Search results for: non-toxique assays

Authors: Lillian Musila, Valerie Oundo, Daniel Erwin, Willie Sang

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Staphylococcus aureus infections are a major cause of nosocomial infections in Kenya. Methicillin resistant S. aureus (MRSA) infections are a significant burden to public health and are associated with considerable morbidity and mortality. At a hospital in Western Kenya two clusters of MRSA cases emerged within short periods of time. In this study we explored whether these clusters represented a nosocomial outbreak by characterizing the isolates using phenotypic and molecular assays and examining epidemiological data to identify possible transmission patterns. Specimens from the site of infection of the subjects were collected, cultured and S. aureus isolates identified phenotypically and confirmed by APIStaph™. MRSA were identified by cefoxitin disk screening per CLSI guidelines. MRSA were further characterized based on their antibiotic susceptibility patterns and spa gene typing. Characteristics of cases with MRSA isolates were compared with those with MSSA isolated around the same time period. Two cases of MRSA infection were identified in the two week period between 21 April and 4 May 2015. A further 2 MRSA isolates were identified on the same day on 7 September 2015. The antibiotic resistance patterns of the two MRSA isolates in the 1st cluster of cases were different suggesting that these were distinct isolates. One isolate had spa type t2029 and the other had a novel spa type. The 2 isolates were obtained from urine and an open skin wound. In the 2nd cluster of MRSA isolates, the antibiotic susceptibility patterns were similar but isolates had different spa types: one was t037 and the other a novel spa type different from the novel MRSA spa type in the first cluster. Both cases in the second cluster were admitted into the hospital but one infection was community- and the other hospital-acquired. Only one of the four MRSA cases was classified as an HAI from an infection acquired post-operatively. When compared to other S. aureus strains isolated within the same time period from the same hospital only one spa type t2029 was found in both MRSA and non-MRSA strains. None of the cases infected with MRSA in the two clusters shared any common epidemiological characteristic such as age, sex or known risk factors for MRSA such as prolonged hospitalization or institutionalization. These data suggest that the observed MRSA clusters were multi strain clusters and not an outbreak of a single strain. There was no clear relationship between the isolates by spa type suggesting that no transmission was occurring within the hospital between these cluster cases but rather that the majority of the MRSA strains were circulating in the community. There was high diversity of spa types among the MRSA strains with none of the isolates sharing spa types. Identification of disease clusters in space and time is critical for immediate infection control action and patient management. Spa gene typing is a rapid way of confirming or ruling out MRSA outbreaks so that costly interventions are applied only when necessary.

Keywords: cluster, Kenya, MRSA, spa typing

Procedia PDF Downloads 330

Authors: Anthony Credoz, Nathalie Nief, Remy Hedacq, Salvador Jordana, Laurent Cazes

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Groundwater monitoring is classically performed in a network of piezometers in industrial sites. Groundwater flow parameters, such as direction, sense and velocity, are deduced from indirect measurements between two or more piezometers. Groundwater sampling is generally done on the whole column of water inside each borehole to provide concentration values for each piezometer location. These flow and concentration values give a global ‘static’ image of potential plume of contaminants evolution in the shallow aquifer with huge uncertainties in time and space scales and mass discharge dynamic. TOTAL R&D Subsurface Environmental team is challenging this classical approach with an innovative dynamic way of characterization of shallow aquifer groundwater. The current study aims at optimizing the tools and methodologies for (i) a direct and multilevel measurement of groundwater velocities in each piezometer and, (ii) a calculation of potential flux of dissolved contaminant in the shallow aquifer. Lab-scale experiments have been designed to test commercial and R&D tools in a controlled sandbox. Multiphysics modeling were performed and took into account Darcy equation in porous media and Navier-Stockes equation in the borehole. The first step of the current study focused on groundwater flow at porous media/piezometer interface. Huge uncertainties from direct flow rate measurements in the borehole versus Darcy flow rate in the porous media were characterized during experiments and modeling. The structure and location of the tools in the borehole also impacted the results and uncertainties of velocity measurement. In parallel, direct-push tool was tested and presented more accurate results. The second step of the study focused on mass flux of dissolved contaminant in groundwater. Several active and passive commercial and R&D tools have been tested in sandbox and reactive transport modeling has been performed to validate the experiments at the lab-scale. Some tools will be selected and deployed in field assays to better assess the mass discharge of dissolved contaminants in an industrial site. The long-term subsurface environmental strategy is targeting an in-situ, real-time, remote and cost-effective monitoring of groundwater.

Keywords: dynamic characterization, groundwater flow, lab-scale, mass flux

Procedia PDF Downloads 167

Authors: Abeer Y. Ibrahim, Faten M. Ibrahim, Samah A. El-Newary, Saber F. Hendawy

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Objective: Appreciation of in-vitro anti-inflammatory and antioxidant characters of Vitex agnus-castus berries alcoholic extract and fractions, as well as in-vivo antitumor ability of alcoholic extract and chloroform fraction against Ehrlich ascites carcinoma is the aim of this study. Material and methods: Antioxidant properties of crude alcoholic extract of vitex berries as well as petroleum ether, chloroform, ethyl acetate and butanol fractions were evaluated, in-vitro assessments, as compared with standard materials, l-ascorbic acid (vitamin C) and butylated hydroxyl toluene(BHT). The anti-inflammatory activity was investigated in cyclooxygenase (COX)-1 and COX-2 inhibition assays. Moreover, in-vivo antitumor effect of vitex berries alcoholic and chloroform extracts were evaluated using Ehrlich ascites carcinoma model. Data were presented as mean±SE, and data were analyzed by one-way analysis of variance test. Results and conclusion: Berries crude extract showed potent antioxidant activity followed with its fractions ethyl acetate and chloroform as compared with standard (V.C and BHT). Ethyl acetate fraction showed good reduction capability, metal ion chelation, hydrogen peroxide scavenging, nitric oxide scavenging and superoxide anion scavenging. Meanwhile, chloroform fraction produced the highest free radical scavenging activity and total antioxidant capacity. In respectable of lipid peroxidation inhibition, crude alcoholic extract and its fractions cleared weak inhibition in comparing with standard materials. Anti-inflammatory activity of V. agnus-castus berries chloroform fraction of vitex was best COX-2 inhibitor (IC₅₀, 135.41 µg/ ml) as compared to vitex alcoholic extract or ethyl acetate fraction with weak inhibitory effect on COX-1 (IC50, 778.432 µg/ ml), where the lowest effect on COX-1 was recorded with alcoholic extract. Alcoholic extract and its fractions showed weak COX-1 inhibition activity, whereas COX-2 was inhibited (100%), compared with celecoxib drug (72% at 1000ppm). The crude alcoholic and chloroform extracts of V. agnus-castus barries significantly reduced the viable Ehrlich cell count and increased nonviable count with amelioration of all hematological parameters. This amelioration was reflected on increasing median survival time and significant increase (P < 0.05) in lifespan.

Keywords: anti-inflammatory, antioxidants, ehrlich ascites carcinoma, Vitex agnus-castus

Procedia PDF Downloads 146

Authors: Randa M. T. Mohamed

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Spices have long been used as traditional ingredients in the kitchen for seasoning, coloring, aromatic and food preservative properties. Besides, spices are equally used for therapeutic purposes. The objective of this study was to survey and document the medicinal properties of spices commonly used in the Sudanese kitchen for different food preparations. Also, extracts from reported spices were screened for the presence of secondary metabolites as well as their antioxidant and beta-lactamase inhibitory properties. This study was conducted in the Rekabbya Quartier in Omdurman, Khartoum State, Sudan. Information was collected by carrying out semi-structured interviews. All informants (30) in the present study were women. Spices were purchased from Attareen shop in Omdurman. Essential oils from spices were extracted by hydrodistillation, and ethanolic extracts by maceration. Phytochemical screening was performed by thin-layer chromatography (TLC). The antioxidant capacity of essential oils and ethanolic extracts was investigated through TLC bioautography. Beta lactamase inhibitory activity was performed by the acidimetric test. Ethnobotany study showed that a total of 16 spices were found to treat 36 ailments belonging to 10 categories. The most frequently claimed medicinal uses were for the digestive system diseases treated by 14 spices and respiratory system diseases treated by 8 spices. Gynecological problems were treated with 4 spices. Dermatological diseases were cured by 5 spices, while infections caused by tapeworms and other microbes causing dysentery were treated by 3 spices. 4 spices were used to treat bad breath, bleeding gum and toothache. Headache, eyes infection, cardiac stimulation and epilepsy were treated with one spice each. Other health problems like fatigue and loss of appetite, and low breast milk production were treated by 1, 3 and 2 spices, respectively. The majority (69%, 11/16) of spices were exported from different countries like India, China, Indonesia, Ethiopia, Egypt and Nigeria, while 31% (5/16) was cultivated in Sudan. Essential oils of all spices were rich in terpenes, while ethanolic extracts contained variable classes of secondary metabolites. Both essential oils and ethanolic extracts of all spices exerted considerable antioxidant activity. Only one extract, Syzygium aromaticum, possessed beta-lactamase inhibitory activity. In conclusion, this study could contribute to conserving information on traditional medicinal uses of spices in Sudan. Also, the results demonstrated the potential of some of these spices to exert beneficial antimicrobial and antioxidant effects. Detailed phytochemical and biological assays of these spices are recommended.

