Search results for: extracellular vesicles

Authors: Yu Wen Wang, Shyh Ming Kuo, Hsia Ying Cheng, Yu Chiuan Wu

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Liver fibrosis is caused by the activation of hepatic stellate cells in the liver to secrete excessive and deposition of extracellular matrix. In recent years, many treatment strategies have been developed to reduce the activation of hepatic stellate cells and therefore to increase the decomposition of extracellular matrix. Bacopa monnieri, an herbaceous plant of the scrophulariaceae, containing saponins and glycosides, which with antioxidant, anti-inflammation, pain relief and free radical scavenging characteristics. This study was to evaluate the inhibition of hepatic stellate cell activity by Bacopa monnieri extract and its therapeutic potential in treating thioacetamide-induced liver fibrosis in rats. The results showed that the IC50 of Bacopa monnieri extract was 0.39 mg/mL. Bacopa monnieri extract could effectively reduce H2O2-induced hepatic stellate cells inflammation. In the TAA-induced liver fibrosis animal studies, albumin secretion recovered to normal level after treated with Bacopa monnieri extract for 2-w, and fibrosis related proteins, α-SMA and TGF-1levels decreased indicating the extract exerted therapeutic effect on the liver fibrosis. However, inflammatory factors TNF- obviously decreased after 4-w treatment. In summary, we could successfully extract the main component-Bacopaside I from the plant and acquired a potential therapy using this component in treating TAA-induced liver fibrosis in rat.

Keywords: anti-inflammatory, Bacopa monnieri, fibrosis, hepatic stellate cells, water extract

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Authors: A. Y. Tsidulko, T. M. Pankova, E. V. Grigorieva

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High-grade gliomas are the most frequent and most aggressive brain tumors which are characterized by active invasion of tumor cells into the surrounding brain tissue, where the extracellular matrix (ECM) plays a crucial role. Disruption of ECM can be involved in anticancer drugs effectiveness, side-effects and also in tumor relapses. The anti-inflammatory agent dexamethasone is a common drug used during high-grade glioma treatment for alleviating cerebral edema. Although dexamethasone is widely used in the clinic, its effects on normal brain tissue ECM remain poorly investigated. It is known that proteoglycans (PGs) are a major component of the extracellular matrix in the central nervous system. In our work, we studied the effects of dexamethasone on the ECM proteoglycans (syndecan-1, glypican-1, perlecan, versican, brevican, NG2, decorin, biglican, lumican) using RT-PCR in the experimental animal model. It was shown that proteoglycans in rat brain have age-specific expression patterns. In early post-natal rat brain (8 days old rat pups) overall PGs expression was quite high and mainly expressed PGs were biglycan, decorin, and syndecan-1. The overall transcriptional activity of PGs in adult rat brain is 1.5-fold decreased compared to post-natal brain. The expression pattern was changed as well with biglycan, decorin, syndecan-1, glypican-1 and brevican becoming almost equally expressed. PGs expression patterns create a specific tissue microenvironment that differs in developing and adult brain. Dexamethasone regimen close to the one used in the clinic during high-grade glioma treatment significantly affects proteoglycans expression. It was shown that overall PGs transcription activity is 1.5-2-folds increased after dexamethasone treatment. The most up-regulated PGs were biglycan, decorin, and lumican. The PGs expression pattern in adult brain changed after treatment becoming quite close to the expression pattern in developing brain. It is known that microenvironment in developing tissues promotes cells proliferation while in adult tissues proliferation is usually suppressed. The changes occurring in the adult brain after dexamethasone treatment may lead to re-activation of cell proliferation due to signals from changed microenvironment. Taken together obtained data show that dexamethasone treatment significantly affects the normal brain ECM, creating the appropriate microenvironment for tumor cells proliferation and thus can reduce the effectiveness of anticancer treatment and promote tumor relapses. This work has been supported by a Russian Science Foundation (RSF Grant 16-15-10243)

Keywords: dexamthasone, extracellular matrix, glioma, proteoglycan

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Authors: Abinaya Balasubramanian, Satyabrata Ghosh, Satyahari Dey

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Non-Digestible Oligosaccharides(NDOs) are low molecular weight carbohydrates with degree of polymerization (DP) 3-20, that are delivered intact to the large intestine. NDOs are gaining attention as effective prebiotic molecules that facilitate prevention and treatment of several chronic diseases. Recently, NDOs are being obtained by cleaving complex polysaccharides as it results in high yield and also as the former tend to display greater bioactivity. Thelebolus sp. IITKGP BT-12, a recently identified psychrophilic, Ascomycetes fungus has been reported to produce a bioactive extracellular polysaccharide(EPS). The EPS has been proved to possess strong prebiotic activity and anti- proliferative effects. The current study is an attempt to identify and optimise the most suitable method for hydrolysis of the above mentioned novel EPS into NDOs, and further purify and characterise the same. Among physical, chemical and enzymatic methods, enzymatic hydrolysis was identified as the best method and the optimum hydrolysis conditions obtained using response surface methodology were: reaction time of 24h, β-(1,3) endo-glucanase concentration of 0.53U and substrate concentration of 10 mg/ml. The NDOs were purified using gel filtration chromatography and their molecular weights were determined using MALDI-TOF. The major fraction was found to have a DP of 7,8. The monomeric units of the NDOs were confirmed to be glucose using TLC and GCMS-MS analysis. The obtained oligosaccharides proved to be non-digestible when subjected to gastric acidity, salivary and pancreatic amylases and hence could serve as efficient prebiotics.

Keywords: characterisation, enzymatic hydrolysis, non-digestible oligosaccharides, response surface methodology

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Authors: Alexander A. Osmolovskiy, Anastasia V. Orekhova, Daria M. Bednenko, Yelyzaveta Boiko

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Micromycetes from Aspergillus genus can produce proteases capable of promoting proteolysis of hemostasis proteins or, along with hydrolytic activity, to show the ability to convert proenzymes of this system activating them into an active form. At the same time, practical medicine needs specific activators for quantitation of the level of some plasma enzymes, especially protein C and factor X, the lack of which leads to the development of thromboembolic diseases. Thus, some micromycetes of the genus Aspergillus were screened for the ability to synthesize extracellular proteases with promising activity for designing anti-thrombotic and diagnostic preparations. Such standard methods like salting out, electrophoresis, isoelectrofocusing were used for isolation, purification and study of physicochemical properties of proteases. Enzyme activity was measured spectrophotometrically fibrin as a substrate of the reaction and chromogenic peptide substrates of different proteases of the human hemostasis system. As a result of the screening, four active producers were selected: Aspergillus janus 301, A. flavus 1, A. terreus 2, and A. ochraceus L-1. The enzyme of A. janus 301 showed the greatest fibrinolytic activity (around 329.2 μmol Tyr/(ml × min)). The protease produced by A. terreus 2 had the highest plasmin-like activity (54.1 nmol pNA/(ml × min)), but fibrinolytic activity was lower than A. janus 301 demonstrated (25.2 μmol Tyr/(ml × min)). For extracellular protease of micromycete A. flavus a high plasmin-like activity was also shown (39.8 nmol pNA / (ml × min)). Moreover, according to our results proteases one of the fungi - A. terreus 2 were able to activate protein C of human plasma - the key factor of the human anticoagulant hemostasis system. This type of activity was 39.8 nmol pNA/(ml × min)). It was also shown that A. ochraceus L-1 could produce extracellular proteases with protein C and factor X activator activities (65.9 nmol pNA/(ml × min) and 34.6 nmol pNA/(ml × min) respectively). The maximum accumulation of the proteases falls on the 4th day of cultivation. Using isoelectrofocusing was demonstrated that the activation of both proenzymes might proceed via limited proteolysis induced by proteases of A. ochraceus L-1. The activatory activity of A. ochraceus L-1 proteases toward essential hemostatic proenzymes, protein C and X factor may be useful for practical needs. It is well known that similar enzymes, activators of protein C and X factor isolated from snake venom, South American copperhead Agkistrodon contortrix contortrix and Russell’s viper Daboia russelli russeli, respectively, are used for the in vitro diagnostics of the functional state of these proteins in blood plasma. Thus, the proteases of Aspergillus genus can be used as cheap components for enzyme thrombolytic preparations.

