Search results for: top-down activity

Authors: Priyanka Sharma, Ranjita Das, Mohan C. Kalita, Debajit Thakur

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Background: Actinomycetes are the richest source of novel bioactive secondary metabolites such as antibiotics, enzymes and other therapeutically useful metabolites with diverse biological activities. The present study aims at the antimicrobial potential and genetic diversity of culturable Actinomycetes isolated from protected forest ecosystems of Assam which includes Kaziranga National Park (26°30˝-26°45˝N and 93°08˝-93°36˝E), Pobitora Wildlife Sanctuary (26º12˝-26º16˝N and 91º58˝-92º05˝E) and Gibbon Wildlife Sanctuary (26˚40˝-26˚45˝N and 94˚20˝-94˚25˝E) which are located in the North-eastern part of India. Northeast India is a part of the Indo-Burma mega biodiversity hotspot and most of the protected forests of this region are still unexplored for the isolation of effective antibiotic-producing Actinomycetes. Thus, there is tremendous possibility that these virgin forests could be a potential storehouse of novel microorganisms, particularly Actinomycetes, exhibiting diverse biological properties. Methodology: Soil samples were collected from different ecological niches of the protected forest ecosystems of Assam and Actinomycetes were isolated by serial dilution spread plate technique using five selective isolation media. Preliminary screening of Actinomycetes for an antimicrobial activity was done by spot inoculation method and the secondary screening by disc diffusion method against several test pathogens, including multidrug resistant Staphylococcus aureus (MRSA). The strains were further screened for the presence of antibiotic synthetic genes such as type I polyketide synthases (PKS-I), type II polyketide synthases (PKS-II) and non-ribosomal peptide synthetases (NRPS) genes. Genetic diversity of the Actinomycetes producing antimicrobial metabolites was analyzed through 16S rDNA-RFLP using Hinf1 restriction endonuclease. Results: Based on the phenotypic characterization, a total of 172 morphologically distinct Actinomycetes were isolated and screened for antimicrobial activity by spot inoculation method on agar medium. Among the strains tested, 102 (59.3%) strains showed activity against Gram-positive bacteria, 98 (56.97%) against Gram-negative bacteria, 92 (53.48%) against Candida albicans MTCC 227 and 130 (75.58%) strains showed activity against at least one of the test pathogens. Twelve Actinomycetes exhibited broad spectrum antimicrobial activity in the secondary screening. The taxonomic identification of these twelve strains by 16S rDNA sequencing revealed that Streptomyces was found to be the predominant genus. The PKS-I, PKS-II and NRPS genes detection indicated diverse bioactive products of these twelve Actinomycetes. Genetic diversity by 16S rDNA-RFLP indicated that Streptomyces was the dominant genus amongst the antimicrobial metabolite producing Actinomycetes. Conclusion: These findings imply that Actinomycetes from the protected forest ecosystems of Assam, India, are a potential source of bioactive secondary metabolites. These areas are as yet poorly studied and represent diverse and largely unscreened ecosystem for the isolation of potent Actinomycetes producing antimicrobial secondary metabolites. Detailed characterization of the bioactive Actinomycetes as well as purification and structure elucidation of the bioactive compounds from the potent Actinomycetes is the subject of ongoing investigation. Thus, to exploit Actinomycetes from such unexplored forest ecosystems is a way to develop bioactive products.

Keywords: Actinomycetes, antimicrobial activity, forest ecosystems, RFLP

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Authors: Emmanuel Jaluchimike Iloputaife, Kelechi Nkechinyere Mbah-Omeje

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This study was designed to determine the antimicrobial activities and minimum inhibitory concentration of three different batches (Fresh, Oven dried and Sundried) of Ass Hay extracted with water, ethanol and methanolagainst selected human pathogenic microorganisms (Escherichia coli, Klebsiella Pneumonia, Staphylococcus aureus, Aspergillus niger and Candidaalbicans). All extracts were reconstituted with peptone water and tested for antimicrobial activity. The antimicrobial activity, the Minimum Inhibitory Concentration and Minimum Bactericidal/Fungicidal concentrations were determined by agar well diffusion methodagainst test organismsin which aseptic conditions were observed. The antimicrobial activities of the different batches of Ass Hay on the test organisms varied considerably. The highest inhibition zone diameter at 200 mg/ml for the different batches of Ass Hay was recorded by sundried methanol extract against Escherichia coli at 36.4 ± 0.2 mm while fresh methanol extract inhibited Klebsiela pneumonia with the least inhibition zone diameter at 20.1 ± 0.1mm. At 100 mg/ml the highest inhibition zone diameter was recorded by oven dried water extract against Escherichia coli at 30.3 ± 0.3 mm while sun dried water extract inhibited Staphylococcus aureus with the least inhibition zone diameter at 15.1 ± 0.1 mm. At 50mg/ml, the highest inhibition zone diameter was recorded by fresh water extract against Escherichia coli at 25.9 ± 0.1 mm while oven dried water extract inhibited Klebsiela pneumonia with least inhibition zone diameter at 12.1 ± 0.2 mm. At 25mg/ml, the highest inhibition zone diameter was recorded by fresh water extract against Escherichia coli at 18.3 ± 0.2 mm while sun dried ethanol extract inhibited Escherichia coli with least inhibition zone diameter at 10.1 ± 0.1 mm. The MIC and MBC result of ethanol extract of fresh Ass Hay showed a uniform value of 6.25 mg/ml and 12.5 mg/ml respectively for all test bacterial isolates. The Minimum Inhibitory concentration and Minimum bactericidal concentration results of Oven dried ethanol Ass Hay extract showed a uniform value of 3.125 mg/ml and 6.25 mg/ml respectively for all test bacterial isolates and Minimum fungicidal concentration value of 12.5 mg/ml for Aspergillus niger. Statistical analysis showed there is significant difference in mean zone inhibition diameter of the products at p < 0.05, p = 0.019. This study has shown there is antimicrobial potential in Ass Hay and at such there is need to further exploit Donkey Ass Hay in order to maximize the potential.