Keywords: spices, enthnobotany, antioxidant, betalactamase inhibition

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Authors: Johannes Mertl

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Our surrounding air contains various particles. Besides typical representatives of inorganic dust, such as soot and ash, also particles originating from animals, microorganisms or plants are floating through the air, so-called bioaerosols. The group of bioaerosols consists of a broad spectrum of particles of different size, including fungi, bacteria, viruses, spores, or tree, flower and grass pollen that are of high relevance for allergy sufferers. In dependence of the environmental climate and the actual season, these allergenic particles can be found in enormous numbers in the air and are inhaled by humans via the respiration tract, with a potential for inflammatory diseases of the airways, such as asthma or allergic rhinitis. As a consequence air filter systems of ventilation and air conditioning devices are required to meet very high standards to prevent, or at least lower the number of allergens and airborne germs entering the indoor air. Still, filter systems are merely classified for their separation rates using well-defined mineral test dust, while no appropriate sufficiently standardized test methods for bioaerosols exist. However, determined separation rates for mineral test particles of a certain size cannot simply be transferred to bioaerosols, as separation efficiency of particularly fine and respirable particles (< 10 microns) is dependent not only on their shape and particle diameter, but also defined by their density and physicochemical properties. For this reason, the OFI developed a test method, which directly enables a testing of filters and filter media for their separation rates on bioaerosols, as well as a classification of filters. Besides allergens from an intact or fractured tree or grass pollen, allergenic proteins bound to particulates, as well as allergenic fungal spores (e.g. Cladosporium cladosporioides), or bacteria can be used to classify filters regarding their separation rates. Allergens passing through the filter can then be detected by highly sensitive immunological assays (ELISA) or in the case of fungal spores by microbiological methods, which allow for the detection of even one single spore passing the filter. The test procedure, which is carried out in laboratory scale, was furthermore validated regarding its sufficiency to cover real life situations by upscaling using air conditioning devices showing great conformity in terms of separation rates. Additionally, a clinical study with allergy sufferers was performed to verify analytical results. Several different air conditioning filters from the car industry have been tested, showing significant differences in their separation rates.

Keywords: airborne germs, allergens, classification of filters, fine dust

Procedia PDF Downloads 253

Authors: Randa M. T. Mohamed

Abstract:

Spices have long been used as traditional ingredients in the kitchen for seasoning, coloring, aromatic and food preservative properties. Besides, spices are equally used for therapeutic purposes. The objective of this study was to survey and document the medicinal properties of spices commonly used in the Sudanese kitchen for different food preparations. Also, extracts from reported spices were screened for the presence of secondary metabolites as well as their antioxidant and beta-lactamase inhibitory properties. This study was conducted in the Rekabbya Quartier in Omdurman, Khartoum State, Sudan. Information was collected by carrying out semi-structured interviews. All informants (30) in the present study were women. Spices were purchased from Attareen shop in Omdurman. Essential oils from spices were extracted by hydrodistillation and ethanolic extracts by maceration. Phytochemical screening was performed by thin layer chromatography (TLC). The antioxidant capacity of essential oils and ethanolic extracts was investigated through TLC bioautography. Beta lactamase inhibitory activity was performed by the acidimetric test. Ethnobotany study showed that a total of 16 spices were found to treat 36 ailments belonging to 10 categories. The most frequently claimed medicinal uses were for the digestive system diseases treated by 14 spices and respiratory system diseases treated by 8 spices. Gynaecological problems were treated by 4 spices. Dermatological diseases were cured by 5 spices while infections caused by tapeworms and other microbes causing dysentery were treated by 3 spices. 4 spices were used to treat bad breath, bleeding gum and toothache. Headache, eyes infection, cardiac stimulation and epilepsy were treated by one spice each. Other health problem like fatigue and loss of appetite and low breast milk production were treated by 1, 3 and 2 spices respectively. The majority (69%, 11/16) of spices were exported from different countries like India, China, Indonesia, Ethiopia, Egypt and Nigeria while 31% (5/16) was cultivated in Sudan. Essential oils of all spices were rich in terpenes while ethanolic extracts contained variable classes of secondary metabolites. Both essential oils and ethanolic extracts of all spices exerted considerable antioxidant activity. Only one extract, Syzygium aromaticum, possessed beta lactamase inhibitory activity. In conclusion, this study could contribute in conserving information on traditional medicinal uses of spices in Sudan. Also, the results demonstrated the potential of some of these spices to exert beneficial antimicrobial and antioxidant effect. Detailed phytochemical and biological assays of these spices are recommended.

Keywords: spices, ethnobotany, phytoconstituents, antioxidant, beta lactamase inhibition

Procedia PDF Downloads 79

Authors: Achiron Anat, Mathilda Mandel, Mayust Sue, Achiron Reuven, Gurevich Michael

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Introduction: As coronavirus disease 2019 (COVID-19) vaccination is currently spreading around the world, it is of importance to assess the ability of multiple sclerosis (MS) patients to mount an appropriate immune response to the vaccine in the context of disease-modifying treatments (DMT’s). Objectives: Evaluate immunity generated following COVID-19 vaccination in MS patients, and assess factors contributing to protective humoral and cellular immune responses in MS patients vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) virus infection. Methods: Review our recent data related to (1) the safety of PfizerBNT162b2 COVID-19 mRNA vaccine in adult MS patients; (2) the humoral post-vaccination SARS-CoV2 IgG response in MS vaccinees using anti-spike protein-based serology; and (3) the cellular immune response of memory B-cells specific for SARS-CoV-2 receptor-binding domain (RBD) and memory T-cells secreting IFN-g and/or IL-2 in response to SARS-CoV2 peptides using ELISpot/Fluorospot assays in MS patients either untreated or under treatment with fingolimod, cladribine, or ocrelizumab; (4) covariate parameters related to mounting protective immune responses. Results: COVID-19 vaccine proved safe in MS patients, and the adverse event profile was mainly characterised by pain at the injection site, fatigue, and headache. Not any increased risk of relapse activity was noted and the rate of patients with acute relapse was comparable to the relapse rate in non-vaccinated patients during the corresponding follow-up period. A mild increase in the rate of adverse events was noted in younger MS patients, among patients with lower disability, and in patients treated with DMTs. Following COVID-19 vaccination protective humoral immune response was significantly decreased in fingolimod- and ocrelizumab- treated MS patients. SARS-CoV2 specific B-cell and T-cell cellular responses were respectively decreased. Untreated MS patients and patients treated with cladribine demonstrated protective humoral and cellular immune responses, similar to healthy vaccinated subjects. Conclusions: COVID-19 BNT162b2 vaccine proved as safe for MS patients. No increased risk of relapse activity was noted post-vaccination. Although COVID-19 vaccination is new, accumulated data demonstrate differences in immune responses under various DMT’s. This knowledge can help to construct appropriate COVID-19 vaccine guidelines to ensure proper immune responses for MS patients.

Keywords: covid-19, vaccination, multiple sclerosis, IgG

Procedia PDF Downloads 139

Authors: Engy Shokry, Giancarlo La Marca, Maria Luisa Della Bona

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Newborn screening for Congenital Adrenal Hyperplasia (CAH) relies on quantification of 17α-hydroxyprogesterone using enzyme immunoassays. These assays, in spite of being rapid, readily available and easy to perform, its reliability was found questionable due to lack of selectivity and specificity resulting in large number of false-positives, consequently family anxiety and associated hospitalization costs. To improve specificity of conventional 17α-hydroxyprogesterone screening which may experience false transient elevation in preterm, low birth weight or acutely ill neonates, steroid profiling by LC-MS/MS as a second-tier test was implemented. Unlike the previously applied LC-MS/MS methods, with the disadvantage of requiring a relatively high number of blood drops. Since newborn screening tests are increasing, it is necessary to minimize the sample volume requirement to make the maximum use of blood samples collected on filter paper. The proposed new method requires just one 3.2 mm dried blood spot (DBS) punch. Extraction was done using methanol: water: formic acid (90:10:0.1, v/v/v) containing deuterium labelled internal standards. Extracts were evaporated and reconstituted in 10 % acetone in water. Column switching strategy for on-line sample clean-up was applied to improve the chromatographic run. The first separative step retained the investigated steroids and passed through the majority of high molecular weight impurities. After the valve switching, the investigated steroids are back flushed from the POROS® column onto the analytical column and separated using gradient elution. Found quantitation limits were 5, 10 and 50 nmol/L for 17α-hydroxyprogesterone, androstenedione and cortisol respectively with mean recoveries of between 98.31-103.24 % and intra-/ inter-assay CV% < 10 % except at LLOQ. The method was validated using standard addition calibration and isotope dilution strategies. Reference ranges were determined by analysing samples from 896 infants of various ages at the time of sample collection. The method was also applied on patients with confirmed CAH. Our method represents an attractive combination of low sample volume requirement, minimal sample preparation time without derivatization and quick chromatography (5 min). The three steroid profile and the concentration ratios (17OHP + androstenedione/cortisol) allowed better screening outcomes of CAH reducing false positives, associated costs and anxiety.