Keywords: anti-trombotic drugs, fibrinolysis, diagnostics, proteases, micromycetes

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Authors: Emily Schlebes, Christian Hundhausen, Jens W. Fischer

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The glycosaminoglycan hyaluronan (HA) is a major component of the extracellular matrix, typically produced by fibroblasts of the connective tissue but also by immune cells. Here, we investigated the capacity of human peripheral blood CD8 T cells from healthy donors to produce HA and to express HA receptors as well as HA degrading enzymes. Further, we evaluated the effect of pharmacological HA inhibition on CD8 T cell function. Using immunocytochemistry together with quantitative PCR analysis, we found that HA synthesis is rapidly induced upon antibody-induced T cell receptor (TCR) activation and almost exclusively mediated by HA synthase 3 (HAS3). TCR activation also resulted in the upregulation of HA receptors CD44, hyaluronan-mediated motility receptor (HMMR), and layilin (LAYN), although kinetics and strength of expression varied greatly between subjects. The HA-degrading enzymes HYAL1 and HYAL2 were detected at low levels and induced by cell activation in some individuals. Interestingly, expression of HAS3, HA receptors, and hyaluronidases were modulated by the proinflammatory cytokines IL-6 and IL-1bβ in most subjects. To assess the functional role of HA in CD8 T cells, we performed carboxyfluorescein succinimidyl ester (CFSE) based proliferation assays and cytokine analysis in the presence of the HA inhibitor 4- Methylumbelliferone (4-MU). Despite significant inter-individual variation with regard to the effective dose, 4-MU resulted in the inhibition of CD8 T cell proliferation and reduced release of TNF-α and IFN-γ. Collectively, these data demonstrate that human CD8 T cells respond to TCR stimulation with a synthesis of HA and expression of HA-related genes. They further suggest that HA inhibition may be helpful in interfering with pathogenic T cell activation in human disease.

Keywords: CD8 T cells, extracellular matrix, hyaluronan, hyaluronan synthase 3

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Authors: I. Karagjozova, B. Dejanova, J. Pluncevic, S. Petrovska, V. Antevska, L. Todorovska

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Introduction: Medical checking assessment is important in sports medicine. To follow the health condition in subjects who perform sports, body composition parameters, such as intracellular water, extracellular water, protein and mineral content, muscle and fat mass might be useful. The aim of the study was to show available parameters and to compare them to manual assessment. Material and methods: A number of 20 subjects (14 male and 6 female) at age of 20±2 years were determined in the study, 5 performed recreational sports, while others were professional ones. The mean height was 175±7 cm, the mean weight was 72±9 cm, and the body mass index (BMI) was 23±2 kg/m2. The measured compartments were as following: intracellular water (IW), extracellular water (EW), protein component (PC), mineral component (MC), skeletal muscle mass (SMM) and body fat mass (BFM). Lean balance were examined for right and left arm (LA), trunk (T), right leg (RL) and left leg (LL). The comparison was made between the calculation derived by manual made measurements, using Matejka formula and parameters obtained by body composition analyzer (BCA) - Inbody 720 BCA Biospace. Used parameters for the comparison were muscle mass (SMM), body fat mass (BFM). Results: BCA obtained values were for: IW - 22.6±5L, EW - 13.5±2 L, PC - 9.8±0.9 kg, MC - 3.5±0.3, SMM - 27±3 kg, BFM - 13.8±4 kg. Lean balance showed following values for: RA - 2.45±0.2 kg, LA - 2.37±0.4, T - 20.9±5 kg, RL - 7.43±1 kg, and LL - 7.49 ±1.5 kg. SMM showed statistical difference between manual obtained value, 51±01% to BCA parameter 45.5±3% (p<0.001). Manual obtained values for BFM was lower (17±2%) than BCA obtained one, 19.5±5.9% (p<0.02). Discussion: The obtained results showed appropriate values for the examined age, regarding to all examined parameters which contribute to overview the body compartments, important for sport performing. Due to comparison between the manual and BCA assessment, we may conclude that manual measurements may differ from the certain ones, which is confirmed by statistical significance.

Keywords: athletes, body composition, bio electrical impedance, sports medicine

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Authors: MyungGu Yeo, JongHan Ha, Gi-Hoon Yang, JaeYoon Lee, SeungHyun Ahn, Hyeongjin Lee, HoJun Jeon, YongBok Kim, Minseong Kim, GeunHyung Kim

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Tissue engineering is a rapidly growing interdisciplinary research area that may provide options for treating damaged tissues and organs. As a promising technique for regenerating various tissues, this technology requires biomedical scaffolds, which serve as an artificial extracellular matrix (ECM) to support neotissue growth. Electrospun micro/nanofibers have been used widely in tissue engineering because of their high surface-area-to-volume ratio and structural similarity to extracellular matrix. However, low mechanical sustainability, low 3D shape-ability, and low cell infiltration have been major limitations to their use. In this work, we propose new hybrid scaffolds interlayered with cell-laden electrospun micro/nano fibers and poly(caprolactone) microstructures. Also, we applied various concentrations of alginate and electric field strengths to determine optimal conditions for the cell-electrospinning process. The combination of cell-laden bioink (2 ⅹ 10^5 osteoblast-like MG63 cells/mL, 2 wt% alginate, 2 wt% poly(ethylene oxide), and 0.7 wt% lecithin) and a 0.16 kV/mm electric field showed the highest cell viability and fiber formation in this process. Using these conditions and PCL microstructures, we achieved mechanically stable hybrid scaffolds. In addition, the cells embedded in the fibrous structure were viable and proliferated. We suggest that the cell-embedded hybrid scaffolds fabricated using the cell-electrospinning process may be useful for various soft- and hard-tissue regeneration applications.

Keywords: bioink, cell-laden scaffold, micro/nanofibers, poly(caprolactone)

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Authors: Daniel Todorov, Anton Hinkov, Petya Angelova, Kalina Shishkova, Venelin Tsvetkov, Stoyan Shishkov

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Human herpes viruses (HHV) are ubiquitous pathogens with a pandemic spread across the globe. HHV type 1 is the main causative agent of cold sores and fever blisters around the mouth and on the face, whereas HHV type 2 is generally responsible for genital herpes outbreaks. The treatment of both viruses is more or less successful with antivirals from the nucleoside analogues group. Their wide application increasingly leads to the emergence of resistant mutants In the past, medicinal plants have been used to treat a number of infectious and non-infectious diseases. Their diversity and ability to produce the vast variety of secondary metabolites according to the characteristics of the environment give them the potential to help us in our warfare with viral infections. The variable chemical characteristics and complex composition is an advantage in the treatment of herpes since the emergence of resistant mutants is significantly complicated. The screening process is difficult due to the lack of standardization. That is why it is especially important to follow the mechanism of antiviral action of plants. On the one hand, it may be expected to interact with its compounds, resulting in enhanced antiviral effects, and the most appropriate environmental conditions can be chosen to maximize the amount of active secondary metabolites. During our study, we followed the activity of various plant extracts on the viral replication cycle as well as their effect on the extracellular virion. We obtained our results following the logical sequence of the experimental settings - determining the cytotoxicity of the extracts, evaluating the overall effect on viral replication and extracellular virion.During our research, we have screened a variety of plant extracts for their antiviral activity against both virus replication and the virion itself. We investigated the effect of the extracts on the individual stages of the viral replication cycle - viral adsorption, penetration and the effect on replication depending on the time of addition. If there are positive results in the later experiments, we had studied the activity over viral adsorption, penetration and the effect of replication according to the time of addition. Our results indicate that some of the extracts from the Lamium album have several targets. The first stages of the viral life cycle are most affected. Several of our active antiviral agents have shown an effect on extracellular virion and adsorption and penetration processes. Our research over the last decade has shown several curative antiviral plants - some of which are from the Lamiacea family. The rich set of active ingredients of the plants in this family makes them a good source of antiviral preparation.

