Degradation of type IV collagen by neoplastic human skin fibroblasts

International Nuclear Information System (INIS)

Sheela, S.; Barrett, J.C.

1985-01-01

An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labelled by reductive alkylation. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. A cell-associated assay was performed by growing either normal or transformed cells in the presence of radioactively labelled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen whereas Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity, whereas protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. These results suggest that the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion

Urocanic acid isomers are good hydroxyl radical scavengers: a comparative study with structural analogues and with uric acid

NARCIS (Netherlands)

Kammeyer, A.; Eggelte, T. A.; Bos, J. D.; Teunissen, M. B.

1999-01-01

UV-exposure of the epidermis leads to the isomerisation of trans-UCA into cis-UCA as well as to the generation of hydroxyl radicals. This study shows by means of the deoxyribose degradation test that UCA isomers are more powerful hydroxyl radical scavengers than the other 4-(5-)substituted imidazole

Assays of residual antibiotics after treatment of γ-ray and UV irradiation

International Nuclear Information System (INIS)

Shin, Ji Hye; Nam, Ji Hyun; Lee, Dong Hun; Yu, Seung Ho; Lee, Myun Joo

2010-01-01

The pollution of antibiotics is a major cause of spreading antibiotics resistant bacteria in the environment. Applications of ozonation, UV, and γ-ray irradiations have been introduced to remove antibiotics in the effluents from wastewater treatment system. In this study, we compared the chemical (HPLC) and biological (antimicrobial susceptibility test, AMS) assays in measuring of the concentrations of residual antibiotics after γ-ray and UV irradiation. Most samples were degraded by γ-ray irradiation (1 ∼ 2 kGy). However, lincomycin and tetracycline were not degraded by UV irradiation. The concentration of residual antibiotics, that was treated with γ-ray and UV irradiation, measuring by bioassay was similar to HPLC. The concentrations of γ-ray irradiated cephradine measured by AMS test were 2 times higher than of HPLC assay, indicating AMS test is more sensitive than HPLC assay. These results indicate that γ-ray irradiation technique is more useful than UV irradiation, and biological assay is more useful to detect the antibiotics and toxic intermediates in antibiotics degradation

Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process.

Science.gov (United States)

Vieira, Gabriela A L; Magrini, Mariana Juventina; Bonugli-Santos, Rafaella C; Rodrigues, Marili V N; Sette, Lara D

2018-05-03

Pyrene and benzo[a]pyrene (BaP) are high molecular weight polycyclic aromatic hydrocarbons (PAHs) recalcitrant to microbial attack. Although studies related to the microbial degradation of PAHs have been carried out in the last decades, little is known about degradation of these environmental pollutants by fungi from marine origin. Therefore, this study aimed to select one PAHs degrader among three marine-derived basidiomycete fungi and to study its pyrene detoxification/degradation. Marasmiellus sp. CBMAI 1062 showed higher levels of pyrene and BaP degradation and was subjected to studies related to pyrene degradation optimization using experimental design, acute toxicity, organic carbon removal (TOC), and metabolite evaluation. The experimental design resulted in an efficient pyrene degradation, reducing the experiment time while the PAH concentration applied in the assays was increased. The selected fungus was able to degrade almost 100% of pyrene (0.08mgmL -1 ) after 48h of incubation under saline condition, without generating toxic compounds and with a TOC reduction of 17%. Intermediate metabolites of pyrene degradation were identified, suggesting that the fungus degraded the compound via the cytochrome P450 system and epoxide hydrolases. These results highlight the relevance of marine-derived fungi in the field of PAH bioremediation, adding value to the blue biotechnology. Copyright © 2018. Published by Elsevier Editora Ltda.

Enzymatic degradation of aliphatic nitriles by Rhodococcus rhodochrous BX2, a versatile nitrile-degrading bacterium.

Science.gov (United States)

Fang, Shumei; An, Xuejiao; Liu, Hongyuan; Cheng, Yi; Hou, Ning; Feng, Lu; Huang, Xinning; Li, Chunyan

2015-06-01

Nitriles are common environmental pollutants, and their removal has attracted increasing attention. Microbial degradation is considered to be the most acceptable method for removal. In this work, we investigated the biodegradation of three aliphatic nitriles (acetonitrile, acrylonitrile and crotononitrile) by Rhodococcus rhodochrous BX2 and the expression of their corresponding metabolic enzymes. This organism can utilize all three aliphatic nitriles as sole carbon and nitrogen sources, resulting in the complete degradation of these compounds. The degradation kinetics were described using a first-order model. The degradation efficiency was ranked according to t1/2 as follows: acetonitrile>trans-crotononitrile>acrylonitrile>cis-crotononitrile. Only ammonia accumulated following the three nitriles degradation, while amides and carboxylic acids were transient and disappeared by the end of the assay. mRNA expression and enzyme activity indicated that the tested aliphatic nitriles were degraded via both the inducible NHase/amidase and the constitutive nitrilase pathways, with the former most likely preferred. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24.

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Sang-Yeop Lee

Full Text Available Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs, including benzene, toluene, and xylene (BTX, as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

Science.gov (United States)

Yun, Sung Ho; Choi, Chi-Won; Yi, Yoon-Sun; Kim, Jonghyun; Chung, Young-Ho; Park, Edmond Changkyun; Kim, Seung Il

2016-01-01

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX. PMID:27124467

Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits (α and β) biosynthesis and degradation (in newborn brain)

International Nuclear Information System (INIS)

Tse, C.F.

1978-01-01

A DEAE-cellulose filter assay, measuring [ 3 H]colchicine bound to colchicine binding protein (CBP) absorbed on filter discs, has been modified to include lM sucrose in the incubation medium for complexing colchicine to CBP in samples before applying the samples to filter discs (single point assay). Due to the much greater stability of colchicine binding capacity in the presence of lM sucrose, multiple time-point assays and least squares linear regression analysis were not necessary for accurate determination of CBP in hybrid mouse brain at different stages of development. The highest concentrations of CBP were observed in the 160,000g supernatant and pellet of newborn brain homogenate. Further studies of the modified filter assay documented that the assay has an overall counting efficiency of 27.3%, that DEAE-cellulose filters bind and retain all tubulin in the assay samples, and that one molecule of colchicine binds approximately one molecule of tubulin dimer. Therefore, millimoles of colchicine bound per milligram total protein can be used to calculate tubulin content. With this technique tubulin content of brain supernatant was found to be 11.9% for newborn, and 7.15% for 11 month old mice. Quantitative densitometry was also used to measure mouse brain supernatant actin content for these two stages. In vivo synthesis and degradation rates of tubulin α and β subunits of two day mouse brain 100,000g supernatant were studied after intracerebral injection of [ 3 H]leucine. Quantitative changes of the ratio of tritium specific activities of tubulin α and β subunits with time were determined. The pattern of change was biphasic. During the first phase the ratio decreased; during the second phase the ratio increased continuously. An interpretation consistent with all the data in this study is that the α subunit is synthesized at a more rapid rate than the β subunit

Microbiological assay for the determination of meropenem in pharmaceutical dosage form.

