Search results for: real-time rt-PCR assay

Authors: Xian-Peng Jiang, Catherine C. Baucom, Toby Jiang, Robert L. Elliott

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HLA-G binds to the inhibitory receptors of uterine NK cells and plays an important role in protection of fetal cells from maternal NK lysis. HLA-G also mediates tumor escape, but the immunosuppressive role is often neglected. These studies have focused on the examination of HLA-G expression in human breast carcinoma and HLA-G immunosuppressive role in NK cytolysis. We examined HLA-G expression in breast cell lines by real time PCR, ELISA and immunofluorescent staining. We treated the breast cancer cell lines with anti-human HLA-G antibody or progesterone. Then, NK cytolysis was measured by using MTT assay. We find that breast carcinoma cell lines increase the expression of HLA-G mRNA and protein, compared to normal cells. Blocking HLA-G of the breast cancer cells by the antibody increases NK cytolysis. Progesterone upregulates HLA-G mRNA and protein of human breast cancer cell lines. The increased HLA-G expression suppresses NK cytolysis. In summary, human breast carcinoma overexpress HLA-G immunosuppressive molecules. Blocking HLA-G protein by antibody improves NK cytolysis. In contrast, upregulation of HLA-G expression by progesterone impairs NK cytolytic function. Thus, HLA-G is a new immunosuppressive checkpoint and potential cancer immunotherapeutic target.

Keywords: HLA-G, Breast carcinoma, NK cells, Immunosuppressive checkpoint

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Authors: Bourenane Bouhafs Naziha

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ARTEA330EC is a fungicide used to inhibit the growth of many types of fungi on and cereals and rice, it is the single largest selling agrochemical that has been widely detected in surface waters in our area (Northeast Algerian). The studies on long-term genotoxic effects of fugicides in different tissues of fish using genotoxic biomarkers are limited. Therefore, in the present study DNA damage by propiconazole in freshwater fish Gambusia affinis by comet assays was investigated. The LC(50)- 96 h of the fungicide was estimated for the fish in a semi-static system. On this basis of LC(50) value sublethal and nonlethal concentrations were determined (25; 50; 75; and 100 ppm). The DNA damage was measured in erythrocytes as the percentage of DNA in comet tails of fishes exposed to above concentrations the fungicide. In general,non significant effects for both the concentrations and time of exposure were observed in treated fish compared with the controls. However It was found that the highest DNA damage was observed at the highest concentration and the longest time of exposure (day 12). The study indicated comet assay to be sensitive and rapid method to detect genotoxicity of propiconasol and other pesticides in fishes.

Keywords: genotoxicity, fungicide, propiconazole, freshwater, Gambusia affinis, alkaline single-cell gel electrophoresis

Procedia PDF Downloads 297

Authors: Faezeh Zaefarian1, Sajedeh Golmohammadzadeh, Mohammad Rezvani

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Weed management decisions for weed species can be derived from knowledge of seed germination biology. Experiments were conducted in laboratory and greenhouse to determine the effects of light, temperature, salt and water stress, seed burial depth on seed germination and seedling emergence of Papaver rhoeas and P.dubium and to assay the response of these species to commonly available POST herbicides. Germination of the Papaver seeds was influenced by the tested temperatures (day/night temperatures of 20 and 25 °C) and light. The concentrations of sodium chloride, ranging from 0 to 80 mM, influence germination of seeds. The osmotic potential required for 50% inhibition of maximum germination of P. rhoeas was -0.27 MPa and for P. dubium species was 0.25 MPa. Seedling emergence was greatest for the seeds placed at 1 cm and emergence declined with increased burial depth in the soil. No seedlings emerged from a burial depth of 6 cm. The herbicide 2,4-D at 400 g ai ha-1 provided excellent control of both species when applied at the four-leaf and six-leaf stages. However, at the six-leaf stage, percent control was reduced. The information gained from this study could contribute to developing components of integrated weed management strategies for Papaver species.

Keywords: germination, papaver species, planting depth, POST herbicides

Procedia PDF Downloads 243

Authors: Zi-Han Li, Zi-Jun Sun, Dong-Yuan Chang, Li Zhu, Min Chen, Ming-Hui Zhao

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Aims: A genome-wide association study (GWAS) reported that patients with the rs55703767 minor allele in collagen type IV α3 chain encoding gene COL4A3 showed protection against diabetic kidney disease (DKD) in type 1 diabetes mellitus (T1DM). However, the role of rs55703767 in type 2 DKD has not been elucidated. The aim of the current study was to investigate the association between COL4A3 variant rs55703767 and DKD risk in Chinese patients with type 2 diabetes mellitus (T2DM). Methods: This nested case-control study was performed on 1311 patients who had T2DM for at least 10 years, including 580 with DKD and 731 without DKD. We detected the genotypes of all patients by TaqMan SNP Genotyping Assay and analyzed the association between COL4A3 variant rs55703767 and DKD risk. Results: Genetic analysis revealed that there was no significant difference between T2DM patients with DKD and those without DKD regarding allele or genotype frequencies of rs55703767, and the effect of this variant was not hyperglycemia specific. Conclusion: Our findings suggested that there was no detectable association between the COL4A3 variant rs55703767 and the susceptibility to DKD in the Chinese T2DM population.

