Search results for: Ames Salmonella assay

Authors: Kristina Karapetyan, Flora Tkhruni, Tsovinar Balabekyan, Arevik Israyelyan, Tatyana Khachatryan

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The ability of the lactic acid bacteria (LAB) to prevent and cure a variety of diseases, their protective role against infections and colonization of pathogenic microorganisms in the digestive tract, has lead to the coining of the term probiotics or pro-life. LAB inhibiting the growth of pathogenic and food spoilage microorganisms, maintaining the nutritive quality and improving the shelf life of foods. They have also been used as flavor and texture producers. Enterococcus strains have been used for treatment of diseases such as diarrhea or antibiotic associated diarrhea, inflammatory pathologies that affect colon such as irritable bowel syndrome, or immune regulation, diarrhea caused by antibiotic treatments. The obtaining and investigation of biological properties of proteinoceous antibiotics, on the basis of probiotic LAB shown, that bacteriocins, metabiotics, and peptides of LAB represent bactericides have a broad range of activity and are excellent candidates for development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics. The genotyping by 16S rRNA sequencing for LAB were used. Cell free culture broth (CFC) broth was purified by the Gel filtration method on the Sephadex Superfine G 25 resin. Antimicrobial activity was determined by spot-on-lawn method and expressed in arbitrary units (AU/ml). The diversity of multidrug-resistance (MDR) of pathogenic strains to antibiotics, most widely used for treatment of human diseases in the Republics of Armenia and Nagorno Karabakh were examined. It was shown, that difference of resistance of pathogens to antibiotics depends on their isolation sources. The influences of partially purified antimicrobial preparations (AMP), obtained from the different strains of Enterococcus genus on the growth of MDR pathogenic bacteria were investigated. It was shown, that bacteriocin containing partially purified preparations, obtained from different strains of Enterococcus faecium and durans species, possess bactericidal or bacteriostatic activity against antibiotic resistant intestinal, spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, E. coli, and Salmonella. Endemic strains of LAB, isolated from Matsoni made from donkey, buffalo and goat milk, shown broad spectrum of activity against food spoiling microorganisms, moulds and fungi, such as Salmonella sp., Esherichia coli, Aspergillus and Penicillium species. Highest activity against MDR pathogens shown bacteria, isolated from goat milk products. High stability of the investigated strains of the genus Enerococcus, isolated from samples of matsun from different regions of Nagorno-Karabakh (NKR) to the antibiotics was shown. The obtained data show high stability of the investigated different strains of the genus Enerococcus. The high genetic diversity in Enterococcus group suggests adaptations for specific mutations in different environments. Thus, endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms isolated from different sources and belong to different taxonomic group. Prospect of the use of certain antimicrobial preparations against pathogenic strains is obvious. These AMP can be applied for long term use against different etiology antibiotic resistant pathogens for prevention or treatment of infectional diseases as an alternative to antibiotics.

Keywords: antimicrobial biopreparation, endemic lactic acid bacteria, intra-species diversity, multidrug resistance of pathogens

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Authors: Shadia Al-Bahlani, Ruqaya Al-Rashdi, Shadia Al-Sinawi, Maya Al-Bahri

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Background: Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer, which is defined by the absence of Estrogen (ER), Progesterone (PR) and Human epidermal growth factor (Her-2) receptors. The calpain system plays an important role in many cellular processes including apoptosis, necrosis, cell signaling and proliferation. The role of clapins in pathogenesis and tumor progression has been studied in certain cancer types; however, its definite role is not yet established in breast cancer especially in the TNBC subtype. Objectives: This study aims to measure calpain-1 expression and correlate this measurement with the proliferating/apoptotic index as well with the prognostic factors in TNBC patients’ tissue. Materials and Methods: Thirty nine paraffin blocks from patients diagnosed with TNBC were used to measure the expression of calpain-1 and Ki-67 (proliferating marker) proteins using immunohistochemistry. Apoptosis was assessed morphological and biochemically using conventional Haematoxylin and Eosin (H&E) staining method and terminal deoxynucleotidyl transferase-mediate dUTP nick and labeling (TUNEL) assay respectively. Data was statistically analyzed using Pearson X2 test of association. Results: Calpain-1 content was visualized in the nucleus of the TNBC cells and its expression varied from low to high among the patients tissue. Calpain expression showed no significant correlation with the proliferating/apoptotic index as well with the clinicopathological variables. Apoptotic counts quantified by H&E staining showed significant association with the apoptotic TUNEL assay, validating both approaches. Conclusion: Although calpain-1 expression showed no significant association with the clinical outcome, its variable level of expression might indicate a hidden role in breast cancer tissue. Larger number of samples and different mode of assessments are needed to fully investigate such role. Exploring the involvement of calpain-1 in cancer progression might help in considering it as a biomarker of breast cancer.

Keywords: breast cancer, calpain, apoptosis, prognosis

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Authors: M. N. Boukhatem, M. A. Ferhat, K. Mansour

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Bovine raw milk represents a favorable environment for the growth of several food-spoilage strains and some pathogens. It must meet stringent standards to ensure the highest microbiological and toxicological qualities.In order to assess the microbiological risks associated with the consumption of this food, we conducted this study to determine the microbiological quality of bovine raw milk (54 samples) commercialized at the state of Blida (Algeria). The samples analyzed were unsatisfactory in terms of total flora where 61.11% of samples were considered as non acceptable in terms of quality standards, fecal coliforms (40.74%), fecal streptococci (55.55%) and staphylococci (74.07%). Salmonella and Clostridium strains were not detected in all the samples. Furthermore, antibiotic residues were found in 26% of analysed samples. These results reflect non-compliance with the rules of good hygiene practices at milking, storage, transportatio, and sale of milk. Bovine raw milk consumed presents a serious health risk to the population of the study areas.The livestock coaching actors and dissemination of good hygiene practices throughout the production chain are needed to improve the quality of local milk.

Keywords: bovine raw milk, microbiological quality, fecal coliforms, antibiotic residue, Blida state

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Authors: K. Karapetyan, F. Tkhruni, A. Aghajanyan, T. S. Balabekyan, L. Arstamyan

