showed the increased expression and accumulation of nephrin mRNA. The confocal photomicrographs also supported the reduction of Bax and a simultaneous increase in Bcl2 and nephrin in glomerular podocytes. Hence, our finding suggests that the nephroprotective role of PTW is mediated via restoration of nephrin thus prevents the podocytes apoptosis and ameliorates diabetic nephropathy. The clinical trial of PTW would prove to be a potential food supplement/ drug of alternative medicine for patients with diabetic nephropathy in early stage.

Keywords: Pueraria tuberosa, diabetic nephropathy, anti-apoptosis, nephrin

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Authors: Shin-Yi Mao, Jiashing Yu

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Decellularized adipose tissue (DAT) has received lots of attention as biological scaffolds recently. DAT that extracted from the extracellular matrix (ECM) of adipose tissues holds great promise as a xenogeneic biomaterial for tissue engineering and regenerative medicine. In our study, 2-D DATsol film was fabricated to enhance cell adhesion, proliferation, and differentiation of ASCs in vitro. DAT was also used to modify alginate for improvement of cell adhesion. Alginate microspheres modified with DAT were prepared by Nisco. These microspheres could provide a highly supportive 3-D environment for ASCs. In our works, ASCs were immobilized in alginate microspheres modified with DAT to promoted cell adhesion and adipogenic differentiation. Accordingly, we hypothesize that tissue regeneration in vivo could be promoted with the aid of modified microspheres in future.

Keywords: adipose stem cells, decellularize adipose tissue, Alginate, microcarries

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Authors: Dimitra C. Banti, Gesthimani Liona, Petros Samaras, Manasis Mitrakas

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Municipal and industrial wastewaters are often treated biologically, by the activated sludge process (ASP). The ASP not only requires large aeration and sedimentation tanks, but also generates large quantities of excess sludge. An alternative technology is the membrane bioreactor (MBR), which replaces two stages of the conventional ASP—clarification and settlement—with a single, integrated biotreatment and clarification step. The advantages offered by the MBR over conventional treatment include reduced footprint and sludge production through maintaining a high biomass concentration in the bioreactor. Notwithstanding these advantages, the widespread application of the MBR process is constrained by membrane fouling. Fouling leads to permeate flux decline, making more frequent membrane cleaning and replacement necessary and resulting to increased operating costs. In general, membrane fouling results from the interaction between the membrane material and the components in the activated sludge liquor. The latter includes substrate components, cells, cell debris and microbial metabolites, such as Extracellular Polymeric Substances (EPS) and Sludge Microbial Products (SMPs). The challenge for effective MBR operation is to minimize the rate of Transmembrane Pressure (TMP) increase. This can be achieved by several ways, one of which is the addition of specific additives, that enhance the coagulation and flocculation of compounds, which are responsible for fouling, hence reducing biofilm formation on the membrane surface and limiting the fouling rate. In this project the effectiveness of a non-commercial composite coagulant was studied as an agent for fouling control in a lab scale MBR system consisting in two aerated tanks. A flat sheet membrane module with 0.40 um pore size was submerged into the second tank. The system was fed by50 L/d of municipal wastewater collected from the effluent of the primary sedimentation basin. The TMP increase rate, which is directly related to fouling growth, was monitored by a PLC system. EPS, MLSS and MLVSS measurements were performed in samples of mixed liquor; in addition, influent and effluent samples were collected for the determination of physicochemical characteristics (COD, BOD5, NO3-N, NH4-N, Total N and PO4-P). The coagulant was added in concentrations 2, 5 and 10mg/L during a period of 2 weeks and the results were compared with the control system (without coagulant addition). EPS fractions were extracted by a three stages physical-thermal treatment allowing the identification of Soluble EPS (SEPS) or SMP, Loosely Bound EPS (LBEPS) and Tightly Bound EPS (TBEPS). Proteins and carbohydrates concentrations were measured in EPS fractions by the modified Lowry method and Dubois method, respectively. Addition of 2 mg/L coagulant concentration did not affect SEPS proteins in comparison with control process and their values varied between 32 to 38mg/g VSS. However a coagulant dosage of 5mg/L resulted in a slight increase of SEPS proteins at 35-40 mg/g VSS while 10mg/L coagulant further increased SEPS to 44-48mg/g VSS. Similar results were obtained for SEPS carbohydrates. Carbohydrates values without coagulant addition were similar to the corresponding values measured for 2mg/L coagulant; the addition of mg/L coagulant resulted to a slight increase of carbohydrates SEPS to 6-7mg/g VSS while a dose of 10 mg/L further increased carbohydrates content to 9-10mg/g VSS. Total LBEPS and TBEPS, consisted of proteins and carbohydrates of LBEPS and TBEPS respectively, presented similar variations by the addition of the coagulant. Total LBEPS at 2mg/L dose were almost equal to 17mg/g VSS, and their values increased to 22 and 29 mg/g VSS during the addition of 5 mg/L and 10 mg/L of coagulant respectively. Total TBEPS were almost 37 mg/g VSS at a coagulant dose of 2 mg/L and increased to 42 and 51 mg/g VSS at 5 mg/L and 10 mg/L doses, respectively. Therefore, it can be concluded that coagulant addition could potentially affect microorganisms activities, excreting EPS in greater amounts. Nevertheless, EPS increase, mainly SEPS increase, resulted to a higher membrane fouling rate, as justified by the corresponding TMP increase rate. However, the addition of the coagulant, although affected the EPS content in the reactor mixed liquor, did not change the filtration process: an effluent of high quality was produced, with COD values as low as 20-30 mg/L.