Keywords: collagen type IV α3 chain, gene polymorphism, type 2 diabetes, diabetic kidney disease

Procedia PDF Downloads 108

Authors: Renu Yadav, Sanjeev Kumar

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In northern India, chickpea (Cicer arietinum L.) come across with terminal high-temperature stress during reproductive stage which leads to reduced yield. Hence, stable production of chickpea will depend on the development of new methods like ‘priming’ which allow improved adaptation to the drought and heat stress. In the present experiment, 11-day chickpea seedling was primed with mild drought stress and put on recovery stage by irrigating and finally 30-day seedlings were exposed to heat stress 38°C (4 hours), 35°C (8 hours) and 32°C (12 hours). To study the effect of combinatorial stress, heat and drought stress was applied simultaneously. Analyses of various physiological parameters like membrane damage assay, photosynthetic pigments, antioxidative enzyme, total sugars were estimated at all stages. To study the effect of heat stress on the metabolites of the plants, GC-MS and HPLC were performed, while at transcriptional level Real-Time PCR of predicted heat stress-related genes was done. It was concluded that the heat stress significantly affected the chickpea plant at physiological and molecular level in all the five varieties. Results also show less damaging effect in primed plants by increasing the activity of antioxidative enzymes and increased expression of heat shock proteins and heat shock factors.

Keywords: chickpea, combinatorial stress, heat stress, oxidative stress, priming, RT-PCR

Procedia PDF Downloads 159

Authors: Jeanne Phyre Lagare, Kirstin Rhys Pueblos

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This study was conducted to evaluate the possible anthelmintic activities of the decoction and the nonpolar constituents of the aerial part of Ruellia tuberosa Linn. against Eudrilus eugeniae or African Night Crawler earthworms as test organism which are of anatomic and physiological resemblance to the intestinal roundworm parasites of human beings. The in vitro anthelmintic assay of each extract was done by determining the time of paralysis and death of the test organisms at three concentrations (3, 25, 50 mg/mL). The hexane-soluble extract (RTH) showed better results compared to the decoction (RTD) at all concentrations employed. All the fractions of RTH showed significantly higher anthelmintic activities (111.43, 48.19, and 62.3 minutes, respectively) compared to their mother extract (164.56 minutes) at 3-mg/mL concentration. Moreover, RTH5 showed a comparable activity with the positive control mebendazole at 3-mg/mL concentration. Remarkably, fraction RTH4 exhibited the best anthelmintic activity at 3-mg/mL concentration for it showed the strongest anthelmintic activity than the rest of the test solutions tested. The study demonstrated the promising anthelmintic activity of the nonpolar constituent of the ethanolic extract of R. tuberosa Linn.

Keywords: anthelmintic activity, Eudrillus eugenia, mebendazole, Ruellia tuberosa Linn

Procedia PDF Downloads 198

Authors: Asif Jamal, Asad Ali, Muhammad Ishtiaq Ali

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The agriculture productivity losses due to microbial pathogens have been a serious issue in Pakistan and rest of the world. Present work was designed to isolate soil borne microorganisms having the antagonistic ability against notorious phytopathogens. From the initial collection of 23 bacterial isolates, two potent strains of Bacillus were screened on the basis of their comparative efficacy against devastating fungal pathogens. The strains AK-1 and AK-5 showed excellent inhibitory indexes against the majority of tested fungal strains. It was noted that both strains of Bacillus showed significant biocontrolling activity against Aspergillus flavus, Fusarium moniliforme, Colletotricum falcatum, Botrytis cinerea, Aspergillus niger, Fusarium oxysporum, Phythopthora capsici and Rhizopus oryzae. The strain AK-1 was efficient to suppress Aspergillus species and Rhizopus oryzae while AK-5 expressed significant antagonistic activity against Fusarium, Botrytis, and Colletotricum species. On the basis of in vitro assay, it can be postulated that the Bacillus strains AK-1 and AK-5 can be used as a bio-protective agent against various plant diseases. In addition, their applications as natural pesticides could be very helpful to prevent the adverse effects of chemical pesticides.

Keywords: biological control, Bacillus spp, fungal pathogens, agriculture

Procedia PDF Downloads 270

Authors: Marek Halenár, Eva Tvrdá, Simona Baldovská, Ľubomír Ondruška, Peter Massányi, Adriana Kolesárová

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This study aimed to assess the in vitro effects of different concentrations of the Viscum album extract on the motility, viability, and reactive oxygen species (ROS) production by rabbit spermatozoa during different time periods (0, 2, and 8h). Spermatozoa motility was assessed by using the CASA (Computer aided sperm analysis) system. Cell viability was evaluated by using the metabolic activity MTT assay, and the luminol-based luminometry was applied to quantify the ROS formation. The CASA analysis revealed that low Viscum concentrations were able to prevent a rapid decline of spermatozoa motility, especially in the case of concentrations ranging between 1 and 5 µg/mL (P<0.05 with respect to time 8h). At the same time, concentrations ranging between 1 and 100 µg/mL of the extract led to a significant preservation of the cell viability (P<0.05 in case of 5, 50 and 100 µg/mL; P<0.01 with respect to 1 and 10 µg/mL, time 8h). 1 and 5 µg/mL of the extract exhibited antioxidant characteristics, translated into a significant reduction of the ROS production, particularly notable at time 8h (P<0.01). The results indicate that the Viscum extract is capable of delaying the damage inflicted to the spermatozoon by the in vitro environment.