Keywords: human herpes virus, antiviral activity, Lamium album, Nepeta nuda

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Authors: Hung-Chih Hsu, Pey-Jium Chang, Ching-Hsein Chen, Jer-Liang Andrew Yeh

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Osteoarthritis (OA) is a common disorder in rehabilitation clinic. The main characteristics include joint pain, localized tenderness and enlargement, joint effusion, cartilage destruction, loss of adhesion of perichondrium, synovium hyperplasia. Synoviocytes inflammation might be a cause of local tenderness and effusion. Inflammation cytokines might also play an important role in joint pain, cartilage destruction, decrease adhesion of perichondrium to the bone. Treatments of osteoarthritis include non-steroid anti-inflammation drugs (NSAID), glucosamine supplementation, hyaluronic acid, arthroscopic debridement, and total joint replacement. Total joint replacement is commonly used in patients with severe OA who failed respond to pharmacological treatment. However, some patients received surgery had serious adverse events, including instability of the implants due to insufficient adhesion to the adjacent bony tissue or synovial inflammation. We tried to develop ideal nano-topographic oxidized silicon nanosponges by using with various chemicals to produce thickness difference in nanometers in order to study more about the cell-environment interactions in vitro like the alterations of cell adhesion, morphology, extracellular matrix secretions in the pathogenesis of osteoarthritis. Cytokines studies like growth factor, reactive oxygen species, reactive inflammatory materials (Like nitrous oxide and prostaglandin E2), extracellular matrix (ECM) degradation enzymes, and synthesis of collagen will also be observed and discussed. Extracellular and intracellular expression transforming growth factor beta (TGF-β) will be studied by reverse transcription-polymerase chain reaction (RT-PCR). The degradation of ECM will be observed by the bioactivity ratio of matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase by ELISA (Enzyme-linked immunosorbent assay). When rabbit synoviocytes were cultured on these nano-topographic structures, they demonstrate better cell adhesion rate, decreased expression of MMP-2,9 and PGE2, and increased expression of TGF-β when cultured in nano-topographic oxidized silicon nanosponges than in the planar oxidized silicon ones. These results show cell behavior, cytokine production can be influenced by physical characteristics from different nano-topographic structures. Our study demonstrates the possibility of manipulating cell behavior in these nano-topographic biomaterials.

Keywords: osteoarthritis, synoviocyte, oxidized silicon surfaces, reactive oxygen species

Procedia PDF Downloads 356

Authors: Chun Hsien Wu, Guan Xuan Wu, Hsia Ying Cheng, Shyh Ming Kuo

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Articular cartilage is composed of chondrocytes and extracellular matrix, such as collagen fibers, glycosaminoglycans, etc., which play an important role in lubricating and cushion joint activities. The phenotypic expression and metabolic activity of chondrocytes are extremely important in maintaining the functions of articular cartilage. In in vitro passaged culture of chondrocytes, chondrocytes gradually lose their original cell phenotype and morphology, which is called dedifferentiation. After continuous passaged culture of chondrocytes or induction by inflammatory factor IL-1, chondrocytes changed their phenotype and morphology. Also, the extracellular matrix type II collagen and GAG secretion were significantly reduced, while type I and X collagen were synthesized. Farnesol is an anti-inflammatory and antioxidant sesquiterpene compound that has the specific property of promoting collagen production. The purpose of this study was to investigate whether farnesol could restore the original type II collagen synthesis and, furthermore, the mechanisms of farnesol on the synthesis of type II collagen from the de-differentiated chondrocytes. The obtained results showed that the de-differentiated chondrocytes significantly restored to secret type II collagen and GAG (2.5-folds increases), and the secretion of collagen I and X and PGE2 synthesis were also significantly reduced after being treated with farnesol, indicating that farnesol had a restoration/re-differentiation effect on de-differentiated chondrocytes. The de-differentiated chondrocytes exhibited decreased expression of PPAR-γ and upregulated TGF-β expression to increase the MMP-13 expression. Higher expression of MMP-13 caused chondrocytes to secret type X collagen. On the contrary, increasing the expression of PPAR-γ would benefit the production of type II collagen. As shown, the PPAR-γ expression increased, and MMP-13 expression decreased after being treated with farnesol, indicating a possible signal pathway of farnesol to restore the production of type II collagen. However, more detailed mechanisms still need to evaluate.

Keywords: chondrocytes, de-differentiation, farnesol, re-differentiation

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Authors: John Justo Ambuchi, Zhaohan Zhang, Yujie Feng

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Quick development and usage of nanotechnology have resulted to massive use of various nanoparticles, such as iron oxide nanoparticles (IONPs) and multi-wall carbon nanotubes (MWCNTs). Thus, this study investigated the role of IONPs and MWCNTs in enhancing bioenergy recovery. Results show that IONPs at a concentration of 750 mg/L and MWCNTs at a concentration of 1500 mg/L induced faster substrate utilization and biogas production rates than the control. IONPs exhibited higher carbon oxygen demand (COD) removal efficiency than MWCNTs while on the contrary, MWCNT performance on biogas generation was remarkable than IONPs. Furthermore, scanning electron microscopy (SEM) investigation revealed extracellular polymeric substances (EPS) excretion from AGS had an interaction with nanoparticles. This interaction created a protective barrier to microbial consortia hence reducing their cytotoxicity. Microbial community analyses revealed genus predominance of bacteria of Anaerolineaceae and Longilinea. Their role in biodegradation of the substrate could have highly been boosted by nanoparticles. The archaea predominance of the genus level of Methanosaeta and Methanobacterium enhanced methanation process. The presence of bacteria of genus Geobacter was also reported. Their presence might have significantly contributed to direct interspecies electron transfer in the system. Exposure of AGS to nanoparticles promoted direct interspecies electron transfer among the anaerobic fermenting bacteria and their counterpart methanogens during the anaerobic digestion process. This results provide useful insightful information in understanding the response of microorganisms to IONPs and MWCNTs in the complex natural environment.

Keywords: anaerobic granular sludge, extracellular polymeric substances, iron oxide nanoparticles, multi-wall carbon nanotubes

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Authors: Moreblessings Shoko

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Polymersomes are materials which are considered as artificial counterparts of natural vesicles. The nanotechnology of such smart nanovesicles is very useful to enhance the efficiency of many therapeutic and diagnostic drugs. Those compounds show a higher stability, flexibility, and mechanical strength to the membrane compared to natural liposomes. Also, they can be designed in detail, the permeability of the membrane can be controlled by different stimuli, and the surface can be functionalized with different biological molecules to facilitate monitoring and target. For this purpose, this study demonstrates the formation of multifunctional and pH sensitive polymersomes and their functionalization with different reactive groups or biomolecules inside and outside of polymersomes´ membrane providing by crossing the membrane and docking/undocking processes for biomedical applications. Overall, they are highly versatile and thus present new opportunities for the design of targeted and selective recognition systems, for example, in mimicking cell functions and in synthetic biology.