Keywords: congenital adrenal hyperplasia (CAH), 17α-hydroxyprogesterone, androstenedione, cortisol, LC-MS/MS

Procedia PDF Downloads 439

Authors: Claudio Lamilla, Andrés Santos, Vicente Llanquinao, Jocelyn Hermosilla, Leticia Barrientos

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The industry in general is very interested in improving and optimizing industrial processes in order to reduce the costs involved in obtaining raw materials and production. Thus, an interesting and cost-effective alternative is the incorporation of bioactive metabolites in such processes, being an example of this enzymes which catalyze efficiently a large number of enzymatic reactions of industrial and biotechnological interest. In the search for new sources of these active metabolites, Antarctica is one of the least explored places on our planet where the most drastic cold conditions, salinity, UVA-UVB and liquid water available are present, features that have shaped all life in this very harsh environment, especially bacteria that live in different Antarctic ecosystems, which have had to develop different strategies to adapt to these conditions, producing unique biochemical strategies. In this work the production of cellulolytic enzymes of seven bacterial strains isolated from marine sediments at different sites in the Antarctic was evaluated. Isolation of the strains was performed using serial dilutions in the culture medium at M115°C. The identification of the strains was performed using universal primers (27F and 1492R). The enzyme activity assays were performed on R2A medium, carboxy methyl cellulose (CMC)was added as substrate. Degradation of the substrate was revealed by adding Lugol. The results show that four of the tested strains produce enzymes which degrade CMC substrate. The molecular identifications, showed that these bacteria belong to the genus Streptomyces and Pseudoalteromonas, being Streptomyces strain who showed the highest activity. Only some bacteria in marine sediments have the ability to produce these enzymes, perhaps due to their greater adaptability to degrade at temperatures bordering zero degrees Celsius, some algae that are abundant in this environment and have cellulose as the main structure. The discovery of new enzymes adapted to cold is of great industrial interest, especially for paper, textiles, detergents, biofuels, food and agriculture. These enzymes represent 8% of industrial demand worldwide and is expected to increase their demand in the coming years. Mainly in the paper and food industry are required in extraction processes starch, protein and juices, as well as the animal feed industry where treating vegetables and grains helps improve the nutritional value of the food, all this clearly puts Antarctic microorganisms and their enzymes specifically as a potential contribution to industry and the novel biotechnological applications.

Keywords: antarctic, bacteria, biotechnological, cellulolytic enzymes

Procedia PDF Downloads 297

Authors: Pukhrambam Helena Chanu, Janardan Yadav

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This study investigates the potential of various fungal isolates to solubilize zinc and counteract rice pathogens, with the aim of mitigating zinc deficiency and disease prevalence in rice farming. Soil samples from the rhizosphere were collected, and zinc-solubilizing fungi were isolated and purified. Molecular analysis identified Talaromyces sp, Talaromyces versatilis, Talaromyces pinophilus, and Aspergillus terreus as effective zinc solubilizers. Through qualitative and quantitative assessments, it was observed that solubilization efficiencies varied among the isolates over time, with Talaromyces versatilis displaying the highest capacity for solubilization. This variability in solubilization rates may be attributed to differences in fungal metabolic activity and their ability to produce organic acids that facilitate zinc release from insoluble sources in the soil. In inhibition assays against rice pathogens, the fungal isolates exhibited antagonistic properties, with Talaromyces versatilis demonstrating the most significant inhibition rates. This antagonistic activity may be linked to the production of secondary metabolites, such as antibiotics or lytic enzymes by fungi, which inhibit the growth of rice pathogens. The ability of Talaromyces versatilis to outperform other isolates in both zinc solubilization and pathogen inhibition highlights its potential as a multifunctional biocontrol agent in rice cultivation systems. These findings emphasize the potential of fungi as natural solutions for enhancing zinc uptake and managing diseases in rice cultivation. Utilizing indigenous zinc-solubilizing fungi offers a sustainable and environmentally friendly approach to addressing zinc deficiency in soils, reducing the need for chemical fertilizers. Moreover, harnessing the antagonistic activity of these fungi can contribute to integrated disease management strategies, minimizing reliance on synthetic pesticides and promoting ecological balance in agroecosystems. Additionally, the study included the evaluation of dipping time under different concentrations, viz.,10 ppm, 20 ppm, and 30 ppm of biosynthesized nano ZnO on rice seedlings. This investigation aimed to optimize the application of nano ZnO for efficient zinc uptake by rice plants while minimizing potential risks associated with excessive nanoparticle exposure. Evaluating the effects of varying concentrations and dipping durations provides valuable insights into the safe and effective utilization of nano ZnO as a micronutrient supplement in rice farming practices.

Keywords: biosynthesized nano ZnO, rice, root dipping, zinc solubilizing fungi.

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Authors: Carolina Gutierrez-Cortes, Hector Suarez, Gustavo Buitrago

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Bacteriocins are antimicrobial peptides produced by bacteria as a competitive strategy for substrate and habitat. Those peptides have a potential use as food biopreservatives due to their antimicrobial activity against foodborne pathogens, avoiding the use of additives that can be harmful to consumers. The industrial production of bacteriocins is currently expensive; one of the options to be competitive is the development of economic culture media, for example, with the use of agro-industrial wastes such as whey. This study evaluated the growth and production of bacteriocins from four strains: Pediococcus pentosaceus 63, Pediococcus pentosaceus 145, Pediococcus pentosaceus 146 and Pediococcus pentosaceus 147 isolated from ‘minas cheese’ (artisanal cheese made from raw milk in the state of Minas Gerais, Brazil) in order to select a strain with growth at high rates and higher antimicrobial activity against Listeria monocytogenes 104 after incubation on the culture medium designed with whey and other components. The media used were: MRS broth, modified MRS broth (using different sources of carbon and nitrogen and different amounts of micronutrients) and a culture medium designed by a factorial design using whey and other components. The final biomass concentrations of the four strains in MRS broth after 24 hours of incubation were very similar 9.25, 9.33, 9.25 and 9.22 (log CFU/mL) for P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146 and P. pentosaceus 147 respectively. In the same assays, antimicrobial activity of 3200 AU/mL for the first three and of 12800 AU/mL for P. pentosaceus 147 were obtained. Culture of P. pentosaceus 63 on modified MRS broth, showed the effect of some sources of carbon on the activity of bacteriocin, obtaining 12800 AU/mL with dextrose and 25600 AU/mL with maltose. Cultures of P. pentosaceus 145, 146 and 147 with these same sugars presented activity of 12800 AU/mL. It was observed that the modified MRS medium using whey increased the antimicrobial activity of the strains at 16000, 6400, 16000 and 19200 AU/mL for each strain respectively, keeping the biomass at values close to 9 log units. About nitrogen sources, it was observed that the combination of peptone (10 g /L), meat extract (10 g/L) and yeast extract (5 g/L) promoted the highest activity (12800 AU/mL), and in all cases MgSO4, MnSO4, K2HPO4 and ammonium citrate at low concentrations adversely affected bacteriocin production. Because P. pentosaceus 147 showed the highest antimicrobial activity in the presence of whey, it was used to evaluate the culture medium (peptone (10 g/L), meat extract (8 g/L), yeast extract (2 g/L), Tween® 80 (1 g/L), ammonium citrate (2 g/L), sodium acetate (5 g/L), MgSO4 (0.2 g/L), MnSO4 (0.04 g/L)). With the designed medium added with whey, 9.34 log units of biomass concentration and 19200 AU/mL were achieved for P. pentosaceus 147. The above suggest that the new medium promotes the antimicrobial activity of P. pentosaceus 147 allowing the use of an economic medium using whey.

Keywords: antimicrobial activity, bacteriocins, pediococcus, whey

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Authors: Chrislaura Carmo, Luca Deiana, Mafalda Laranjo, Abilio Sobral, Armando Cordova

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Photodynamic therapy (PDT) is currently used for the treatment of malignancies and premalignant tumors. It is based on the capture of a photosensitizing molecule (PS) which, when excited by light at a certain wavelength, reacts with oxygen and generates oxidizing species (radicals, singlet oxygen, triplet species) in target tissues, leading to cell death. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indaceno) derivatives are emerging as important candidates for photosensitizer in photodynamic therapy of cancer cells due to their high triplet quantum yield. Today these dyes are relevant molecules in photovoltaic materials and fluorescent sensors. In this study, it will be demonstrated the possibility that BODIPY can be covalently linked to thioglycolic acid through the click reaction. Thiol−ene click chemistry has become a powerful synthesis method in materials science and surface modification. The design of biobased allyl-terminated precursors with high renewable carbon content for the construction of the thiol-ene polymer networks is essential for sustainable development and green chemistry. The work aims to synthesize the BODIPY (10-(4-(allyloxy) phenyl)-2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2] diazaborinin-4-ium-5-uide) and to click reaction with Thioglycolic acid. BODIPY was synthesized by the condensation reaction between aldehyde and pyrrole in dichloromethane, followed by in situ complexation with BF3·OEt2 in the presence of the base. Then it was functionalized with allyl bromide to achieve the double bond and thus be able to carry out the click reaction. The thiol−ene click was performed using DMPA (2,2-Dimethoxy-2-phenylacetophenone) as a photo-initiator in the presence of UV light (320–500 nm) in DMF at room temperature for 24 hours. Compounds were characterized by standard analytical techniques, including UV-Vis Spectroscopy, 1H, 13C, 19F NMR and mass spectroscopy. The results of this study will be important to link BODIPY to polymers through the thiol group offering a diversity of applications and functionalization. This new molecule can be tested as third-generation photosensitizers, in which the dye is targeted by antibodies or nanocarriers by cells, mainly in cancer cells, PDT and Photodynamic Antimicrobial Chemotherapy (PACT). According to our studies, it was possible to visualize a click reaction between allyl BODIPY and thioglycolic acid. Our team will also test the reaction with other thiol groups for comparison. Further, we will do the click reaction of BODIPY with a natural polymer linked with a thiol group. The results of the above compounds will be tested in PDT assays on various lung cancer cell lines.