Keywords: CASA, mistletoe, mitochondrial activity, motility, reactive oxygen species, rabbits, spermatozoa, Viscum album

Procedia PDF Downloads 392

Authors: R. Reshma srilakshmi, S. Shalini, P. Yogeeswari, D. Sriram

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Lysine aminotransferase is a crucial enzyme for dormancy in M. tuberculosis. It is involved in persistence and antibiotic resistance. In present work, we attempted to develop benzthiazole derivatives as lysine aminotransferase inhibitors. In our attempts, we also unexpectedly arrived at an interesting compound 21 (E)-4-(5-(2-(benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)benzoic acid which even though has moderate activity against persistent phase of mycobacterium, it has significant potency against active phase. In the entire series compound 22 (E)-4-(5-(2-(benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)isophthalic acid emerged as potent molecule with LAT IC50 of 2.62 µM. It has a significant log reduction of 2.9 and 2.3 fold against nutrient starved and biofilm forming mycobacteria. It was found to be inactive in MABA assay and M.marinum induced zebra fish model. It is also devoid of cytotoxicity. Compound 22 was also found to possess bactericidal effect which is independent of concentration and time. It was found to be effective in combination with Rifampicin in 3D granuloma model. The results are very encouraging as the hit molecule shows activity against active as well as persistent forms of tuberculosis. The identified hit needs further more pharmacokinetic and dynamic screening for development as new drug candidate.

Keywords: benzothiazole, latent tuberculosis, LAT, nutrient starvation

Procedia PDF Downloads 329

Authors: Mansur Abdulrasheed, Ibrahim I. Hussein, Ahmed M. Mubarak, Ahmed F. Umar

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This study was aimed at evaluating the phytochemical constituents and the antimicrobial activity of the seed oil of Moringa oleifera and garlic against some selected food-borne microorganisms (Staphylococcus aureus, Escherichia coli, Salmonella spp and Pseudomonas aeruginosa) using disc diffusion method. The results of the phytochemical screening revealed differences in the presence of the phytochemicals among the extracts. Saponins were detected in both Moringa oleifera and garlic seed oil, while alkaloid and tannins were observed in seed oil of garlic. Furthermore, the antibacterial assay results show that the seed oil of Moringa oleifera was inactive against all the tested organisms, even at 100 % concentration. In contrast, garlic oil was found to be active against all the tested organisms. The highest inhibition was observed in E. coli (12 mm) at 100 % concentration, while at 20 % concentration, Salmonella Sp and P. aeruginosa showed the least inhibiton (6 mm). The antimicrobial activity of the seed oil of garlic may be attributed to its phytochemicals components which were not detected in the seed oil of Moringa oleifera. The results of this study have shown the potentials of the seed oil of garlic as an antimicrobial agent more especially in foods, by inhibiting the growth of the test organisms, which range from food-borne pathogens to food spoilage organisms.

Keywords: antimicrobial, garlic, Moringa oleifera, food borne pathogens

Procedia PDF Downloads 505

Authors: D. Jablonskytė-Raščė, A. Mankevičienė, S. Supronienė, I. Kerienė, S. Maikštėnienė, S. Bliznikas, R. Česnulevičienė

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Organic farming does not allow the use of conventional mineral fertilizers and crop protection products. As a result, in our experiments we chose to grow different species of cereals and to see how cereal species affects mycotoxin accumulation. From the phytopathological and entomological viewpoint, the glumes of spelt grain perform a positive role since they protect grain from the infection of pathogenic microorganisms. On the background of the above-mentioned infection, there were more Fusarium–affected grains of spelt than of common wheat. It can be assumed that spelt is more susceptible to the Fusarium fungi infection than common wheat. This study describes the occurrence of DON, ZEA and T2/HT2 toxin in a survey of spelt and common wheat and their bran as well as flour. The analysis was conducted using the enzyme-linked immunosorbent assay (ELISA) method. The concentrations of DON, ZEA, and T2/HT2 in Triticum spelta and Triticum aestivum are influenced by species, cereal type and year interaction. The highest concentration of mycotoxin was found in spelt grain with glumes. The obtained results indicate the significantly higher concentrations of Fusarium toxins in glumes than in dehulled grain which implicate the possible protective effect of spelt wheat glumes. The lowest DON, ZEA, and T2/HT2 concentration was determined in spelt grain without glumes.

Keywords: Fusarium mycotoxins, organic farming, spelt

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Authors: A. Panacek, M. Kilianova, R. Prucek, V. Husickova, R. Vecerova, M. Kolar, L. Kvitek, R. Zboril

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In this work we describe the preparation of NanoSilver/methylcellulose hydrogel containing silver nanoparticles (NPs) for topical bactericidal applications. Highly concentrated dispersion of silver NPs as high as of 5g/L of silver with diameter of 10nm was prepared by reduction of AgNO3 via strong reducing agent NaBH4. Silver NPs were stabilized by addition of sodium polyacrylate in order to prevent their aggregation at such high concentration. This way synthesized silver NPs were subsequently incorporated into methylcellulose suspension at elevated temperature resulting in formation of NanoSilver/methylcellulose hydrogel when temperature cooled down to laboratory conditions. In vitro antibacterial activity assay proved high bactericidal and fungicidal efficiency of silver NPs alone in the form of dispersion as well as in the form of hydrogel against broad spectrum of bacteria and yeasts including highly multiresistant strains such as methicillin-resistant Staphylococcus aureus. A very low concentrations of silver as low as 0.84mg/L Ag in as-prepared dispersion gave antibacterial performance. NanoSilver/methylcellulose hydrogel showed antibacterial action at the lowest used silver concentration equal to 25mg/L. Such prepared NanoSilver/methylcellulose hydrogel represent promising topical antimicrobial formulation for treatment of burns and wounds.