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Introduction: Globalization of the food supply has created the conditions favorable for the emergence and spread of food-borne and especially dangerous pathogens (EDP) in developing countries. The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the processed foods product. Antimicrobial compounds of lactic acid bacteria (LAB) possess bactericidal or bacteriostatic activity against intestinal pathogens, spoilage organisms and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella. Endemic strains of LAB were isolated. The strains, showing broad spectrum of antimicrobial activity against food spoiling microorganisms, were selected. The genotyping by 16S rRNA sequencing, GS-PCR, RAPD PCR methods showed that they were presented by Lactobacillus rhamnosus109, L.plantarum 65, L.plantarum 66 and Enterococcus faecium 64 species. LAB are deposited in "Microbial Depository Center" (MDC) SPC "Armbiotechnology". Methods: LAB strains were isolated from different dairy products from rural households from the highland regions of Armenia. Serially diluted samples were spread on MRS (Merck, Germany) and hydrolyzed milk agar (1,2 % w/v). Single colonies from each LAB were individually inoculated in liquid MRS medium and incubated at 37oC for 24 hours. Culture broth with biomass was centrifuged at 10,000 g during 20 min for obtaining of cell free culture broth (CFC). The antimicrobial substances from CFC broth were purified by the combination of adsorption-desorption and ion-exchange chromatography methods. Separation of bacteriocins was performed using a HPLC method on "Avex ODS" C18 column. Mass analysis of peptides recorded on the device API 4000 in the electron ionization mode. The spot-on-lawn method on the test culture plated in the solid medium was applied. The antimicrobial activity is expressed in arbitrary units (AU/ml). Results. Purification of CFC broth of LAB allowed to obtain partially purified antimicrobial preparations which contains bacteriocins with broad spectrum of antimicrobial activity. Investigation of their main biochemical properties shown, that inhibitory activity of preparations is partially reduced after treatment with proteinase K, trypsin, pepsin, suggesting a proteinaceous nature of bacteriocin-like substances containing in CFC broth. Preparations preserved their activity after heat treatment (50-121 oC, 20 min) and were stable in the pH range 3–8. The results of SDS PAAG electrophoresis show that L.plantarum 66 and Ent.faecium 64 strains have one bacteriocin (BCN) with maximal antimicrobial activity with approximate molecular weight 2.0-3.0 kDa. From L.rhamnosus 109 two BCNs were obtained. Mass spectral analysis indicates that these bacteriocins have peptide bonds and molecular weight of BCN 1 and BCN 2 are approximately 1.5 kDa and 700 Da. Discussion: Thus, our experimental data shown, that isolated endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms of different taxonomic group, such as Salmonella sp., Esherichia coli, Bacillus sp., L.monocytogenes, Proteus mirabilis, Staph. aureus, Ps. aeruginosa. Obtained results proved the perspectives for use of endemic strains in the preservation of foodstuffs. Acknowledgments: This work was realized with financial support of the Project Global Initiatives for Preliferation Prevention (GIPP) T2- 298, ISTC A-1866.

Keywords: antimicrobial activity, bacteriocins, endemic strains, food safety

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Authors: Vera Lukasova, Eva Filova, Jana Dankova, Vera Sovkova, Matej Daniel, Michala Rampichova

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Proper bone implants should promote fast adhesion of cells, stimulate cell differentiation and support the formation of bone tissue. Nowadays titanium is used as a biocompatible material capable of bone tissue integration. This study was focused on comparison of bioactive properties of two titanium alloys - beta titanium alloy Ti36Nb6Ta and standard medical titanium alloy Ti6A14V. The advantage of beta titanium alloy Ti36Nb6Ta is mainly that this material does not contain adverse elements like vanadium or aluminium. Titanium alloys were sterilized in ethanol, placed into 48 well plates and seeded with porcine mesenchymal stem cells. Cells were cultivated for 14 days in standard growth cultivation media with osteogenic supplements. Cell metabolic activity was quantified using MTS assay (Promega). Cell adhesion on day 1 and cell proliferation on further days were verified immunohistochemically using beta-actin monoclonal antibody and secondary antibody conjugated with AlexaFluor®488. Differentiation of cells was evaluated using alkaline phosphatase assay. Additionally, gene expression of collagen I was measured by qRT-PCR. Porcine mesenchymal stem cells adhered and spread well on beta titanium alloy Ti36Nb6Ta on day 1. During the 14 days’ time period the cells were spread confluently on the surface of the beta titanium alloy Ti36Nb6Ta. The metabolic activity of cells increased during the whole cultivation period. In comparison to standard medical titanium alloy Ti6A14V, we did not observe any differences. Moreover, the expression of collagen I gene revealed no statistical differences between both titanium alloys. Therefore, a beta titanium alloy Ti36Nb6Ta promotes cell adhesion, metabolic activity, proliferation and collagen I expression equally to standard medical titanium alloy Ti6A14V. Thus, beta titanium is a suitable material that provides sufficient biocompatible properties. This project was supported by the Czech Science Foundation: grant No. 16-14758S.

Keywords: beta titanium alloy, biocompatibility, differentiation, mesenchymal stem cells

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Authors: Elena I. Oprita, Oana Craciunescu, Rodica Tatia, Teodora Ciucan, Reka Barabas, Orsolya Raduly, Anca Oancea

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Healing of osteochondral defects requires repair of the damaged articular cartilage, the underlying subchondral bone and the interface between these tissues (the functional calcified layer). For this purpose, developing a single monophasic scaffold that can regenerate two specific lineages (cartilage and bone) becomes a challenge. The aim of this work was to develop variants of biodegradable cross-linked composite hydrogel based on natural polypeptides (gelatin), polysaccharides components (chondroitin-4-sulphate and hyaluronic acid), in a ratio of 2:0.08:0.02 (w/w/w) and mixed with Si-hydroxyapatite (Si-Hap), in two ratios of 1:1 and 2:1 (w/w). Si-Hap was synthesized and characterized as a better alternative to conventional Hap. Subsequently, both composite hydrogel variants were cross-linked with (N, N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC) and enriched with a small bioactive molecule (icariin). The small molecule icariin (Ica) (C33H40O15) is the main active constituent (flavonoid) of Herba epimedium used in traditional Chinese medicine to cure bone- and cartilage-related disorders. Ica enhances osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), facilitates matrix calcification and increases the specific extracellular matrix (ECM) components synthesis by chondrocytes. Afterward, the composite hydrogels were characterized for their physicochemical properties in terms of the enzymatic biodegradation in the presence of type I collagenase and trypsin, the swelling capacity and the degree of crosslinking (TNBS assay). The cumulative release of Ica and real-time concentration were quantified at predetermined periods of time, according to the standard curve of standard Ica, after hydrogels incubation in saline buffer at physiological parameters. The obtained cross-linked composite hydrogels enriched with small-molecule Ica were also characterized for morphology by scanning electron microscopy (SEM). Their cytocompatibility was evaluated according to EN ISO 10993-5:2009 standard for medical device testing. Thus, analyses regarding cell viability (Live/Dead assay), cell proliferation (Neutral Red assay) and cell adhesion to composite hydrogels (SEM) were performed using NCTC clone L929 cell line. The final results showed that both cross-linked composite hydrogel variants enriched with Ica presented optimal physicochemical, structural and biological properties to be used as a natural scaffold able to repair osteochondral defects. The data did not reveal any toxicity of composite hydrogels in NCTC stabilized cell lines within the tested range of concentrations. Moreover, cells were capable of spreading and proliferating on both composite hydrogel surfaces. In conclusion, the designed biodegradable cross-linked composites enriched with Si and Ica are recommended for further testing as natural temporary scaffolds, which can allow cell migration and synthesis of new extracellular matrix within osteochondral defects.

Keywords: composites, gelatin, osteochondral defect, small molecule

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Authors: Anna Höfer, Johannes Herrmann, Patrick Meybohm, Christopher Lotz