Keywords: bodipy, click reaction, thioglycolic acid, allyl, thiol-ene click

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Authors: Yunjia Yang, Peng Li, Gordon Xu, Timothy Mahony, Bing Zhang, Neena Mitter, Karishma Mody

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Sheep flystrike is one of the most economically important diseases affecting the Australian sheep and wool industry (>356M/annually). Currently, control of Lucillia cuprina relies almost exclusively on chemicals controls, and the parasite has developed resistance to nearly all control chemicals used in the past. It is, therefore, critical to develop an alternative solution for the sustainable control and management of flystrike. RNA interference (RNAi) technologies have been successfully explored in multiple animal industries for developing parasites controls. This research project aims to develop a RNAi based biological control for sheep blowfly. Double-stranded RNA (dsRNA) has already proven successful against viruses, fungi, and insects. However, the environmental instability of dsRNA is a major bottleneck for successful RNAi. Bentonite polymer (BenPol) technology can overcome this problem, as it can be tuned for the controlled release of dsRNA in the gut challenging pH environment of the blowfly larvae, prolonging its exposure time to and uptake by target cells. To investigate the potential of BenPol technology for dsRNA delivery, four different BenPol carriers were tested for their dsRNA loading capabilities, and three of them were found to be capable of affording dsRNA stability under multiple temperatures (4°C, 22°C, 40°C, 55°C) in sheep serum. Based on stability results, dsRNA from potential targeted genes was loaded onto BenPol carriers and tested in larvae feeding assays, three genes resulting in knockdowns. Meanwhile, a primary blowfly embryo cell line (BFEC) derived from L. cuprina embryos was successfully established, aim for an effective insect cell model for testing RNAi efficacy for preliminary assessments and screening. The results of this study establish that the dsRNA is stable when loaded on BenPol particles, unlike naked dsRNA rapidly degraded in sheep serum. The stable nanoparticle delivery system offered by BenPol technology can protect and increase the inherent stability of dsRNA molecules at higher temperatures in a complex biological fluid like serum, providing promise for its future use in enhancing animal protection.

Keywords: lucilia cuprina, primary cell line establishment, RNA interference, insect cell transfection

Procedia PDF Downloads 73

Authors: Sugandha Asthana, Geetika Vajpayee, Pratibha Kumari, Shanthy Sundaram

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An economically important disease of wheat in North Western region of India is Karnal Bunt caused by smut fungus Tilletia indica. This fungal pathogen spreads by air, soil and seed borne sporodia at the time of flowering, which ultimately leads to partial bunting of wheat kernels with fishy odor and taste to wheat flour. It has very serious effects due to quarantine measures which have to be applied for grain exports. Chemical fungicides such as mercurial compounds and Propiconazole applied to the control of Karnal bunt have been only partially successful. Considering the harmful effects of chemical fungicides on man as well as environment, many countries are developing biological control as the superior substitute to chemical control. Repeated use of fungicides can be responsible for the development of resistance in fungal pathogens against certain chemical compounds. The present investigation is based on the isolation and evaluation of antifungal properties of some isolated (from natural manure) and commercial bacterial strains against Tilletia indica. Total 23 bacterial isolates were obtained and antagonistic activity of all isolates and commercial bacterial strains (Bacillus subtilis MTCC8601, Bacillus pumilus MTCC 8743, Pseudomonas aeruginosa) were tested against T. indica by dual culture plate assay (pour plate and streak plate). Test for the production of antifungal volatile organic compounds (VOCs) by antagonistic bacteria was done by sealed plate method. Amongst all s1, s3, s5, and B. subtilis showed more than 80% inhibition. Production of extracellular hydrolytic enzymes such as protease, beta 1, 4 glucanase, HCN and ammonia was studied for confirmation of antifungal activity. s1, s3, s5 and B. subtilis were found to be the best for protease activity and s5 and B. subtilis for beta 1, 4 glucanase activity. Bacillus subtilis was significantly effective for HCN whereas s3, s5 and Bacillus subtilis for ammonia production. Isolates were identified as Pseudomonas aeruginosa (s1) and B. licheniformis (s3, s5) by various biochemical assays and confirmed by16s rRNA sequencing. Use of microorganisms or their secretions as biocontrol agents to avoid plant diseases is ecologically safe and may offer long term of protection to crop. The above study reports the promising effects of these strains in better pathogen free crop production and quality maintenance as well as prevention of the excessive use of synthetic fungicides.

Keywords: antagonistic, antifungal, biocontrol, Karnal bunt

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Authors: Lorena C. Cintra, Izadora M. De Oliveira, Amanda G. Fernandes, Francieli Colussi, Rosália S. A. Jesuíno, Fabrícia P. Faria, Cirano J. Ulhoa

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Xylan is the main component of hemicellulose and for its complete degradation is required cooperative action of a system consisting of several enzymes including endo-xylanases (XYN), β-xylosidases (XYL) and α-L-arabinofuranosidases (ABF). The recombinant hemicellulolytic enzymes an endoxylanase (HXYN2), β-xylosidase (HXYLA), and an α-L-arabinofuranosidase (ABF3) were used in hydrolysis tests. These three enzymes are produced by filamentous fungi and were expressed heterologously and produced in Pichia pastoris previously. The aim of this work was to evaluate the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of sugarcane bagasse (SCB). The interaction between the three recombinant enzymes during SCB pre-treated by steam explosion hydrolysis was performed with different concentrations of HXYN2, HXYLA and ABF3 in different ratios in according to a central composite rotational design (CCRD) 23, including six axial points and six central points, totaling 20 assays. The influence of the factors was assessed by analyzing the main effects and interaction between the factors, calculated using Statistica 8.0 software (StatSoft Inc. Tulsa, OK, USA). The Pareto chart was constructed with this software and showed the values of the Student’s t test for each recombinant enzyme. It was considered as response variable the quantification of reducing sugars by DNS (mg/mL). The Pareto chart showed that the recombinant enzyme ABF3 exerted more significant effect during SCB hydrolysis, with higher concentrations and with the lowest concentration of this enzyme. It was performed analysis of variance according to Fisher method (ANOVA). In ANOVA for the release of reducing sugars (mg/ml) as the variable response, the concentration of ABF3 showed significance during hydrolysis SCB. The result obtained by ANOVA, is in accordance with those presented in the analysis method based on the statistical Student's t (Pareto chart). The degradation of the central chain of xylan by HXYN2 and HXYLA was more strongly influenced by ABF3 action. A model was obtained, and it describes the performance of the interaction of all three enzymes for the release of reducing sugars, and can be used to better explain the results of the statistical analysis. The formulation capable of releasing the higher levels of reducing sugars had the following concentrations: HXYN2 with 600 U/g of substrate, HXYLA with 11.5 U.g-1 and ABF3 with 0.32 U.g-1. In conclusion, the recombinant enzyme that has a more significant effect during SCB hydrolysis was ABF3. It is noteworthy that the xylan present in the SCB is arabinoglucoronoxylan, due to this fact debranching enzymes are important to allow access of enzymes that act on the central chain.

Keywords: experimental design, hydrolysis, recombinant enzymes, sugar cane bagasse

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Authors: Jesus Salmeron, Carmen De Vega, Maria Soledad Vicente, Mireia Olabarria, Olaia Martinez

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Dysphagia or difficulty in swallowing affects mostly elder people: 56-78% of the institutionalized and 44% of the hospitalized. Liquid food thickening is a necessary measure in this situation because it reduces the risk of penetration-aspiration. Until now, and as proposed by the American Dietetic Association in 2002, possible consistencies have been categorized in three groups attending to their viscosity: nectar (50-350 mPa•s), honey (350-1750 mPa•s) and pudding (>1750 mPa•s). The adequate viscosity level should be identified for every patient, according to her/his impairment. Nevertheless, a systematic review on dysphagia diet performed recently indicated that there is no evidence to suggest that there is any transition of clinical relevance between the three levels proposed. It was also stated that other physical properties of the bolus (slipperiness, density or cohesiveness, among others) could influence swallowing in affected patients and could contribute to the amount of remaining residue. Texture parameters need to be evaluated as possible alternative to viscosity. The aim of this study was to evaluate the instrumental extrusion-compression test as a possible tool to characterize changes along time in water thickened with various products and in the three theoretical consistencies. Six commercial thickeners were used: NM® (NM), Multi-thick® (M), Nutilis Powder® (Nut), Resource® (R), Thick&Easy® (TE) and Vegenat® (V). All of them with a modified starch base. Only one of them, Nut, also had a 6,4% of gum (guar, tara and xanthan). They were prepared as indicated in the instructions of each product and dispensing the correspondent amount for nectar, honey and pudding consistencies in 300 mL of tap water at 18ºC-20ºC. The mixture was stirred for about 30 s. Once it was homogeneously spread, it was dispensed in 30 mL plastic glasses; always to the same height. Each of these glasses was used as a measuring point. Viscosity was measured using a rotational viscometer (ST-2001, Selecta, Barcelona). Extrusion-compression test was performed using a TA.XT2i texture analyzer (Stable Micro Systems, UK) with a 25 mm diameter cylindrical probe (SMSP/25). Penetration distance was set at 10 mm and a speed of 3 mm/s. Measurements were made at 1, 5, 10, 20, 30, 40, 50 and 60 minutes from the moment samples were mixed. From the force (g)–time (s) curves obtained in the instrumental assays, maximum force peak (F) was chosen a reference parameter. Viscosity (mPa•s) and F (g) showed to be highly correlated and had similar development along time, following time-dependent quadratic models. It was possible to predict viscosity using F as an independent variable, as they were linearly correlated. In conclusion, compression-extrusion test could be an alternative and a useful tool to assess physical characteristics of thickened liquids.