Keywords: antimicrobial, burn, hydrogel, silver NPs

Procedia PDF Downloads 449

Authors: Wasi Khan, Suboohi Shervani, Swaleha Naseem, Mohd. Shoeb, J. A. Khan, B. R. Singh, A. H. Naqvi

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We have successfully synthesized ZnO/TiO2 nanocomposite using a two-step solochemical synthesis method. The influence of annealing temperature on microstructural, optical, anticandidal, photocatalytic activities and dielectric properties were investigated. X-ray diffraction (XRD) and scanning electron microscopy (SEM) show the formation of nanocomposite and uniform surface morphology of all samples. The UV-Vis spectra indicate decrease in band gap energy with increase in annealing temperature. The anticandidal activity of ZnO/TiO2 nanocomposite was evaluated against MDR C. albicans 077. The in-vitro killing assay revealed that the ZnO/TiO2 nanocomposite efficiently inhibit the growth of the C. albicans 077. The nanocomposite also exhibited the photocatalytic activity for the degradation of methyl orange as a function of time at 465 nm wavelength. The electrical behaviour of composite has been studied over a wide range of frequencies at room temperature using complex impedance spectroscopy. The dielectric constants, dielectric loss and ac conductivity (σac) were studied as the function of frequency, which have been explained by ‘Maxwell Wagner Model’. The data reveals that the dielectric constant and loss (tanδ) exhibit the normal dielectric behavior and decreases with the increase in frequency.

Keywords: ZnO/TiO2 nanocomposites, SEM, photocatalytic activity, dielectric properties

Procedia PDF Downloads 404

Authors: Mansur Abdulrasheed, Ibrahim I. Hussein, Ahmed M. Mubarak, Ahmed F. Umar

Abstract:

This study was aimed at evaluating the phytochemical constituents and the antimicrobial activity of the seed oil of Moringa oleifera and garlic against some selected food-borne microorganisms (Staphylococcus aureus, Escherichia coli, Salmonella spp and Pseudomonas aeruginosa) using disc diffusion method. The results of the phytochemical screening revealed differences in the presence of the phytochemicals among the extracts. Saponins were detected in both Moringa oleifera and garlic seed oil, while alkaloid and tannins were observed in seed oil of garlic. Furthermore, the antibacterial assay results show that the seed oil of Moringa oleifera was inactive against all the tested organisms, even at 100 % concentration. In contrast, garlic oil was found to be active against all the tested organisms. The highest inhibition was observed in E. coli (12 mm)at 100 % concentration, while at 20 % concentration, Salmonella Sp and P. aeruginosa showed the least inhibit on (6 mm). The antimicrobial activity of the seed oil of garlic may be attributed to its phytochemicals components which were not detected in the seed oil of Moringa oleifera. The results of this study have shown the potentials of the seed oil of garlic as an antimicrobial agent more especially in foods, by inhibiting the growth of the test organisms, which range from food-borne pathogens to food spoilage organisms.

Keywords: antimicrobial, garlic, Moringa oleifera, food borne pathogens

Procedia PDF Downloads 413

Authors: Leah Ghebreberhan, Liya Abraham, John Issac, Atul Kaushik

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Medicinal plants are used for various indications in Eritrea according to traditional systems of medicine. Safety concerns, however, are dubious since some medicinal plants have toxic effects indeed. The medicinal plants under study (Commicarpus pedunculosis, Steganotaenia araliaceae, Boscia angustifolia, Solanum incanum, and Calpurnia aurea) are used in the treatment of various diseases. Thus, safety studies must be performed prior to usage since they are rich in phytoconstituents like saponins. Saponns are natural glycosides with several pharmacologic activities including hemolysis. The study was done to assess the level of saponin and toxic effects (hemolysis) of medicinal plants used in folk medicine. The plant extracts were subject to phytochemical analysis, foam test, and hemolytic assay. Regarding the Fh value, Solanam incanum consisted highest Fh value (20mm), whereas Boscia angustifolia showed the lowest Fh value (10mm). The level of hemolysis of all the plant extracts ranged between 9.0 to 20.2 %. All the plant extracts were suitable for treatment with respect to saponin level since they exhibited minimal hemolytic effect against erythrocytes.

Keywords: Boscia angustifolia, Calpurnia aurea, Commicarpus pedunculosis, hemolysis, saponin, Solanum incanum, Steganotaenia araliaceae

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Authors: Naouelle Azzag, Nadia Haddad, Benoit Durand, Elisabeth Petit, Ali Ammouche, Bruno Chomel, Henri J. Boulouis

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Bartonella henselae is a small, gram negative, arthropod-borne bacterium that has been shown to cause multiple clinical manifestations in humans including cat scratch disease, bacillary angiomatosis, endocarditis, and bacteremia. In this research, we report the results of a cross sectional study of Bartonella henselae bacteremia in stray cats from Algiers. Whole blood of 227 stray cats from Algiers was tested for the presence of Bartonella species by culture and for the evaluation of the genetic diversity of B. henselae strains by multi-locus variable number of tandem repeats assay (MLVA). Bacteremia prevalence was 17% and only B. henselae was identified. Type I was the predominant type (64%). MLVA typing of 259 strains from 30 bacteremic cats revealed 52 different profiles. 51 of these profiles were specific to Algerian cats/identified for the first time. 20/30 cats (67%) harbored 2 to 7 MLVA profiles simultaneously. The similarity of MLVA profiles obtained from the same cat, neighbor-joining clustering and structure-neighbor clustering showed that such a diversity likely results from two different mechanisms occurring either independently or simultaneously independent infections and genetic drift from a primary strain.