Science.gov (United States)

Mendez, Andreas S L; Weisheimer, Vanessa; Oppe, Tércio P; Steppe, Martin; Schapoval, Elfrides E S

2005-04-01

Meropenem is a highly active carbapenem antibiotic used in the treatment of a wide range of serious infections. The present work reports a microbiological assay, applying the cylinder-plate method, for the determination of meropenem in powder for injection. The validation method yielded good results and included linearity, precision, accuracy and specificity. The assay is based on the inhibitory effect of meropenem upon the strain of Micrococcus luteus ATCC 9341 used as the test microorganism. The results of assay were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9999) in the range of 1.5-6.0 microg ml(-1), precise (intra-assay: R.S.D.=0.29; inter-assay: R.S.D.=0.94) and accurate. A preliminary stability study of meropenem was performed to show that the microbiological assay is specific for the determination of meropenem in the presence of its degradation products. The degraded samples were also analysed by the HPLC method. The proposed method allows the quantitation of meropenem in pharmaceutical dosage form and can be used for the drug analysis in routine quality control.

High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

2016-01-01

for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

Optimization of Forced Degradation Using Experimental Design and Development of a Stability-Indicating Liquid Chromatographic Assay Method for Rebamipide in Bulk and Tablet Dosage Form

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Sandeep SONAWANE

2016-09-01

Full Text Available A novel stability-indicating RP-HPLC assay method was developed and validated for quantitative determination of rebamipide in bulk and tablet dosage form. Rebamipide (drug and drug product solutions were exposed to acid and alkali hydrolysis, thermal stress, oxidation by hydrogen peroxide and photodegradation. Experimental design has been used during forced degradation to determine significant factors responsible for degradation and to obtain optimal degradation conditions. In addition, acid and alkali hydrolysis was performed using a microwave oven. The chromatographic method employed the HiQ sil C-18HS (250 × 4.6 mm; 5 μm column with mobile phase consisting of 0.02 M potassium phosphate (pH adjusted to 6.8 and methanol (40:60, v/v and the detection was performed at 230 nm. The procedure was validated for specificity, linearity, accuracy, precision and robustness. There was no interference observed of excipients and degradation products in the determination of the active pharmaceutical ingredient. The method showed good accuracy and precision (intra and inter day and the response was linear in a range from 0.5 to 5 μg mL−1. The method was found to be simple and fast with less trial and error experimentation by making use of experimental design. Also, it proved that microwave energy can be used to expedite hydrolysis of rebamipide.

Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

Science.gov (United States)

Helmerhorst, Eva J; Wei, Guoxian

2014-05-05

Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits ( α and β) biosynthesis and degradation (in newborn brain)

Energy Technology Data Exchange (ETDEWEB)

Tse, Cek-Fyne [Univ. of Rochester, NY (United States)

1978-01-01

A DEAE-cellulose filter assay, measuring (³H)colchicine bound to colchicine binding protein (CBP) absorbed on filter discs, has been modified to include lM sucrose in the incubation medium for complexing colchicine to CBP in samples before applying the samples to filter discs (single point assay). Due to the much greater stability of colchicine binding capacity in the presence of lM sucrose, multiple time-point assays and least squares linear regression analysis were not necessary for accurate determination of CBP in hybrid mouse brain at different stages of development. The highest concentrations of CBP were observed in the 160,000g supernatant and pellet of newborn brain homogenate. Further studies of the modified filter assay documented that the assay has an overall counting efficiency of 27.3%, that DEAE-cellulose filters bind and retain all tubulin in the assay samples, and that one molecule of colchicine binds approximately one molecule of tubulin dimer. Therefore, millimoles of colchicine bound per milligram total protein can be used to calculate tubulin content. With this technique tubulin content of brain supernatant was found to be 11.9% for newborn, and 7.15% for 11 month old mice. Quantitative densitometry was also used to measure mouse brain supernatant actin content for these two stages. In vivo synthesis and degradation rates of tubulin ..cap alpha.. and ..beta.. subunits of two day mouse brain 100,000g supernatant were studied after intracerebral injection of (³H)leucine. Quantitative changes of the ratio of tritium specific activities of tubulin ..cap alpha.. and ..beta.. subunits with time were determined. The pattern of change was biphasic. During the first phase the ratio decreased; during the second phase the ratio increased continuously. An interpretation consistent with all the data in this study is that the ..cap alpha.. subunit is synthesized at a more rapid rate than the ..beta.. subunit. (ERB)

Degradation of endothelial basement membrane by human breast cancer cell lines

International Nuclear Information System (INIS)

Yee, C.; Shiu, R.P.

1986-01-01

During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of [35S]methionine-labeled and [3H]proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer

Comet assay as a cold chain control tool

International Nuclear Information System (INIS)

Duarte, Renato Cesar

2009-01-01

Bearing in mind an ever more demanding market regarding the quality of food, it has been necessary to develop processes that meet the demands of consumers. Within the existing processes the cold chain and irradiation stand out. The cold chain comprises all the stages of conserving food from production, cooling, freezing, storing and transportation to the final consumer. Irradiation, as a means of conserving food, prolongs the shelf life, inhibits budding and reduces pathogenic contamination among other benefits. Is very important the identification of food degradation in function of failure on the processes which they were subjected. The comet assay is a screening test widely studied, considerate fast and with low cost. By the fact of the test identify breaks on the DNA, may be possible use the comet test on the control of cold chain failures that degrade de food. The labels and stamp, do not consider the previous food situation and indicate failures from the moment where they be placed in contact with the product. With the comet assay is possible to check the degradation that has occurred in liver chicken samples until the moment of comet's test realization. (author)

Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

Science.gov (United States)

Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

2016-05-01

Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved.

In vitro glutathione peroxidase mimicry of ebselen is linked to its oxidation of critical thiols on key cerebral suphydryl proteins - A novel component of its GPx-mimic antioxidant mechanism emerging from its thiol-modulated toxicology and pharmacology.

Science.gov (United States)

Kade, I J; Balogun, B D; Rocha, J B T

2013-10-25

The antioxidant mechanism of ebselen in rats brain is largely linked with its glutathione peroxidase (GPx) rather than its peroxiredoxin mimicry ability. However, the precise molecular dynamics between the GPx-mimicry of ebselen and thiol utilization is yet to be fully clarified and thus still open. Herein, we investigated the influence of dithiothreitol (DTT) on the antioxidant action of ebselen against oxidant-induced cerebral lipid peroxidation and deoxyribose degradation. Furthermore, the critical inhibitory concentrations of ebselen on the activities of sulphydryl enzymes such as cerebral sodium pump, δ-aminolevulinic acid dehydratase (δ-ALAD) and lactate dehydrogenase (LDH) were also investigated. We observe that ebselen (at ≥42 μM) markedly inhibited lipid peroxidation in the presence and absence of DTT, whereas it inhibited deoxyribose degradation only in the presence of DTT. Furthermore, under in vitro conditions, ebselen inhibited the thiol containing enzymes; cerebral sodium pump (at ≥40 μM), δ-ALAD (≥10 μM) and LDH (≥1 μM) which were either prevented or reversed by DTT. However, the inhibition of the activities of these sulphydryl proteins in diabetic animals was prevented by ebselen. Summarily, it is apparent that the effective in vitro inhibitory doses of ebselen on the activity of the sulphydryl proteins are far less than its antioxidant doses. In addition, the presence of DTT is evidently a critical requirement for ebselen to effect its antioxidant action against deoxyribose degeradation and not lipid peroxidation. Consequently, we conclude that ebselen possibly utilizes available thiols on sulphydryl proteins to effect its GPx mimicry antioxidant action against lipid peroxidation in rat brain homogenate. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri

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Moisés Martínez-Castillo

2015-01-01

Full Text Available Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C employing conditioned medium (CM and total crude extracts (TCEs of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

Science.gov (United States)

Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

2015-01-01

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme

Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

Directory of Open Access Journals (Sweden)

George Janssen

Full Text Available Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP.Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1 enzyme assays and 2 mass spectrometric identification. Gluten epitope degradation was monitored by 1 R5 ELISA, 2 mass spectrometric analysis of the degradation products and 3 T cell proliferation assays.The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.Currently available digestive enzyme supplements are ineffective in

Detection of Toluene Degradation in Bacteria Isolated from Oil Contaminated Soils

International Nuclear Information System (INIS)

Ainon Hamzah; Tavakoli, A.; Amir Rabu

2011-01-01

Toluene (C 7 H 8 ) a hydrocarbon in crude oil, is a common contaminant in soil and groundwater. In this study, the ability to degrade toluene was investigated from twelve bacteria isolates which were isolated from soil contaminated with oil. Out of 12 bacterial isolates tested, most of Pseudomonas sp. showed the capability to grow in 1 mM of toluene compared with other isolates on the third day of incubation. Based on enzyme assays towards toluene monooxygenase, Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were shown to have the highest ability to degrade toluene. The toluene monooxygenase activity was analysed by using two calorimetric methods, Horseradish peroxidase (HRP) and indole-indigo. Both of the methods measured the production of catechol by the enzymatic reaction of toluene monooxygenase. In the HRP assay, the highest enzyme activity was 0.274 U/ mL, exhibited by Pseudomonas aeruginosa UKMP-14T. However, for indole-indigo assay, Bacillus cereus UKMP-6G produced the highest enzyme activity of 0.291 U/ ml. Results from both experiments showed that Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were able to degrade toluene. (author)

Scavenging capacity of medicinal plants against free radical-induced cellular damage by radiation and photoactivation

Energy Technology Data Exchange (ETDEWEB)

Gadkar, Shalaka [Ruia College, Mumbai (India); Mohan, H [Chemistry Group, Bhabha Atomic Research Centre, Mumbai (India); Kamat, J P [Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai (India)

2004-01-01

The scavenging capacity of medicinal plants. Andrographis paniculata (Ap) and Swertia chirata (Sc) was examined against cellular damage, induced by radiation and photo-activation in sub-cellular membranes. The results demonstrated significant radical scavenging capacity of the extracts. The rate constants as evaluated by deoxyribose degradation studies and the pulse radiolysis studies carried in presence of ABTS radical well supported the antioxidant properties of the extracts. (author)

A multiwell format assay for heparanase.

Science.gov (United States)

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

Subcellular distribution of histone-degrading enzyme activities from rat liver

International Nuclear Information System (INIS)

Heinrich, P.C.; Raydt, G.; Puschendorf, B.; Jusic, M.

1976-01-01

Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity. (orig.) [de

Methods and devices for protein assays

Science.gov (United States)

Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

2009-11-03

Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

Comet assay on tetraploid yeast cells

DEFF Research Database (Denmark)

Rank, Jette; Syberg, Kristian; Jensen, Klara

2009-01-01

Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

Human sperm degradation of zona pellucida proteins contributes to fertilization.

Science.gov (United States)

Saldívar-Hernández, Analilia; González-González, María E; Sánchez-Tusié, Ana; Maldonado-Rosas, Israel; López, Pablo; Treviño, Claudia L; Larrea, Fernando; Chirinos, Mayel

2015-09-02

The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

Endoproteolytic activity assay in malting barley

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Blanca Gómez Guerrero

2013-12-01

Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

Degraded iota-carrageenan can induce apoptosis in human osteosarcoma cells via the Wnt/β-catenin signaling pathway.

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Jin, Zhe; Han, Ya-Xin; Han, Xiao-Rui

2013-01-01

Osteosarcoma (OS) is a high-grade malignant bone tumor. Therefore, using both in vitro and in vivo assays, the effects of degraded iota-Carrageenan (ι-CGN) on a human osteosarcoma cell line, HOS, were examined. Degraded ι-CGN was observed to induce apoptosis and G(1) phase arrest in HOS cells. Moreover, degraded ι-CGN suppressed tumor growth in established xenograft tumor models. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumors treated with native ι-CGN or PBS. In addition, the formation of intratumoral microvessels was inhibited following treatment with degraded ι-CGN. In Western blot assays, degraded ι-CGN was found to inhibit the Wnt/β-catenin signaling pathway. Overall, these studies demonstrate the antitumor activity of degraded ι-CGN toward the OS cell line, HOS. Moreover, valuable insight into the mechanisms mediated by degraded ι-CGN was obtained, potentially leading to the identification of novel treatments for OS. However, additional studies are needed to confirm these results in other cell types, particularly in human umbilical vein endothelial cells.

Assays of D-Amino Acid Oxidase Activity

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Elena Rosini

2018-01-01

Full Text Available D-amino acid oxidase (DAAO is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs. Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

A deadenylase assay by size-exclusion chromatography.

Science.gov (United States)

He, Guang-Jun; Yan, Yong-Bin

2012-01-01

The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3'-end of eukaryotic mRNAs and release 5'-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

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Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

2017-01-01

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

Science.gov (United States)

Cortés-Gutiérrez, Elva I.; López-Fernández, Carmen; Fernández, José Luis; Dávila-Rodríguez, Martha I.; Johnston, Stephen D.; Gosálvez, Jaime

2014-01-01

Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to

Characterizing the collagen stabilizing effect of crosslinked chitosan nanoparticles against collagenase degradation.

Science.gov (United States)

Kishen, Anil; Shrestha, Suja; Shrestha, Annie; Cheng, Calvin; Goh, Cynthia

2016-08-01

Antibacterial and chelating properties of chitosan has been widely studied for various dental applications. To characterize the interaction between chitosan-nanoparticles (CSnp) and collagen, and understand their stabilizing effect against collagenase degradation for dentin matrix stabilization. Phase-1: a single Type I collagen-fibril model was used to study the interaction with CSnp along with carbodiimides crosslinking treatment. Degradation of the crosslinked fibrils was studied with bacterial collagenase enzyme and monitored using Fourier Transform Infrared (FTIR) spectroscopy, turbidity measurement (400nm), ninhydrin assay and Atomic Force Microscopy (AFM). Interaction of CSnp with collagenase and Type I collagen, were evaluated using SDS-PAGE, and proteolytic cleavage potential of a synthetic peptide. Phase-2: degradation of dentin collagen crosslinked with/without CSnp was evaluated using FTIR, ninhydrin assay and Scanning Electron Microscopy (SEM). Glutaraldehyde crosslinking was used as a positive control. Both native collagen-fibrils and dentin collagen after crosslinking showed higher resistance to collagenase degradation, as observed in turbidity measurements and FTIR spectra. AFM images showed the interaction of CSnp with single collagen-fibril and crosslinked collagen resisted collagenase degradation up to 54h. The collagen and collagenase both formed complexes with CSnp resulting in thickening of bands and reduction in collagen degradation. CSnp treated collagenase showed significantly reduced cleavage of the fluorescent peptides. Dentin collagen was coated with CSnp following crosslinking with significant increase in resistance to collagenase degradation. Crosslinked CSnp on collagen stabilized and enhanced the resistance of dentin matrix against bacterial collagenase degradation due to non-specific interaction with both collagen and collagenase. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Drosophila comet assay: insights, uses, and future perspectives

Science.gov (United States)

Gaivão, Isabel; Sierra, L. María

2014-01-01

The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type. PMID:25221574

Isolation, identification and application in lignin degradation of an ...