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Mitochondria are pivotal for energy supply and regulation of cellular functions. Deficiencies of mitochondrial metabolism have been implicated in diverse stressful conditions including infections. Platelets are key mediators for thrombo-inflammation during development and resolution of acute respiratory distress syndrome (ARDS). Previous data point to an exhausted platelet phenotype in critically-ill patients with coronavirus 19 disease (COVID-19) impacting the course of disease. The objective of this work was to characterize platelet mitochondrial metabolism in patients suffering from COVID-19 ARDSA longitudinal analysis of platelet mitochondrial metabolism in 24 patients with COVID-19 induced ARDS compared to 35 healthy controls (ctrl) was performed. Blood samples were analyzed at two time points (t1=day 1; t2=day 5-7 after study inclusion). The activity of mitochondrial citrate synthase was photometrically measured. The impact of oxidative stress on mitochondrial permeability was assessed by a photometric calcium-induced swelling assay and the activity of superoxide dismutase (SOD) by a SOD assay kit. The amount of protein carbonylation and the activity of mitochondria complexes I-IV were photometrically determined. Levels of interleukins (IL)-1α, IL-1β and tumor necrosis factor (TNF-) α were measured by a Multiplex assay kit. Median age was 54 years, 63 % were male and BMI was 29.8 kg/m2. SOFA (12; IQR: 10-15) and APACHE II (27; IQR: 24-30) indicated critical illness. Median Murray Score was 3.4 (IQR: 2.8-3.4), 21/24 (88%) required mechanical ventilation and V-V ECMO support in 14/24 (58%). Platelet counts in ARDS did not change during ICU stay (t1: 212 vs. t2: 209 x109/L). However, mean platelet volume (MPV) significantly increased (t1: 10.6 vs. t2: 11.9 fL; p<0.0001). Citrate synthase activity showed no significant differences between ctrl and ARDS patients. Calcium induced swelling was more pronounced in patients at t1 compared to t2 and to ctrl (50µM; t1: 0.006 vs. ctrl: 0.016 ΔOD; p=0.001). The amount of protein carbonylation as marker for irreversible proteomic modification constantly increased during ICU stay and compared to ctrl., without reaching significance. In parallel, superoxid dismutase activity gradually declined during ICU treatment vs. ctrl (t2: - 29 vs. ctrl.: - 17 %; p=0.0464). Complex I analysis revealed significantly stronger activity in ARDS vs. ctrl. (t1: 0.633 vs. ctrl.: 0.415 ΔOD; p=0.0086). There were no significant differences in complex II, III or IV activity in platelets from ARDS patients compared to ctrl. IL-18 constantly increased during the observation period without reaching significance. IL-1α and TNF-α did not differ from ctrl. However, IL-1β levels were significantly elevated in ARDS (t1: 16.8; t2: 16.6 vs. ctrl.: 12.4 pg/mL; p1=0.0335, p2=0.0032). This study reveals new insights in platelet mitochondrial metabolism during COVID-19 caused ARDS. it data point towards enhanced platelet activity with a pronounced turnover rate. We found increased activity of mitochondria complex I and evidence for enhanced oxidative stress. In parallel, protective mechanisms against oxidative stress were narrowed with elevated levels of IL-1β likely causing a pro-apoptotic environment. These mechanisms may contribute to platelet exhaustion in ARDS.

Keywords: acute respiratory distress syndrome (ARDS), coronavirus 19 disease (COVID-19), oxidative stress, platelet mitochondrial metabolism

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Authors: Wenjie Wu, Peng Cheng, Zehua Zhang, Fei Luo, Feng Wu, Min Zhong, Jianzhong Xu

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Background: There has been limited research into the therapeutic efficacy of rapid diagnosis of spinal tuberculosis complicated with pulmonary tuberculosis. We attempted to discover whether the utilization of a DNA microarray assay to detect multidrug-resistant spinal tuberculosis complicated with pulmonary tuberculosis can improve clinical outcomes. Methods: A prospective study was conducted from February 2006 to September 2015. One hundred and forty-three consecutive culture–confirmed, clinically and imaging diagnosed MDR-TB patients with spinal tuberculosis complicated by pulmonary tuberculosis were enrolled into the study. The initial time to treatment for MDR-TB, the method of infection control, radiological indicators of spinal tubercular infectious foci, culture conversion, and adverse drug reactions were compared with the standard culture methods. Results: Of the total of 143 MDR-TB patients, 68 (47.6%) were diagnosed by conventional culture methods and 75 (52.4%) following the implementation of detection using the DNA microarray. Patients in the microarray group began rational use of the second-line drugs schedule more speedily than sufferers in the culture group (17.3 vs. 74.1 days). Among patients were admitted to a general tuberculosis ward, those from the microarray group spent less time in the ward than those from the culture group (7.8 vs. 49.2 days). In those patients with six months follow-up (n=134), patients in the microarray group had a higher rate of sputum negativity conversion at six months (89% vs. 73%). In the microarray group, the rate of drug adverse reactions was significantly lower (22.2% vs. 67.7%). At the same time, they had a more obvious reduction of the area with spinal tuberculous lesions in radiological examinations (77% vs. 108%). Conclusions: The application of the CapitalBio™ DNA Microarray assay caused noteworthy clinical advances including an earlier time to begin MDR-TB treatment, increased sputum culture conversion, improved infection control measures and better radiographical results

Keywords: tuberculosis, multidrug-resistant, tuberculous spondylitis, DNA microarray, clinical outcomes

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Authors: Zakaria Baka, Halima Alem

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Cancer is a major worldwide health problem that has caused around ten million deaths in 2020. In addition, efforts to develop new anti-cancer drugs still face a high failure rate. This is partly due to the lack of preclinical models that recapitulate in-vivo drug responses. Indeed conventional cell culture approach (known as 2D cell culture) is far from reproducing the complex, dynamic and three-dimensional environment of tumors. To set up more in-vivo-like cancer models, 3D bioprinting seems to be a promising technology due to its ability to achieve 3D scaffolds containing different cell types with controlled distribution and precise architecture. Moreover, the introduction of microfluidic technology makes it possible to simulate in-vivo dynamic conditions through the so-called “cancer-on-chip” platforms. Whereas several cancer types have been modeled through the cancer-on-chip approach, such as lung cancer and breast cancer, only a few works describing ovarian cancer models have been described. The aim of this work is to combine 3D bioprinting and microfluidic technics with setting up a 3D dynamic model of ovarian cancer. In the first phase, alginate-gelatin hydrogel containing SKOV3 cells was used to achieve tumor-like structures through an extrusion-based bioprinter. The desired form of the tumor-like mass was first designed on 3D CAD software. The hydrogel composition was then optimized for ensuring good and reproducible printability. Cell viability in the bioprinted structures was assessed using Live/Dead assay and WST1 assay. In the second phase, these bioprinted structures will be included in a microfluidic device that allows simultaneous testing of different drug concentrations. This microfluidic dispositive was first designed through computational fluid dynamics (CFD) simulations for fixing its precise dimensions. It was then be manufactured through a molding method based on a 3D printed template. To confirm the results of CFD simulations, doxorubicin (DOX) solutions were perfused through the dispositive and DOX concentration in each culture chamber was determined. Once completely characterized, this model will be used to assess the efficacy of anti-cancer nanoparticles developed in the Jean Lamour institute.

Keywords: 3D bioprinting, ovarian cancer, cancer-on-chip models, microfluidic techniques

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Authors: Salman Akrokayan

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Synthetic cDNA (ScDNA) of granulocyte colony-stimulating factor (G-CSF) was constructed using a DNA synthesizer with the aim to increase its expression level. 5' end of the ScDNA of G-CSF coding region was modified by decreasing the GC content without altering the predicted amino acids sequence. The identity of the resulting protein from ScDNA was confirmed by the highly specific enzyme-linked immunosorbent assay. In conclusion, a synthetic G-CSF cDNA in combination with the recombinant DNA protocol offers a rapid and reliable strategy for synthesizing the target protein. However, the commercial utilization of this methodology requires rigorous validation and quality control.