Keywords: compression-extrusion test, dysphagia, texture analyzer, thickener

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Authors: Ying Li, Rui Hu, Shizhen Chen, Xin Zhou, Yunhuang Yang

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Prostate cancer is one of the leading causes of cancer-related death among men. Prostate-specific antigen (PSA), a specific product of prostatic epithelial cells, is an important indicator of prostate cancer. Though PSA is not a specific serum biomarker for the screening of prostate cancer, it is recognized as an indicator for prostate cancer recurrence and response to therapy for patient’s post-prostatectomy. Since radical prostatectomy eliminates the source of PSA production, serum PSA levels fall below 50 pg/mL, and may be below the detection limit of clinical immunoassays (current clinical immunoassay lower limit of detection is around 10 pg/mL). Many clinical studies have shown that intervention at low PSA levels was able to improve patient outcomes significantly. Therefore, ultra-sensitive and precise assays that can accurately quantify extremely low levels of PSA (below 1-10 pg/mL) will facilitate the assessment of patients for the possibility of early adjuvant or salvage treatment. Currently, the commercially available ultra-sensitive ELISA kit (not used clinically) can only reach a detection limit of 3-10 pg/mL. Other platforms developed by different research groups could achieve a detection limit as low as 0.33 pg/mL, but they relied on sophisticated instruments to get the final readout. Herein we report a microfluidic platform for point-of-care (POC) detection of PSA with a detection limit of 0.5 pg/mL and without the assistance of any equipment. This platform is based on a previously reported volumetric-bar-chart chip (V-Chip), which applies platinum nanoparticles (PtNPs) as the ELISA probe to convert the biomarker concentration to the volume of oxygen gas that further pushes the red ink to form a visualized bar-chart. The length of each bar is used to quantify the biomarker concentration of each sample. We devised a long reading channel V-Chip (LV-Chip) in this work to achieve a wide detection window. In addition, LV-Chip employed a unique enzyme-free ELISA probe that enriched PtNPs significantly and owned 500-fold enhanced catalytic ability over that of previous V-Chip, resulting in a significantly improved detection limit. LV-Chip is able to complete a PSA assay for five samples in 20 min. The device was applied to detect PSA in 50 patient serum samples, and the on-chip results demonstrated good correlation with conventional immunoassay. In addition, the PSA levels in finger-prick whole blood samples from healthy volunteers were successfully measured on the device. This completely stand-alone LV-Chip platform enables convenient POC testing for patient follow-up in the physician’s office and is also useful in resource-constrained settings.

Keywords: point-of-care detection, microfluidics, PSA, ultra-sensitive

Procedia PDF Downloads 110

Authors: Zoi Konsoula

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The increasing prevalence of Alzheimer’s disease (AD) and diabetes mellitus (DM) constitutes them major global health problems. Recently, the inhibition of key enzyme activity is considered a potential treatment of both diseases. Specifically, inhibition of acetylcholinesterase (AChE), the key enzyme involved in the breakdown of the neurotransmitter acetylcholine, is a promising approach for the treatment of AD, while inhibition of α-amylase retards the hydrolysis of carbohydrates and, thus, reduces hyperglycemia. Unfortunately, commercially available AChE and α-amylase inhibitors are reported to possess side effects. Consequently, there is a need to develop safe and effective treatments for both diseases. In the present study, pomegranate peel (PP) was extracted using various solvents of increasing polarity, while two extraction methods were employed, the conventional maceration and the ultrasound assisted extraction (UAE). The concentration of bioactive phytoconstituents, such as total phenolics (TPC) and total flavonoids (TFC) in the prepared extracts was evaluated by the Folin-Ciocalteu and the aluminum-flavonoid complex method, respectively. Furthermore, the anti-neurodegenerative and anti-hyperglycemic activity of all extracts was determined using AChE and α-amylase inhibitory activity assays, respectively. The inhibitory activity of the extracts against AChE and α-amylase was characterized by estimating their IC₅₀ value using a dose-response curve, while galanthamine and acarbose were used as positive controls, respectively. Finally, the kinetics of AChE and α-amylase in the presence of the most inhibitory potent extracts was determined by the Lineweaver-Burk plot. The methanolic extract prepared using the UAE contained the highest amount of phytoconstituents, followed by the respective ethanolic extract. All extracts inhibited acetylcholinesterase in a dose-dependent manner, while the increased anticholinesterase activity of the methanolic (IC₅₀ = 32 μg/mL) and ethanolic (IC₅₀ = 42 μg/mL) extract was positively correlated with their TPC content. Furthermore, the activity of the aforementioned extracts was comparable to galanthamine. Similar results were obtained in the case of α-amylase, however, all extracts showed lower inhibitory effect on the carbohydrate hydrolyzing enzyme than on AChE, since the IC₅₀ value ranged from 84 to 100 μg/mL. Also, the α-amylase inhibitory effect of the extracts was lower than acarbose. Finally, the methanolic and ethanolic extracts prepared by UAE inhibited both enzymes in a mixed (competitive/noncompetitive) manner since the Kₘ value of both enzymes increased in the presence of extracts, while the Vmax value decreased. The results of the present study indicate that PP may be a useful source of active compounds for the management of AD and DM. Moreover, taking into consideration that PP is an agro-industrial waste product, its valorization could not only result in economic efficiency but also reduce the environmental pollution.

Keywords: acetylcholinesterase, Alzheimer’s disease, α-amylase, diabetes mellitus, pomegranate

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Authors: Azza El-Medany, Jamila El-Medany

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Residential and agricultural pesticide use is widespread in the world. Their extensive and indiscriminative use, in addition with their ability to interact with biological systems other than their primary targets constitute a health hazards to both humans and animals. The toxic effects of pesticides include alterations in metabolism; there is a lack of knowledge that organophosphates can cause pancreatic toxicity. The primary goal of this work is to study the effects of chronic exposure to Diazinon an organophosphate used in agriculture on pancreatic tissues and evaluate the ameliorating effect of Mesna as antioxidant on the toxicity of Diazinon on pancreatic tissues.40 adult male rats, their weight ranged between 300-350 g. The rats were classified into three groups; control (10 rats) was received corn oil at a dose of 1 0 mg/kg/day by gavage once a day for 2 months. Diazinon (15 rats) was received Diazinon at a dose of 10 mg/kg/day dissolved in corn oil by gavage once a day for 2 months. Treated group (15 rats), were received Mesna 180mg/kg once a week by gavage 15 minutes before administration of Diazinon for 2 months. At the end of the experiment, animals were anesthetized, blood samples were taken by cardiac puncture for glucose and insulin assays and pancreas was removed and divided into 3 portions; first portion for histopathological study; second portion for ultrastructural study; third portion for biochemical study using Elisa Kits including determination of malondialdehyde (MDA), tumor necrosis factor α (TNF-α), myeloperoxidase activity (MPO), interleukin 1β (IL-1β). A significant increase in the levels of MDA, TNF-α, MPO activity, IL-1β, serum glucose levels in the toxicated group with Diazinon were observed, while a significant reduction was noticed in GSH in serum insulin levels. After treatment with Mesna a significant reduction was observed in the previously mentioned parameters except that there was a significant rise in GSH in insulin levels. Histopathological and ultra-structural studies showed destruction in pancreatic tissues and β cells were the most affected cells among the injured islets as compared with the control group. The current study try to spot light about the effects of chronic exposure to pesticides on vital organs as pancreas also the role of oxidative stress that may be induced by them in evoking their toxicity. This study shows the role of antioxidant drugs in ameliorating or preventing the toxicity. This appears to be a promising approach that may be considered as a complementary treatment of pesticide toxicity.