African Journals Online (AJOL)

This study was undertaken to isolate an ascomycete producing ligninolytic enzyme and characterize its lignin degradation capability. Among 20 isolates, GHJ-4 was isolated from decayed wood of Salix matsudana Koidz in Mount Tai, China, by different indicator compounds assay. The taxonomy of the fungi was ...

Enzymatic formulation capable of degrading scrapie prion under mild digestion conditions.

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Emeka A Okoroma

Full Text Available The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrP(Sc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrP(Sc at 65 °C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50 °C over 2 h revealed the progressive attenuation of PrP(Sc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrP(Sc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1 (p-value = 0.008 at 95% confidence interval. This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions.

Different Spectrophotometric Methods for Simultaneous Determination of Trelagliptin and Its Acid Degradation Product

Science.gov (United States)

Hassan, Mostafa A.; Zaghary, Wafaa A.

2018-01-01

New spectrophotometric and chemometric methods were carried out for the simultaneous assay of trelagliptin (TRG) and its acid degradation product (TAD) and applied successfully as a stability indicating assay to recently approved Zafatek® tablets. TAD was monitored using TLC to ensure complete degradation. Furthermore, HPLC was used to confirm dealing with one major acid degradation product. The proposed methods were developed by manipulating zero-order, first-derivative, and ratio spectra of TRG and TAD using simultaneous equation, first-derivative, and mean-centering methods, respectively. Using Spectra Manager II and Minitab v.14 software, the absorbance at 274 nm–260.4 nm, amplitudes at 260.4 nm–274.0 nm, and mean-centered values at 287.6 nm–257.2 nm were measured against methanol as a blank for TRG and TAD, respectively. Linearity and the other validation parameters were acceptable at concentration ranges of 5–50 μg/mL and 2.5–25 μg/mL for TRG and TAD, respectively. Using one-way analysis of variance (ANOVA), the optimized methods were compared and proved to be accurate for the simultaneous assay of TRG and TAD. PMID:29629213

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals

DEFF Research Database (Denmark)

Galuszka, P.; Frebort, I.; Sebela, M.

2001-01-01

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purifi...

Degradation of selected agrochemicals by the white rot fungus Trametes versicolor.

Science.gov (United States)

Mir-Tutusaus, Josep Anton; Masís-Mora, Mario; Corcellas, Cayo; Eljarrat, Ethel; Barceló, Damià; Sarrà, Montserrat; Caminal, Glòria; Vicent, Teresa; Rodríguez-Rodríguez, Carlos E

2014-12-01

Use of agrochemicals is a worldwide practice that exerts an important effect on the environment; therefore the search of approaches for the elimination of such pollutants should be encouraged. The degradation of the insecticides imiprothrin (IP) and cypermethrin (CP), the insecticide/nematicide carbofuran (CBF) and the antibiotic of agricultural use oxytetracycline (OTC) were assayed with the white rot fungus Trametes versicolor. Experiments with fungal pellets demonstrated extensive degradation of the four tested agrochemicals, at rates that followed the pattern IP>OTC>CP>CBF. In vitro assays with laccase-mediator systems showed that this extracellular enzyme participates in the transformation of IP but not in the cases of CBF and OTC. On the other hand, in vivo studies with inhibitors of cytochrome P450 revealed that this intracellular system plays an important role in the degradation of IP, OTC and CBF, but not for CP. The compounds 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCCA) and 3-phenoxybenzoic acid (PBA) were detected as transformation products of CP, as a result of the breakdown of the molecule. Meanwhile, 3-hydroxycarbofuran was detected as a transformation product of CBF; this metabolite tended to accumulate during the process, nonetheless, the toxicity of the system was effectively reduced. Simultaneous degradation of CBF and OTC showed a reduction in toxicity; similarly, when successive additions of OTC were done during the slower degradation of CBF, the fungal pellets were able to degrade both compounds. The simultaneous degradation of the four compounds successfully took place with minimal inhibition of fungal activity and resulted in the reduction of the global toxicity, thus supporting the potential use of T. versicolor for the treatment of diverse agrochemicals. Copyright © 2014 Elsevier B.V. All rights reserved.

In situ generation of a hydroxyl radical by nanoporous activated carbon derived from rice husk for environmental applications: kinetic and thermodynamic constants.

Science.gov (United States)

Karthikeyan, S; Sekaran, G

2014-03-07

The objective of this investigation is to evaluate the hydroxyl radical (˙OH) generation using nanoporous activated carbon (NPAC), derived from rice husk, and dissolved oxygen in water. The in situ production of the ˙OH radical was confirmed through the DMPO spin trapping method in EPR spectroscopy and quantitative determination by a deoxyribose assay procedure. NPAC served as a heterogeneous catalyst to degrade 2-deoxy-d-ribose (a reference compound) using hydroxyl radical generated from dissolved oxygen in water at temperatures in the range 313-373 K and pH 6, with first order rate constants (k = 9.2 × 10(-2) min(-1), k = 1.2 × 10(-1) min(-1), k = 1.3 × 10(-1) min(-1) and k = 1.68 × 10(-1) min(-1)). The thermodynamic constants for the generation of hydroxyl radicals by NPAC and dissolved oxygen in water were ΔG -1.36 kJ mol(-1) at 313 K, ΔH 17.73 kJ mol(-1) and ΔS 61.01 J mol(-1) K(-1).

Assessing cellulolysis in passive treatment systems for mine drainage: a modified enzyme assay.

Science.gov (United States)

McDonald, Corina M; Gould, W Douglas; Lindsay, Matthew B J; Blowes, David W; Ptacek, Carol J; Condon, Peter D

2013-01-01

A modified cellulase enzyme assay was developed to monitor organic matter degradation in passive treatment systems for mine drainage. This fluorogenic substrate method facilitates assessment of exo-(1,4)-β-D-glucanase, endo-(1,4)-β-D-glucanase, and β-glucosidase, which compose an important cellulase enzyme system. The modified method was developed and refined using samples of organic carbon-amended mine tailings from field experiments where sulfate reduction was induced as a strategy for managing water quality. Sample masses (3 g) and the number of replicates ( ≥ 3) were optimized. Matrix interferences within these metal-rich samples were found to be insignificant. Application of this modified cellulase assay method provided insight into the availability and degradation of organic carbon within the amended tailings. Results of this study indicate that cellulase enzyme assays can be applied to passive treatment systems for mine drainage, which commonly contain elevated concentrations of metals. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

International Nuclear Information System (INIS)

Ulrich, F.

1988-01-01

When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [ 3 H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others,suggest that deoxyribose damages DNA

Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism.

Science.gov (United States)

Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

2016-06-01

The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2 (-) scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays. Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2(-) scavenging, DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3',4'-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form.