Keywords: microorganisms, Ass Hay, antimicrobial activity, extracts

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Authors: Morteza Elsa, Amirhossein Moghanian

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The major aim of this study was to evaluate the effect of CaO content on in vitro hydroxyapatite formation, MC3T3 cells cytotoxicity and proliferation as well as antibacterial efficiency of sol-gel derived SiO₂–CaO–P₂O₅ ternary system. For this purpose, first two grades of bioactive glass (BG); BG-58s (mol%: 60%SiO₂–36%CaO–4%P₂O₅) and BG-68s (mol%: 70%SiO₂–26%CaO–4%P₂O₅)) were synthesized by sol-gel method. Second, the effect of CaO content in their composition on in vitro bioactivity was investigated by soaking the BG-58s and BG-68s powders in simulated body fluid (SBF) for time periods up to 14 days and followed by characterization inductively coupled plasma atomic emission spectrometry (ICP-AES), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM) techniques. Additionally, live/dead staining, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity assays were conducted respectively, as qualitatively and quantitatively assess for cell viability, proliferation and differentiations of MC3T3 cells in presence of 58s and 68s BGs. Results showed that BG-58s with higher CaO content showed higher in vitro bioactivity with respect to BG-68s. Moreover, the dissolution rate was inversely proportional to oxygen density of the BG. Live/dead assay revealed that both 58s and 68s increased the mean number live cells which were in good accordance with MTT assay. Furthermore, BG-58s showed more potential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) bacteria. Taken together, BG-58s with enhanced MC3T3 cells proliferation and ALP activity, acceptable bioactivity and significant high antibacterial effect against MRSA bacteria is suggested as a suitable candidate in order to further functionalizing for delivery of therapeutic ions and growth factors in bone tissue engineering.

Keywords: antibacterial, bioactive glass, hydroxyapatite, proliferation, sol-gel processes

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Authors: Nilgün Öztürk, Hakan Sabahtin Ali, Hülya Tuba Kıyan

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The genus Chenopodium belongs to Amaranthaceae, is represented by approximately 250 species in the world and 15 species and three subspecies in Turkey. Chenopodium species are traditionally used to treat chest and abdominal pain, shortness of breath, cough and neurological disorders. Chenopodium quinoa Willd. (Quinoa) is native to Andes region of South America (especially Peru and Bolivia) and cultivated in many countries include also Turkey in the world nowadays. The seeds of quinoa are rich in protein, and the phytochemical composition consists of antioxidant substances such as polyphenolic compounds, flavonoids, vitamins, and minerals; anticancer and neuroprotective compounds such as tocotrienols; anti-inflammatory compounds such as carotenoids and anthocyanins and also saponins and starch. Food products of quinoa such as quinoa cereal bar, pasta and cornflakes are used in the diet made during many disorders like obesity, cardiovascular disorder, hypertension and Celiac disease. Also quinoa seems to have antimicrobial, anti-inflammatory and cholesterol-lowering properties because of its bioactive compounds. In this present study, the aqueous ethanolic extracts of the seeds of three different coloured genotypes of quinoa were investigated for their antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion-chelating effect, ferric-reducing antioxidant power, ABTS radical cation decolorization assays and total phenolic contents using Folin-Ciocalteu assay. Among the three genotypes of quinoa; the aqueous ethanolic extract of the red genotype had the highest total phenolic content (83.54 ± 2.12 mg gallic acid/100 g extract) whereas the extract of the white genotype had the lowest total phenolic content (70.66 ± 0.25 mg gallic acid/100 g). According to the antioxidant activity results; the extracts showed moderate reducing power effect whereas weak ABTS radical cation decolorization and ferrous ion-chelating effect and also too weak DPPH radical scavenging activity when compared to the positive standards.

Keywords: amaranthaceae, antioxidant activity, Chenopodium quinoa willd., total phenolic content

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Authors: Iman Farasat, Howard M. Salis

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The CRISPR/Cas9 system has revolutionized genome engineering by enabling site-directed and high-throughput genome editing, genome insertion, and gene knockdowns in several species, including bacteria, yeast, flies, worms, and human cell lines. This technology has the potential to enable human gene therapy to treat genetic diseases and cancer at the molecular level; however, the current CRISPR/Cas9 system suffers from seemingly sporadic off-target genome mutagenesis that prevents its use in gene therapy. A comprehensive mechanistic model that explains how the CRISPR/Cas9 functions would enable the rational design of the guide-RNAs responsible for target site selection while minimizing unexpected genome mutagenesis. Here, we present the first quantitative model of the CRISPR/Cas9 genome mutagenesis system that predicts how guide-RNA sequences (crRNAs) control target site selection and cleavage activity. We used statistical thermodynamics and law of mass action to develop a five-step biophysical model of cas9 cleavage, and examined it in vivo and in vitro. To predict a crRNA's binding specificities and cleavage rates, we then compiled a nearest neighbor (NN) energy model that accounts for all possible base pairings and mismatches between the crRNA and the possible genomic DNA sites. These calculations correctly predicted crRNA specificity across 5518 sites. Our analysis reveals that cas9 activity and specificity are anti-correlated, and, the trade-off between them is the determining factor in performing an RNA-mediated cleavage with minimal off-targets. To find an optimal solution, we first created a scheme of safe-design criteria for Cas9 target selection by systematic analysis of available high throughput measurements. We then used our biophysical model to determine the optimal Cas9 expression levels and timing that maximizes on-target cleavage and minimizes off-target activity. We successfully applied this approach in bacterial and mammalian cell lines to reduce off-target activity to near background mutagenesis level while maintaining high on-target cleavage rate.

Keywords: biophysical model, CRISPR, Cas9, genome editing

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Authors: Luisa Carrer, Lorenzo De Masi

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In this paper, we exploit the massive amount of information from Facebook to build a measure of gender attitudes in Italy at a previously impossible resolution—the municipal level. We construct our index via a machine learning method to replicate a benchmark region-level measure. Interestingly, we find that most of the variation in our Gender Norms Index (GNI) is across towns within narrowly defined geographical areas rather than across regions or provinces. In a second step, we show how this local variation in norms can be leveraged for identification purposes. In particular, we use our index to investigate whether these differences in norms carry over to the policy activity of politicians elected in the Italian Parliament. We document that females are more likely to sit in parliamentary committees focused on gender-sensitive matters, labor, and social issues, but not if they come from a relatively conservative town. These effects are robust to conditioning the legislative term and electoral district, suggesting the importance of social norms in shaping legislators’ policy activity.