Keywords: Bartonella, cat, MLVA, genetic

Procedia PDF Downloads 147

Authors: Feng Ju Chuang, Yu Wen Wang, Tai Jung Hsieh, Shyh Ming Kuo

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Polylactic acid is a synthetic polymer with good biocompatibility and degradability, is widely used in clinical applications. In this study, we utilized Polylactic acid particles as stimulants for macrophages and the collagen synthesis of co-cultured fibroblasts was evaluated. The results indicated that Polylactic acid particles were nontoxic to cells from 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. No obvious inflammation effect was observed (under the PLLA concentration of 1 mg/mL) after 24-h co-culture of Raw264.7 and NIH3T3 cells (from TNF-α assay). The addition of PLLA particles to the Raw264.7 and NIH3T3 co-cultures increased the synthesis of collagen, the highest collagen synthesis from the fibroblast was the 0.2 mg/mL (approximately 60% increased as compared with without addition Polylactic acid particles). Moreover, a co-axial atomization delivery device was used to percutaneously introduce Polylactic acid particles into the dermis layer and stimulating macrophages to secrete growth factors promoting fibroblasts to produce collagen. The preliminary results demonstrated the synthesis of collagen was increased mildly after the introduction of Polylactic acid particles for 28-d post implantation. The Polylactic acid particles could be successfully introduced into the dermis layer from H&E staining examination, however, the optimum concentration of Polylactic acid particles and the time-period for collagen synthesis still need to be evaluated.

Keywords: collagen synthesis, macrophage, NIH3T3 cells, polylactic acid particles

Procedia PDF Downloads 111

Authors: Muhammad Fiaz, Muhammad Saqlain, Abid Mahmood, S. M. Saqlan Naqvi, Rizwan Aziz Qazi, Ghazala Kaukab Raja

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Background: Metabolic Syndrome is a clustering of multiple risk factors including central obesity, hypertension, dyslipidemia and hyperglycemia. These risk phenotypes of metabolic syndrome (MetS) prevalent world-wide, Therefore we aimed to identify the frequency of risk phenotypes among metabolic syndrome subjects in local adult Pakistani population. Methods: Screening of subjects visiting out-patient department of medicine, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad was performed to assess the occurrence of risk phenotypes among MetS subjects in Pakistani population. The Metabolic Syndrome was defined based on International Diabetes Federation (IDF) criteria. Anthropometric and biochemical assay results were recorded. Data was analyzed using SPSS software (16.0). Results: Our results showed that dyslipidemia (31.50%) and hyperglycemia (30.50%) was most population specific risk phenotypes of MetS. The results showed the order of association of metabolic risk phenotypes to MetS as follows hyperglycemia>dyslipidemia>obesity >hypertension. Conclusion: The hyperglycemia and dyslipidemia were found be the major risk phenotypes among the MetS subjects and have greater chances of deceloping MetS among Pakistani Population.

Keywords: dyslipidemia, hypertention, metabolic syndrome, obesity

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Authors: Peramachi Sathiyamoorthy, Jacop Gopas, Avi Golan Goldhirsh

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Corchorus olitorius is a leafy vegetable and an industrial crop. The herb has antioxidant, anti inflammatory, and anti-cancer properties. To assay the pharmaceutical properties, aqueous extracts of leaves and seeds from C. olitorius were tested against drug resistant melanoma cell line. The test showed LC50 of the extract was 0.08µg/ml. Aqueous seed extract exhibited higher melanoma inhibiting activity than leaf extract. Dialysis of seed extract showed that the active compound is less than 12 KDa. The compound with <3 KDa MW separated by microconcentration of seed extract showed 70.5 % inhibition of melanoma cell growth. Among the two fractions obtained by Gel filtration with G10 column, the first fraction at 1:2000 dilutions exhibited 100% inhibition of melanoma growth. The compound with Rf value 0.86 (MA4) isolated by TLC separation showed about 98% cytotoxicity against melanoma at 1: 1000 dilutions. Furthermore, HPLC separation of MA4 compound with Superdex 75 column resulted in 4 compounds. Out of 4, one compound showed melanoma inhibition. The active compound is identified by reagent methods as Strophanthidin. Further toxicological and clinical studies will lead to the development of a potential drug to treat drug resistant melanoma.

Keywords: corchorus olitorius, melanoma, drug development, strophanthidin

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Authors: Abdul Matin, Salik Nawaz, Suk-Yul Jung

Abstract:

Balamuthia mandrillaris is well known to cause fatal Balamuthia amoebic encephalitis (BAE). Amoebic transmission into the central nervous system (CNS), haematogenous spread is thought to be the prime step, followed by blood-brain barrier (BBB) dissemination. Macrophages are considered to be the foremost line of defense and present in excessive numbers during amoebic infections. The aim of the present investigation was to evaluate the effects of macrophages alone or primed with cytokines on the biological characteristics of Balamuthia in vitro. Using human brain microvascular endothelial cells (HBMEC), which constitutes the BBB, we have shown that Balamuthia demonstrated > 90% binding and > 70% cytotoxicity to host cells. However, macrophages further increased amoebic binding and Balamuthia-mediated cell cytotoxicity. Furthermore, macrophages exhibited no amoebicidal effect against Balamuthia. Zymography assay demonstrated that macrophages exhibited no inhibitory effect on proteolytic activity of Balamuthia. Overall, to our best knowledge, we have shown for the first time macrophages has no inhibitory effects on the biological properties of Balamuthia in vitro. This also strengthened the concept that how and why Balamuthia can cause infections in both immuno-competent and immuno-compromised individuals.