Keywords: extracellular polymeric substances, MBR, membrane fouling, EPS

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Authors: Mohammadhosein Rahimi, Mohammad Raouf Hosseini, Mehran Bakhshi, Alireza Baghbanan

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Silver ions (Ag⁺) and their compounds are consequentially toxic to microorganisms, showing biocidal effects on many species of bacteria. Silver-phosphate (or silver orthophosphate) is one of these compounds, which is famous for its antimicrobial effect and catalysis application. In the present study, a green method was presented to synthesis silver-phosphate nanoparticles using Sporosarcina pasteurii. The composition of the biosynthesized nanoparticles was identified as Ag₃PO₄ using X-ray Diffraction (XRD) and Energy Dispersive Spectroscopy (EDS). Also, Fourier Transform Infrared (FTIR) spectroscopy showed that Ag₃PO₄ nanoparticles was synthesized in the presence of biosurfactants, enzymes, and proteins. In addition, UV-Vis adsorption of the produced colloidal suspension approved the results of XRD and FTIR analyses. Finally, Transmission Electron Microscope (TEM) images indicated that the size of the nanoparticles was about 20 nm.

Keywords: bacteria, biosynthesis, silver-phosphate, Sporosarcina pasteurii, nanoparticle

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Authors: Mohammed Fayez Al Rez

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Nowadays, there is an unmet clinical need for new small-diameter vascular grafts to overcome the drawbacks of traditional methods used for treatment of widespread cardiovascular diseases. Vascular tissue engineering (VTE) is a promising approach that can be utilized to develop viable vascular grafts by in vitro seeding of functional cells onto a scaffold allowing them to attach, proliferate and differentiate. To achieve this purpose, the scaffold should provide cells with the initial necessary extracellular matrix environment and structure until being able to reconstruct the required vascular tissue. Therefore, producing scaffolds with suitable features is crucial for guiding cells properly to develop the desired tissue-engineered vascular grafts for clinical applications. The main objective of this work is fabrication and characterization of tubular small-diameter ( < 6 mm) bilayered scaffolds for VTE. The scaffolds were prepared via mixing electrospinning approach of biodegradable poly(ε-caprolactone) (PCL) polymer – due to its favorable physicochemical properties – to mimic the natural environment-extracellular matrix. Firstly, tubular nanofibrous construct with inner diameter of 3, 4 or 5 mm was electrospun as inner layer, and secondly, microfibrous construct was electrospun as outer layer directly on the first produced inner layer. To improve the biological properties of PCL, a group of the electrospun scaffolds was immersed in type-1 collagen solution. The morphology and structure of the resulting fibrous scaffolds were investigated by scanning electron microscope. The electrospun nanofibrous inner layer contained fibers measuring 219±35 nm in diameter, while the electrospun microfibrous outer layer contained fibers measuring 1011 ± 150 nm. Furthermore, mechanical, thermal and physical tests were conducted with both electrospun bilayered scaffold types where revealed improved properties. Biological investigations using endothelial, smooth muscle and fibroblast cell line showed good biocompatibility of both tested electrospun scaffolds. Better attachment and proliferation were obviously found when cells were cultured on the scaffolds immersed with collagen due to increasing the hydrophilicity of the PCL. The easy, inexpensive and versatile electrospinning approach used in this work was able to successfully produce double layered tubular elastic structures containing both nanofibers and microfibers to imitate the native vascular structure. The PCL – as a suitable and approved biomaterial for many biomedical and tissue engineering applications – can ensure favorable mechanical properties of scaffolds used for VTE. The VTE approach using electrospun bilayered scaffolds offers optimal solutions and holds significant promises for treatment of many cardiovascular diseases.