Keywords: gender equality, gender norms index, Facebook, machine learning, politics

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Authors: Jovana M. Ćirković, Aleksandar M. Radojković, Sanja Z. Perać, Jelena N. Jovanović, Zorica M. Branković, Slobodan D. Milanović, Ivan Lj. Milenković, Jovan N. Dobrosavljević, Nemanja V. Simović, Vanja M. Tadić, Ana R. Žugić, Goran O. Branković

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The protection of forests is a national interest and of strategic importance in every country. The spongy moth (Lymantria dispar) is a damaging invasive pest that can weaken and destroy trees by defoliating them. Chemical pesticides commonly used to protect forests against spongy moths not only have a negative impact on terrestrial and aquatic organisms/ecosystems but also often fail to provide significant protection. Therefore, many eco-friendly alternatives have been considered. Within this research, a new biopesticide was developed based on the method of nanoencapsulation of plant extracts in a biopolymer matrix, which provides a slow release of the active components during a substantial time period. The antifeedant activity of plant extracts of common (Fraxinus excelsior L.), manna (F. ornus L.) ash tree, and the tree of heaven Ailanthus altissima (Mill.) was tested on the spongy moth (Lymantria dispar L, 1758) larvae. To test the antifeedant activity of these compounds, the choice and non-choice tests in laboratory conditions for different plant extract concentrations (0.01, 0.1, 0.5, and 1 % v/v) were carried out. In both cases, the best results showed formulations based on the tree of heaven and common ash for the concentration of 1%, with deterioration indices of 163 and 132, respectively. The main benefit of these formulations is their versatility, effectiveness, prolonged effect, and because they are completely environmentally acceptable. Therefore, they can be considered for suppression of the spongy moth in forest ecosystems.

Keywords: Ailanthus altissima (Mill.), Fraxinus excelsior L., encapsulation, Lymantria dispar

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Authors: Ayesha Selwyn, Annapoorni Chandrashekar, Kumar Ashwarya, Nishant Baghel

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Project-based learning (PBL) activities are typically implemented in technology-enabled schools by highly trained teachers. In rural India, students have limited access to technology and quality education. Implementing typical PBL activities is challenging. This study details how Pratham Education Foundation’s Hybrid Learning model was used to implement two PBL activities related to music in 900 remote Indian villages with 46,000 students aged 10-14. The activities were completed by 69% of groups that submitted a total of 15,000 videos (completed projects). Pratham’s H-Learning model reaches 100,000 students aged 3-14 in 900 Indian villages. The community-driven model engages students in 20,000 self-organized groups outside of school. The students are guided by 6,000 youth volunteers and 100 facilitators. The students partake in learning activities across subjects with the support of community stakeholders and offline digital content on shared Android tablets. A training and implementation toolkit for PBL activities is designed by subject experts. This toolkit is essential in ensuring efficient implementation of activities as facilitators aren’t highly skilled and have limited access to training resources. The toolkit details the activity at three levels of student engagement - enrollment, participation, and completion. The subject experts train project leaders and facilitators who train youth volunteers. Volunteers need to be trained on how to execute the activity and guide students. The training is focused on building the volunteers’ capacity to enable students to solve problems, rather than developing the volunteers’ subject-related knowledge. This structure ensures that continuous intervention of subject matter experts isn’t required, and the onus of judging creativity skills is put on community members. 46,000 students in the H-Learning program were engaged in two PBL activities related to Music from April-June 2019. For one activity, students had to conduct a “musical survey” in their village by designing a survey and shooting and editing a video. This activity aimed to develop students’ information retrieval, data gathering, teamwork, communication, project management, and creativity skills. It also aimed to identify talent and document local folk music. The second activity, “Pratham Idol”, was a singing competition. Students participated in performing, producing, and editing videos. This activity aimed to develop students’ teamwork and creative skills and give students a creative outlet. Students showcased their completed projects at village fairs wherein a panel of community members evaluated the videos. The shortlisted videos from all villages were further evaluated by experts who identified students and adults to participate in advanced music workshops. The H-Learning framework enables students in low resource settings to engage in PBL and develop relevant skills by leveraging community support and using video creation as a tool. In rural India, students do not have access to high-quality education or infrastructure. Therefore designing activities that can be implemented by community members after limited training is essential. The subject experts have minimal intervention once the activity is initiated, which significantly reduces the cost of implementation and allows the activity to be implemented at a massive scale.

Keywords: community supported learning, project-based learning, self-organized learning, education technology

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Authors: Bouaziz H., Soudania N., Essafia M., Ben Amara I., Hakim A., Jamoussi K., Zeghal Km, Zeghal N.

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In this investigation, we have evaluated the effects of arsenic trioxide on hepatic function in pregnant and lactating Swiss albino mice and their suckling pups. Experiments were carried out on female mice given 175 ppm As2O3 in their drinking water from the 14th day of pregnancy until day 14 after delivery. Our results showed a significant decrease in plasma levels of total protein and albumin, cholesterol and triglyceride in As2O3 treated mice and their pups. The hyperbilirubinemia and the increased plasma total alkaline phosphatase activity suggested the presence of cholestasis. Transaminase activities as well as lactate deshydrogenase activity in plasma, known as biomarkers of hepatocellular injury, were elevated indicating hepatic cells’damage after treatment with As2O3. Exposure to arsenic led to an increase of liver thiobarbituric acid reactive substances level along with a concomitant decrease in the activities of superoxide dismutase, catalase and glutathione peroxidase and in glutathione.

Keywords: antioxidant status, arsenic trioxide, hepatotoxicity, mice, oxidative stress

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Authors: Deniz Karagöz

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This study is attempt to comprehend the relationship between the spatial characteristics of cultural structure, activities and the tourism demand in Turkey. The analysis divided into four parts. The first part consisted of a cultural structure and cultural activity (CSCA) index provided by principal component analysis. The analysis determined four distinct dimensions, namely, cultural activity/structure, accessing culture, consumption, and cultural management. The exploratory spatial data analysis employed to determine the spatial models of cultural structure and cultural activities in 81 provinces in Turkey. Global Moran I indices is used to ascertain the cultural activities and the structural clusters. Finally, the relationship between the cultural activities/cultural structure and tourism demand was analyzed. The raw/original data of the study official databases. The data on the cultural structure and activities gathered from the Turkish Statistical Institute and the data related to the tourism demand was provided by the Republic of Turkey Ministry of Culture and Tourism.