Keywords: Balamuthia mandrillaris, macrophages, cytokines, human brain microvascular endothelial cells, Balamuthia amoebic encephalitis

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Authors: Hai Xu, Yiwei Wang, Xi Bao, Bihua Deng, Pengcheng Li, Yu Lu

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T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 10¹⁰ pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac^®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.

Keywords: Gonadotrophin Releasing Hormone (GnRH), Immunocastration, T7 phage, Phage vaccine

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Authors: Ikram Mohamed Eltayeb Elsiddig, Abdel Khalig Muddather, Hiba Abdel Rahman Ali, Saad Mohamed Hussein Ayoub

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Anogeissusleiocarpus (Combretaceae) is well known for its medicinal uses in African traditional medicine, for treating many human diseases mainly skin diseases and infections.Mycetoma disease is a fungal and/ or bacterial skin infection, mainly cause by Madurella mycetomatis fungus.This study was carried out in vitro to investigate the antifungal activity of Anogeissusleiocarpus leaf extracts against the isolated pathogenicMadurellamycetomatis, by using the NCCLS modified method compared to Ketoconazole standard drug and MTT assay. The bioactive fraction was subjected to chemical analysis implementing different chromatographic analytical methods (TLC, HPLC, and LC-MS/MS). The results showed significance antifungal activity of A. leiocarpus leaf extractsagainst the isolated pathogenicM. mycetomatis, compared to negative and positive controls. The chloroform fraction showed the highest antifungal activity.The chromatographic analysis of the chloroform fraction with the highest activity showed the presence of important bioactive compounds such as ellagic and flavellagic acids derivatives, flavonoids and stilbenoid, which are well known for their antifungal activity.

Keywords: Anogeissus leiocarpus, crude extracts and fractions of Anogeissus leiocarpus, in vitrosusceptibility of Madurella mycetomatis, Madurella mycetomatis

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Authors: Demetrio L. Valle Jr., Juliana Janet M. Puzon, Windell L. Rivera

Abstract:

From the various medicinal plants available in the Philippines, crude ethanol extracts of twelve (12) Philippine medicinal plants, namely: Senna alata L. Roxb. (akapulko), Psidium guajava L. (bayabas), Piper betle L. (ikmo), Vitex negundo L. (lagundi), Mitrephora lanotan (Blanco) Merr. (Lanotan), Zingiber officinale Roscoe (luya), Curcuma longa L. (Luyang dilaw), Tinospora rumphii Boerl (Makabuhay), Moringga oleifera Lam. (malunggay), Phyllanthus niruri L. (sampa-sampalukan), Centella asiatica (L.) Urban (takip kuhol), and Carmona retusa (Vahl) Masam (tsaang gubat) were studied. In vitro methods of evaluation against selected Gram-positive and Gram-negative multidrug-resistant (MDR), bacteria were performed on the plant extracts. Although five of the plants showed varying antagonistic activities against the test organisms, only Piper betle L. exhibited significant activities against both Gram-negative and Gram-positive multidrug-resistant bacteria, exhibiting wide zones of growth inhibition in the disk diffusion assay, and with the lowest concentrations of the extract required to inhibit the growth of the bacteria, as supported by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Further antibacterial studies of the Piper betle L. leaf, obtained by three extraction methods (ethanol, methanol, supercritical CO2), revealed similar inhibitory activities against a multitude of Gram-positive and Gram-negative MDR bacteria. Thin layer chromatography (TLC) assay of the leaf extract revealed a maximum of eight compounds with Rf values of 0.92, 0.86, 0.76, 0.53, 0.40, 0.25, 0.13, and 0.013, best visualized when inspected under UV-366 nm. TLC- agar overlay bioautography of the isolated compounds showed the compounds with Rf values of 0.86 and 0.13 having inhibitory activities against Gram-positive MDR bacteria (MRSA and VRE). The compound with an Rf value of 0.86 also possesses inhibitory activity against Gram-negative MDR bacteria (CRE Klebsiella pneumoniae and MBL Acinetobacter baumannii). Gas Chromatography-Mass Spectrometry (GC-MS) was able to identify six volatile compounds, four of which are new compounds that have not been mentioned in the medical literature. The chemical compounds isolated include 4-(2-propenyl)phenol and eugenol; and the new four compounds were ethyl diazoacetate, tris(trifluoromethyl)phosphine, heptafluorobutyrate, and 3-fluoro-2-propynenitrite. Phytochemical screening and investigation of its antioxidant, cytotoxic, possible hemolytic activities, and mechanisms of antibacterial activity were also done. The results showed that the local variant of Piper betle leaf extract possesses significant antioxidant, anti-cancer and antimicrobial properties, attributed to the presence of bioactive compounds, particularly of flavonoids (condensed tannin, leucoanthocyanin, gamma benzopyrone), anthraquinones, steroids/triterpenes and 2-deoxysugars. Piper betle L. is also traditionally known to enhance wound healing, which could be primarily due to its antioxidant, anti-inflammatory and antimicrobial activities. In vivo studies on mice using 2.5% and 5% of the ethanol leaf extract cream formulations in the excised wound models significantly increased the process of wound healing in the mice subjects, the results and values of which are at par with the current antibacterial cream (Mupirocin). From the results of the series of studies, we have definitely proven the value of Piper betle L. as a source of bioactive compounds that could be developed into therapeutic agents against MDR bacteria.