Keywords: synthetic cDNA, recombinant G-CSF, cloning, gene expression

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Authors: Purva Nemavarkar, Badri Narain Pandey, Jitendra Kumar

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Introduction: The objective was to understand the effect of various radioprotectors on DNA damage repair enzyme and survival in gamma-irradiated wild and cdc mutants of S. pombe (fission yeast) cultured under permissive and restrictive conditions. DNA repair process, as influenced by radioprotectors, was measured by activity of DNA polymerase in the cells. The use of single cell gel electrophoresis assay (SCGE) or Comet Assay to follow gamma-irradiation induced DNA damage and effect of radioprotectors was employed. In addition, studying the effect of caffeine at different concentrations on S-phase of cell cycle was also delineated. Materials and Methods: S. pombe cells grown at permissive temperature (250C) and/or restrictive temperature (360C) were followed by gamma-radiation. Percentage survival and activity of DNA Polymerase (yPol II) were determined after post-irradiation incubation (5 h) with radioprotectors such as Caffeine, Curcumin, Disulphiram, and Ellagic acid (the dose depending on individual D 37 values). The gamma-irradiated yeast cells (with and without the radioprotectors) were spheroplasted by enzyme glusulase and subjected to electrophoresis. Radio-resistant cells were obtained by arresting cells in S-phase using transient treatment of hydroxyurea (HU) and studying the effect of caffeine at different concentrations on S-phase of cell cycle. Results: The mutants of S. pombe showed insignificant difference in survival when grown under permissive conditions. However, growth of these cells under restrictive temperature leads to arrest in specific phases of cell cycle in different cdc mutants (cdc10: G1 arrest, cdc22: early S arrest, cdc17: late S arrest, cdc25: G2 arrest). All the cdc mutants showed decrease in survival after gamma radiation when grown at permissive and restrictive temperatures. Inclusion of the radioprotectors at respective concentrations during post irradiation incubation showed increase in survival of cells. Activity of DNA polymerase enzyme (yPol II) was increased significantly in cdc mutant cells exposed to gamma-radiation. Following SCGE, a linear relationship was observed between doses of irradiation and the tail moments of comets. The radioprotection of the fission yeast by radioprotectors can be seen by the reduced tail moments of the yeast comets. Caffeine also exhibited its radio-protective ability in radio-resistant S-phase cells obtained after HU treatment. Conclusions: The radioprotectors offered notable radioprotection in cdc mutants when added during irradiation. The present study showed activation of DNA damage repair enzyme (yPol II) and an increase in survival after treatment of radioprotectors in gamma irradiated wild type and cdc mutants of S. pombe cells. Results presented here showed feasibility of applying SCGE in fission yeast to follow DNA damage and radioprotection at high doses, which are not feasible with other eukaryotes. Inclusion of caffeine at 1mM concentration to S phase cells offered protection and did not decrease the cell viability. It can be proved that at minimal concentration, caffeine offered marked radioprotection.

Keywords: radiation protection, cell cycle, fission yeast, comet assay, s-phase, DNA repair, radioprotectors, caffeine, curcumin, SCGE

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Authors: Hilal Turkoglu Sasmazel, Ozan Ozkan, Seda Surucu

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Tissue engineering is the field of treating defects caused by injuries, trauma or acute/chronic diseases by using artificial scaffolds that mimic the extracellular matrix (ECM), the natural biological support for the tissues and cells within the body. The main aspects of a successful artificial scaffold are (i) large surface area in order to provide multiple anchorage points for cells to attach, (ii) suitable porosity in order to achieve 3 dimensional growth of the cells within the scaffold as well as proper transport of nutrition, biosignals and waste and (iii) physical, chemical and biological compatibility of the material in order to obtain viability throughout the healing process. By hybrid scaffolds where two or more different materials were combined with advanced fabrication techniques into complex structures, it is possible to combine the advantages of individual materials into one single structure while eliminating the disadvantages of each. Adding this to the complex structure provided by advanced fabrication techniques enables obtaining the desired aspects of a successful artificial tissue scaffold. In this study, fibroblast compatibility of poly(ε-caprolactone) (PCL)/chitosan core-shell electrospun hybrid scaffolds with proper mechanical, chemical and physical properties successfully developed in our previous study was investigated. Standard 7-day cell culture was carried out with L929 fibroblast cell line. The viability of the cells cultured with the scaffolds was monitored with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay for every 48 h starting with 24 h after the initial seeding. In this assay, blank commercial tissue culture polystyrene (TCPS) Petri dishes, single electrospun PCL and single electrospun chitosan mats were used as control in order to compare and contrast the performance of the hybrid scaffolds. The adhesion, proliferation, spread and growth of the cells on/within the scaffolds were observed visually on the 3rd and the 7th days of the culture period with confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). The viability assay showed that the hybrid scaffolds caused no toxicity for fibroblast cells and provided a steady increase in cell viability, effectively doubling the cell density for every 48 h for the course of 7 days, as compared to TCPS, single electrospun PCL or chitosan mats. The cell viability on the hybrid scaffold was ~2 fold better compared to TCPS because of its 3D ECM-like structure compared to 2D flat surface of commercially cell compatible TCPS, and the performance was ~2 fold and ~10 fold better compared to single PCL and single chitosan mats, respectively, even though both fabricated similarly with electrospinning as non-woven fibrous structures, because single PCL and chitosan mats were either too hydrophobic or too hydrophilic to maintain cell attachment points. The viability results were verified with visual images obtained with CSLM and SEM, in which cells found to achieve characteristic spindle-like fibroblast shape and spread on the surface as well within the pores successfully at high densities.

Keywords: chitosan, core-shell, fibroblast, electrospinning, PCL

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Authors: Sabah El-Sawalhi, Elie Fayad, Roula M. Abdel-Massih

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Ilex paraguariensis (Yerba Mate) is a medium to large tree commonly consumed by South Americans. Its leaves and stems are associated with different biological activities. The purpose of this study was to evaluate the antibacterial activity of Yerba Mate against Gram-positive and Gram-negative bacterial strains and its action against some resistant bacteria with different resistance profiles. Yerba Mate aqueous extracts were prepared at 70°C for 2 hrs, and the microdilution method was used to determine the minimum inhibitory concentration (MIC). Gram-positive bacteria exhibited a stronger antibacterial activity (MIC ranged between 0.468 mg/mL and 15 mg/mL) than Gram-negative bacteria. Yerba Mate was also extracted with acetone: water (1:1) and then further sub-fractionated with hexane, chloroform, and ethyl acetate. MIC values against Staphylococcus aureus ranged from 0.78 to 2.5 mg/ml for the chloroform fraction, from 1.56 to 3.75 mg/ml for the ethyl acetate fraction, and 0.78 to 1.87 mg/ml for the water fraction. The water fraction also exhibited antibacterial activity against Salmonella species (MIC ranged from 1.56 mg/ml to 3.12 mg/ml). The water fraction exhibited the highest antibacterial activity among all the fractions obtained. More studies are needed to determine the molecule or molecules responsible for this activity.

Keywords: antibacterial activity, bacterial resistance, minimum inhibitory concentration, yerba mate

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Authors: Grzegorz Swiderski, Agata Jablonska-Trypuc, Natalia Popow, Renata Swislocka, Wlodzimierz Lewandowski

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Doxorubicin belongs to the group of anthracycline antitumor antibiotics. Because of the wide spectrum of actions, it is one of the most widely used anthracycline antibiotics, including the treatment of breast, ovary, bladder, lung cancers as well as neuroblastoma, lymphoma, leukemia and myeloid leukemia. Antitumor activity of doxorubicin is based on the same mechanisms as for most anthracyclines. Like the metal ions affect the nucleic acids on many biological processes, so the environment of the metal chelates of antibiotics can have a significant effect on the pharmacological properties of drugs. Complexation of anthracyclines with metal ions may contribute to the production of less toxic compounds. In the framework of this study, the composition of complexes obtained in aqueous solutions of doxorubicin with metal ions (Cu2+ and Zn2+). Complexation was analyzed by spectrophotometric titration in aqueous solution at pH 7.0. The pH was adjusted with 0.02M Tris-HCl buffer. The composition of the complexes found was Cu: doxorubicin (1: 2) and a Zn: doxorubicin (1: 1). The effect of Dox, Dox-Cu and Dox-Zn was examined in MCF-7 breast cancer cell line, which were obtained from American Type Culture Collection (ATCC). The compounds were added to the cultured cells for a final concentration in the range of 0,01µM to 0,5µM. The number of MCF-7 cells with division into living and dead, was determined by direct counts of cells with the use of trypan blue dye using LUNA Logos Biosystems cell counter. ApoTox-Glo Triplex Assay (Promega, Madison, Wisconsin, USA) was used according to the manufacturer’s instructions to measure the MCF-7 cells’ viability, cytotoxicity and apoptosis. We observed a decrease in cells proliferation in a dose-dependent manner. An increase in cytotoxicity and decrease in viability in the ApoTox Triplex assay was also showed for all tested compounds. Apoptosis, showed as caspase 3/7 activation, was observed only in Dox treatment. In Dox-Zn and Dox-Cu caspase 3/7 activation was not observed. This work was financially supported by National Science Centre, Poland, under the research project number 2014/13/B/NZ7/02 352.