Keywords: cultural activities, cultural structure, spatial characteristics, tourism demand, Turkey

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Authors: Vu Thu Trang, Lam Xuan Thanh, Samira Sarter, Tomoko Shimamura, Hiroaki Takeuchi　　

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Antimicrobial activities of aminoreductone (AR), a product formed in the initial stage of Maillard reaction, were screened against pathogenic bacteria. A significant growth inhibition of AR against all 7 isolates (Staphylococcus aureus ATCC® 25923™, Salmonella Typhimurium ATCC® 14028™, Bacillus cereus ATCC® 13061™, Bacillus subtilis ATCC® 11774™, Escherichia coli ATCC® 25922™, Enterococcus faecalis ATCC® 29212™, Listeria innocua ATCC® 33090™) were observed by the standard disc diffusion methods. The inhibition zone for each isolate by AR (2.5 mg) ranged from 15±0 mm to 28.3±0.4 mm in diameter. The minimum inhibitory concentration (MIC) of AR ranging from 20 mM to 26 mM was proven in the seven isolates tested. AR also showed the similar effect of growth inhibition in comparison with antibiotics frequently used for the treatment of infections bacteria, such as amikacin, ciprofloxacin, meropennem, and levofloxacin. The results indicated that foods containing AR are valuable sources of bioactive compounds towards pathogenic bacteria.

Keywords: pathogenic bacteria, aminoreductone, Maillard reaction, antimicrobial activity

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Authors: Mai. M. Toughan, Ahmed A. A. Sallam, Ashraf O. Abd El-Latif

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Termites are eusocial insects that are found on all continents except Antarctica. Termites have serious destructive impact, damaging local huts and crops of poor subsistence. The annual cost of termite damage and its control is determined in the billions globally. In Egypt, most of these damages are due to the subterranean termite species especially the sand termite, P. hypostoma. Pyrethroids became the primary weapon for subterranean termite control, after the use of chlorpyrifos as a soil termiticide was banned. Despite the important role of pyrethroids in termite control, its extensive use in pest control led to the eventual rise of insecticide resistance which may make many of the pyrethroids ineffective. The ability to diagnose the precise mechanism of pyrethroid resistance in any insect species would be the key component of its management at specified location for a specific population. In the present study, detailed toxicological and biochemical studies was conducted on the mechanism of pyrethroid resistance in P. hypostoma. The susceptibility of field populations of P. hypostoma against deltamethrin, α-cypermethrin and ƛ-cyhalothrin was evaluated. The obtained results revealed that the workers of P. hypostoma have developed high resistance level against the tested pyrethroids. Studies carried out through estimation of detoxification enzyme activity indicated that enhanced esterase and cytochrome P450 activities were probably important mechanisms for pyrethroid resistance in field populations. Elevated esterase activity and also additional esterase isozyme were observed in the pyrethroid-resistant populations compared to the susceptible populations. Strong positive correlation between cytochrome P450 activity and pyrethroid resistance was also reported. |Deltamethrin could be recommended as a resistance-breaking pyrethroid that is active against resistant populations of P. hypostoma.

Keywords: Psammotermes hypostoma, pyrethroid resistance, esterase, cytochrome P450

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Authors: Ljerka Kratofil Krehula, Vanja Gilja, Andrea Husak, Sniježana Šuka, Zlata Hrnjak-Murgić

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Zinc oxide (ZnO) can be used as photocatalysts for water purification. However, one particular interest is given on the integration of inorganic ZnO nanoclusters with conducting polymers because the resulting nanocomposites may possess unique properties and enhanced photocatalytic activity in comparison to pure ZnO, using UV and also visible light. It is needed to explore the appropriate structure of polypyrrole that can induce activation of ZnO photocatalyst since the synthesis of organic/inorganic hybrid materials can result in a synergistic and complementary feature, increasing ZnO photocatalytic efficiency. In this paper several different composites of polypyrrole/zinc oxide (ZnO) were studied. Composite samples were characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), cyclic voltammetry (CV) and scanning electron microscopy (SEM). The photocatalytic efficiency of prepared samples was studied as a decomposition of Reactive Red 45 (RR 45) dye, which was monitored by UV-Vis spectroscopy as a change in absorbance of characteristic wavelength at 542 nm. Results show good photocatalytic efficiency of all nanocomposite samples.

Keywords: photocatalysis, polypyrrole, wastewater, zinc oxide

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Authors: S. Mamoucha, N.Tsafantakis, T. Ioannidis, S. Chatzipanagiotou, C. Nikolaou, L. Skaltsounis, N. Fokialakis, N. Christodoulakis

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Introduction: Pistacia lentiscus L. (well known as Mastic tree) is an evergreen sclerophyllous shrub that extensively thrives in the eastern Mediterranean area yet only the trees cultivated in the southern region of the Greek island Chios produces mastic resin. Different parts of P. lentiscus L. var. chia have been used in folk medicine for various purposes, such as tonic, aphrodisiac, antiseptic, antihypertensive and management of dental, gastrointestinal, liver, urinary, and respiratory tract disorders. Several studies have focused on the antibacterial activity of its resin (gum) and its essential oil. However, there is no study combining anatomy of the plant organs, phytochemical profile, and antibacterial screening of the plant. In our attempt to discover novel bioactive metabolites from the mastic tree, we screened its antibacterial activity not only against ATCC strains but also against clinical, resistant strains. Materials-methods: Leaves were investigated using Transmission (ΤΕΜ) and Scanning Εlectron Microscopy (SEM). Histochemical tests were performed on fresh and fixed tissue. Extracts prepared from dried, powdered leaves using 3 different solvents (DCM, MeOH and H2O) the waste water obtained after a hydrodistillation process for essential oil production were screened for their phytochemical content and antibacterial activity. Μetabolite profiling of polar and non-polar extracts was recorded by GC-MS and LC-HRMS techniques and analyzed using in-house and commercial libraries. The antibacterial screening was performed against Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 and against clinical, resistant strains Methicillin-resistant S. aureus (MRSA), Carbapenem-Resistant Metallo-β-Lactamase (carbapenemase) P. aeruginosa (VIM), Klebsiella pneumoniae carbapenemases (KPCs) and Acinetobacter baumanii resistant strains. The antibacterial activity was tested by the Kirby Bauer and the Agar Well Diffusion method. The zone of inhibition (ZI) of each extract was measured and compared with those of common antibiotics. Results: Leaf is compact with inosclereids and numerous idioblasts containing a globular, spiny crystal. The major nerves of the leaf contain a resin duct. Mesophyll cells showed accumulation of osmiophillic metabolites. Histochemical treatments defined secondary metabolites in subcellular localization. The phytochemical investigation revealed the presence of a large number of secondary metabolites, belonging to different chemical groups, such as terpenoids, phenolic compounds (mainly myricetin, kaempferol and quercetin glycosides), phenolic, and fatty acids. Among the extracts, the hydrostillation wastewater achieved the best results against most of the bacteria tested. MRSA, VIM and A. baumanii were inhibited. Conclusion: Extracts from plants have recently been of great interest with respect to their antimicrobial activity. Their use emerged from a growing tendency to replace synthetic antimicrobial agents with natural ones. Leaves of P. lentiscus L. var. chia showed a high antimicrobial activity even against drug - resistant bacteria. Future prospects concern the better understanding of mode of action of the antibacterial activity, the isolation of the most bioactive constituents and the clarification if the activity is related to a single compound or to the synergistic effect of several ones.