Oxidative Damage and Its Possible Mechanism

Directory of Open Access Journals (Sweden)

Tingting Wang

2016-06-01

Full Text Available Purpose: The paper tries to assess the protective effect of fisetin against •OH-induced DNAdamage, then to investigate the possible mechanism.Methods: The protective effect was evaluated based on the content of malondialdehyde(MDA. The possible mechanism was analyzed using various antioxidant methods in vitro,including •OH scavenging (deoxyribose degradation, •O2- scavenging (pyrogallolautoxidation, DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays.Results: Fisetin increased dose-dependently its protective percentages against •OH-inducedDNA damage (IC50 value =1535.00±29.60 μM. It also increased its radical-scavengingpercentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in•OH scavenging, •O2- scavenging, DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays, were 47.41±4.50 μM, 34.05±0.87 μM, 9.69±0.53 μM, 2.43±0.14μM, and 1.49±0.16 μM, respectively.Conclusion: Fisetin can effectively protect DNA against •OH-induced oxidative damagepossibly via reactive oxygen species (ROS scavenging approach, which is assumed to behydrogen atom (H• and/or single electron (e donation (HAT/SET pathways. In the HATpathway, the 3’,4’-dihydroxyl moiety in B ring of fisetin is thought to play an importantrole, because it can be ultimately oxidized to a stable ortho-benzoquinone form.

Aerobic degradation of 4-nitroaniline (4-NA) via novel degradation intermediates by Rhodococcus sp. strain FK48

Energy Technology Data Exchange (ETDEWEB)

Khan, Fazlurrahman; Pandey, Janmejay; Vikram, Surendra; Pal, Deepika; Cameotra, Swaranjit Singh, E-mail: ssc@imtech.res.in

2013-06-15

Highlights: • This study reports isolation of a novel bacterium capable of mineralizing 4-nitroaniline (4-NA). • This bacterium has been identified as Rhodococcus sp. strain FK48. • Strain FK48 degrades 4-NA via a novel aerobic degradation pathway that involves 4-AP and 1,2,4-BT. • Subsequent degradation proceeds via ring fission and formation of maleylacetate. • This is the first report showing elucidation of catabolic pathway for microbial degradation 4-NA. -- Abstract: An aerobic strain, Rhodococcus sp. strain FK48, capable of growing on 4-nitroaniline (4-NA) as the sole source of carbon, nitrogen, and energy has been isolated from enrichment cultures originating from contaminated soil samples. During growth studies with non- induced cells of FK48 catalyzed sequential denitrification (release of NO{sub 2} substituent) and deamination (release of NH{sub 2} substituent) of 4-NA. However, none of the degradation intermediates could be identified with growth studies. During resting cell studies, 4-NA-induced cells of strain FK48 transformed 4-NA via a previously unknown pathway which involved oxidative hydroxylation leading to formation of 4-aminophenol (4-AP). Subsequent degradation involved oxidated deamination of 4-AP and formation of 1,2,4-benzenetriol (BT) as the major identified terminal aromatic intermediate. Identification of these intermediates was ascertained by HPLC, and GC–MS analyses of the culture supernatants. 4-NA-induced cells of strain FK48 showed positive activity for 1,2,4-benzenetriol dioxygenase in spectrophotometric assay. This is the first conclusive study on aerobic microbial degradation of 4-NA and elucidation of corresponding metabolic pathway.

Aerobic degradation of 4-nitroaniline (4-NA) via novel degradation intermediates by Rhodococcus sp. strain FK48

International Nuclear Information System (INIS)

Khan, Fazlurrahman; Pandey, Janmejay; Vikram, Surendra; Pal, Deepika; Cameotra, Swaranjit Singh

2013-01-01

Highlights: • This study reports isolation of a novel bacterium capable of mineralizing 4-nitroaniline (4-NA). • This bacterium has been identified as Rhodococcus sp. strain FK48. • Strain FK48 degrades 4-NA via a novel aerobic degradation pathway that involves 4-AP and 1,2,4-BT. • Subsequent degradation proceeds via ring fission and formation of maleylacetate. • This is the first report showing elucidation of catabolic pathway for microbial degradation 4-NA. -- Abstract: An aerobic strain, Rhodococcus sp. strain FK48, capable of growing on 4-nitroaniline (4-NA) as the sole source of carbon, nitrogen, and energy has been isolated from enrichment cultures originating from contaminated soil samples. During growth studies with non- induced cells of FK48 catalyzed sequential denitrification (release of NO 2 substituent) and deamination (release of NH 2 substituent) of 4-NA. However, none of the degradation intermediates could be identified with growth studies. During resting cell studies, 4-NA-induced cells of strain FK48 transformed 4-NA via a previously unknown pathway which involved oxidative hydroxylation leading to formation of 4-aminophenol (4-AP). Subsequent degradation involved oxidated deamination of 4-AP and formation of 1,2,4-benzenetriol (BT) as the major identified terminal aromatic intermediate. Identification of these intermediates was ascertained by HPLC, and GC–MS analyses of the culture supernatants. 4-NA-induced cells of strain FK48 showed positive activity for 1,2,4-benzenetriol dioxygenase in spectrophotometric assay. This is the first conclusive study on aerobic microbial degradation of 4-NA and elucidation of corresponding metabolic pathway

Fibrin related antigens: assay development, clinical and kinetic studies

Energy Technology Data Exchange (ETDEWEB)

Kruskal, J B

1987-08-01

This thesis describes an assay which is able to measure and to determine the proportions of fibrin- and fibrinogen-related antigens (FRA) present in clinical samples. No assay exists at present which is capable of distinguishing between fibrin and fibrinogen degradation products concurrently and in a clinical setting. The assay may be used as a tool with which to gain further insight to pathophysiology of disorders characterized by activation of the coagulation and fibrinolytic pathways. This study provides and analysis of the FRA profiles in patients with disorders characterised by possible enhanced fibrinolytic activity. Studies have been undertaken on patients with acute and chronic liver diseases, on patients with the various syndromes of coronary artery disease and on patients with insulin-dependent diabetes mellitus with and without evidence of microvascular disease. Certain observations made it evident that further studies were required in order to explain previously undocumented fibrinolytic abnormalities in certain patient groups. Data obtained from patients with liver disease provided information compatible with the activation of their fibrinolytic pathways. The initial scope of this study was then extended to further investigate the deranged haemostatic mechanisms in patients with severe liver diseases. Kinetic studies were performed which required the development of specific technology to be able to measure certain previously undertermined parameters. Mathematical models describing the rates of fibrin formation and lysis were developed for human studies. Fibrin-derived D-dimer was radiolabelled and its validity as and intravenous tracer and maker of fibrin degradation established.

Degradation of textile dyes by cyanobacteria.

Science.gov (United States)

Dellamatrice, Priscila Maria; Silva-Stenico, Maria Estela; Moraes, Luiz Alberto Beraldo de; Fiore, Marli Fátima; Monteiro, Regina Teresa Rosim

Dyes are recalcitrant compounds that resist conventional biological treatments. The degradation of three textile dyes (Indigo, RBBR and Sulphur Black), and the dye-containing liquid effluent and solid waste from the Municipal Treatment Station, Americana, São Paulo, Brazil, by the cyanobacteria Anabaena flos-aquae UTCC64, Phormidium autumnale UTEX1580 and Synechococcus sp. PCC7942 was evaluated. The dye degradation efficiency of the cyanobacteria was compared with anaerobic and anaerobic-aerobic systems in terms of discolouration and toxicity evaluations. The discoloration was evaluated by absorption spectroscopy. Toxicity was measured using the organisms Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. The three cyanobacteria showed the potential to remediate textile effluent by removing the colour and reducing the toxicity. However, the growth of cyanobacteria on sludge was slow and discoloration was not efficient. The cyanobacteria P. autumnale UTEX1580 was the only strain that completely degraded the indigo dye. An evaluation of the mutagenicity potential was performed by use of the micronucleus assay using Allium sp. No mutagenicity was observed after the treatment. Two metabolites were produced during the degradation, anthranilic acid and isatin, but toxicity did not increase after the treatment. The cyanobacteria showed the ability to degrade the dyes present in a textile effluent; therefore, they can be used in a tertiary treatment of effluents with recalcitrant compounds. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Improving shuffler assay accuracy

International Nuclear Information System (INIS)

Rinard, P.M.