Keywords: electrospinning, poly(ε-caprolactone) (PCL), tissue-engineered vascular graft, tubular bilayered scaffolds, vascular cells

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Authors: Mahmut Sozmen, Alparslan Kadir Devrim, Yonca Betil Kabak, Tuba Devrim

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Acute myocardial infarction is the leading cause of deaths in the worldwide. Mature cardiomyocytes do not have the ability to regenerate instead fibrous tissue proliferate and granulation tissue to fill out. Periostin is an extracellular matrix protein from fasciclin family and it plays an important role in the cell adhesion, migration, and growth of the organism. Periostin prevents apoptosis while stimulating cardiomyocytes. The main objective of this project is to investigate the effects of the recombinant murine periostin peptide administration for the cardiomyocyte regeneration in a rat model of acute myocardial infarction. The experiment was performed on 84 male rats (6 months old) in 4 group each contains 21 rats. Saline applied subcutaneously (1 ml/kg) two times with 24 hours intervals to the rats in control group (Group 1). Recombinant periostin peptide (1 μg/kg) dissolved in saline applied intraperitoneally in group 2 on 1, 3, 7, 14 and 21. days on same dates in group 4. Isoproterenol dissolved in saline applied intraperitoneally (85mg/kg/day) two times with 24 hours intervals to the groups 3 and 4. Rats in group 4 further received recombinant periostin peptide (1 μg/kg) dissolved in saline intraperitoneally starting one day after the final isoproterenol administration on days 1, 3, 7, 14 and 21. Following the final application of periostin rats continued to feed routinely with pelleted chow and water ad libitum for further seven days. At the end of 7th day rats sacrificed, blood and heart tissue samples collected for the immunohistochemical and biochemical analysis. Angiogenesis in response to tissue damage, is a highly dynamic process regulated by signals from the surrounding extracellular matrix and blood serum. In this project, VEGF, ANGPT, bFGF, TGFβ are the key factors that contribute to cardiomyocyte regeneration were investigated. Additionally, the relationship between mitosis and apoptosis (Bcl-2, Bax, PCNA, Ki-67, Phopho-Histone H3), cell cycle activators and inhibitors (Cyclin D1, D2, A2, Cdc2), the origin of regenerating cells (cKit and CD45) were examined. Present results revealed that periostin stimulated cardiomyocye cell-cycle re-entry in both normal and MCA damaged cardiomyocytes and increased angiogenesis. Thus, periostin contributes to cardiomyocyte regeneration during the healing period following myocardial infarction which provides a better understanding of its role of this mechanism, improving recovery rates and it is expected to contribute the lack of literature on this subject. Acknowledgement: This project was financially supported by Turkish Scientific Research Council- Agriculture, Forestry and Veterinary Research Support Group (TUBİTAK-TOVAG; Project No: 114O734), Ankara, TURKEY.