Keywords: antibacterial screening, leaf anatomy, phytochemical profile, Pistacia lentiscus var. chia

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Authors: Sevil Arabaci Tamer, Gonul Gurol, Ibrahim Tekeoglu, Halil Harman, Ihsan Hakki Ciftci

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Neuroserpin (NSP) is a serine protease inhibitor and member of the serpin family. It is expressed in developing and adult nervous systems, and acts as an inhibitor of protease tissue plasminogen activator (tPA) and a regulator of neuronal growth and plasticity. Also NSP displays anti-inflammatory activity. But, its role in rheumatoid arthritis had never been studied before. So, the aim of the present study was to investigate the effect of neuroserpin in patients with RA. A total of 50 frozen (-20 ºC) serum samples 40 of them belonged to patients with RA, and 10 sample belonged to healthy subjects, were enrolled prospectively. We used DAS-28 to evaluate disease activity. The following clinical data gathered from the original patients' charts. Serum neuroserpin levels were measured by enzyme-linked immunosorbent assay. Our preliminary study results demonstrate, for the first time, that NSP levels are significantly different in RA patients relative to healthy subjects (P = 0.014). So, NSP contribute to pathological condition of RA. Thus, we believe that serum NSP levels can be as a marker in patients with RA. However other inflammatory diseases should be further investigated.

Keywords: neuroserpin, rheumatoid arthritis, tPA, tPA inhibitor

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Authors: Alireza Arab, Morteza Elsa, Amirhossein Moghanian

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In this study, experiments were carried out to achieve a promising multifunctional and modified silicate based bioactive glass (BG). The main aim of the study was investigating the effect of lithium (Li) and magnesium (Mg) substitution, on in vitro bioactivity of substituted-58S BG. Moreover, it is noteworthy to state that modified BGs were synthesized in 60SiO₂–(36-x)CaO–4P₂O₅–(x)Li₂O and 60SiO₂–(36-x)CaO–4P₂O₅–(x)MgO (where x = 0, 5, 10 mol.%) quaternary systems, by sol-gel method. Their performance was investigated through different aspects such as biocompatibility, antibacterial activity as well as their effect on alkaline phosphatase (ALP) activity, and proliferation of MC3T3 cells. The antibacterial efficiency was evaluated against methicillin-resistant Staphylococcus aureus bacteria. To do so, CaO was substituted with Li₂O and MgO up to 10 mol % in 58S-BGs and then samples were immersed in simulated body fluid up to 14 days and then, characterized by X-ray diffraction, Fourier transform infrared spectroscopy, inductively coupled plasma atomic emission spectrometry, and scanning electron microscopy. Results indicated that this modification led to a retarding effect on in vitro hydroxyapatite (HA) formation due to the lower supersaturation degree for nucleation of HA compared with 58s-BG. Meanwhile, magnesium revealed further pronounced effect. The 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) and ALP analysis illustrated that substitutions of both Li₂O and MgO, up to 5 mol %, had increasing effect on biocompatibility and stimulating proliferation of the pre-osteoblast MC3T3 cells in comparison to the control specimen. Regarding to bactericidal efficiency, the substitution of either Li or Mg for Ca in the 58s BG composition led to statistically significant difference in antibacterial behaviors of substituted-BGs. Meanwhile, the sample containing 5 mol % CaO/Li₂O substitution (BG-5L) was selected as a multifunctional biomaterial in bone repair/regeneration due to the improved biocompatibility, enhanced ALP activity and antibacterial efficiency among all of the synthesized L-BGs and M-BGs.

Keywords: alkaline, alkaline earth, bioactivity, biomedical applications, sol-gel processes

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Authors: Yeng Min Yi, Rosli Md Illias, Salehhuddin Hamdan

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Industrial biocatalysis is mainly based on the use of cell free or intracellular enzyme systems. However, the expensive cost and relatively lower operational stability of free enzymes limit practical use in industries. Cell surface display system can be used as a cost-efficient alternative to overcome the laborious purification and substrate transport limitation. In this research, TibA autotransporter from E. coli was used to display Aspergillus fumigatus xylanase (xyn). The amplified xyn was fused in between N-terminal signal peptide and C-terminal β-barrel of TibA. The cloned was transformed and expressed in E. coli BL21 (DE3). Outer membrane localization of TibA-xyn fusion protein was confirmed by SDS PAGE and western blot with expected size of 62.5 kDa. Functional display of xyn was examined by activity assay. Cell surface displayed xyn exhibited the highest activity at 37 °c, 0.3 mM IPTG. As a summary, TibA displaying system has the potential for further industrial applications. Moreover, this is the first report of the display of xylanase using TibA on the surface of E. coli.