1995-01-01

Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year

ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator.

Science.gov (United States)

Ranjan, Rajeev; Priyanka, B S; Thakur, M S

2014-07-01

The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5-10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures.

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders.

Science.gov (United States)

Kristensen, J H; Larsen, L; Dasgupta, B; Brodmerkel, C; Curran, M; Karsdal, M A; Sand, J M B; Willumsen, N; Knox, A J; Bolton, C E; Johnson, S R; Hägglund, P; Svensson, B; Leeming, D J

2015-11-01

Elastin is a signature protein of the lungs. Matrix metalloproteinase-7 (MMP-7) is important in lung defence mechanisms and degrades elastin. However, MMP-7 activity in regard to elastin degradation has never been quantified serologically in patients with lung diseases. An assay for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. Monoclonal antibodies (mABs) were raised against eight carefully selected MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n=123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n=40) and compared with age- and sex-matched controls. The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, pelastin fragments can be quantified in serum and may reflect pathological lung tissue turnover in several important lung diseases. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Volatile fatty acid degradation kinetics in anaerobic process; Cinetica de la degradacion de acidos grasos volatiles en procesos anaerobios

Energy Technology Data Exchange (ETDEWEB)

Riscado, S.; Osuna, B.; Iza, J.; Ruiz, E. [Universidad del Pais Vasco. Bilbao (Spain)

1998-10-01

While searching for the optimal substrate load for anaerobic toxicity assays, the inhibition caused by the propionic acid has been addressed. Lab scale experiments have been carried out to assess the effects of different loads and acid ratios. Results bad been subjected to kinetic analysis and show the degradation follows a first order kinetic, and acetic is easier to degrade than propionic acid. The optimal load for a 100 ml vial assay is composed of 158 mg COD of the 3:1:1 HAc:HPr:HBu mixture. (Author) 9 refs.

Degradation of a chiral nonylphenol isomer in two agricultural soils

International Nuclear Information System (INIS)

Zhang Haifeng; Spiteller, Michael; Guenther, Klaus; Boehmler, Gabriele; Zuehlke, Sebastian

2009-01-01

The degradation of a chiral nonylphenol isomer, 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP 112 ), in two agricultural soils from Monheim and Dortmund, Germany has been studied. The degradation of NP 112 and the formation of a nitro-nonylphenol metabolite were determined by means of GC-MS analysis. The degradation followed bi-exponential order kinetics, with half-life of less than 5 days in both soils. The nitro-metabolite was found at different concentration levels in the two soils. The nitro-metabolite of NP 112 was more persistent than its parent compound. After 150 days about 13% of the initially applied NP 112 remained in the Monheim soil as its nitro-metabolite. Results of the E-screen assay revealed that the nitro-NP 112 has oestrogenic potency of 85% of that of NP 112 . Furthermore, the results of chiral GC-MS analysis revealed that no chiral degradation of NP 112 occurred in this study. - The degradation of a chiral nonylphenol isomer in agricultural soils followed bi-exponential order kinetics resulting in a more persistent nitro-metabolite.

The Prestige oil spill: bacterial community dynamics during a field biostimulation assay

Energy Technology Data Exchange (ETDEWEB)

Jimenez, N; Solanas, A M [Barcelona Univ. (Spain). Dept. of Microbiology; Vinas, M [GIRO Technological Centre, Mollet del Valles (Spain); Bayona, J M; Albaiges, J [IIQAB-CSIC, Barcelona (Spain). Dept. of Environmental Chemistry

2007-12-15

A field bioremediation assay using the oleophilic fertilizer S200 was carried out 12 months after the Prestige heavy fuel-oil spill on a beach on the Cantabrian coast (north Spain). This assay showed that S200-enhanced oil degradation, particularly of high-molecular-weight n-alkanes and alkylated PAHs, suggesting an increase in the microbial bioavailability of these compounds. The bacterial community structure was determined by cultivation-independent analysis of polymerase chain reaction-amplified 16S rDNA by denaturing gradient gel electrophoresis. Bacterial community was mainly composed of {alpha}-Proteobacteria (Rhodobacteriaceae and Sphingomonadaceae). Representatives of {gamma}-Proteobacteria (Chromatiales, Moraxellaceae, and Halomonadaceae), Bacteroidetes (Flavobacteriaceae), and Actinobacteria group (Nocardiaceae and Corynebacteriaceae) were also found. The addition of the fertilizer led to the appearance of the bacterium Mesonia algae in the early stages, with a narrow range of growth substrates, which has been associated with the common alga Achrosiphonia sonderi. The presence of Mesonia algae may be attributable to the response of the microbial community to the addition of N and P rather than indicating a role in the biodegradation process. The Rhodococcus group appeared in both assay plots, especially at the end of the experiment. It was also found at another site on the Galician coast that had been affected by the same spill. This genus has been associated with the degradation of n-alkanes up to C{sub 36}. Taking into account the high content of heavy alkanes in the Prestige fuel, these microorganisms could play a significant role in the degradation of such fuel. A similar bacterial community structure was observed at another site that showed a similar degree of fuel weathering. (orig.)

Furfural and 5-hydroxymethyl-furfural degradation using recombinant manganese peroxidase.

Science.gov (United States)

Yee, Kelsey L; Jansen, Lauren E; Lajoie, Curtis A; Penner, Michael H; Morse, Lettie; Kelly, Christine J

2018-01-01

Biomass pretreatment-derived degradation compounds, such as furfural and 5-hydroxymethyl-furfural (HMF), inhibit the growth of fermentation microorganisms that utilize biomass to produce fuels and chemicals. Here we report that recombinant manganese peroxidase (rMnP) produced from the yeast Pichia pastoris can degrade furfural and HMF making them less toxic to microorganisms. Treatment with rMnP or manganese(III) acetate reduced furfural and HMF concentrations in a dose-dependent manner. Furfural disappearance was accompanied by malonate disappearance and accumulation of four distinct degradation products. Furfural was more readily degraded by rMnP and manganese(III) acetate than HMF. Growth assays using Saccharomyces cerevisiae indicated that rMnP treatment reduced the toxicity of furfural and HMF. This work provides an avenue to use rMnP to increase the growth of fermentation microorganisms that are inhibited by toxic compounds derived from pretreatment of biomass. Copyright © 2017 Elsevier Inc. All rights reserved.

A fluorescence-based rapid screening assay for cytotoxic compounds

International Nuclear Information System (INIS)

Montoya, Jessica; Varela-Ramirez, Armando; Estrada, Abril; Martinez, Luis E.; Garza, Kristine; Aguilera, Renato J.

2004-01-01

A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

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Asther Marcel

2010-07-01

Full Text Available Abstract Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol, it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the

Radioligand purification prior to routine receptor assays

International Nuclear Information System (INIS)

Le Goff, J.-M.; Berthois, Y.; Martin, P.-M.