Keywords: Diazinon, reduced glutathione, myeloperoxidase activity, tumor necrosis factor α, Mesna

Procedia PDF Downloads 242

Authors: Paola Modesto, Floriana Fruscione, Isabella Martini, Simona Perga, Federica Riccardo, Mariateresa Camerino, Davide Giacobino, Cecilia Gola, Luca Licenziato, Elisabetta Razzuoli, Katia Varello, Lorella Maniscalco, Elena Bozzetta, Angelo Ferrari

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Canine and human melanoma, osteosarcoma (OSA), and mammary carcinomas are aggressive tumors with common characteristics making dogs a good model for comparative oncology. Novel therapeutic strategies against these tumors could be useful to both species. In humans, chondroitin sulphate proteoglycan 4 (CSPG4) is a marker involved in tumor progression and could be a candidate target for immunotherapy. The anti-CSPG4 DNA electrovaccination has shown to be an effective approach for canine malignant melanoma (CMM) [1]. An immunohistochemistry evaluation of CSPG4 expression in tumour tissue is generally performed prior to electrovaccination. To assess the possibility to perform a rapid molecular evaluation and in order to validate these spontaneous canine tumors as the model for human studies, we investigate the CSPG4 gene expression by RT qPCR in CMM, OSA, and canine mammary tumors (CMT). The total RNA was extracted from RNAlater stored tissue samples (CMM n=16; OSA n=13; CMT n=6; five paired normal tissues for CMM, five paired normal tissues for OSA and one paired normal tissue for CMT), retro-transcribed and then analyzed by duplex RT-qPCR using two different TaqMan assays for the target gene CSPG4 and the internal reference gene (RG) Ribosomal Protein S19 (RPS19). RPS19 was selected from a panel of 9 candidate RGs, according to NormFinder analysis following the protocol already described [2]. Relative expression was analyzed by CFX Maestro™ Software. Student t-test and ANOVA were performed (significance set at P<0.05). Results showed that gene expression of CSPG4 in OSA tissues is significantly increased by 3-4 folds when compared to controls. In CMT, gene expression of the target was increased from 1.5 to 19.9 folds. In melanoma, although an increasing trend was observed, no significant differences between the two groups were highlighted. Immunohistochemistry analysis of the two cancer types showed that the expression of CSPG4 within CMM is concentrated in isles of cells compared to OSA, where the distribution of positive cells is homogeneous. This evidence could explain the differences in gene expression results.CSPG4 immunohistochemistry evaluation in mammary carcinoma is in progress. The evidence of CSPG4 expression in a different type of canine tumors opens the way to the possibility of extending the CSPG4 immunotherapy marker in CMM, OSA, and CMT and may have an impact to translate this strategy modality to human oncology.

Keywords: canine melanoma, canine mammary carcinomas, canine osteosarcoma, CSPG4, gene expression, immunotherapy

Procedia PDF Downloads 174

Authors: Mmbulaheni Ramulondi, Sandy Van Vuuren, Helene De Wet

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The use of plant combinations to treat various medical conditions is not a new concept, and it is known that traditional people do not only rely on a single plant extract for efficacy but often combine various plant species for treatment. The knowledge of plant combinations is transferred from one generation to the other in the belief that combination therapy may enhance efficacy, reduce toxicity, decreases adverse effects, increase bioavailability and result in lower dosages. However, combination therapy may also be harmful when the interaction is antagonistic, since it may result in increasing toxicity. Although a fair amount of research has been done on the toxicity of medicinal plants, there is very little done on the toxicity of medicinal plants in combination. The aim of the study was to assess the toxicity potential of 19 plant combinations which have been documented as treatments of hypertension in northern KwaZulu-Natal by lay people. The aqueous extracts were assessed using two assays; the Brine shrimp assay (Artemia franciscana) and the Ames test (Mutagenicity). Only one plant combination (Aloe marlothii with Hypoxis hemerocallidea) in the current study has been previously assessed for toxicity. With the Brine shrimp assay, the plant combinations were tested in two concentrations (2 and 4 mg/ml), while for mutagenicity tests, they were tested at 5 mg/ml. The results showed that in the Brine shrimp assay, six combinations were toxic at 4 mg/ml. The combinations were Albertisia delagoensis with Senecio serratuloides (57%), Aloe marlothii with Catharanthus roseus (98%), Catharanthus roseus with Hypoxis hemerocallidea (66%), Catharanthus roseus with Musa acuminata (89%), Catharanthus roseus with Momordica balsamina (99%) and Aloe marlothii with Trichilia emetica and Hyphaene coriacea (50%). However when the concentration was reduced to 2 mg/ml, only three combinations were toxic which were Aloe marlothii with Catharanthus roseus (76%), Catharanthus roseus with Musa acuminata (66%) and Catharanthus roseus with Momordica balsamina (73%). For the mutagenicity assay, only the combinations between Catharanthus roseus with Hypoxis hemerocallidea and Catharanthus roseus with Momordica balsamina were mutagenic towards the Salmonella typhimurium strains TA98 and TA100. Most of the combinations which were toxic involve C. roseus which was also toxic when tested singularly. It is worth noting that C. roseus was one of the most frequently used plant species both to treat hypertension singularly and in combination and some of the individuals have been using this for the last 20 years. The mortality percentage of the Brine shrimp showed a significant correlation between dosage and toxicity thus toxicity was dosage dependant. A combination which is worth noting is the combination between A. delagoensis and S. serratuloides. Singularly these plants were non-toxic towards Brine shrimp, however their combination resulted in antagonism with the mortality rate of 57% at the total concentration of 4 mg/ml. Low toxicity was mostly observed, giving some validity to combined use, however the few combinations showing increased toxicity demonstrate the importance of analysing plant combinations.

Keywords: dosage, hypertension, plant combinations, toxicity

Procedia PDF Downloads 353

Authors: Iulianna Taritsa, Kuldeep Neote, Eric Fossel

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Lysosome-induced immunogenic cell death (LIICD) is a powerful mechanism of targeting cancer cells that kills circulating malignant cells and primes the host’s immune cells against future remission. Current immunotherapies for cancer are limited in preventing recurrence – a gap that can be bridged by training the immune system to recognize cancer neoantigens. Lysosomal leakage can be induced therapeutically to traffic antigens from dying cells to dendritic cells, which can later present those tumorigenic antigens to T cells. Previous research has shown that oxidative agents administered in the tumor microenvironment can initiate LIICD. We generated a fusion protein between an oxidative agent known as xanthine oxidase (XO) and a mini-antibody specific for EGFR/HER2-sensitive breast tumor cells. The anti-EGFR single domain antibody fragment is uniquely sourced from llama, which is functional without the presence of a light chain. These llama micro-antibodies have been shown to be better able to penetrate tissues and have improved physicochemical stability as compared to traditional monoclonal antibodies. We demonstrate that the fusion protein created is stable and can induce early markers of immunogenic cell death in an in vitro human breast cancer cell line (SkBr3). Specifically, we measured overall cell death, as well as surface-expressed calreticulin, extracellular ATP release, and HMGB1 production. These markers are consensus indicators of ICD. Flow cytometry, luminescence assays, and ELISA were used respectively to quantify biomarker levels between treated versus untreated cells. We also included a positive control group of SkBr3 cells dosed with doxorubicin (a known inducer of LIICD) and a negative control dosed with cisplatin (a known inducer of cell death, but not of the immunogenic variety). We looked at each marker at various time points after cancer cells were treated with the XO/antibody fusion protein, doxorubicin, and cisplatin. Upregulated biomarkers after treatment with the fusion protein indicate an immunogenic response. We thus show the potential for this fusion protein to induce an anticancer effect paired with an adaptive immune response against EGFR/HER2+ cells. Our research in human cell lines here provides evidence for the success of the same therapeutic method for patients and serves as the gateway to developing a new treatment approach against breast cancer.

Keywords: apoptosis, breast cancer, immunogenic cell death, lysosome

Procedia PDF Downloads 199

Authors: S.Kauroo, J. Govinden Soulange, D. Marie

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Psiadia species from the Asteraceae are traditionally used in the folk medicine of Mauritius to treat cutaneous and bronchial infections. The present study aimed at validating the phytomedicinal properties of the selected species from the Asteraceae family, namely Psiadia arguta, Psiadia viscosa, Psiadia lithospermifolia, and Distephanus populifolius. Dried hexane, ethyl acetate, and methanol leaf extracts were studied for their antioxidant properties using the DPPH (1, 1-diphenyl-2-picryl-hydrazyl), FRAP (Ferric Reducing Ability of Plasma), and Deoxyribose assays. Antibacterial activity against human pathogenic bacteria namely Escherichia coli (ATCC 27853), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Klebsiella pneumonia (ATCC27853), Pseudomonas aeruginosa (ATCC 27853), and Bacillus cereus (ATCC 11778) was measured using the broth microdilution assay. Qualitative phytochemical screening using standard methods revealed the presence of coumarins, tannins, leucoanthocyanins, and steroids in all the tested extracts. The measured phenolics level of the selected plant extracts varied from 24.0 to 231.6 mg GAE/g with the maximum level in methanol extracts in all four species. The highest flavonoids and proanthocyanidins content was noted in Psiadia arguta methanolic extracts with 65.7±1.8 mg QE/g and 5.1±0.0 mg CAT/g dry weight (DW) extract, respectively. The maximum free radical scavenging activity was measured in Psiadia arguta methanol and ethyl acetate extracts with IC50 11.3±0.2 and 11.6± 0.2 µg/mL, respectively and followed by Distephanus populifolius methanol extracts with an IC50 of 11.3± 0.8 µg/mL. The maximum ferric reducing antioxidant potential was noted in Psiadia lithospermifolia methanol extracts with a FRAP value of 18.8 ± 0.4 µmol Fe2+/L/g DW. The antioxidant capacity based on DPPH and Deoxyribose values were negatively related to total phenolics, flavonoid and proanthocyanidins content while the ferric reducing antioxidant potential were strongly correlated to total phenolics, flavonoid and proanthocyanidins content. All four species exhibited antimicrobial activity against the tested bacteria (both Gram-negative and Gram-positive). Interestingly, the hexane and ethyl acetate extracts of Psiadia viscosa and Psiadia lithospermifolia were more active than the control antibiotic Chloramphenicol. The Minimum inhibitory concentration (MIC) values for hexane and ethyl acetate extracts of Psiadia viscosa and Psiadia lithospermifolia against the tested bacteria ranged from (62.5 to 500 µg/ml). These findings validate the use of these tested Asteraceae in the traditional medicine of Mauritius and also highlight their pharmaceutical potential as prospective phytomedicines.