Keywords: biocatalysis, cell surface display, Escherichia coli, TibA autotransporter

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Authors: L. K. Nivedha, V. Maruthapandian, R. Kothandaraman

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Rechargeable zinc-air batteries (ZAB) are gaining significant research attention owing to their high energy density and copious zinc resources worldwide. However, the unsolved obstacles such as dendrites, passivation, depth of discharge and the lack of an efficient cathode catalyst restrict their practical application1. By and large, non-noble transition metal-based catalysts are well-reputed materials for catalysing oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) with greater stability in alkaline medium2. Herein, we report the synthesis and application of Mn₃O4-NiFeLDH/Carbon composite as a cathode catalyst for rechargeable ZAB. The synergetic effects of the mixed transition metals (Mn/Ni/Fe) have aided in catalysing ORR and OER in alkaline electrolyte with a shallow potential gap of 0.7 V. The composite, by its distinctive physicochemical characteristics, shows an excellent OER activity with a current density of 1.5 mA cm⁻² at a potential of 1.6 V and a superior ORR activity with an onset potential of 0.8 V when compared with their counterparts. Nevertheless, the catalyst prefers a two-electron pathway for the electrochemical reduction of oxygen which results in a limiting current density of 2.5 mA cm⁻². The bifunctional activity of the Mn₃O₄-NiFeLDH/Carbon composite was utilized in developing rechargeable ZAB. The fully fabricated ZAB delivers an open circuit voltage of 1.4 V, a peak power density of 70 mW cm⁻², and a specific capacity of 800 mAh g⁻¹ at a current density of 20 mA cm⁻² with an average discharge voltage of 1 V and the cell is operable upto 50 mA cm-2. Rechargeable ZAB demonstrated over 110 h at 10 mA cm⁻². Further, the cause for the diminished charge-discharge performance experienced beyond the 100th cycle was investigated, and carbon corrosion was testified using Infrared spectroscopy.

Keywords: rechargeable zinc-air battery, oxygen evolution reaction, bifunctional catalyst, alkaline medium

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Authors: Khaled Alshammari

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A facile approach to synthesizing highly active and stable Au/NiO catalysts for the hydrogenation of nitro-aromatics is reported. Preformed gold nanoparticles have been immobilized onto NiO using a colloidal method. In this article, the reduction of 4-nitrophenol with NaBH4 has been used as a model reaction to investigate the catalytic activity of synthesized Au/NiO catalysts. In addition, we report a systematic study of the reduction kinetics and the influence of specific reaction parameters such as (i) temperature, (ii) stirring rate, (iii) sodium borohydride concentration and (iv) substrate/metal molar ratio. The reaction has been performed at a substrate/metal molar ratio of 7.4, a ratio significantly higher than previously reported. The reusability of the catalyst has been examined, with little to no decrease in activity observed over 5 catalytic cycles. Systematic variation of Au loading reveals the successful synthesis of low-cost and efficient Au/NiO catalysts at very low Au content and using high substrate/metal molar ratios.

Keywords: nonochemistry, catalyst, nanoparticles supported, characterization of materials, colloidal nanoparticles

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Authors: H. Hanine, H. H'ssaini, M. Ibno Alaoui, A. Nablousi, H. Zahir, S. Ennahli, H. Latrache, H. Zine Abidine

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Biochemical and pomological variability among 14 cultivars of Prunus dulcis planted in a germoplasm collection site in Morocco were evaluated. Almond samples from six local and eight foreign cultivars (France, Italy, Spain, and USA) were characterized. Biochemical and pomological data revealed significant genetic variability among the 14 cultivars; local cultivars exhibited higher total polyphenol content. Oil content ranged from 35 to 57% among cultivars; both Texas and Toundout genotypes recorded the highest oil content. Total protein concentration from select cultivars ranged from 50 mg/g in Ferraduel to 105 mg/g in Rizlane1 cultivars. Antioxidant activity of almond samples was examined by a DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging assay; the antioxidant activity varied significantly within the cultivars, with IC50 (the half-maximal inhibitory concentration) values ranging from 2.25 to 20 mg/ml. Autochthonous cultivars originated from the Oujda region exhibited higher tegument total polyphenol and amino acid content compared to others. The genotype Rizlane2 recorded the highest flavonoid content. Pomological traits revealed a large variability within the almond germplasms. The hierarchical clustering analysis of all the data regarding pomological traits distinguished two groups with some particular genotypes as distinct cultivars, and groups of cultivars as polyclone varieties. These results strongly exhibit a potential use of Moroccan-originated almonds as potential clones for future selection due to their nutritional values and pomological traits compared to well-established cultivars.

Keywords: antioxidant activity, DDPH, Moroccan almonds, Prunus dulcis

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Authors: M. Masar, P. Janota, J. Sedlak, M. Machovsky, I. Kuritka

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Zinc oxide (ZnO) nano crystals assembled porous architecture was prepared by thermal decomposition of zinc oxalate precursor at various temperatures ranging from 400-900°C. The effect of calcining temperature on structure and morphology was examined by scanning electron microscopy (SEM), X-ray diffractometry, thermogravimetry, and BET adsorption analysis. The porous nano crystalline ZnO morphology was developed due to the release of volatile precursor products, while the overall shape of ZnO micro crystals was retained as a legacy of the precursor. The average crystallite size increased with increasing temperature of calcination from approximately 21 nm to 79 nm, while the specific surface area decreased from 30 to 1.7 m2g-1. The photo catalytic performance of prepared ZnO powders was evaluated by degradation of methyl violet 2B, a model compound. The significantly highest photo catalytic activity was achieved with powder calcined at 500°C. This may be attributed to the sufficiently well-developed crystalline arrangement, while the specific surface area is still high enough.

Keywords: ZnO, porous structure, photodegradation, methyl violet

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Authors: W. Mazari, K. Boucherit, Z. Boucherit-Otmani, M. N. Rahmoun, M. Benabdallah

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The hospital or any other establishment of care can be considered as an ecosystem where the patient comes into contact with a frightening microbial universe and a risk to contract infection that is referred to as nosocomial or health care-associated. In these last years, the incidence of these infections has risen sharply. Several microorganisms are the cause of these nosocomial infections and the emergence of resistance of the microbial strains against antibiotics creates a danger to public health. The search for new antimicrobial agents to overcome this problem has produced interesting compounds through chemical synthesis, which plays a very important role in the research and discovery of new drugs. It is in this framework that our study was conducted at our laboratory and it involves evaluating the antibacterial activity of thirteen 2-pyridone derivatives synthesized by two methods, the diffusion disc method and the dilution method against eight Gram negative bacterial strains. The results seem interesting especially for two products that have shown the best activities against Escherichia coli ATCC 25922 and Enterobacter cloacae ATCC 13047 with CMI of 512µg/ml.