Keywords: women, geomatics, challenges, capacity building

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Authors: Bezhan Tutberidze

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The Neogene-Quaternary volcanic rocks of the Javakheti Highland are products of post-collisional continental magmatism and are related to divergent and convergent margins of Eurasian-Afroarabian lithospheric plates. The studied area constitutes an integral part of the volcanic province of Central South Georgia. Three cycles of volcanic activity are identified here: 1. Late Miocene-Early Pliocene, 2. Late Pliocene-Early /Middle/ Pleistocene and 3. Late Pleistocene. An intense basic dolerite magmatic activity occurred within the time span of the Late Pliocene and lasted until at least Late /Middle/ Pleistocene. The age of the volcanogenic and volcanogenic-sedimentary formation was dated by geomorphological, paleomagnetic, paleontological and geochronological methods /1.7-1.9 Ma/. The volcanic area of the Javakheti Highland contains multiple dolerite Plateaus: Akhalkalaki, Gomarethi, Dmanisi, and Tsalka. Petrographic observations of these doleritic rocks reveal fairly constant mineralogical composition: olivine / Fo₈₇.₆₋₈₂.₇ /, plagioclase / Ab₂₂.₈ An₇₅.₉ Or₁.₃; Ab₄₅.₀₋₃₂.₃ An₅₂.₉₋₆₂.₃ Or₂.₁₋₅.₄/. The pyroxene is an augite and may exhibit a visible zoning: / Wo 39.7-43.1 En 43.5-45.2 Fs 16.8-11.7/. Opaque minerals /magnetite, titanomagnetite/ is abundant as inclusions within olivine and pyroxene crystals. The texture of dolerites exhibits intergranular, holocrystalline to ophitic to sub ophitic granular. Dolerites are most common vesicular rocks. Vesicles range in shape from spherical to elongated and in size from 0.5 mm to than 1.5-2 cm and makeup about 20-50 % of the volume. The dolerites have been subjected to considerable alteration. The secondary minerals in the geothermal field are: zeolite, calcite, chlorite, aragonite, clay-like mineral /dominated by smectites/ and iddingsite –like mineral; rare quartz and pumpellyite are present. These vesicles are filled by secondary minerals. In the chemistry, dolerites are the calc-alkalic transition to sub-alkaline with a predominance of Na₂O over K₂O. Chemical analyses indicate that dolerites of all plateaus of the Javakheti Highland have similar geochemical compositions, signifying that they were formed from the same magmatic source by crystallization of olivine basalis magma which less differentiated / ⁸⁷Sr \ ⁸⁶Sr 0.703920-0704195/. There is one argument, which is less convincing, according to which the dolerites/basalts of the Javakheti Highland are considered to be an activity of a mantle plume. Unfortunately, there does not exist reliable evidence to prove this. The petrochemical peculiarities and eruption nature of the dolerites of the Javakheti Plateau point against their plume origin. Nevertheless, it is not excluded that they influence the formation of dolerite producing primary basaltic magma.

Keywords: calc-alkalic, dolerite, Georgia, Javakheti Highland

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Authors: Lynette Lincoln, Sunil S. More

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With the rising demand of sugar used today, it is proposed that world sugar is expected to escalate up to 203 million tonnes by 2021. Hydrolysis of sucrose (table sugar) into glucose and fructose equimolar mixture is catalyzed by β-D-fructofuranoside fructohydrolase (EC 3.2.1.26), commonly called as invertase. For fluid filled center in chocolates, preparation of artificial honey, as a sweetener and especially to ensure that food stuffs remain fresh, moist and soft for longer spans invertase is applied widely and is extensively being used. From an industrial perspective, properties such as increased solubility, osmotic pressure and prevention of crystallization of sugar in food products are highly desired. Screening for invertase does not involve plate assay/qualitative test to determine the enzyme production. In this study, we use a three-step screening strategy for identification of a novel bacterial isolate from soil which is positive for invertase production. The primary step was serial dilution of soil collected from sugarcane fields (black soil, Maddur region of Mandya district, Karnataka, India) was grown on a Czapek-Dox medium (pH 5.0) containing sucrose as the sole C-source. Only colonies with the capability to utilize/breakdown sucrose exhibited growth. Bacterial isolates released invertase in order to take up sucrose, splitting the disaccharide into simple sugars. Secondly, invertase activity was determined from cell free extract by measuring the glucose released in the medium at 540 nm. Morphological observation of the most potent bacteria was examined by several identification tests using Bergey’s manual, which enabled us to know the genus of the isolate to be Bacillus. Furthermore, this potent bacterial colony was subjected to 16S rDNA PCR amplification and a single discrete PCR amplicon band of 1500 bp was observed. The 16S rDNA sequence was used to carry out BLAST alignment search tool of NCBI Genbank database to obtain maximum identity score of sequence. Molecular sequencing and identification was performed by Xcelris Labs Ltd. (Ahmedabad, India). The colony was identified as Bacillus sp. BAB-3434, indicating to be the first novel strain for extracellular invertase production. Molasses, a by-product of the sugarcane industry is a dark viscous liquid obtained upon crystallization of sugar. An enhanced invertase production and optimization studies were carried out by one-factor-at-a-time approach. Crucial parameters such as time course (24 h), pH (6.0), temperature (45 °C), inoculum size (2% v/v), N-source (yeast extract, 0.2% w/v) and C-source (molasses, 4% v/v) were found to be optimum demonstrating an increased yield. The findings of this study reveal a simple screening method of an extracellular invertase from a rapidly growing Bacillus sp., and selection of best factors that elevate enzyme activity especially utilization of molasses which served as an ideal substrate and also as C-source, results in a cost-effective production under submerged conditions. The invert mixture could be a replacement for table sugar which is an economic advantage and reduce the tedious work of sugar growers. On-going studies involve purification of extracellular invertase and determination of transfructosylating activity as at high concentration of sucrose, invertase produces fructooligosaccharides (FOS) which possesses probiotic properties.

Keywords: Bacillus sp., invertase, molasses, screening, submerged fermentation

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Authors: M. I. Tyletc, O. I. Podgornya, T. G. Shaposhnikova, S. V. Shabelnikov, A. G. Mittenberg, M. A. Daugavet

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Ascidiacea is the most numerous class of the Tunicata subtype. These chordates' distinctive feature of the anatomical structure is a tunic consisting of cellulose fibrils, protein molecules, and single cells. The mechanisms of the tunic formation are not known in detail; tunic formation could be used as the model system for studying the interaction of cells with the extracellular matrix. Our model species is the ascidian Styela rustica, which is prevalent in benthic communities of the White Sea. As previously shown, the tunic formation involves morula blood cells, which contain the major 48 kDa protein p48. P48 participation in the tunic formation was proved using antibodies against the protein. The nature of the protein and its function remains unknown. The current research aims to determine the amino acid sequence of p48, as well as to clarify its role in the tunic formation. The peptides that make up the p48 amino acid sequence were determined by mass spectrometry. A search for peptides in protein sequence databases identified sequences homologous to p48 in Styela clava, Styela plicata, and Styela canopus. Based on sequence alignment, their level of similarity was determined as 81-87%. The correspondent sequence of ascidian Styela canopus was used for further analysis. The Styela rustica p48 sequence begins with a signal peptide, which could indicate that the protein is secretory. This is consistent with experimentally obtained data: the contents of morula cells secreted in the tunic matrix. The isoelectric point of p48 is 9.77, which is consistent with the experimental results of acid electrophoresis of morula cell proteins. However, the molecular weight of the amino acid sequence of ascidian Styela canopus is 103 kDa, so p48 of Styela rustica is a shorter homolog. The search for conservative functional domains revealed the presence of two Ca-binding EGF-like domains, thrombospondin (TSP1) and tyrosinase domains. The p48 peptides determined by mass spectrometry fall into the region of the sequence corresponding to the last two domains and have amino acid substitutions as compared to Styela canopus homolog. The tyrosinase domain (pfam00264) is known to be part of the phenoloxidase enzyme, which participates in melanization processes and the immune response. The thrombospondin domain (smart00209) interacts with a wide range of proteins, and is involved in several biological processes, including coagulation, cell adhesion, modulation of intercellular and cell-matrix interactions, angiogenesis, wound healing and tissue remodeling. It can be assumed that the tyrosinase domain in p48 plays the role of the phenoloxidase enzyme, and TSP1 provides a link between the extracellular matrix and cell surface receptors, and may also be responsible for the repair of the tunic. The results obtained are consistent with experimental data on p48. The domain organization of protein suggests that p48 is an enzyme involved in the tunic tunning and is an important regulator of the organization of the extracellular matrix.