1988-01-01

The need to repurify the commercially available radioligands [ 3 H]estradiol and [ 3 H]testosterone before use in routine assays was investigated. Storage of these products for 2 months after delivery led to appreciable degradation of [ 3 H]estradiol compared to [ 3 H]testosterone. Unexpectedly, TLC and even HPLC procedures were ineffective in completely restoring the purity of [ 3 H]-estradiol and the unremoved polar products induced important variations in our estrogen receptor assays. An increase in non-specific binding and a concomitant decrease in total binding were observed resulting in an underestimation of specific binding sites and of the affinity constant. In some cases Scatchard analysis was not possible. The authors therefore strongly recommend the repurification of low-stability radioligands and propose an economic time-saving procedure for the purification of [ 3 H]estradiol by solvent differential partition which requires no high-cost investment in apparatus. (author)

Use of statistical analysis to validate ecogenotoxicology findings arising from various comet assay components.

Science.gov (United States)

Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, Khalid Abdullah; Masoud, Muhammad Shahreef; Mahboob, Shahid

2018-04-01

Cirrhinus mrigala, Labeo rohita, and Catla catla are economically important fish for human consumption in Pakistan, but industrial and sewage pollution has drastically reduced their population in the River Chenab. Statistics are an important tool to analyze and interpret comet assay results. The specific aims of the study were to determine the DNA damage in Cirrhinus mrigala, Labeo rohita, and Catla catla due to chemical pollution and to assess the validity of statistical analyses to determine the viability of the comet assay for a possible use with these freshwater fish species as a good indicator of pollution load and habitat degradation. Comet assay results indicated a significant (P comet head diameter, comet tail length, and % DNA damage. Regression analysis and correlation matrices conducted among the parameters of the comet assay affirmed the precision and the legitimacy of the results. The present study, therefore, strongly recommends that genotoxicological studies conduct appropriate analysis of the various components of comet assays to offer better interpretation of the assay data.

Plant exudates promote PCB degradation by a rhodococcal rhizobacteria

Energy Technology Data Exchange (ETDEWEB)

Toussaint, Jean-Patrick; Pham, Thi Thanh My; Barriault, Diane; Sylvestre, Michel [Instiut National de la Recherche Scientifique INRS, Laval, QC (Canada). Inst. Armand-Frappier

2012-09-15

Rhodococcus erythropolis U23A is a polychlorinated biphenyl (PCB)-degrading bacterium isolated from the rhizosphere of plants grown on a PCB-contaminated soil. Strain U23A bphA exhibited 99% identity with bphA1 of Rhodococcus globerulus P6. We grew Arabidopsis thaliana in a hydroponic axenic system, collected, and concentrated the plant secondary metabolite-containing root exudates. Strain U23A exhibited a chemotactic response toward these root exudates. In a root colonizing assay, the number of cells of strain U23A associated to the plant roots (5.7 x 105 CFU g{sup -1}) was greater than the number remaining in the surrounding sand (4.5 x 104 CFU g{sup -1}). Furthermore, the exudates could support the growth of strain U23A. In a resting cell suspension assay, cells grown in a minimal medium containing Arabidopsis root exudates as sole growth substrate were able to metabolize 2,3,4'- and 2,3',4-trichlorobiphenyl. However, no significant degradation of any of congeners was observed for control cells grown on Luria-Bertani medium. Although strain U23A was unable to grow on any of the flavonoids identified in root exudates, biphenyl-induced cells metabolized flavanone, one of the major root exudate components. In addition, when used as co-substrate with sodium acetate, flavanone was as efficient as biphenyl to induce the biphenyl catabolic pathway of strain U23A. Together, these data provide supporting evidence that some rhodococci can live in soil in close association with plant roots and that root exudates can support their growth and trigger their PCB-degrading ability. This suggests that, like the flagellated Gram-negative bacteria, non-flagellated rhodococci may also play a key role in the degradation of persistent pollutants. (orig.)

Determination of protective properties of Bardejovske Kuple spa curative waters by rotational viscometry and ABTS assay

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TOPOLSKA Dominika

2014-02-01

Full Text Available Mineral waters from Bardejovske Kupele spa are natural, strongly mineralized, with healing effects. They are classified as hydrocarbonic - containing chloride and sodium - carbonic, cold, hypotonic, with a relatively high boric acid content. Potential anti-oxidative effects of curative waters from Bardejovske Kupele were investigated against the hyaluronan (HA degradation. High-molar-mass HA was exposed to the action of ascorbate and cupric ions, which initiate free-radical chain degradation. Time-dependent changes of dynamic viscosity (h of the HA solutions were monitored by rotational viscometry. The radical scavenging capacity of curative waters was determined by the ABTS assay. Despite a significantly high content of transition metal ions, especially iron, remarkable protective effects of the two curative spa waters were found, namely Alzbeta and Klara. Even though “Alzbeta´s“ iron content was 3.5-fold higher than “Klara´s“, “Alzbeta“ was shown to have better protective properties against the HA degradation compared to “Klara“. Bolus addition of ferric ions to the reaction system instead of the natural iron-containing curative water caused a significant HA degradation. The ABTS decolorization assay revealed that the curative spa waters were proven as poorly effective scavengers of the ABTS·+ cation radical.

Application of the comet assay in studies of programmed cell death (PCD in plants

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Maria Charzyńska

2014-01-01

Full Text Available Programmed cell death (PCD in plants is an intensively investigated process. One of the main characteristics of PCD in both animal and plant organisms is the non-random, internucleosomal fragmentation of nuclear DNA, usually analysed using total DNA gel electrophoresis or TUNEL method. In this paper we present application of the "comet assay" (Single Cell Gel Electrophoresis for detection of nDNA degradation in studies of PCD during plant life cycle. We analyzed three types of tissue: anther tapetum, endosperm and mesophyll which were prepared in different ways to obtain a suspension of viable cells (without cell walls. The comet assay gives a possibility of examination of the nDNA degradation in individual cell. This method is significant for studies of the plant tissue differentiation and senescence especially in the cases when it is not possible to isolate large number of cells at the same developmental stage.

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples.

Science.gov (United States)

Westen, Antoinette A; Matai, Anuska S; Laros, Jeroen F J; Meiland, Hugo C; Jasper, Mandy; de Leeuw, Wiljo J F; de Knijff, Peter; Sijen, Titia

2009-09-01

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.

Purple Corn (Zea mays L.) Phenolic Compounds Profile and Its Assessment as an Agent Against Oxidative Stress in Isolated Mouse Organs

Science.gov (United States)

Ramos-Escudero, Fernando; Muñoz, Ana María; Alvarado-Ortíz, Carlos; Alvarado, Ángel

2012-01-01

Abstract This study was designed to determine the contents of total polyphenols, flavonoids, flavonols, flavanols, and anthocyanins of purple corn (Zea mays L.) extracts obtained with different methanol:water concentrations, acidified with 1% HCl (1 N). Another objective was to determine the antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and deoxyribose assay, individual phenolic compounds by high-performance liquid chromatography (HPLC), and endogenous antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and total peroxidase [TPX]) activity and lipid peroxidation activity (thiobarbituric acid–reactive substances [TBARS] assay) in isolated mouse organs. Overall, the highest total content of polyphenols, anthocyanins, flavonoids, flavonols, and flavanols was obtained with the 80:20 methanol:water extract, acidified with 1% HCl (1 N). The 50% inhibitory concentration values obtained by the DPPH and ABTS assays with this extract were 66.3 μg/mL and 250 μg/mL, respectively. The antioxidant activity by the FRAP assay was 26.1 μM Trolox equivalents/g, whereas the deoxyribose assay presented 93.6% inhibition. Because of these results, the 80:20 methanol:water extract, acidified with 1% HCl (1 N), was used for the remaining tests. Eight phenolic compounds were identified by HPLC: chlorogenic acid, caffeic acid, rutin, ferulic acid, morin, quercetin, naringenin, and kaempferol. Furthermore, it was observed that the purple corn extract was capable of significantly reducing lipid peroxidation (lower malondialdehyde [MDA] concentrations by the TBARS assay) and at the same time increasing endogenous antioxidant enzyme (CAT, TPX, and SOD) activities in isolated mouse kidney, liver, and brain. On the basis of the results, it was concluded that the purple corn extract contained various bioactive phenolic compounds that exhibited

Purple corn (Zea mays L.) phenolic compounds profile and its assessment as an agent against oxidative stress in isolated mouse organs.