Keywords: heterocyclic derivatives, chemical synthesis, antimicrobial activity, biotechnology

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Authors: Apurva Singh, Naseem Ahmed

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Dual functional manganese complex compelling with 2-hydrazineylpyridine moiety as ligand relevant to the activity of galactose oxidase enzyme is synthesized. Single crystal XRD and EPR studies showed both Manganese-centers are in +2 oxidation states with μ-Chlorido-bridged between them. The catalysts have maintained stability even at 300°C temperature, as demonstrated through TGA analysis. PXRD and XPS studies were used to further characterize the catalyst structure. The catalyst (Mn-C₁) is highly efficient in the selective dehydrogenative oxidation of alcohols in the presence of oxidant 30 % aq. H₂O₂ through a radical pathway. Likewise, the catalysts (Mn-C₁ and Mn-C₄) were efficiently used in the synthesis of β-hydroxy carbonyl compounds (aldol products) and chalcones up to 90% and 95% yield, respectively, at different temperatures via C-H bond activation. Both benzylic and aliphatic substrates are explored, having functional group tolerance.

Keywords: manganese-based 2-hydrazineylpyridine complex, β-hydroxy carbonyl product, free-radical reaction, dehydrogenative oxidation, Lewis acidity

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Authors: Walaa H. Salama, Azza M. Abdel-Aty, Afaf S. Fahmy

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Background: Egyptian Cobra; Naja haje (Elapidae) is one of most common snakes, widely distributed in Egypt and its envenomation causes multi-organ failure leading to rapid death. Thus, Different medicinal plants showed a protective effect against venom toxicity and may complement the conventional antivenom therapy. Aim: The present study was designed to assess both the antioxidant capacity of methanolic extract of rosemary leaves and evaluate the neutralizing ability of the extract against hepatotoxicity induced by Naja haje venom. Methods: The total phenolic and flavonoid contents and the antioxidant capacity of the methanolic rosemary extract were estimated by DPPH and ABTS Scavenging methods. In addition, the rosemary extract were assessed for anti-venom properties under in vitro and in vivo standard assays. Results: The rosemary extract had high total phenolic and flavonoid content as 12 ± 2 g of gallic acid equivalent per 100 gram of dry weight (g GAE/100g dw) and 5.5 ± 0.8 g of catechin equivalent per 100 grams of dry weight (g CE/100g dw), respectively. In addition, the rosemary extract showed high antioxidant capacity. Furthermore, The rosemary extract were inhibited in vitro the enzymatic activities of phospholipase A₂, L-amino acid oxidase, and hyaluronidase of the venom in a dose-dependent manner. Moreover, indirect hemolytic activity, hepatotoxicity induced by venom were completely neutralized as shown by histological studies. Conclusion: The phenolic compounds of rosemary extract with potential antioxidant activity may be considered as a promising candidate for future therapeutics in snakebite therapy.

Keywords: antioxidant activity, neutralization, phospholipase A₂ enzyme, snake venom

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Authors: Joudia Akil, Stephane Siffert, Laurence Pirault-Roy, Renaud Cousin, Christophe Poupin

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Oxyfuel combustion is a promising method that enables to obtain a CO2 rich stream, with water vapor ( ̴10%), unburned components such as CO and NO, which must be cleaned before the use of CO2. Our objective is then the final treatment of CO and NO by catalysis. Three-way catalysts are well-developed material for simultaneous conversion of NO, CO and hydrocarbons. Pt and/or Rh ensure a quasi-complete removal of NOx, CO and HC and there is also a growing interest in partly replacing Pt with less-expensive Pd. The use of alumina and ceria as support ensures, respectively, the stabilization of such species in active state and discharging or storing oxygen to control the oxidation of CO and HC and the reduction of NOx. In this work, we will compare different metals (Pd, Rh and Pt) supported on Al2O3 and CeO2, for CO2 purification from oxyfuel combustion. The catalyst must reduce NO by CO in an oxidizing environment, in the presence of CO2 rich stream and resistant to water. In this study, Al2O3 and CeO2 were used as support materials of the catalysts. 1wt% M/Support where M = Pd, Rh or Pt catalysts were obtained by wet impregnation on supports with a precursor of palladium [Pd(acac)2], rhodium [Rh(NO3)3] and platinum [Pt(NO2)2(NO3)2]. Materials were characterized by BET surface area, H2 chemisorption, and TEM. Catalytic activity was evaluated in CO2 purification which is carried out in a fixed-bed flow reactor containing 150 mg of catalyst at atmospheric pressure. The flow of the reactant gases is composed of: 20% CO2, 10% O2, 0.5% CO, 0.02% NO and 8.2% H2O (He as eluent gas) with a total flow of 200 mL.min−1, with same GHSV (2.24x104 h-1). The catalytic performances of the samples were investigated with and without water. It shows that the total oxidation of CO occurred over the different materials. This study evidenced an important effect of the nature of the metals, supports and the presence or absence of H2O during the reduction of NO by CO in oxyfuel combustions conditions. Rh based catalysts show that the addition of water has a very positive influence especially on the Rh catalyst on CeO2. Pt based catalysts keep a good activity despite the addition of water on the both supports studied. For the NO reduction, addition of water act as a poison with Pd catalysts. The interesting results of Rh based catalysts with water can be explained by a production of hydrogen through the water gas shift reaction. The produced hydrogen acts as a more effective reductant than CO for NO removal. Furthermore, in TWCs, Rh is the main component responsible for NOx reduction due to its especially high activity for NO dissociation. Moreover, cerium oxide is a promotor for WGSR.