Keywords: cardiotoxicity, immunohistochemistry, isoproterenol, periostin

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Authors: G. C. Santini, C. Potrich, L. Lunelli, L. Vanzetti, S. Marasso, M. Cocuzza, C. Pederzolli

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The relevance of circulating miRNAs as non-invasive biomarkers for several pathologies is nowadays undoubtedly clear, as they have been found to have both diagnostic and prognostic value able to add fundamental information to patients’ clinical picture. The availability of these data, however, relies on a time-consuming process spanning from the sample collection and processing to the data analysis. In light of this, strategies which are able to ease this procedure are in high demand and considerable effort have been made in developing Lab-on-a-chip (LOC) devices able to speed up and standardise the bench work. In this context, a very promising polydimethylsiloxane (PDMS)-based microdevice which integrates the processing of the biological sample, i.e. purification of extracellular miRNAs, and reverse transcription was previously developed in our lab. In this study, we aimed at the improvement of the miRNA extraction performances of this micro device by increasing the ability of its surface to absorb extracellular miRNAs from biological samples. For this purpose, we focused on the modulation of two properties of the material: roughness and charge. PDMS surface roughness was modulated by casting with several templates (terminated with silicon oxide coated by a thin anti-adhesion aluminum layer), followed by a panel of curing conditions. Atomic force microscopy (AFM) was employed to estimate changes at the nanometric scale. To introduce modifications in surface charge we functionalized PDMS with different mixes of positively charged 3-aminopropyltrimethoxysilanes (APTMS) and neutral poly(ethylene glycol) silane (PEG). The surface chemical composition was characterized by X-ray photoelectron spectroscopy (XPS) and the number of exposed primary amines was quantified with the reagent sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate (s-SDTB). As our final end point, the adsorption rate of all these different conditions was assessed by fluorescence microscopy by incubating a synthetic fluorescently-labeled miRNA. Our preliminary analysis identified casting on thermally grown silicon oxide, followed by a curing step at 85°C for 1 hour, as the most efficient technique to obtain a PDMS surface roughness in the nanometric scaleable to trap miRNA. In addition, functionalisation with 0.1% APTMS and 0.9% PEG was found to be a necessary step to significantly increase the amount of microRNA adsorbed on the surface, therefore, available for further steps as on-chip reverse transcription. These findings show a substantial improvement in the extraction efficiency of our PDMS microdevice, ultimately leading to an important step forward in the development of an innovative, easy-to-use and integrated system for the direct purification of less abundant circulating microRNAs.

Keywords: circulating miRNAs, diagnostics, Lab-on-a-chip, polydimethylsiloxane (PDMS)

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Authors: Masoumeh Ghasemi, Afshin Farahbakhsh, Arman Farahbakhsh, Ali Asghar Safari

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In this study, lipase production has been investigated using submerge fermentation by Aspergillus niger in Kilka fish oil as main substrate. The Taguchi method with an L9 orthogonal array design was used to investigate the effect of parameters and their levels on lipase productivity. The optimum conditions for Kilka fish oil concentration, incubation temperature and pH were obtained 3 gr./ml 35°C and 7, respectively. The amount of lipase activity in optimum condition was obtained 4.59IU/ml. By comparing this amount with the amount of productivity in the olive oil medium based on the cost of each medium, it was that using Kilka fish oil is 84% economical. Therefore Kilka fish oil can be used as an economical and suitable substrate in the lipase production and industrial usages.

Keywords: lipase, Aspergillus niger, Kilka fish oil, submerge fermentation method

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Authors: Yogesh D. Bansod, Jiri Bursa

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The quantitative study of cell mechanics is of paramount interest since it regulates the behavior of the living cells in response to the myriad of extracellular and intracellular mechanical stimuli. The novel experimental techniques together with robust computational approaches have given rise to new theories and models, which describe cell mechanics as a combination of biomechanical and biochemical processes. This review paper encapsulates the existing continuum-based computational approaches that have been developed for interpreting the mechanical responses of living cells under different loading and boundary conditions. The salient features and drawbacks of each model are discussed from both structural and biological points of view. This discussion can contribute to the development of even more precise and realistic computational models of cell mechanics based on continuum approaches or on their combination with microstructural approaches, which in turn may provide a better understanding of mechanotransduction in living cells.