Keywords: ascidian, p48, thrombospondin, tyrosinase, tunic, tunning

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Authors: Sijie Chen, Xiaofang Chen, Juan Wang, Yingying Zhang, Yu Hong, Wanyu Zhuang, Xinxin Huang, Ping Ou, Longsheng Huang

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Purpose: Autistic symptom improvement can be observed in children treated with acupuncture, but the mechanism is still being explored. In the present study, we used scalp acupuncture to treat autism rat model, and then their improvement in the abnormal behaviors and specific mechanisms behind were revealed by detecting animal behaviors, analyzing the RNA sequencing of the prefrontal cortex(PFC), and observing the ultrastructure of PFC neurons under the transmission electron microscope. Methods: On gestational day 12.5, Wistar rats were given valproic acid (VPA) by intraperitoneal injection, and their offspring were considered to be reliable rat models of autism. They were randomized to VPA or VPA-acupuncture group (n=8). Offspring of Wistar pregnant rats that were simultaneously injected with saline were randomly selected as the wild-type group (WT). VPA_acupuncture group rats received acupuncture intervention at 23 days of age for 4 weeks, and the other two groups followed without intervention. After the intervention, all experimental rats underwent behavioral tests. Immediately afterward, they were euthanized by cervical dislocation, and their prefrontal cortex was isolated for RNA sequencing and transmission electron microscopy. Results: The main results are as follows: 1. Animal behavioural tests: VPA group rats showed more anxiety-like behaviour and repetitive, stereotyped behaviour than WT group rats. While VPA group rats showed less spatial exploration ability, activity level, social interaction, and social novelty preference than WT group rats. It was gratifying to observe that acupuncture indeed improved these abnormal behaviors of autism rat model. 2. RNA-sequencing: The three groups of rats differed in the expression and enrichment pathways of multiple genes related to synaptic function, neural signal transduction, and circadian rhythm regulation. Our experiments indicated that acupuncture can alleviate the major symptoms of ASD by improving these neurological abnormalities. 3. Under the transmission electron microscopy, several lysosomes and mitochondrial structural abnormalities were observed in the prefrontal neurons of VPA group rats, which were manifested as atrophy of the mitochondrial membran, blurring or disappearance of the mitochondrial cristae, and even vacuolization. Moreover, the number of synapses and synaptic vesicles was relatively small. Conversely, the mitochondrial structure of rats in the WT group and VPA_acupuncture was normal, and the number of synapses and synaptic vesicles was relatively large. Conclusion: Acupuncture effectively improved the abnormal behaviors of autism rat model and the ultrastructure of the PFC neurons, which might worked by improving their abnormal synaptic function, synaptic plasticity and promoting neuronal signal transduction.

Keywords: autism spectrum disorder, acupuncture, animal behavior, RNA sequencing, transmission electron microscope

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Authors: Anna Lierova, Jitka Kasparova, Marcela Jelicova, Lucie Korecka, Zuzana Bilkova, Zuzana Sinkorova

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Hyaluronic acid (HA) is a simple linear, unbranched polysaccharide with a lot of exceptional physiological and chemical properties such as high biocompatibility and biodegradability, strong hydration and viscoelasticity that depend on the size of the molecule. It plays the important role in a variety of molecular events as tissue hydration, mechanical protection of tissues and as well as during inflammation, leukocyte migration, and extracellular matrix remodeling. Also, HA-based biomaterials, including HA scaffolds, hydrogels, thin membranes, matrix grafts or nanoparticles are widely use in various biomedical applications. Our goal is to determine the radioprotective effect of hyaluronic acid nanoparticles (HA NPs). We are investigating effect of ionizing radiation on stability of HA NPs, in vitro relative toxicity of nanoscale as well as effect on cell lines and specific surface receptors and their response to ionizing radiation. An exposure to ionizing radiation (IR) can irreversibly damage various cell types and may thus have implications for the level of the whole tissue. Characteristic manifestations are formation of over-granulated tissue, remodeling of extracellular matrix (ECM) and abortive wound healing. Damages are caused by either direct interaction with DNA and IR proteins or indirectly by radicals formed during radiolysis of water Accumulation and turnover of ECM are a hallmark of radiation induces lung injury, characterized by inflammation, repair or remodeling health pulmonary tissue. HA is a major component of ECM in lung and plays an important role in regulating tissue injury, accelerating tissue repair, and controlling disease outcomes. Due to that, HA NPs were applied to in vivo model (C57Bl/6J mice) before total body or partial thorax irradiation. This part of our research is targeting on effect of exogenous HA on the development and/or mitigating acute radiation syndrome and radiation induced lung injuries.

Keywords: hyaluronic acid, ionizing radiation, nanoparticles, radiation induces lung damages

Procedia PDF Downloads 132

Authors: Silvia Moreno, Banu Iyisan, Hannes Gumz, Brigitte Voit, Dietmar Appelhans

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Polymersomes are materials which are considered as artificial counterparts of natural vesicles. The nanotechnology of such smart nanovesicles is very useful to enhance the efficiency of many therapeutic and diagnostic drugs. Those compounds show a higher stability, flexibility, and mechanical strength to the membrane compared to natural liposomes. In addition, they can be designed in detail, the permeability of the membrane can be controlled by different stimuli, and the surface can be functionalized with different biological molecules to facilitate monitoring and target. For this purpose, this study demonstrates the formation of multifunctional and pH sensitive polymersomes and their functionalization with different reactive groups or biomolecules inside and outside of polymersomes´ membrane providing by crossing the membrane and docking/undocking processes for biomedical applications. Overall, they are highly versatile and thus present new opportunities for the design of targeted and selective recognition systems, for example, in mimicking cell functions and in synthetic biology.

Keywords: multifunctionalized, pH stimulus, controllable release, cellular uptake

Procedia PDF Downloads 294

Authors: Noura El-Ahmady El-Naggar

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Among the antitumour drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological and biochemical properties, together with 16S rDNA sequence as Streptomyces olivaceus NEAE-119 and sequencing product(1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production by Streptomyces olivaceus NEAE-119 using Plackett–Burman experimental design and response surface methodology was carried out. Fifteen nutritional variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO4.7H2O, NaCl and FeSO4. 7H2O) were screened using Plackett–Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age and agitation speed) were further optimized by the central composite face-centered design -response surface methodology. As a result, a medium of the following formula is the optimum for producing an extracellular L-asparaginase in the culture filtrate of Streptomyces olivaceus NEAE-119: Dextrose 3g, starch 20g, L-asparagine 10g, KNO3 1g, K2HPO4 1g, MgSO4.7H2O 0.1g, NaCl 0.1g, pH 7, temperature 37°C, agitation speed 200 rpm/min, inoculum size 4%, v/v, inoculum age 72 h and fermentation period 5 days.