Keywords: carbon dioxide, environmental chemistry, heterogeneous catalysis

Procedia PDF Downloads 182

Authors: Przemyslaw J. Jodlowski, Roman J. Jedrzejczyk, Damian K. Chlebda, Anna Dziedzicka, Lukasz Kuterasinski, Anna Gancarczyk, Maciej Sitarz

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The aim of this study was to obtain highly active catalysts based on non-noble metal oxides supported on zirconia prepared via a sonochemical method. In this study, the influence of the stabilizers addition during the preparation step was checked. The final catalysts were characterized by using such characterization methods as X-ray Diffraction (XRD), nitrogen adsorption, X-ray fluorescence (XRF), scanning electron microscopy (SEM) equipped with energy dispersive X-ray spectrometer (EDS), transmission electron microscopy (TEM) and µRaman. The proposed preparation method allowed to obtain uniformly dispersed metal-oxide nanoparticles at the support’s surface. The catalytic activity of prepared catalyst samples was measured in a methane combustion reaction. The activity of the catalysts prepared by the sonochemical method was considerably higher than their counterparts prepared by the incipient wetness method.

Keywords: methane catalytic combustion, nanoparticles, non-noble metals, sonochemistry

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Authors: Bekan Bogale, Tsegaye Girma Asere, Tilahun Yai, Fekadu Melak

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Aims: To study photocatalytic degradation of methylene blue dye on cuprous oxide/graphene nanocomposite. Background: Cuprous oxide (Cu2O) nanoparticles are among the metal oxides that demonstrated photocatalytic activity. However, the stability of Cu2O nanoparticles due to the fast recombination rate of electron/hole pairs remains a significant challenge in their photocatalytic applications. This, in turn, leads to mismatching of the effective bandgap separation, tending to reduce the photocatalytic activity of the desired organic waste (MB). To overcome these limitations, graphene has been combined with cuprous oxides, resulting in cuprous oxide/graphene nanocomposite as a promising photocatalyst. Objective: In this study, Cu2O/graphene nanocomposite was synthesized and evaluated for its photocatalytic performance of methylene blue (MB) dye degradation. Method: Cu2O/graphene nanocomposites were synthesized from graphite powder and copper nitrate using the facile sol-gel method. Batch experiments have been conducted to assess the applications of the nanocomposites for MB degradation. Parameters such as contact time, catalyst dosage, and pH of the solution were optimized for maximum MB degradation. The prepared nanocomposites were characterized by using UV-Vis, FTIR, XRD, and SEM. The photocatalytic performance of Cu2O/graphene nanocomposites was compared against Cu2O nanoparticles for cationic MB dye degradation. Results: Cu2O/graphene nanocomposite exhibits higher photocatalytic activity for MB degradation (with a degradation efficiency of 94%) than pure Cu2O nanoparticles (67%). This has been accomplished after 180 min of irradiation under visible light. The kinetics of MB degradation by Cu2O/graphene composites can be demonstrated by the second-order kinetic model. The synthesized nanocomposite can be used for more than three cycles of photocatalytic MB degradation. Conclusion: This work indicated new insights into Cu2O/graphene nanocomposite as high-performance in photocatalysis to degrade MB, playing a great role in environmental protection in relation to MB dye.

Keywords: methylene blue, photocatalysis, cuprous oxide, graphene nanocomposite

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Authors: Fatemeh Moradian, Razieh Ghorbani, Poria Biparva

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Growing resistance of microorganisms to potent antibiotics has renewed a great interest towards investigating bactericidal properties of nanoparticles and their Nano composites as an alternative. The use of metal nanoparticles to combat bacterial infections is one of the most wide spread applications of nanotechnology in the field of antibacterial. Nanomaterials have unique properties compared to their bulk counterparts. In this report, we demonstrate the antimicrobial activity of zerovalent Iron(ZVI) and Ag(silver) nanoparticles against Gram-negative bacteria E.coli(DH5α) and Pseudomonas aeruginosa. At first ZVI and Ag nanoparticles were synthesized by chemical reduction method and using scanning electron microscopy (SEM) the nanoparticle size determined. Different concentrations of Ag and ZVI nanoparticles were added to bacteria on nutrient agar medium. Minimum inhibitory concentration (MIC) of Ag and Fe nanoparticles for P. aeruginosa were 5µM and 1µg as well as for E.coli were 6µM. and 10 µg, respectively. Among the two nanoparticles, ZVI showed that the greatest antimicrobial activity against E.coli and Ag nanoparticle on P.aeruginosa. Results suggested that the bactericidal effect of metal nanoparticles has been attributed to their small size as well as high surface to volume ratio and NPs could be used as an effective antibacterial material.

Keywords: bactericidal properties, MIC, nanoparticle, SEM

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Authors: Mohamed A. Morsy, Azza A. El-Sheikh, Abdulla Y. Al-Taher

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Resveratrol (RES) is a well-known polyphenol antioxidant. We have previously shown that testicular protective effect of RES against the anticancer drug methotrexate (MTX)-induced toxicity involves transporter-mediated mechanisms. Here, we investigated the effect of RES on MTX-induced nephrotoxicity. Rats were administered RES (10 mg/kg/day) for 8 days, with or without a single MTX dose (20 mg/kg i.p.) at day 4 of the experiment. MTX induced nephrotoxicity evident by significantly increase in serum blood urea nitrogen and creatinine compared to control, as well as distortion of kidney microscopic structure. MTX also significantly increased renal nitric oxide level, with induction of inducible nitric oxide synthase expression. MTX also significantly up-regulated fas ligand and caspase 3. Administering RES prior to MTX significantly improved kidney function and microscopic picture, as well as significantly decreased nitrosative and apoptotic markers compared to MTX alone. RES, but not MTX, caused significant increase in expression of breast cancer resistance protein (BCRP), an apical efflux renal transporter that participates in urinary elimination of both MTX and RES. Interestingly, concomitant MTX and RES caused further up-regulation of renal Bcrp compared to RES alone. Using Human BCRP ATPase assay, both RES and MTX exhibited dose-dependent increase in ATPase activity, with Km values of 0.52 ± 0.03 and 30.9 ± 4.2 µM, respectively. Furthermore, combined RES and MTX caused ATPase activity which was significantly less than maximum ATPase activity attained by the positive control; sulfasalazine (12.5 µM). In conclusion, RES exerted nephro-protection against MTX-induced toxicity through anti-nitrosative and anti-apoptotic effects, as well as via up-regulation of renal Bcrp.

Keywords: methotrexate, resveratrol, nephrotoxicity, breast cancer resistance protein

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

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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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