Keywords: cell mechanics, computational models, continuum approach, mechanical models

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Authors: Muhasin Koyiloth, Sathyanarayana N. Gummadi

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Biological membranes are ordered association of lipids, proteins, and carbohydrates. Lipids except sterols possess asymmetric distribution across the bilayer. Eukaryotic membranes possess a group of lipid translocators called scramblases that disrupt phospholipid asymmetry. Their action is implicated in cell activation during wound healing and phagocytic clearance of apoptotic cells. Cholesterol is one of the major membrane lipids distributed evenly on both the leaflet and can directly influence the membrane fluidity through the ordering effect. The fluidity has an impact on the activity of several membrane proteins. The palmitoylated phospholipid scramblases localized to the lipid raft which is characterized by a higher number of sterols. Here we propose that cholesterol can interact with scramblases through putative CRAC motif and can modulate their activity. To prove this, we reconstituted phospholipid scramblase 1 of C. elegans (SCRM-1) in proteoliposomes containing different amounts of cholesterol (Liquid ordered/Lo). We noted that the presence of cholesterol reduced the scramblase activity of wild-type SCRM-1. The interaction between SCRM-1 and cholesterol was confirmed by fluorescence spectroscopy using NBD-Chol. Also, we observed loss of such interaction when one of I273 in the CRAC motif mutated to Asp. Interestingly, the point mutant has partially retained scramblase activity in Lo vesicles. The current study elucidated the important interaction between cholesterol and SCRM-1 to fine-tune its activity in artificial membranes.

Keywords: artificial membranes, CRAC motif, plasma membrane, PL scramblase

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Authors: N. Kotabin, Y. Tahara, K. Issakul, O. Chunhachart

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Cadmium (II) (Cd) is one of the major toxic elemental pollutants which is hazardous for humans, animals and plants. γ-Polyglutamic acid (γ-PGA) is an extracellular biopolymer produced by several species of Bacillus which has been reported to be an effective biosorbent for metal ions. The effect of γ-PGA on growth of rice grown under laboratory conditions was investigated. Rice seeds were germinated and then grown at 30±1°C on filter paper soaked with Cd solution and γ-PGA for 7 days. The result showed that Cd significantly inhibited the growth of roots and shoots by reducing root and shoot lengths. Fresh and dry weights also decreased compared with control; however, the addition of 500 mg•L-1 γ-PGA alleviated rice seedlings from the adverse effects of Cd. The analysis of physiological traits revealed that Cd caused a decrease in the total chlorophyll and soluble protein contents and amylase activities in all treatments. The Cd content in seedling tissues increased for the Cd 250 μM treatment (P < 0.05) but the addition of 500 mg•L-1 γ-PGA resulted in a noticeable decrease in Cd (P < 0.05).

Keywords: polyglutamic acid, cadmium, rice, bacillus subtilis

Procedia PDF Downloads 270

Authors: Lingfeng Wang, Zhipeng Chen, Shuang Qiu, Shijian Ge

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The effects of wastewater, with four different nutrient loadings, from synthetic centrate on biomass production of Chlorella vulgaris, nutrient removal, microalgal settling, and lipid production were investigated in photobioreactors under both batches and, subsequently, semi-continuous operations. At higher centrate concentration factors (17.2% and 36.2%), hydraulic retention time and pH adjustments could be employed to sustain acceptable microalgal growth rates and wastewater treatment. Similar nutrient removals efficiencies (>95%) and biomass production (0.42-0.51 g/L) were observed for the four centrate concentrations. Both the lipid productivity and lipid content decreased with increasing nutrient loading in the wastewater. The results also demonstrated that the mass ratio of carbohydrate to protein could provide a good indication of microalgal settling performance, rather than sole component composition or total extracellular polymeric substances.

Keywords: lipid production, microalgae, nutrient removal, wastewater

Procedia PDF Downloads 194

Authors: Sourita Ghosh, Falguni Pati, Suhanya Duraiswamy

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Spheroid, a simple three-dimensional cellular aggregate, allows us to simulate the in-vivo complexity of cellular signaling and interactions in greater detail than traditional 2D cell culture. It can be used as an in-vitro model for drug toxicity testing, tumor modeling and many other such applications specifically for cancer. Our work is focused on the development of an affordable, user-friendly, robust, reproducible, high throughput microfluidic device for water in oil droplet production, which can, in turn, be used for spheroids manufacturing. Here, we have investigated the droplet breakup between two non-Newtonian fluids, viz. silicone oil and decellularized liver matrix, which acts as our extra cellular matrix (ECM) for spheroids formation. We performed some biochemical assays to characterize the liver ECM, as well as rheological studies on our two fluids and observed a critical dependence of capillary number (Ca) on droplet breakup and homogeneous drop formation