Keywords: Streptomyces olivaceus NEAE-119, glutaminase free L-asparaginase, production, Plackett-Burman design, central composite face-centered design, 16S rRNA, scanning electron microscope

Procedia PDF Downloads 335

Authors: Safee Chaudhary, Mahnoor Naseer Gondal, Hira Anees Awan, Abdul Rehman, Ammar Arif, Risham Hussain, Huma Khawar, Zainab Arshad, Muhammad Faizyab Ali Chaudhary, Waleed Ahmed, Muhammad Umer Sultan, Bibi Amina, Salaar Khan, Muhammad Moaz Ahmad, Osama Shiraz Shah, Hadia Hameed, Muhammad Farooq Ahmad Butt, Muhammad Ahmad, Sameer Ahmed, Fayyaz Ahmed, Omer Ishaq, Waqar Nabi, Wim Vanderbauwhede, Bilal Wajid, Huma Shehwana, Muhammad Tariq, Amir Faisal

Abstract:

Cancer is a manifestation of multifactorial deregulations in biomolecular pathways. These deregulations arise from the complex multi-scale interplay between cellular and extracellular factors. Such multifactorial aberrations at gene, protein, and extracellular scales need to be investigated systematically towards decoding the underlying mechanisms and orchestrating therapeutic interventions for patient treatment. In this work, we propose ‘TISON’, a next-generation web-based multiscale modeling platform for clinical systems oncology. TISON’s unique modeling abstraction allows a seamless coupling of information from biomolecular networks, cell decision circuits, extra-cellular environments, and tissue geometries. The platform can undertake multiscale sensitivity analysis towards in silico biomarker identification and drug evaluation on cellular phenotypes in user-defined tissue geometries. Furthermore, integration of cancer expression databases such as The Cancer Genome Atlas (TCGA) and Human Proteome Atlas (HPA) facilitates in the development of personalized therapeutics. TISON is the next-evolution of multiscale cancer modeling and simulation platforms and provides a ‘zero-code’ model development, simulation, and analysis environment for application in clinical settings.

Keywords: systems oncology, cancer systems biology, cancer therapeutics, personalized therapeutics, cancer modelling

Procedia PDF Downloads 182

Authors: Pelin Balcik Ercin

Abstract:

Introduction: ZEB transcription factor family member ZEB2, has a role in epithelial to mesenchymal transition during development and metastasis. The altered circulating extracellular miRNAs expression is observed in diseases, and extracellular miRNAs have an important role in cancer cell microenvironment. In ChIP-Seq study, the expression of miR-2110 was found to be regulated by ZEB2. In this study, the effects of miR2110 on cell proliferation and migration of hepatocellular carcinoma (HCC) cells were examined. Material and Methods: SNU398 cells transfected with mimic miR2110 (20nM) (HMI0375, Sigma-Aldrich) and negative control miR (HMC0002, Sigma-Aldrich). MicroRNA isolation was accomplished with miRVANA isolation kit according to manufacturer instructions. cDNA synthesis was performed expression, respectively, and calibrated with Ct of controls. The real-time quantitative PCR (RT-qPCR) reaction was performed using the TaqMan Fast Advanced Master Mix (Thermo Sci.). Ct values of miR2110 were normalized to miR-186-5p and miR16-5p for the intracellular gene. Cell proliferation analysis was analyzed with the xCELLigence RTCA System. Wound healing assay was analyzed with the ImageJ program and relative fold change calculated. Results: The mimic-miR-2110 transfected SNU398 cells nearly nine-fold (log2) more miR-2110 expressed compared to negative control transfected cells. The mimic-miR-2110 transfected HCC cell proliferation significantly inhibited compared to the negative control cells. Furthermore, miR-2110-SNU398 cell migration capacity was relatively four-fold decreased compared to negative control-miR-SNU398 cells. Conclusion: Our results suggest the miR-2110 inhibited cell proliferation and also miR-2110 negatively affect cell migration compared to control groups in HCC cells. These data suggest the complexity of microRNA EMT transcription factors regulation. These initial results are pointed out the predictive biomarker capacity of miR-2110 in HCC.

Keywords: epithelial to mesenchymal transition, EMT, hepatocellular carcinoma cells, micro-RNA-2110, ZEB2

Procedia PDF Downloads 91

Authors: Carol N. Flores-Fernández, Guadalupe Espilco, Cynthia Esquerre, Amparo I. Zavaleta

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Saline environments represent a valuable source of enzymes with novel properties and particular features for application in food, pharmaceutical and chemical industry. This study focuses on the isolation and screening of hydrolase-producing bacteria from Chilca salterns and the evaluation of their biotechnological potential. Soil samples were collected from Chilca salterns in Peru. For the isolation, medium containing 0.2 % of yeast extract, 5 % of NaCl and 10 % of the soil sample was used. After 72 h of incubation at 37 °C, serial dilutions were made up to 10−12 dilutions, spread on agar plates with 0.5 % of yeast extract and 5 % of NaCl, and incubated at 37 °C for 48 h. Screening of hydrolase-producing bacteria was carried out for cellulases, amylases, lipases, DNase, and proteases on specific media. Moreover, protease-producing bacteria were tested using protein extracted from the following legumes as substrate: Glycine max, Lupinus mutabilis, Pisum sativum, Erythrina edulis, Cicer arietinum, Phaseolus vulgaris and Vicia faba. A total of 16 strains were isolated from soil samples. On the screening media; 75, 44, 81 and 50 % were cellulase, amylase, DNase and protease producers, respectively. Also, 19 % of the isolates produced all the hydrolytic enzymes above mentioned. Lipase producers were not found. The 37 % and 12 % of the strains grew at 20 % and 30 % of salt concentration, respectively. In addition, 75 % of the strains grew at pH range between 5 and 10. From the total of protease-producing bacteria, 100 % hydrolyzed Glycine max, Lupinus mutabilis, and Pisum sativum protein, while 87 % hydrolyzed Erythrina edulis and Cicer arietinum protein. Finally, 75 % and 50 % of the strains hydrolyzed Phaseolus vulgaris and Vicia faba protein, respectively. Hydrolase-producing bacteria isolated from Chilca salterns in Peru grew at high salt concentrations and wide range of pH. In addition, protease-producing bacteria hydrolyzed protein from different sources such as leguminous. These enzymes have great biotechnological potential and could be used for different industrial processes and applications.

Keywords: bacteria, extracellular, hydrolases, Peru, salterns

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Authors: Yeseul Park

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Biopolymers produced by organisms highly contribute to the production of metal sulfides, both in extracellular and intracellular biomineralization. We discovered a new type of intracellular biomineral composed of copper sulfide in the periplasm of a sulfate-reducing bacterium. We suggest that the structural features of biomineral composed of 1-2 nm subgrains are based on biopolymer-based capping agents and an organic compartment. We further compare with other types of metal sulfide biominerals.