Keywords: antibacterial, antioxidant, DPPH, flavonoids, FRAP, Psiadia spp

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Authors: Aarti Sharma, Sarita Kumar, Pushplata Tripathi

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Aedes aegypti L. is one of the most important insect vectors in the world transmitting several diseases of concern; dengue fever, dengue haemorrhagic fever and yellow fever. Though since ages the control of dengue vector is primarily relied upon the use of synthetic chemical insecticides, the continued and indiscriminate use of insecticides for their control has received wide public apprehension because of multifarious problems including insecticide resistance, resurgence of pest species, environmental pollution, toxic hazards to humans and non-target organisms. These problems have necessitated the need to explore and develop alternative strategies using eco-friendly and bio-degradable plant products. Bio-insecticides, despite being the focus of research nowadays, have not been investigated much regarding their physiological effects on the mosquitoes. Thus, the present studies were carried out to investigate the anti-mosquito potential of the leaf and stem hexane extracts of Achyranthes aspera against early fourth instars of Aedes aegypti L and their effects on the histological architecture of their midgut. The larvicidal bioassays conducted with the A. aspera leaf hexane extracts revealed the respective LC30, LC50 and LC90 values of 66.545 ppm, 82.555 ppm, 139.817 ppm while the assays with stem hexane extracts resulted in respective values of 54.982 ppm, 68.133 ppm, 115.075 ppm. The studies clearly indicate the efficacy of extracts as larvicidal agents against Ae. aegypti, the stem extracts being found more effective than the leaf extracts. When the larvae assayed with extracts were investigated for the modifications in the histo-architecture of the midgut, the studies showed significant damage, shrinkage, distortion and vacuolization of gut tissues and peritrophic membrane causing disintegration of epithelial cells and cytoplasmic organelles; extent of toxicity and damage varied depending upon the concentration and exposure time period. These changes revealed appreciable stomach poison potential of A. aspera extracts against Ae. aegypti larvae, which may have also caused adverse impact on the growth and development of larvae. These effects were also found to be more pronounced with the stem extract than the leaf extract. Our findings may prove significant suggesting the use of A. aspera extract as a bio-insecticide against early fourth instar larvae of Ae. aegypti. Further studies are needed to identify the bioactive component in the extracts and to ascertain the use of component in the fields as anti-mosquito control agent.

Keywords: Achyranthes aspera, Aedes aegypti, histological architecture, larvicidal, midgut, stomach poison

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Authors: W. Mohammed Saeed, A. J. Mcbain, S. M. Cruickshank, C. A. O’Neill

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In the gut, probiotics have been shown to protect epithelial cells from pathogenic bacteria through a number of mechanisms: 1-Increasing epithelial barrier function, 2-Modulation of the immune response especially innate immune response, 3-Inhibition of pathogen adherence and down regulation of virulence factors. Since probiotics have positive impacts on the gut, their potential effects on other body tissues, such as skin have begun to be investigated. The purpose of this project is to characterize the potential of probiotic bacteria lysate as therapeutic agent for preventing or reducing the S. aureus infection. Normal human primary keratinocytes (KCs) were exposed to S. aureus (106/ml) in the presence or absence of L. rhamnosus GG lysate (extracted from 108cfu/ml). The viability of the KCs was measured after 24 hours using a trypan blue exclusion assay. When KCs were treated with S aureus alone, only 25% of the KCs remained viable at 24 hours post infection. However, in the presence of L. rhamnosus GG lysate the viability of pathogen infected KCs increased to 58% (p=0.008, n=3). Furthermore, when KCs co-exposed, pre- exposed or post-exposed to L. rhamnosus GG lysate, the viability of the KCs increased to ≈60%, the L. rhamnosus GG lysate was afforded equal protection in different conditions. These data suggests that two possible separate mechanisms are involved in the protective effects of L. rhamnosus GG such as reducing S. aureus growth, or inhibiting of pathogenic adhesion. Interestingly, a lysate of L rhamnosus GG provided significant reduction in S. aureus growth and adhesion of S. aureus that being viable following 24 hours incubation with S aureus. Therefore, a series of Liquid Chromatography (RP-LC) methods were adopted to partially purify the lysate in combination with functional assays to elucidate in which fractions the efficacious molecules were contained. In addition, the Mass Spectrometry-based protein sequencing was used to identify putative proteins in the fractions. The data presented from purification process demonstrated that L. rhamnosus GG lysate has the potential to protect keratinocytes from the toxic effects of the skin pathogen, S. aureus. Three potential mechanisms were identified: inhibition of pathogen growth; competitive exclusion; and displacement of the pathogen from keratinocyte binding sites. In this study, ‘moonlight’ proteins were identified in the current study’s MS/MS data for L. rhamnosus GG lysate, which could elucidate the ability of lysate in the competitive exclusion and displacement of S. aureus from keratinocyte binding sites. Taken together, it can be speculated that L. rhamnosus GG lysate utilizes different mechanisms to protect keratinocytes from S. aureus toxicity. The present study indicates that the proteinaceous substances are involved in anti-adhesion activity. This is achieved by displacing the pathogen and preventing the severity of pathogen infection and the moonlight proteins might be involved in inhibiting the adhesion of pathogens.

Keywords: lysate, fractions, adhesion, L. rhamnosus GG, S. aureus toxicity

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Authors: Parimelazhagan Thangaraj

Abstract:

Natural products serves human kind as a source of all drugs and higher plants provide most of these therapeutic agents. These products are widely recognized in the pharmaceutical industry for their broad structural diversity as well as their wide range of pharmacological activities. Euphorbiaceae is one of the important families with significant pharmacological activities, of which many species has been used traditionally for the treatment of various ailments. Breynia retusa belongs to the family Euphorbiaceae is used to cure ailments like body pain, skin inflammation, hyperglycaemia, diarrhoea, dysentery and toothache. Flowers and young leaves of B. retusa are cooked and eaten, roots are used for meningitis. The juice of the stem is used in conjunctivtis and leaves as poultice to hasten suppuration. Based on the strong evidences of traditional uses of Breynia retusa, the present study was focused on neutraceuticals evaluation of the species with special reference to oxidative stress and diabetes. Both leaves and stem of B. retusa were extracted with different solvents and analyzed for radical scavenging ability wherein ABTS.+ (8396.95±1529.01 µM TEAC/g extract), phosphomolybdenum (17.34±0.08 g AAE/100 g extract) and FRAP (6075.66±414.28 µM Fe (II) E/mg extract) assays showed good radical scavenging activity in stem. Furthermore, leaf extracts showed good radical inhibition in DPPH (2.4 µg/mL), metal ion (27.44±0.09 mg EDTAE/g extract) scavenging methods. The α-amylase and α-glucosidase inhibitors are currently used for diabetic treatment as oral hypoglycemic agents. The inhibitory effects of the B. retusa leaf and stem ethyl acetate extracts showed good inhibition on α-amylase (96.25% and 95.69 respectively) and α-glucosidase (54.50% and 50.87% respectively) enzymes compared to standard acarbose. The proximate composition analysis of B. retusa leaves contains higher amount of total carbohydrates (14.08 g Glucose equivalents/100 g sample), ash (19.04 %) and crude fibre (0.52 %). The examination of mineral profile explored that the leaves was rich in calcium (1891 ppm), sulphur (1406 ppm), copper (2600 ppm) and magnesium (778 ppm). Leaves sample revealed very minimal amount of anti-nutrient contents like trypsin (14.08±0.03 TIU/mg protein) and tannin (0.011±0.001 mg TAE/g sample). The low anti nutritional factors may not pose any serious nutritional problems when these leaves are consumed. In conclusion, it is very clear that dietary compounds from B. retusa are suitable and promising for the development of safe food products and natural additives. Based on the studies, it may be concluded that nutritional composition, antioxidant and anti-diabetic activities this species can be used as future therapeutic medicine.