Keywords: Philippine herbal medicine, multidrug-resistant bacteria, Piper betle, TLC-bioautography

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Authors: M. Lizzy Madiwani, Ignatious Ncube, Evelyn Madoroba

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This study was designed to determine the distribution of pathogenic bacteria in household raised chickens and study their virulence and antibiotic profiles. For this purpose, 40 chickens were purchased from families in the Capricorn district and sacrificed for sampling. Tissues were cultured on different bacteriological media followed by biotyping using Matrix-assisted Laser Desorption Ionization-time of Flight (MALDI-TOF). Disk diffusion test was performed to determine the antibiotic susceptibility profiles of these bacteria. Out of a total of 160 tissue samples evaluated, E. coli and Salmonella were detected in these tissues. Furthermore, determination of the pathogenic E. coli and Salmonella strains at species level using primer sets that target selected genes of interest in the polymerase chain reaction (PCR) assay was employed. The invA gene, a confirmatory gene of Salmonella was detected in all the Salmonella isolates. The study revealed that there is a high distribution of Salmonella and pathogenic E. coli in these chickens. Therefore, further studies on identification at the species level are highly recommended to provide management and sanitation practices to lower this prevalence. The antimicrobial susceptibly data generated from this study can be a valuable reference to veterinarians for treating bacterial diseases in poultry.

Keywords: antimicrobial, Escherichia coli, pathogens, Salmonella

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Authors: Asmaa Mandour, Ramzia El-Bagary, Asmaa El-Zaher, Ehab Elkady

Abstract:

Background: An UHPLC (ultra high performance liquid chromatography) method for the simultaneous determination of norfloxacin (NOR), metronidazole (MET) and tinidazole (TNZ) using monolithic column is presented. Purpose: The method is considered an environmentally friendly method with relatively low organic composition of the mobile phase. Methods: The chromatographic separation was performed using Phenomenex® Onyex Monolithic C18 (50mmx 20mm) column. An elution program of mobile phase consisted of 0.5% aqueous phosphoric acid : methanol (85:15, v/v). Where elution of all drugs was completed within 3.5 min with 1µL injection volume. The UHPLC method was applied for the stability indication of NOR in the presence of its acid degradation product ND. Results: Retention times were 0.69, 1.19 and 3.23 min for MET, TNZ and NOR, respectively. While ND retention time was 1.06 min. Linearity, accuracy, and precision were acceptable over the concentration range of 5-50µg mL-1for all drugs. Conclusions: The method is simple, sensitive and suitable for the routine quality control and dosage form assay of the three drugs and can also be used for the stability indication of NOR in the presence of its acid degradation product.

Keywords: antibacterial, monolithic cilumn, simultaneous determination, UHPLC

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Authors: Mandeep Kataria, Ankita Thakur

Abstract:

Glycyrrhiza glabra grows in the sub- tropical and warm temperate regions of the world, in Mediterranean countries and China, America, Europe, Asia and Australia. It grows in areas with sunny, dry and hot climates. It has numerous medicinal properties like it is used to cure Peptic Ulcers, Canker sores, Eczema, Indigestion and Upper Respiratory Infections. Biosynthetic methods such as plant extract have emerged as a simple and viable alternative to more complex chemical synthetic procedures to obtain nanomaterials. Extract from plant may act both as reducing and capping agents in silver nanoparticles synthesis. In the present work, Green Silver nanoparticles were successfully formulated from bioreduction of silver nitrate solutions using Glycyrrhiza glabra root extract. These Green Silver nanoparticles have been appropriately characterized using Visible spectroscopy, colour change. The Antimicrobial activity was done by Agar disc diffusion assay. AgNPs were developed by using aqueous root extract of Glycyrrhiza glabra, which acts as a reducing as well as stabilizing agent. The green synthetic method is a fast, low cost and eco-friendly process in the field of nanotechnology. The study revealed that the green-synthesized silver nanoparticle provides a promising approach for antimicrobial activity.

Keywords: Glycyrrhiza glabra, nanoparticles, antimicrobial activity, aqueous extract

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Authors: Meriche Hacene

Abstract:

Introduction: Allergy to animal hair is hypersensitivity to animal appendages to look for in front of any rhinoconjunctivitis or asthma. An anamnesis associated with the prick-tests makes it possible to guide the diagnosis, which will be supplemented in case of doubt by specific immunoglobulin E (IgE) assays. The objective of our study is to study the characteristics of patients sensitized to animal hair. Patients and methods: Retrospective study conducted on 105 adult patients and 69 children over a period of 3 years, including patients who received a specific IgE assay (respiratory panel and pediatric panel) by immunodot method. Result: 105 adult patients, including 74 women and 31 men, with an average age of 41 years, of which 8.5% had sensitization to animal hair (5 men and 4 women), namely: cat (5%), horse (4.7%) and dog (3.8%). For the 69 children, a slight female predominance was noted (56%), with an average age of 7.5 years, of which (13%) are sensitized to animal hair (5 girls and 4 boys): cat (10%), while awareness of dog and horse hair was less frequent with an identical prevalence of (4.34%). The dominant symptoms are rhinorrhea and sneezing for both categories, respectively (40% and 26.6% in adults and 23% for both symptoms in children). Cross-sensitization was observed in the 2 series: 1 single cat-dog and cat-horse case and 2 dog-horse cases in adults. In children, 100% of patients with sensitization to dog hair had cross-sensitization to cat hair, only 1 case was observed for cat-horse cross-reactivity. Conclusion This work shows that allergy to animal hair is common. Studies on more representative samples are recommended.