Keywords: biomineralization, copper sulfide, metal sulfide, biopolymer, capping agent

Procedia PDF Downloads 85

Authors: J. Desbrieres, M. Popa, C. Peptu, S. Bacaita

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Because of their favorable properties (non-toxicity, biodegradability, mucoadhesivity etc.), polysaccharides were studied as biomaterials and as pharmaceutical excipients in drug formulations. These formulations may be produced in a wide variety of forms including hydrogels, hydrogel based particles (or capsules), films etc. In these formulations, the polysaccharide based materials are able to provide local delivery of loaded therapeutic agents but their delivery can be rapid and not easily time-controllable due to, particularly, the burst effect. This leads to a loss in drug efficiency and lifetime. To overcome the consequences of burst effect, systems involving liposomes incorporated into polysaccharide hydrogels may appear as a promising material in tissue engineering, regenerative medicine and drug loading systems. Liposomes are spherical self-closed structures, composed of curved lipid bilayers, which enclose part of the surrounding solvent into their structure. The simplicity of production, their biocompatibility, the size and similar composition of cells, the possibility of size adjustment for specific applications, the ability of hydrophilic or/and hydrophobic drug loading make them a revolutionary tool in nanomedicine and biomedical domain. Drug delivery systems were developed as hydrogels containing chitosan or carboxymethylcellulose (CMC) as polysaccharides and gelatin (GEL) as polypeptide, and phosphatidylcholine or phosphatidylcholine/cholesterol liposomes able to accurately control this delivery, without any burst effect. Hydrogels based on CMC were covalently crosslinked using glutaraldehyde, whereas chitosan based hydrogels were double crosslinked (ionically using sodium tripolyphosphate or sodium sulphate and covalently using glutaraldehyde). It has been proven that the liposome integrity is highly protected during the crosslinking procedure for the formation of the film network. Calcein was used as model active matter for delivery experiments. Multi-Lamellar vesicles (MLV) and Small Uni-Lamellar Vesicles (SUV) were prepared and compared. The liposomes are well distributed throughout the whole area of the film, and the vesicle distribution is equivalent (for both types of liposomes evaluated) on the film surface as well as deeper (100 microns) in the film matrix. An obvious decrease of the burst effect was observed in presence of liposomes as well as a uniform increase of calcein release that continues even at large time scales. Liposomes act as an extra barrier for calcein release. Systems containing MLVs release higher amounts of calcein compared to systems containing SUVs, although these liposomes are more stable in the matrix and diffuse with difficulty. This difference comes from the higher quantity of calcein present within the MLV in relation with their size. Modeling of release kinetics curves was performed and the release of hydrophilic drugs may be described by a multi-scale mechanism characterized by four distinct phases, each of them being characterized by a different kinetics model (Higuchi equation, Korsmeyer-Peppas model etc.). Knowledge of such models will be a very interesting tool for designing new formulations for tissue engineering, regenerative medicine and drug delivery systems.

Keywords: controlled and delayed release, hydrogels, liposomes, polysaccharides

Procedia PDF Downloads 197

Authors: Noura El-Ahmady El-Naggar

Abstract:

L-asparaginase is an important enzyme as therapeutic agents used in combination therapy with other drugs in the treatment of acute lymphoblastic leukemia in children. L-asparaginase producing actinomycete strain, NEAE-K, was isolated from soil sample and identified on the basis of morphological, cultural, physiological and biochemical properties, together with 16S rDNA sequence as new subsp. Streptomyces rochei subsp. chromatogenes NEAE-K and sequencing product (1532 bp) was deposited in the GenBank database under accession number KJ200343. The study was conducted to screen parameters affecting the production of L-asparaginase by Streptomyces rochei subsp. chromatogenes NEAE-K on solid state fermentation using Plackett–Burman experimental design. Sixteen different independent variables including incubation time, moisture content, inoculum size, temperature, pH, soybean meal+ wheat bran, dextrose, fructose, L-asparagine, yeast extract, KNO3, K2HPO4, MgSO4.7H2O, NaCl, FeSO4. 7H2O, CaCl2, and three dummy variables were screened in Plackett–Burman experimental design of 20 trials. The most significant independent variables affecting enzyme production (dextrose, L-asparagine and K2HPO4) were further optimized by the central composite design. As a result, a medium of the following formula is the optimum for producing an extracellular L-asparaginase by Streptomyces rochei subsp. chromatogenes NEAE-K from solid state fermentation: g/L (soybean meal+ wheat bran 15, dextrose 3, fructose 4, L-asparagine 8, yeast extract 2, KNO3 1, K2HPO4 2, MgSO4.7H2O 0.5, NaCl 0.1, FeSO4. 7H2O 0.02, CaCl2 0.01), incubation time 7 days, moisture content 50%, inoculum size 3 mL, temperature 30°C, pH 8.5.

Keywords: streptomyces rochei subsp. chromatogenes neae-k, 16s rrna, identification, solid state fermentation, l-asparaginase production, plackett-burman design, central composite design

Procedia PDF Downloads 381

Authors: Tayyaba Bari, Muhammad Hamza Anjum, Samra Kanwal, Fakhera Ikram

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Osteoarthritis, a degenerative condition, affects more than 213 million individuals globally. Since articular cartilage has no or limited vessels, therefore, after deteriorating, it is unable to rejuvenate. Traditional approaches for cartilage repair, like autologous chondrocyte implantation, microfracture and cartilage transplantation are often associated with postoperative complications and lead to further degradation. Decellularized human umbilical cord has gained interest as a viable treatment for cartilage repair. Decellularization removes all cellular contents as well as debris, leaving a biologically active 3D network known as extracellular matrix (ECM). This matrix is biodegradable, non-immunogenic and provides a microenvironment for homeostasis, growth and repair. UC derived bioink function as 3D scaffolding material, not only mediates cell-matrix interactions but also adherence, proliferation and propagation of cells for 3D organoids. This study comprises different physical, chemical and biological approaches to optimize the decellularization of human umbilical cord (UC) tissues followed by the solubilization of these tissues to bioink formation. The decellularization process consisted of two cycles of freeze thaw where the umbilical cord at -20˚C was thawed at room temperature followed by dissection in small sections from 0.5 to 1cm. Similarly decellularization with ionic and non-ionic detergents Sodium dodecyl sulfate (SDS) and Triton-X 100 revealed that both concentrations of SDS i.e 0.1% and 1% were effective in complete removal of cells from the small UC tissues. The results of decellularization was further confirmed by running them on 1% agarose gel. Histological analysis revealed the efficacy of decellularization, which involves paraffin embedded samples of 4μm processed for Hematoxylin-eosin-safran and 4,6-diamidino-2-phenylindole (DAPI). ECM preservation was confirmed by Alcian Blue, and Masson’s trichrome staining on consecutive sections and images were obtained. Sulfated GAG’s content were determined by 1,9-dimethyl-methylene blue (DMMB) assay, similarly collagen quantification was done by hydroxy proline assay. This 3D bioengineered scaffold will provide a typical atmosphere as in the extracellular matrix of the tissue, which would be seeded with the mesenchymal cells to generate the desired 3D ink for in vitro and in vivo cartilage regeneration applications.

Keywords: umbilical cord, 3d printing, bioink, tissue engineering, cartilage regeneration

Procedia PDF Downloads 58

Authors: Sunil Kamboj, Vipin Saini, Suman Bala, Gaurav Sharma

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The main aim of the present research was to encapsulate mefenamic acid in niosomes and incorporate the prepared niosomes in the carbopol gel base for sustained therapeutic action. Mefenamic acid loaded niosomes were prepared by thin film hydration technique and evaluated for entrapment efficiency, vesicular size and zeta potential. The entrapment efficiency of the prepared niosomes was found to increase with decreasing the HLB values of surfactants and vesicle size was found to increase with increasing the cholesterol concentration. Niosomal vesicles with good entrapment efficiencies were incorporated in carbopol gel base to form the niosomal gel. The prepared niosomal gel was evaluated for pH, viscosity, spreadability, extrudability and skin permeation study across the rat skin.The results of permeation study revealed that the gel formulated with span 60 niosomes sustained the drug release for 12 h. Further the in vivo study showed the good inhibition of inflammation by the gel prepared with span 60 niosomes.