Keywords: chip less, droplets, extracellular matrix, liver spheroid

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Authors: Lakshmi Sirisha Kotikalapudi

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Atenolol, the widely used antihypertensive drug is ionisable and degrades in the acidic environment of the GIT lessening the bioavailability. Transdermal route may be selected as an alternative to enhance the bioavailability. Half-life of the drug is 6-7 hours suggesting the requirement of prolonged release of the drug. The present work of transdermal niosomal gel aims to extend release of the drug and increase the bioavailability. Ethanol injection method was used for the preparation of niosomes using span-60 and cholesterol at different molar ratios following central composite design. The prepared niosomes were characterized for size, zeta-potential, entrapment efficiency, drug content and in-vitro drug release. Optimized formulation was selected by statistically analyzing the results obtained using the software Stat-Ease Design Expert. The optimized formulation also showed high drug retention inside the vesicles over a period of three months at a temperature of 4 °C indicating stability. Niosomes separated as a pellet were dried and incorporated into the hydrogel prepared using chitosan a natural polymer as a gelling agent. The effect of various chemical permeation enhancers was also studied over the gel formulations. The prepared formulations were characterized for viscosity, pH, drug release using Franz diffusion cells, and skin irritation test as well as in-vivo pharmacological activities. Atenolol niosomal gel preparations showed the prolonged release of the drug and pronounced antihypertensive activity indicating the suitability of niosomal gel for topical and systemic delivery of atenolol.

Keywords: atenolol, chitosan, niosomes, transdermal

Procedia PDF Downloads 248

Authors: Yousuf M. Al Suleimani, Robin Hiley

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The endogenous phospholipid lysophosphatidylinositol (LPI) was recently identified as a novel ligand for the G protein-coupled receptor 55 (GPR55) and an inducer of intracellular Ca2+ [Ca2+]i release. This study attempts to characterize Ca2+ signals provoked by LPI in HEK293 cells engineered to stably express human GPR55 and to test cannabinoid ligand activity at GPR55. The study shows that treatment with LPI stimulates a sustained, oscillatory Ca2+ release. The response is characterized by an initial rapid rise, which is mediated by the Gαq-PLC-IP3 pathway, and this is followed by prolonged oscillations that require RhoA activation. Ca2+ oscillations are initiated by intracellular mechanisms and extracellular Ca2+ is only required to replenish Ca2+ lost from the cytoplasm. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoid anandamide, however, rimonabant and the CB1 receptor antagonist AM251 evoked GPR55-mediated [Ca2+]i. Thus, LPI is likely to be a key plasma membrane mediator of signaling events and changes in gene expression through GPR55 activation.

Keywords: lysophosphatidylinositol, calcium, GPR55, cannabinoid

Procedia PDF Downloads 328

Authors: Deepak Mohan, Sushma Sharma

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Diclofenac sodium, a member of the acetic acid family of non-steroidal anti-inflammatory drugs, is used to retard inflammation, arthritis pain and ankylosing spondylitis. The drug is known to cause severe injury in different tissues due to formation of reactive oxygen species. The present study is focused on the effect of different doses of diclofenac (4 mg/kg/body weight and 14 mg/kg/body weight on histoarchitecture of the liver from 7-28 days of the investigation. Diclofenac administration resulted in distorted hepatic degeneration and formation of wide areas in the form of sinusoidal gaps. Hepatic fibrosis noticed in different stages of investigation could be attributed to chronic inflammation and reactive oxygen species which results in deposition of extracellular matrix proteins. The abrupt degenerative changes observed during later stages of the experiment showed maximum damage to the liver, and there was enlargement of sinusoidal gaps accompanied by maximum necrosis in the tissues.

Keywords: arthritis, diclofenac, histoarchitecture, sinusoidal

Procedia PDF Downloads 240

Authors: Federico Cevoli

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Purinergic P2X7 receptors (P2X7R) are ligand-gated non-selective cation channels involved in several physiological and pathological processes. They are particularly promising pharmacological targets as they are present in an increasing number of different cells types. P2X7R activation is triggered following elevated concentrations of extracellular ATP, similarly to those observed in tissues injury, chronic inflammation and T-cell activation, as well as in the scrambling of phospholipids leading to membrane blebbing and apoptosis. Another hallmark of P2X7 is cell permeabilization, commonly known as “macropore” formation allowing the passage of nanometer-sized molecules up to 900Da. Recently, full-length P2X7 Cryo-EM structures revealed unique functional sites, including two cytoplasmic domains - the cytoplasmic "anchor" and "ballast". To date, the molecular units/complex by which P2X7R exerts its pathophysiological functions are unknown. Using custom-made cell-penetrating HIV-1 TAT peptides, we show for the first-time potential implications of P2X7 cytoplasmic anchor in the regulation of caspase3/7 activation as well as TNFα regulation.