Keywords: anticancer properties, anthracycline antibiotic, doxorubicine, metal complexes

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Authors: E. Kilicay, Z. Karahaliloglu, B. Hazer, E. B. Denkbas

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In this study, we have prepared concanavaline A (ConA) functionalized curcumin (CUR) loaded PHBHHx (poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)) nanoparticles as a novel and efficient drug delivery system. CUR is a promising anticancer agent for various cancer types. The aim of this study was to evaluate therapeutic potential of curcumin loaded PHBHHx nanoparticles (CUR-NPs) and concanavaline A conjugated curcumin loaded NPs (ConA-CUR NPs) for ovarian cancer treatment. ConA was covalently connected to the carboxylic group of nanoparticles by EDC/NHS activation method. In the ligand attachment experiment, the binding capacity of ConA on the surface of NPs was found about 90%. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis showed that the prepared nanoparticles were smooth and spherical in shape. The size and zeta potential of prepared NPs were about 228±5 nm and −21.3 mV respectively. ConA-CUR NPs were characterized by FT-IR spectroscopy which confirmed the existence of CUR and ConA in the nanoparticles. The entrapment and loading efficiencies of different polymer/drug weight ratios, 1/0.125 PHBHHx/CUR= 1.25CUR-NPs; 1/0.25 PHBHHx/CUR= 2.5CUR-NPs; 1/0.5 PHBHHx/CUR= 5CUR-NPs, ConA-1.25CUR NPs, ConA-2.5CUR NPs and ConA-5CUR NPs were found to be ≈ 68%-16.8%; 55%-17.7 %; 45%-33.6%; 70%-15.7%; 60%-17%; 51%-30.2% respectively. In vitro drug release showed that the sustained release of curcumin was observed from CUR-NPs and ConA-CUR NPs over a period of 19 days. After binding of ConA, the release rate was slightly increased due to the migration of curcumin to the surface of the nanoparticles and the matrix integrities was decreased because of the conjugation reaction. This functionalized nanoparticles demonstrated high drug loading capacity, sustained drug release profile, and high and long term anticancer efficacy in human cancer cell lines. Anticancer activity of ConA-CUR NPs was proved by MTT assay and reconfirmed by apoptosis and necrosis assay. The anticancer activity of ConA-CUR NPs was measured in ovarian cancer cells (SKOV-3) and the results revealed that the ConA-CUR NPs had better tumor cells decline activity than free curcumin. The nacked nanoparticles have no cytotoxicity against human ovarian carcinoma cells. Thus the developed functionalized nanoformulation could be a promising candidate in cancer therapy.

Keywords: curcumin, curcumin-PHBHHx nanoparticles, concanavalin A, concanavalin A-curcumin PHBHHx nanoparticles, PHBHHx nanoparticles, ovarian cancer cell

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Authors: Syahidah N. Zulkifli, Herlina A. Rahim, Nurul A. M. Subha

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Sustaining water quality is critical in order to avoid any harmful health consequences for end-user consumers. The detection of microbial impurities at the household level is the foundation of water security. Water quality is now monitored only at water utilities or infrastructure, such as water treatment facilities or reservoirs. This research provides a first-hand scientific understanding of microbial composition presence in Malaysia’s household point-of-use (POUs) water supply influenced by seasonal fluctuations, standstill periods, and flow dynamics by using the NIR-Raman spectroscopic technique. According to the findings, 20% of water samples were contaminated by pathogenic bacteria, which are Legionella and Salmonella cells. A comparison of the spectra reveals significant signature peaks (420 cm⁻¹ to 1800 cm⁻¹), including species-specific bands. This demonstrates the importance of regularly monitoring POUs water quality to provide a safe and clean water supply to homeowners. Conventional Raman spectroscopy, up-to-date, is no longer suited for real-time monitoring. Therefore, this study introduced an alternative micro-spectrometer to give a rapid and sustainable way of monitoring POUs water quality. Assessing microbiological threats in water supply becomes more reliable and efficient by leveraging IoT protocol.

Keywords: microbial contaminants, water quality, water monitoring, Raman spectroscopy

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Authors: Jovana Bogojeski, Dusan Cocic, Nenad Jankovic, Angelina Petrovic

Abstract:

Kinetically-inert transition metal complexes, such as Rh(III) and Os(III) complexes, attract increasing attention as leading scaffolds for the development of potential pharmacological agents due to their inertness and stability. Therefore, we have designed and fully characterized a few novel rhodium(III) and osmium(III) complexes with a tridentate nitrogen−donor chelate system. For some complexes, the crystal X-ray structure analysis was performed. Reactivity of the newly synthesized complexes towards small biomolecules, such as L-methionine (L-Met), guanosine-5’-monophosphate (5’-GMP), and glutathione (GSH) has been examined. Also, the reactivity of these complexes towards the DNA/RNA (Ribonucleic acid) duplexes was investigated. Obtained results show that the newly synthesized complexes exhibit good affinity towards the studied ligands. Results also show that the complexes react faster with the RNA duplex than with the DNA and that in the DNA duplex reaction is faster with 15mer GG than with the 22mer GG. The UV-Vis (Ultraviolet-visible spectroscopy) is absorption spectroscopy, and the EB (Ethidium bromide) displacement studies were used to examine the interaction of these complexes with CT-DNA and BSA (Bovine serum albumin). All studied complex showed good interaction ability with both the DNA and BSA. Furthermore, the DFT (Density-functional theory) calculation and docking studies were performed. The impact of the metal complex on the cytotoxicity was tested by MTT assay (a colorimetric assay for assessing cell metabolic activity) on HCT-116 lines (human colon cancer cell line). In addition, all these tests were repeated in the presence of several water-soluble biologically active ionic liquids. Attained results indicate that the ionic liquids increase the activity of the investigated complexes. All obtained results in this study imply that the introduction of different spectator ligand can be used to improve the reactivity of rhodium(III) and osmium(III) complexes. Finally, these results indicate that the examined complexes show reactivity characteristics needed for potential anti-tumor agents, with possible targets being both the DNA and proteins. Every new contribution in this field is highly warranted due to the current lack of clinically used Metallo-based alternatives to cisplatin.