Keywords: mefenamic acid, niosomal gel, nonionic surfactants, sustained release

Procedia PDF Downloads 375

Authors: Priyanka Mokashi, Aparna Khanna, Nancy Pandita

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Diabetes mellitus is a chronic metabolic disorder which will be the 7th leading cause of death by 2030. The current line of treatment for the diabetes mellitus is oral antidiabetic drugs (biguanides, sulfonylureas, meglitinides, thiazolidinediones and alpha-glycosidase inhibitors) and insulin therapy depending upon the type 1 or type 2 diabetes mellitus. But, these treatments have their disadvantages, ranging from the developing of resistance to the drugs and adverse effects caused by them. Alternative to these synthetic agents, natural products provides a new insight for the development of more efficient and safe drugs due to their therapeutic values. Enicostema littorale blume (A. Raynal) is a traditional Indian plant belongs to the Gentianaceae family. It is widely distributed in Asia, Africa, and South America. There are few reports on Swrtiamarin, major component of this plant for its antidiabetic activity. However, the antidiabetic activity of flavonoids from E. littorale and their mechanism of action have not yet been elucidated. Flavonoids have a positive relationship with disease prevention and can act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, adipocytes, hepatocytes and skeletal myofibers. They may exert beneficial effects in diabetes by (i) improving hyperglycemia through regulation of glucose metabolism in hepatocytes; (ii) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells; (iii) increasing glucose uptake in hepatocytes, skeletal muscle and white adipose tissue (iv) reducing insulin resistance, inflammation and oxidative stress. Therefore, we have isolated four flavonoid rich fractions, Fraction A (FA), Fraction B (FB), Fraction C (FC), Fraction D (FD) from crude alcoholic hot (AH) extract from E. littorale, identified by LC/MS. Total eight flavonoids were identified on the basis of fragmentation pattern. Flavonoid FA showed the presence of swertisin, isovitexin, and saponarin; FB showed genkwanin, quercetin, isovitexin, FC showed apigenin, swertisin, quercetin, 5-O-glucosylswertisin and 5-O-glucosylisoswertisin whereas FD showed the presence of swertisin. Further, these fractions were assessed for their antidiabetic activity on stimulating glucose uptake in insulin-resistant HepG2 cell line model (IR/HepG2). The results showed that FD containing C-glycoside Swertisin has significantly increased the glucose uptake rate of IR/HepG2 cells at the concentration of 10 µg/ml as compared to positive control Metformin (0.5mM) which was determined by glucose oxidase- peroxidase method. It has been reported that enhancement of glucose uptake of cells occurs due the translocation of Glut4 vesicles to cell membrane through IR/IRS1/AKT pathway. Therefore, we have studied expressions of three genes IRS1, AKT and Glut4 by real-time PCR to evaluate whether they follow the same pathway or not. It was seen that the glucose uptake rate has increased in FD treated IR/HepG2 cells due to the activation of insulin receptor substrate-1 (IRS1) followed by protein kinase B (AKT) through phosphoinositide 3-kinase (PI3K) leading to translocation of Glut 4 vesicles to cell membrane, thereby enhancing glucose uptake and insulin sensitivity of insulin resistant HepG2 cells. Hence, the up-regulation indicated the mechanism of action through which FD (Swertisin) acts as antidiabetic candidate in the treatment of type 2 diabetes mellitus.

Keywords: E. littorale, glucose transporter, glucose uptake rate, insulin resistance

Procedia PDF Downloads 287

Authors: Pauline Nyssen, Ange Mouithys-Mickalad, Maryse Hoebeke

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Inflammation is a complex physiological phenomenon involving chemical and enzymatic mechanisms. Polymorphonuclear neutrophil leukocytes (PMNs) play an important role by producing reactive oxygen species (ROS) and releasing myeloperoxidase (MPO), a pro-oxidant enzyme. Released both in the phagolysosome and the extracellular medium, MPO produces during its peroxidase and halogenation cycles oxidant species, including hypochlorous acid, involved in the destruction of pathogen agents, like bacteria or viruses. Inflammatory pathologies, like rheumatoid arthritis, atherosclerosis induce an excessive stimulation of the PMNs and, therefore, an uncontrolled release of ROS and MPO in the extracellular medium, causing severe damages to the surrounding tissues and biomolecules such as proteins, lipids, and DNA. The treatment of chronic inflammatory pathologies remains a challenge. For many years, MPO has been used as a target for the development of effective treatments. Numerous studies have been focused on the design of new drugs presenting more efficient MPO inhibitory properties. However, some designed inhibitors can be toxic. An alternative consists of assessing the potential inhibitory action of clinically-known molecules, having antioxidant activity. Propofol, 2,6-diisopropyl phenol, which is used as an intravenous anesthetic agent, meets these requirements. Besides its anesthetic action employed to induce a sedative state during surgery or in intensive care units, propofol and its injectable form Diprivan indeed present antioxidant properties and act as ROS and free radical scavengers. A study has also evidenced the ability of propofol to inhibit the formation of the neutrophil extracellular traps fibers, which are important to trap pathogen microorganisms during the inflammation process. The aim of this study was to investigate the potential inhibitory action mechanism of propofol and Diprivan on MPO activity. To go into the anti-inflammatory action of propofol in-depth, two of its oxidative derivatives, 2,6-diisopropyl-1,4-p-benzoquinone (PPFQ) and 3,5,3’,5’-tetra isopropyl-(4,4’)-diphenoquinone (PPFDQ), were studied regarding their inhibitory action. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling have evidenced the low anti-catalytic action of propofol. Stopped-flow absorption spectroscopy and direct MPO activity analysis have proved that propofol acts as a reversible MPO inhibitor by interacting as a reductive substrate in the peroxidase cycle and promoting the accumulation of redox compound II. Overall, Diprivan exhibited a weaker inhibitory action than the active molecule propofol. In contrast, PPFQ seemed to bind and obstruct the enzyme active site, preventing the trigger of the MPO oxidant cycles. PPFQ induced a better chlorination cycle inhibition at basic and neutral pH in comparison to propofol. PPFDQ did not show any MPO inhibition activity. The three interest molecules have also demonstrated their inhibition ability on an important step of the inflammation pathway, the PMNs superoxide anions production, thanks to EPR spectroscopy and chemiluminescence. In conclusion, propofol presents an interesting immunomodulatory activity by acting as a reductive substrate in the peroxidase cycle of MPO, slowing down its activity, whereas PPFQ acts more as an anti-catalytic substrate. Although PPFDQ has no impact on MPO, it can act on the inflammation process by inhibiting the superoxide anions production by PMNs.

Keywords: Diprivan, inhibitor, myeloperoxidase, propofol, spectroscopy

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Authors: Ashish Kumar Jain, Deepak Mishra

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The objective of the present investigation was to prepare and evaluate a vesicular dual drug delivery system for effective management of mucosal ulcer. Inner encapsulating and Double liposomes were prepared by glass bead and reverse phase evaporation method respectively. The formulation consisted of inner liposomes bearing Ranitidine Bismuth Citrate (RBC) and outer liposomes encapsulating Amoxicillin trihydrate (AMOX). The optimized inner liposomes and double liposomes were extensively characterized for vesicle size, morphology, zeta potential, vesicles count, entrapment efficiency and in vitro drug release. In vitro, the double liposomes demonstrated a sustained release of AMOX and RBC viz 91.4±1.8% and 77.2±2.1% respectively at the end of 72 hr. Furthermore binding specificity and targeting propensity toward H. pylori (SKP-56) was confirmed by agglutination and in situ adherence assay. Reduction of the absolute alcohol induced ulcerogenic index from 3.01 ± 0.25 to 0.31 ± 0.09 and 100% H. pylori clearance rate was observed. These results suggested that double liposomes are potential vector for the development of dual drug delivery for effective treatment of H. pylori-associated peptic ulcer.

Keywords: double liposomes, H. pylori targeting, PE liposomes, glass-beads method, peptic ulcers

Procedia PDF Downloads 420