Keywords: P2X7R, immunology, TAT-peptide, cell death

Procedia PDF Downloads 97

Authors: Kyoung Lee

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Numerous aerobic bacteria have the ability to form multicellular communities on the surface layer of the air-liquid (A-L) interface as a biofilm called a pellicle. Pellicles occupied at the A-L interface will benefit from the utilization of oxygen from air and nutrient from liquid. Buoyancy of cells can be obtained by high surface tension at the A-L interface. Thus, formation of pellicles is an adaptive advantage in utilization of excess nutrients in the standing culture where oxygen depletion is easily set up due to rapid cell growth. In natural environments, pellicles are commonly observed on the surface of lake or pond contaminated with pollutants. Previously, we have shown that when cultured in standing LB media an alkylphenol-degrading bacteria Pseudomonas alkylphenolia KL28 forms pellicles in a diameter of 0.3-0.5 mm with a thickness of ca 40 µm. The pellicles have unique features for possessing flatness and unusual rigidity. In this study, the biogenesis of the circular pellicles has been investigated by observing the cell organization at early stages of pellicle formation and cell arrangements in pellicle, providing a clue for highly organized cellular arrangement to be adapted to the air-liquid niche. Here, we first monitored developmental patterns of pellicle from monolayer to multicellular organization. Pellicles were shaped by controlled growth of constituent cells which accumulate extracellular polymeric substance. The initial two-dimensional growth was transited to multilayers by a constraint force of accumulated self-produced extracellular polymeric substance. Experiments showed that pellicles are formed by clonal growth and even with knock-out of genes for flagella and pilus formation. In contrast, the mutants in the epm gene cluster for alginate-like polymer biosynthesis were incompetent in cell alignment for initial two-dimensional growth of pellicles. Electron microscopic and confocal laser scanning microscopic studies showed that the fully matured structures are highly packed by matrix-encased cells which have special arrangements. The cells on the surface of the pellicle lie relatively flat and inside longitudinally cross packed. HPLC analysis of the extrapolysaccharide (EPS) hydrolysate from the colonies from LB agar showed a composition with L-fucose, L-rhamnose, D-galactosamine, D-glucosamine, D-galactose, D-glucose, D-mannose. However, that from pellicles showed similar neutral and amino sugar profile but missing galactose. Furthermore, uronic acid analysis of EPS hydrolysates by HPLC showed that mannuronic acid was detected from pellicles not from colonies, indicating the epm-derived polymer is critical for pellicle formation as proved by the epm mutants. This study verified that for the circular pellicle architecture P. alkylphenolica KL28 cells utilized EPS building blocks different from that used for colony construction. These results indicate that P. alkylphenolica KL28 is a clever architect that dictates unique cell arrangements with selected EPS matrix material to construct sophisticated building, circular biofilm pellicles.