Keywords: biomolecules, ionic liquids, osmium(III), rhodium(III)

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Authors: Holm Zerbe, Laura Macias, Hans-Joachim Schuberth, Wolfram Petzl

Abstract:

Mastitis is among the most important production diseases in cows. It accounts for large parts of antimicrobial drug use in the dairy industry worldwide. Due to the imminent normative to reduce the use of antimicrobial drugs in livestock, new ways for therapy and prophylaxis of mastitis are needed. Recently epigenetic regulation of inflammation by chromatin modifications has increasingly drawn attention. Currently, some epigenetic modifiers have already been approved for the use in humans, however little is known about their actions in the bovine system. The aim of our study was to investigate whether three selected epigenetic modifiers (Vitamin D3, SAHA and S2101) influence the initial immune response towards mastitis pathogens in bovine udder tissue in vitro. Tissue explants of the teat cistern and udder parenchyma were collected from 21 cows and were incubated for 36 hours in the absence and presence of epigenetic modifiers. Additionally, the tissue was stimulated with heat-inactivated particles of Escherichia coli and Staphylococcus aureus, which are regarded as two of the most important mastitis pathogens. After incubation, the explants were tested by RT-qPCR for transcript abundances of immune-related candidate genes. Gene expression was validated in culture supernatants by an AlphaLISA assay. Furthermore, the culture supernatants were analyzed for their chemotactic capacity through a chemotaxis assay. Statistical analysis of data was performed with the program ‘R’ version 3.2.3. Vitamin D3 had no effect on the immune response of udder tissue in vitro after stimulation with mastitis pathogens. The epigenetic modifiers SAHA and S2101 however significantly blocked the pathogen-induced upregulation of CXCL8, TNFα, S100A9 and LAP (P < 0.05). The regulation of IL10 was not affected by treatment with SAHA and S2101. Transcript abundances for CXCL8 were reflected by IL8 contents and chemotactic activity in culture supernatants. In conclusion, these data show the potential of epigenetic modifiers (SAHA and S2101) to block overshooting inflammation in the udder. Thus epigenetic modifiers may serve in future as immune modulators for the treatment and/or prophylaxis of clinical mastitis. (Funded by Deutsche Forschungsgemeinschaft PE 1495/2-1).

Keywords: mastitis, cattle, epigenetics, immunomodulation

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Authors: Nima Samie, Batoul Sadat Haerian, Sekaran Muniandy, M. S. Kanthimathi

Abstract:

Colon and breast cancers are categorized as the most prevalent types of cancer worldwide. Recently, the broad clinical application of metal complex compounds has led to the discovery of potential therapeutic drugs. The aim of this study was to evaluate the cytotoxic action of a selected nickel complex compound (NCC) against human colon and breast cancer cells. In this context, we determined the potency of the compound in the induction of apoptosis, cell cycle arrest, and cytoskeleton rearrangement. HT-29, WiDr, CCD-18Co, MCF-7 and Hs 190.T cell lines were used to determine the IC50 of the compound using the MTT assay. Analysis of apoptosis was carried out using immunofluorescence, acridine orange/ propidium iodide double staining, Annexin-V-FITC assay, evaluation of the translocation of NF-kB, oxygen radical antioxidant capacity, quenching of reactive oxygen species content , measurement of LDH release, caspase-3/-7, -8 and -9 assays and western blotting. The cell cycle arrest was examined using flowcytometry and gene expression was assessed using qPCR array. Results showed that our nickel complex compound displayed a potent suppressive effect on HT-29, WiDr, MCF-7 and Hs 190.T after 24 h of treatment with IC50 value of 2.02±0.54, 2.13±0.65, 3.76±015 and 3.14±0.45 µM respectively. This cytotoxic effect on normal cells was insignificant. Dipping in the mitochondrial membrane potential and increased release of cytochrome c from the mitochondria indicated induction of the intrinsic apoptosis pathway by the nickel complex compound. Activation of this pathway was further evidenced by significant activation of caspase 9 and 3/7.The nickel complex compound (NCC) was also shown activate the extrinsic pathways of apoptosis by activation of caspase-8 which is linked to the suppression of NF-kB translocation to the nucleus. Cell cycle arrest in the G1 phase and up-regulation of glutathione reductase, based on excessive ROS production were also observed. The results of this study suggest that the nickel complex compound is a potent anti-cancer agent inducing both intrinsic and extrinsic pathways as well as cell cycle arrest in colon and breast cancer cells.

Keywords: nickel complex, apoptosis, cytoskeletal rearrangement, colon cancer, breast cancer

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Authors: Ghebremedhin Tsegay, Weldu Tesfagaber, Yuanmao Zhu, Xijun He, Wan Wang, Zhenjiang Zhang, Encheng Sun, Jinya Zhang, Yuntao Guan, Fang Li, Renqiang Liu, Zhigao Bu, Dongming Zhao*

Abstract:

African swine fever (ASF) is a highly infectious viral disease of pigs, resulting in significant economic loss worldwide. As there is no approved vaccines and treatments, the control of ASF entirely depends on early diagnosis and culling of infected pigs. Thus, highly specific and sensitive diagnostic assays are required for accurate and early diagnosis of ASF virus (ASFV). Currently, only a few recombinant proteins have been tested and validated for use as reagents in ASF diagnostic assays. The most promising ones for ASFV antibody detection were p72, p30, p54, and pp62. So far, three ELISA kits based on these recombinant proteins have been commercialized. Due to the complex nature of the virus and variety forms of the disease, robust serodiagnostic assays are still required. ASFV p22 protein, encoded by KP177R gene, is located in the inner membrane of viral particle and appeared transiently in the plasma membrane early after virus infection. The p22 protein interacts with numerous cellular proteins, involved in processes of phagocytosis and endocytosis through different cellular pathways. However, p22 does not seem to be involved in virus replication or swine pathogenicity. In this study, E.coli expressed recombinant p22 protein was used to generate a monoclonal antibody (mAb), and its potential use for the development of blocking ELISA (bELISA) was evaluated. A total of 806 pig serum samples were tested to evaluate the bELISA. Acording the ROC (Reciever operating chracteristic) analysis, 100% sensitivity and 98.10% of specificity was recorded when the PI cut-off value was set at 47%. The novel assay was able to detect the antibodies as early as 9 days post infection. Finaly, a highly sensitive, specific and rapid novel p22-mAb based bELISA assay was developed, and optimized for detection of antibodies against genotype I and II ASFVs. It is a promising candidate for an early and acurate detection of the antibodies and is highly expected to have a valuable role in the containment and prevention of ASF.

Keywords: ASFV, blocking ELISA, diagnosis, monoclonal antibodies, sensitivity, specificity

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Authors: Hawraa Zbeeb, Hala Khalifeh, Mohamad Khalil, Francesca Storace, Francesca Baldini, Giulio Lupidi, Laura Vergani

Abstract:

Sarcopoterium spinosum, a widely distributed spiny shrub belonging to the Rosaceae family, is rich in essential and beneficial constituents. In fact, S. spinosum fruits and roots are traditionally used as herbal medicine in the eastern Mediterranean landscape, and this shrub is mentioned as a medicinal plant in a large number of ethnobotanical surveys. Aqueous root extracts from S. spinosum are used by traditional medicinal practitioners for weight loss treatment of diabetes and pain. Moreover, the anti-diabetic activity of S. spinosum root extract has been reported in different studies, but the beneficial effects of aerial parts, especially fruits, have not been elucidated yet. The aim of the present study was to investigate the in vitro antioxidant and lipid-lowering properties of an ethanolic extract from S. spinosum fruits using both hepatic (FaO) and endothelial (HECV) cells in an attempt to evaluate its possible employment as a nutraceutical supplement. First of all, in vitro spectrophotometric assays were employed to characterize the extract. The total phenol content (TPC) was evaluated by Folin–Ciocalteu spectrophotometric method and the radical scavenging activity was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. After that, the beneficial effects of the extract were tested on cells. FaO cells treated for 3 hours with 0.75 mM oleate/palmitate mix (1:2 molar ratio) mimic in vitro a moderate hepato-steatosis. HECV cells exposed for 1 hour to 100 µM H₂O₂ mimic an oxidative insult leading to oxidative stress conditions. After the metabolic and oxidative insult, both cell lines were treated with increasing concentrations of the S. spinosum extract (1, 10, 25 µg/mL) for 24 hours. The results showed the S. spinosum ethanolic extract is rather rich in phenols (TPC of 18.6 mgGAE/g dry extracts). Moreover, the extract showed a good scavenging ability in vitro (IC₅₀ 15.9 µg/ml and 10.9 µg/ml measured by DPPH and ABTS assays, respectively). When the extract was tested on cells, the results showed that it could ameliorate some markers of cell dysfunction. The three concentrations of the extract led to a significant decrease in the intracellular triglyceride (TG) content in steatotic FaO cells measured by spectrophotometric assay. On the other hand, HECV cells treated with increasing concentrations of the extract did not result in a significant decrease in both lipid peroxidation measured by the Thiobarbituric Acid Reactive Substances (TBARS) assay, and in reactive oxygen species (ROS) production measured by fluorometric analysis after DCF staining. Interestingly, the ethanolic extract was able to accelerate the wound repair of confluent HECV cells with respect to H₂O₂-insulted cells as measured by T-scratch assay. Taken together, these results seem to indicate that the ethanol extract from S. spinosum fruits is rich in phenol compounds and plays considerable lipid-lowering activity in vitro on steatotic hepatocytes and accelerates wound healing repair on endothelial cells. In light of that, the ethanolic extract from S. spinosum fruits could be a potential candidate for nutraceutical applications.

Keywords: antioxidant activity, ethanolic extract, lipid-lowering activity, phenolic compounds, Sarcopoterium spinosum fruits

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Authors: Mansi Patel, Priti Mehta

Abstract:

Background: Ionizing radiations have detrimental effects on humans, and the growing technological encroachment has increased human exposure to it enormously. So, the safety issues have emphasized researchers to develop radioprotector from natural resources having minimal toxicity. A substance having anti-inflammatory, antioxidant, and immunomodulatory activity can be a potential candidate for radioprotection. One such plant with immense potential i.e. Bamboo was selected for the present study. Purpose: The study aims to evaluate the potential of Indian bamboo leaves for protection against the clastogenic effect of gamma radiation. Methods: The protective effect of bamboo leaf extract against gamma radiation-induced genetic damage in human peripheral blood lymphocytes (HPBLs) was evaluated in vitro using Cytokinesis blocked micronuclei assay (CBMN). The blood samples were pretreated with varying concentration of extract 30 min before the radiation exposure (4Gy & 6Gy). The reduction in the frequency of micronuclei was observed for the irradiated and control groups. The effect of various concentration of bamboo leaf extract (400,600,800 mg/kg) on the development of radiation induced sickness and altered mortality in mice exposed to 8 Gy of whole-body gamma radiation was studied. The developed symptoms were clinically scored by multiple endpoints for 30 days. Results: Treatment of HPBLs with varying concentration of extract before exposure to a different dose of γ- radiation resulted in significant (P < 0.0001) decline of radiation induced micronuclei. It showed dose dependent and concentration driven activity. The maximum protection ~ 70% was achieved at nine µg/ml concentration. Extract treated whole body irradiated mice showed 50%, 83.3% and 100% survival for 400, 600, and 800mg/kg with 1.05, 0.43 and 0 clinical score respectively when compared to Irradiated mice having 6.03 clinical score and 0% survival. Conclusion: Our findings indicate bamboo leaf extract reduced the radiation induced cytogenetic damage. It has also increased the survival ratio and reduced the radiation induced sickness and mortality when exposed to a lethal dose of gamma radiation.

Keywords: bamboo leaf extract, Cytokinesis blocked micronuclei (CBMN) assay, ionizing radiation, radio protector

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Authors: Maria Nejjari, Michel Cloutier, Guylaine Talbot, Martin Lanthier

Abstract:

The fluorescence in situ hybridization (FISH) is a staining technique that allows the identification, detection and quantification of microorganisms without prior cultivation by means of epifluorescence and confocal laser scanning microscopy (CLSM). Oligonucleotide probes have been used to detect bacteria and archaea that colonize the cattle and swine digestive systems. These bacterial strains have been obtained from fecal samples issued from cattle manure and swine slurry. The collection of these samples has been done at 3 different pit’s levels A, B and C with same height. Two collection depth levels have been taken in consideration, one collection level just under the pit’s surface and the second one at the bottom of the pit. Cells were fixed and FISH was performed using oligonucleotides of 15 to 25 nucleotides of length associated with a fluorescent molecule Cy3 or Cy5. The double hybridization using Cy3 probe targeting bacteria (Cy3-EUB338-I) along with a Cy5 probe targeting Archaea (Gy5-ARCH915) gave a better signal. The CLSM images show that there are more bacteria than archaea in swine slurry. However, the choice of fluorescent probes is critical for getting the double hybridization and a unique signature for each microorganism. FISH technique is an easy way to detect pathogens like E. coli O157, Listeria, Salmonella that easily contaminate water streams, agricultural soils and, consequently, food products and endanger human health.

Keywords: archaea, bacteria, detection, FISH, fluorescence

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Authors: Ahmed Jibrin Uttu, Maimuna Waziri

Abstract:

The root bark of Glossonema boveanum (Decne), a member of Apocynaceae family, is used by traditional medicine practitioner to treat urinary and respiratory tract infections, bacteremia, typhoid fever, bacillary dysentery, diarrhea and stomach pain. This present study aims to validate the medicinal claims ascribed to the root bark of the plant. Preliminary phytochemical study of the root bark extracts (n-hexane, ethyl acetate, chloroform and methanol extracts) showed the presence of alkaloids, carbohydrates, steroids, triterpenes, cardiac glycosides, saponins, tannins and flavonoids. Antimicrobial study of the extracts showed activities against Staphylococus aureus, Bacillus subtilis, Salmonella typhii, Shigella dysenteriae, Escherichia coli, Enterobacter cloacae, Streptococcus agalactiae and Candida albicans while Micrococcus luteus, Pseudomonas aeruginosa and Klebsiella Pneumoniae showed resistance to all the extracts. The inhibitory effect was compared with the standard drug ciprofloxacin and fluconazole. MIC and MBC for both extracts were also determined using the tube dilution method. This study concluded that the root bark of G. boveanum, used traditionally as a medicinal plant, has antimicrobial activities against some causative organisms.

Keywords: Glossonema boveanum (Decne.), phytochemical, antimicrobial, minimum inhibitory concentration, minimum bactericidal concentration

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Authors: Siddig H. Hamad, Salah M. Al-Eid, Fahad M. Al-Jassas

Abstract:

Composite samples of minced chicken were vacuum-packaged and pressure treated at 300, 400, 450 and 500 MPa in a Stansted 'FOOD-LAB' model S-FL-850-9-W high hydrostatic pressure research apparatus (Stansted Fluid Power Ltd., Stansted, UK). Treated and untreated samples were then stored at 3°C, and microbial content as well as some chemical and physical properties monitored. The microbial load of the untreated samples reached the spoilage level of 107 cfu/g in about one week, resulting in bad smell and dark brown color. The pressure treatments reduced total bacterial counts by about 1.8 to 3.2 log10 cycles and reduced counts of Enterobacteriaceae and Salmonella to non-detectable levels. The color of meat was slightly affected, but pH, moisture content and the oxidation products of lipids were not substantially changed. The treatment killed mainly gram negative bacteria but also caused sub-lethal injury to part of the population resulting in prolonged lag phase. The population not killed by the 350 to 450 MPa treatments grew relatively slowly during storage, and its loads reached spoilage level in 4 to 6 weeks, while the load of the population treated at 500 MPa did not reach this level till the end of a storage period of 9 weeks.