Keywords: Breynia retusa, nutraceuticals, antioxidant, anti diabetic

Procedia PDF Downloads 331

Authors: Martyna Chotomska, Aleksandra Studzinska, Marta Lisowska, Justyna Szubert, Aleksandra Tabis, Jacek Bania, Arkadiusz Miazek

Abstract:

Objectives: Assessing antigen-specific T cell responses is of utmost importance for the pre-clinical testing of prototype vaccines against intracellular pathogens and tumor antigens. Mainly two types of in vitro assays are used for this purpose 1) enzyme-linked immunospot (ELISpot) and 2) intracellular cytokine staining (ICS). Both are time-consuming, relatively expensive, and require manual dexterity. Here, we assess if a straightforward detection of luciferase activity in blood samples of transgenic reporter mice expressing a secreted Lucia luciferase under the transcriptional control of IFN-γ promoter parallels the sensitivity of IFNγ ELISpot assay. Methods: IFN-γ-LUCIA mouse strain carrying multiple copies of Lucia luciferase transgene under the transcriptional control of IFNγ minimal promoter were generated by pronuclear injection of linear DNA. The specificity of transgene expression and mobilization was assessed in vitro using transgenic splenocytes exposed to various mitogens. The IFN-γ-LUCIA mice were immunized with 50mg of ovalbumin (OVA) emulsified in incomplete Freund’s adjuvant three times every two weeks by subcutaneous injections. Blood samples were collected before and five days after each immunization. Luciferase activity was assessed in blood serum. Peripheral blood mononuclear cells were separated and assessed for frequencies of OVA-specific IFNγ-secreting T cells. Results: We show that in vitro cultured splenocytes of IFN-γ-LUCIA mice respond by 2 and 3 fold increase in secreted luciferase activity to T cell mitogens concanavalin A and phorbol myristate acetate, respectively but fail to respond to B cell-stimulating E.coli lipopolysaccharide. Immunization of IFN-γ-LUCIA mice with OVA leads to over 4 fold increase in luciferase activity in blood serum five days post-immunization with a barely detectable increase in OVA-specific, IFNγ-secreting T cells by ELISpot. Second and third immunizations, further increase the luciferase activity and coincidently also increase the frequencies of OVA-specific T cells by ELISpot. Conclusions: We conclude that minimally invasive monitoring of luciferase secretions in blood serum of IFN-γ-LUCIA mice constitutes a sensitive method for evaluating primary and memory Th1 responses to protein antigens. As such, this method may complement existing methods for rapid immunogenicity assessment of prototype vaccines.

Keywords: ELISpot, immunogenicity, interferon-gamma, reporter mice, vaccines

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Authors: Tomás Sobrino, Lara García-Varela, Marta Aramburu-Núñez, Mónica Castro, Noemí Gómez-Lado, Mariña Rodríguez-Arrizabalaga, Antía Custodia, Juan Manuel Pías-Peleteiro, José Manuel Aldrey, Daniel Romaus-Sanjurjo, Ángeles Almeida, Pablo Aguiar, Alberto Ouro

Abstract:

Introduction: Alzheimer’s disease (AD) and other tauopathies are primary causes of dementia, causing progressive cognitive deterioration that entails serious repercussions for the patients' performance of daily tasks. Currently, there is no effective approach for the early diagnosis and treatment of AD and tauopathies. This study suggests a theragnostic approach based on the importance of phosphorylated tau protein (p-Tau) in the early pathophysiological processes of AD. We have developed a novel theragnostic monoclonal antibody (mAb) to provide both diagnostic and therapeutic effects. Methods/Results: We have developed a p-Tau mAb, which was doped with deferoxamine for radiolabeling with Zirconium-89 (89Zr) for PET imaging, as well as fluorescence dies for immunofluorescence assays. The p-Tau mAb was evaluated in vitro for toxicity by MTT assay, LDH activity, propidium iodide/Annexin V assay, caspase-3, and mitochondrial membrane potential (MMP) assay in both mouse endothelial cell line (bEnd.3) and cortical primary neurons cell cultures. Importantly, non-toxic effects (up to concentrations of p-Tau mAb greater than 100 ug/mL) were detected. In vivo experiments in the tauopathy model mice (PS19) show that the 89Zr-pTau-mAb and 89Zr-Fragments-pTau-mAb are stable in circulation for up to 10 days without toxic effects. However, only less than 0.2% reached the brain, so further strategies have to be designed for crossing the Brain-Blood-Barrier (BBB). Moreover, an intraparenchymal treatment strategy was carried out. The PS19 mice were operated to implement osmotic pumps (Alzet 1004) at two different times, at 4 and 7 months, to stimulate the controlled release for one month each of the B6 antibody or the IgG1 control antibody. We demonstrated that B6-treated mice maintained their motor and memory abilities significantly compared with IgG1 treatment. In addition, we observed a significant reduction in p-Tau deposits in the brain. Conclusions /Discussion: A theragnostic pTau-mAb was developed. Moreover, we demonstrated that our p-Tau mAb recognizes very-early pathology forms of p-Tau by non-invasive techniques, such as PET. In addition, p-Tau mAb has non-toxic effects, both in vitro and in vivo. Although the p-Tau mAb is stable in circulation, only 0.2% achieve the brain. However, direct intraventricular treatment significantly reduces cognitive impairment in Alzheimer's animal models, as well as the accumulation of toxic p-Tau species.

Keywords: alzheimer's disease, theragnosis, tau, PET, immunotherapy, tauopathies

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Authors: Sarah Crawford, Gerry Lesley

Abstract:

This research is a pre-clinical assessment of anti-cancer effects of novel non-steroidal diethyl stilbestrol-like estrogen analogs in estrogen-receptor positive/ progesterone-receptor positive human breast cancer microtumors of MCF 7 cell line. Tamoxifen analog formulation (Tam A1) was used as a single agent or in combination with therapeutic concentrations of 4-hydroxytamoxifen, currently used as a long-term treatment for the prevention of breast cancer recurrence in women with estrogen receptor positive/ progesterone receptor positive malignancies. At concentrations ranging from 30-50 microM, Tam A1 induced microtumor disaggregation and cell death. Incremental cytotoxic effects correlated with increasing concentrations of Tam A1. Live tumor microscopy showed that microtumos displayed diffuse borders and substrate-attached cells were rounded-up and poorly adherent. A complete cytotoxic effect was observed using 40-50 microM Tam A1 with time course kinetics similar to 4-hydroxytamoxifen. Combined treatment with TamA1 (30-50 microM) and 4-hydroxytamoxifen (10-15 microM) induced a highly cytotoxic, synergistic combined treatment response that was more rapid and complete than using 4-hydroxytamoxifen as a single agent therapeutic. Microtumors completely dispersed or formed necrotic foci indicating a highly cytotoxic combined treatment response. Moreover, breast cancer microtumors treated with both 4-hydroxytamoxifen and Tam A1 displayed lower levels of long-term post-treatment regrowth, a critical parameter of primary drug resistance, than observed for 4-hydroxytamoxifen when used as a single agent therapeutic. Tumor regrowth at 6 weeks post-treatment with either single agent 4-hydroxy tamoxifen, Tam A1 or a combined treatment was assessed for the development of drug resistance. Breast cancer cells treated with both 4-hydroxytamoxifen and Tam A1 displayed significantly lower levels of post-treatment regrowth, indicative of decreased drug resistance, than observed for either single treatment modality. The preclinical data suggest that combined treatment involving the use of tamoxifen analogs may be a novel clinical approach for long-term maintenance therapy in patients with estrogen-receptor positive/progesterone-receptor positive breast cancer receiving hormonal therapy to prevent disease recurrence. Detailed data on time-course, IC50 and tumor regrowth assays post- treatment as well as a proposed mechanism of action to account for observed synergistic drug effects will be presented.

Keywords: 4-hydroxytamoxifen, tamoxifen analog, drug-resistance, microtumors

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Authors: S. H. Gasa, L. Singh, B. Pillay, A. O. Olaniran

Abstract:

Wastewater treatment plants (WWTPs) have been implicated as the leading reservoir for antibiotic resistant bacteria (ARB), including Enterococci spp. and antibiotic resistance genes (ARGs), worldwide. Enterococci are a group of clinically significant bacteria that have gained much attention as a result of their antibiotic resistance. They play a significant role as the principal cause of nosocomial infections and dissemination of antimicrobial resistance genes in the environment. The main objective of this study was to ascertain the role of WWTPs in Durban, South Africa as potential reservoirs for antibiotic resistant Enterococci (ARE) and their related ARGs. Furthermore, the antibiogram and resistance gene profile of Enterococci species recovered from treated wastewater effluent and receiving surface water in Durban were also investigated. Using membrane filtration technique, Enterococcus selective agar and selected antibiotics, ARE were enumerated in samples (influent, activated sludge, before chlorination and final effluent) collected from two WWTPs, as well as from upstream and downstream of the receiving surface water. Two hundred Enterococcus isolates recovered from the treated effluent and receiving surface water were identified by biochemical and PCR-based methods, and their antibiotic resistance profiles determined by the Kirby-Bauer disc diffusion assay, while PCR-based assays were used to detect the presence of resistance and virulence genes. High prevalence of ARE was obtained at both WWTPs, with values reaching a maximum of 40%. The influent and activated sludge samples contained the greatest prevalence of ARE with lower values observed in the before and after chlorination samples. Of the 44 vancomycin and high-level gentamicin-resistant isolates, 11 were identified as E. faecium, 18 as E. faecalis, 4 as E. hirae while 11 are classified as “other” Enterococci species. High-level aminoglycoside resistance for gentamicin (39%) and vancomycin (61%) was recorded in species tested. The most commonly detected virulence gene was the gelE (44%), followed by asa1 (40%), while cylA and esp were detected in only 2% of the isolates. The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(3')-IIIa, and ant(6')-Ia detected in 43%, 45% and 41% of the isolates, respectively. Positive correlation was observed between resistant phenotypes to high levels of aminoglycosides and presence of all aminoglycoside resistance genes. Resistance genes for glycopeptide: vanB (37%) and vanC-1 (25%), and macrolide: ermB (11%) and ermC (54%) were detected in the isolates. These results show the need for more efficient wastewater treatment and disposal in order to prevent the release of virulent and antibiotic resistant Enterococci species and safeguard public health.

Keywords: antibiogram, enterococci, gentamicin, vancomycin, virulence signatures

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