Keywords: biofilm, matrix, pellicle, pseudomonas

Procedia PDF Downloads 132

Authors: Meitham Amereh, Mohsen Akbari, Ben Nadler

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Cancer has been recognized as one of the most challenging problems in biology and medicine. Aggressive tumors are a lethal type of cancers characterized by high genomic instability, rapid progression, invasiveness, and therapeutic resistance. Their behavior involves complicated molecular biology and consequential dynamics. Although tremendous effort has been devoted to developing therapeutic approaches, there is still a huge need for new insights into the dark aspects of tumors. As one of the key requirements in better understanding the complex behavior of tumors, mathematical modeling and continuum physics, in particular, play a pivotal role. Mathematical modeling can provide a quantitative prediction on biological processes and help interpret complicated physiological interactions in tumors microenvironment. The pathophysiology of aggressive tumors is strongly affected by the extracellular cues such as stresses produced by mechanical forces between the tumor and the host tissue. During the tumor progression, the growing mass displaces the surrounding extracellular matrix (ECM), and due to the level of tissue stiffness, stress accumulates inside the tumor. The produced stress can influence the tumor by breaking adherent junctions. During this process, the tumor stops the rapid proliferation and begins to remodel its shape to preserve the homeostatic equilibrium state. To reach this, the tumor, in turn, upregulates epithelial to mesenchymal transit-inducing transcription factors (EMT-TFs). These EMT-TFs are involved in various signaling cascades, which are often associated with tumor invasiveness and malignancy. In this work, we modeled the tumor as a growing hyperplastic mass and investigated the effects of mechanical stress from surrounding ECM on tumor invasion. The invasion is modeled as volume-preserving inelastic evolution. In this framework, principal balance laws are considered for tumor mass, linear momentum, and diffusion of nutrients. Also, mechanical interactions between the tumor and ECM is modeled using Ciarlet constitutive strain energy function, and dissipation inequality is utilized to model the volumetric growth rate. System parameters, such as rate of nutrient uptake and cell proliferation, are obtained experimentally. To validate the model, human Glioblastoma multiforme (hGBM) tumor spheroids were incorporated inside Matrigel/Alginate composite hydrogel and was injected into a microfluidic chip to mimic the tumor’s natural microenvironment. The invasion structure was analyzed by imaging the spheroid over time. Also, the expression of transcriptional factors involved in invasion was measured by immune-staining the tumor. The volumetric growth, stress distribution, and inelastic evolution of tumors were predicted by the model. Results showed that the level of invasion is in direct correlation with the level of predicted stress within the tumor. Moreover, the invasion length measured by fluorescent imaging was shown to be related to the inelastic evolution of tumors obtained by the model.

Keywords: cancer, invasion, mathematical modeling, microfluidic chip, tumor spheroids

Procedia PDF Downloads 86

Authors: Ben Amar Cheba, Taha Ibrahim Zaghloul, Mohamad Hisham El-Massry, Ahmad Rafik El-Mahdy

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Seventy two bacterial strains with ability to degrade chitin were isolated during a screening program. One of the most potent isolates (strain R2) was identified as Bacillus sp. using conventional methods as well as 16S rRNA technique and submitted in the Gen Bank sequence database as Bacillus sp. R2 with a given accession number DQ 923161. This strain was able to produce high levels of extracellular chitinase. The chitinase of Bacillus sp. R2 hydrolyzed several chitinous substrates preferentially and showed a maximum activity toward the β chitin such as Calmar pen and squid bone chitins with the folds 1.47 and 1.23 respectively. The enzyme also exhibited a substrate binding capacity of more than 70% for squid chitin, shrimp shell colloidal chitin, chitosan and prawn shell chitin. The chitinase showed a moderate antifungal activity against many phytopathogenic fungi such as Aspergillus niger, A. flavus, Penicillium degitatum and Fusarium calmorum.This strain could be a suitable candidate for chitinase production on an industrial scale for using as promising antifungal biopestecide.

Keywords: antifungal activity, Bacillus sp. R2, chitinase, substrate specificity

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Authors: Nor Jannah Mohd Sebri, Khairul Anuar Mat Amin

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Dication cross-linked gellan gum hydrogel loaded with Ibuprofen with excellent mechanical properties had been synthesized as potential candidate for non-toxic biocompatible polymer material in tissue engineering. The gellan gum hydrogel with 5% Ibuprofen had produced a slow release profile with total drug release time of 25 hours as a resulting low swelling value recorded at 22+0.5%. Its compressive strength, 200.13+21 kPa was highest of all other hydrogel ratio of 0.5% and 1.0% Ibuprofen incorporation. Young’s Modulus of the hydrogel with 5% Ibuprofen was recorded at 1.8+0.01 MPa, indicating good gel strength in which it is capable of withstanding a fair amount of subjected force during topical wound dressing application. Excellent mechanical properties, together with slow release profile, make the ibuprofen-loaded hydrogel a prospect candidate as biocompatible extracellular matrices in wound management.

Keywords: gellan gum, ibuprofen, slow drug release, hydrogel

Procedia PDF Downloads 363