Keywords: chicken, cold storage, microbial spoilage, high hydrostatic pressure

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Authors: M. Vijaya Bhaskar Reddy

Abstract:

The air-dried and powdered methanol solvent extraction of the rhizomes of Alpinia galangal is subjected to bio-assay guided fractionation and isolation yielded a known compound namely, 1'-S-1'-Acetoxychavicol acetate (1). The isolated known compound has been identified based on the physical, spectral data (IR, ¹H, ¹³C, NMR and mass spectroscopy) and comparison with an authentic sample. Finally isolated 1'-S-1'-Acetoxychavicol acetate (1) was confirmed by synthesis. The crude methanol extract and identified known compound (1) were tested for antidandruff property against Malassezia furfur showed with MIC 1000 µg/mL and 7.81 µg/mL, respectively.

Keywords: Alpinia galanga, isolation, 1'-S-1'-Acetoxychavicol acetate, antidandruff activity, Malassezia furfur

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Authors: Muhammad Zaheer Awan, Adnan Qadir, Zeeshan Anjum

Abstract:

Housefly, Musca domestica Linnaeus is ubiquitous and hazardous for Homo sapiens and livestock in sundry venerations. Musca domestica cart 100 different pathogens, such as typhoid, salmonella, bacillary dysentery, tuberculosis, anthrax and parasitic worms. The flies in rural areas usually carry more pathogens. Houseflies feed on liquid or semi-liquid substances besides solid materials which are softened by saliva. Neem botanically known as Azadirachta indica belongs to the family Meliaceae and is an indigenous tree to Pakistan. The neem tree is also one such tree which has been revered by the Pakistanis and Kashmiris for its medicinal properties. Present study showed neem seed extract has potentially toxic ability that affect Total Haemocyte Count (THC) and Differential Haemocytes Count (DHC) in insect’s blood cells, of the housefly. A significant variation in haemolymph density was observed just after application, 30 minutes and 60 minutes post treatment in term of THC and DHC in comparison with novastar. The study strappingly acclaim use of neem seed extract as insecticide as compare to artificial insecticides.

Keywords: neem, Azadirachta indica, Musca domestica, differential haemocyte count (DHC), total haemocytes count (DHC), novastar

Procedia PDF Downloads 179

Authors: Rabah Iratni, Husain El Hasasna, Khawlah Athamneh, Halima Al Sameri, Nehla Benhalilou, Asma Al Rashedi

Abstract:

Background: Cancer therapies have witnessed great advances in the recent past, however, cancer continues to be a leading cause of death, with colorectal cancer being the fourth cause of cancer-related deaths. Colorectal cancer affects both sexes equally with poor survival rate once it metastasizes. Phytochemicals, which are plant derived compounds, have been on a steady rise as anti-cancer drugs due to the accumulation of evidences that support their potential. Here, we investigated the anticancer effect of Rhus coriaria on colon cancer cells. Material and Method: Human colon cancer HT-29 cell line was used. Protein expression and protein phosphorylation were examined using Western blotting. Transcription activity was measure using Quantitative RT-PCR. Human tumoral clonogenic assay was used to assess cell survival. Senescence was assessed by the senescence-associated beta-galactosidase assay. Results: Rhus coriaria extract (RCE) was found to significantly inhibit the viability and colony growth of human HT-29 colon cancer cells. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, p16, downregulation of cyclin D1, p27, c-myc and expression of Senescence-associated-β-Galactosidase activity. Moreover, RCE induced non-canonical beclin-1independent autophagy and subsequent apoptotic cell death through activation of activation caspase 8 and caspase 7. The blocking of autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death. Further, RCE induced DNA damage, reduced mutant p53 protein level and downregulated phospho-AKT and phospho-mTOR, events that preceded autophagy. Mechanistically, we found that RCE inhibited the AKT and mTOR pathway, a regulator of autophagy, by promoting the proteasome-dependent degradation of both AKT and mTOR proteins. Conclusion: Our findings provide strong evidence that Rhus coriaria possesses strong anti-colon cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against colon cancer.

Keywords: autophagy, proteasome degradation, senescence, mTOR, apoptosis, Beclin-1

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Authors: G. Disciglio, G. Gatta, A. Libutti, A. Tarantino, L. Frabboni, E. Tarantino

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Results of a field study carried out at Trinitapoli (Puglia region, southern Italy) on the irrigation of an artichoke crop with three types of water (secondary-treated wastewater, SW; tertiary-treated wastewater, TW; and freshwater, FW) are reported. Physical, chemical and microbiological analyses were performed on the irrigation water, and on soil and yield samples. The levels of most of the chemical parameters, such as electrical conductivity, total suspended solids, Na+, Ca2+, Mg+2, K+, sodium adsorption ratio, chemical oxygen demand, biological oxygen demand over 5 days, NO3 –N, total N, CO32, HCO3, phenols and chlorides of the applied irrigation water were significantly higher in SW compared to GW and TW. No differences were found for Mg2+, PO4-P, K+ only between SW and TW. Although the chemical parameters of the three irrigation water sources were different, few effects on the soil were observed. Even though monitoring of Escherichia coli showed high SW levels, which were above the limits allowed under Italian law (DM 152/2006), contamination of the soil and the marketable yield were never observed. Moreover, no Salmonella spp. were detected in these irrigation waters; consequently, they were absent in the plants. Finally, the data on the quantitative-qualitative parameters of the artichoke yield with the various treatments show no significant differences between the three irrigation water sources. Therefore, if adequately treated, municipal wastewater can be used for irrigation and represents a sound alternative to conventional water resources.

Keywords: artichoke, soil chemical characteristics, fecal indicators, treated municipal wastewater, water recycling

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Authors: Adeleye O. O., T. M. Obuotor, A. O. Kolawole, I. O. Opowoye, M. I. Olasoju, L. T. Egbeyale, R. A. Ajadi

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This study investigated the effect of Lagenaria breviflora on broiler poultry birds, including its effect on the microbial count of the poultry droppings. A total of 240-day-old broiler chicks were randomly assigned to six groups, with four replicates per group. The first group was the control, while the other four groups were fed water containing 300g/L and 500g/L concentrations of Lagenaria breviflora twice and thrice daily. The microbial load was determined using the plate count method. The results showed that the administration of Lagenaria breviflora in the water of broiler birds significantly improved their growth performance with an average weight gain range of 1.845g - 2.241g. Mortality rate was at 0%. The study also found that Lagenaria breviflora had a significant effect on the microbial count of the poultry droppings with colony count values from 3.5 x 10-7 - 9.9 x10-7CFU/ml, The total coliforms (Escherichia coli, and Salmonella sp.) was obtained as 1 x 10 -5CFU/ml. The reduction in microbial counts of the poultry droppings could be attributed to the antimicrobial properties of Lagenaria breviflora, which contain phytochemicals reported to possess antimicrobial activity. Therefore, the inclusion of Lagenaria breviflora in the diets of broiler poultry could be an effective strategy for improving growth performance and immune function and reducing the microbial load of poultry droppings, which can help to mitigate the risk of disease transmission to humans and other animals.

Keywords: gut microbes, bacterial count, lagenaria breviflora, coliforms

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