Keywords: specific IgE, allergy, cat, fed1

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Authors: Ayodeji O. Falade, Leonard V. Mabinya, Uchechukwu U. Nwodo, Anthony I. Okoh

Abstract:

Given the high-utility of peroxidase, its production in large amount is of utmost importance. Over the years, actinomycetes have been the major peroxidase-producing bacteria. Consequently, other classes of bacteria with peroxidase production potentials are underexplored. This study, therefore, sought to enhance peroxidase production by a Raoultella species, a new ligninolytic proteobacteria strain, by determining the optimum culture conditions (initial pH, incubation temperature and agitation speed) for peroxidase production under submerged fermentation using the classical process of one variable at a time and supplementing the fermentation medium with some lignin model and inorganic nitrogen compounds. Subsequently, the time-course assay was carried out under optimized conditions. Then, some agricultural residues were valorized for peroxidase production under solid state fermentation. Peroxidase production was optimal at initial pH 5, incubation temperature of 35 °C and agitation speed of 150 rpm with guaiacol and ammonium chloride as the best inducer and nitrogen supplement respectively. Peroxidase production by the Raoultella species was optimal at 72 h with specific productivity of 16.48 ± 0.89 U mg⁻¹. A simultaneous production of a non-peroxide dependent extracellular enzyme which suggests probable laccase production was observed with specific productivity of 13.63 ± 0.45 U mg⁻¹ while sawdust gave the best peroxidase yield under solid state fermentation. In conclusion, peroxidase production by the Raoultella species was increased by 3.40-fold.

Keywords: enzyme production, ligninolytic bacteria, peroxidase, proteobacteria

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Authors: Merouani Nawel, Belhattab Rachid

Abstract:

Aristolochia longa L. (Aristolochiacea) is a native plant of Algeria used in traditional medicine. This study was devoted to the determination of polyphenols, flavonoids, and condensed tannins contents of Aristolochia longa L. after their extraction by using various solvents with different polarities (methanol, acetone and distilled water). These extracts were prepared from stem, leaves, fruits and rhizome. The antioxidant activity was determined using three in vitro assays methods: scavenging effect on DPPH, the reducing power assay and ẞ-carotene bleaching inhibition (CBI). The results obtained indicate that the acetone extracts from the aerial parts presented the highest contents of polyphenols. The results of The antioxidant activity showed that all extracts of Aristolochia longa L., prepared using different solvent, have diverse antioxidant capacities. However, the aerial parts methanol extract exhibited the highest antioxidant capacity of DPPH and reducing power (Respectively 55,04ug/ml±1,29 and 0,2 mg/ml±0,019 ). Nevertheless, the aerial parts acetone extract showed the highest antioxidant capacity in the test of ẞ-carotene bleaching inhibition with 57%. These preliminary results could be used to justify the traditional use of this plant and their bioactive substances could be exploited for therapeutic purposes such as antioxidant and antimicrobial.

Keywords: aristolochia longa l., polyphenols, flavonoids, condensed tannins, antioxidant activity

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Authors: Hatem A. El-Mezayen, Hossam Darwesh

Abstract:

Background/Aim: Hepatocellular carcinoma (HCC) is often diagnosed at advanced stage where effective therapies are lacking. Identification of new scoring system is needed to discriminate HCC patients from those with chronic liver disease. Based on the link between vascular endothelial growth factor (VEGF) and HCC progression, we aimed to develop a novel score based on combination of VEGF and routine laboratory tests for early prediction of HCC. Methods: VEGF was assayed for HCC group (123), liver cirrhosis group (210) and control group (50) by Enzyme Linked Immunosorbent Assay (ELISA). Data from all groups were retrospectively analyzed including α feto protein (AFP), international normalized ratio (INR), albumin and platelet count, transaminases, and age. Areas under ROC curve were used to develop the score. Results: A novel index named hepatocellular carcinoma-vascular endothelial growth factor score (HCC-VEGF score)=1.26 (numerical constant) + 0.05 ×AFP (U L-1)+0.038 × VEGF(ng ml-1)+0.004× INR –1.02 × Albumin (g l-1)–0.002 × Platelet count × 109 l-1 was developed. HCC-VEGF score produce area under ROC curve of 0.98 for discriminating HCC patients from liver cirrhosis with sensitivity of 91% and specificity of 82% at cut-off 4.4 (ie less than 4.4 considered cirrhosis and greater than 4.4 considered HCC). Conclusion: Hepatocellular carcinoma-VEGF score could replace AFP in HCC screening and follow up of cirrhotic patients.

Keywords: Hepatocellular carcinoma, cirrhosis, HCV, diagnosis, tumor markers

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