Science.gov (United States)

Ramos-Escudero, Fernando; Muñoz, Ana María; Alvarado-Ortíz, Carlos; Alvarado, Ángel; Yáñez, Jaime A

2012-02-01

This study was designed to determine the contents of total polyphenols, flavonoids, flavonols, flavanols, and anthocyanins of purple corn (Zea mays L.) extracts obtained with different methanol:water concentrations, acidified with 1% HCl (1 N). Another objective was to determine the antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and deoxyribose assay, individual phenolic compounds by high-performance liquid chromatography (HPLC), and endogenous antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and total peroxidase [TPX]) activity and lipid peroxidation activity (thiobarbituric acid-reactive substances [TBARS] assay) in isolated mouse organs. Overall, the highest total content of polyphenols, anthocyanins, flavonoids, flavonols, and flavanols was obtained with the 80:20 methanol:water extract, acidified with 1% HCl (1 N). The 50% inhibitory concentration values obtained by the DPPH and ABTS assays with this extract were 66.3 μg/mL and 250 μg/mL, respectively. The antioxidant activity by the FRAP assay was 26.1 μM Trolox equivalents/g, whereas the deoxyribose assay presented 93.6% inhibition. Because of these results, the 80:20 methanol:water extract, acidified with 1% HCl (1 N), was used for the remaining tests. Eight phenolic compounds were identified by HPLC: chlorogenic acid, caffeic acid, rutin, ferulic acid, morin, quercetin, naringenin, and kaempferol. Furthermore, it was observed that the purple corn extract was capable of significantly reducing lipid peroxidation (lower malondialdehyde [MDA] concentrations by the TBARS assay) and at the same time increasing endogenous antioxidant enzyme (CAT, TPX, and SOD) activities in isolated mouse kidney, liver, and brain. On the basis of the results, it was concluded that the purple corn extract contained various bioactive phenolic compounds that exhibited considerable in vitro

Pyrene degradation by yeasts and filamentous fungi.

Science.gov (United States)

Romero, M Cristina; Salvioli, Mónica L; Cazau, M Cecilia; Arambarri, A M

2002-01-01

The saprotrophic soil fungi Fusarium solani (Mart.) Sacc., Cylindrocarpon didymum (Hartig) Wollenw, Penicillium variabile Sopp. and the yeasts Rhodotorula glutinis (Fresenius) Harrison and Rhodotorula minuta (Saito) Harrison were cultured in mineral medium with pyrene. The remaining pyrene concentrations were periodically determined during 20 incubation days, using HPLC. To assess the metabolism of pyrene degradation we added 0.1 microCi of [4,5,9,10] 14C-pyrene to each fungi culture and measured the radioactivity in the volatile organic substances, extractable, aqueous phase, biomass and 14CO2 fractions. The assays demonstrated that F. solani and R. glutinis metabolized pyrene as a sole source of carbon. Differences in their activities at the beginning of the cultures disappeared by the end of the experiment, when 32 and 37% of the original pyrene concentration was detected, for the soil fungi and yeasts, respectively. Among the filamentous fungi, F. solani was highly active and oxidized pyrene; moreover, small but significant degradation rates were observed in C. didymum and P. variahile cultures. An increase in the 14CO2 evolution was observed at the 17th day with cosubstrate. R. glutinis and R. minuta cultures showed similar ability to biotransform pyrene, and that 35% of the initial concentration was consumed at the end of the assay. The same results were obtained in the experiments with or without glucose as cosubstrate.

Non-covalent interaction between polyubiquitin and GTP cyclohydrolase 1 dictates its degradation.

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Yu Zhao

Full Text Available GTP cyclohydrolase 1 (GTPCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4. GTPCH1 protein degradation has been reported in animal models of several diseases, including diabetes mellitus and hypertension. However, the molecular mechanisms by which GTPCH1 is degraded remain uncharacterized. Here we report a novel non-covalent interaction between polyubiquitin and GTPCH1 in vitro and in vivo. The non-covalent binding of GTPCH1 to polyubiquitin via an ubiquitin-binding domain (UBD results in ubiquitination and degradation. Ectopic expression of ubiquitin in cultured cells accelerated GTPCH1 degradation. In cultured cells and in vitro assays, Lys48-linked ubiquitin chains, but not Lys63-linked chains, interacted with GTPCH1 and targeted it for degradation. Consistently, proteasome inhibition attenuated GTPCH1 degradation. Finally, direct mutagenesis of an isoleucine (Ile131 in the hydrophobic patch of the GTPCH1 UBD affected its ubiquitin binding and the enzyme stability. Taken together, we conclude that GTPCH1 non-covalently interacts with polyubiquitin via an ubiquitin-binding domain. The polyubiquitin binding directs GTPCH1 ubiquitination and proteasome degradation.

Research of the degradation products of chitosan's angiogenic function

International Nuclear Information System (INIS)

Wang Jianyun; Chen Yuanwei; Ding Yulong; Shi Guoqi; Wan Changxiu

2008-01-01

Angiogenesis is of great importance in tissue engineering and has gained large attention in the past decade. But how it will be influenced by the biodegradable materials, especially their degradation products, remains unknown. Chitosan (CS) is a kind of naturally occurred polysaccharide which can be degraded in physiological environment. In order to gain some knowledge of the influences of CS degradation products on angiogenesis, the interaction of vascular endothelial cells with the degradation products was investigated in the present study. The CS degradation products were prepared by keeping CS sample in physiological saline aseptically at 37 deg. C for 120 days. Endothelial cells were co-cultured with the degradation products and the angiogenic cell behaviors, including cell proliferation, migration and tube-like structure (TLS) formation, were tested by MTT assay, cell migration quantification method (CMQM), and tube-like structure quantification method (TLSQM) respectively. Furthermore, mRNA expressions of vascular endothelial growth factor (VEGF) and matrix metallo proteinase (MMP-2) were determined by real-time reverse transcriptional polymerase chain reaction (RT-PCR). Physiological saline served as a negative control. As the results showed, the degradation products obtained from 20th to 60th day significantly inhibited the proliferation, migration, and TLS formation of endothelial cells. However, degradation products of the first 14 days and the last 30 days were found to be proangiogenic. At the molecular level, the initial results indicated that the mRNA expressions of VEGF and MMP-2 were increased by the degradation products of 7th day, but were decreased by the ones of 60th day. According to all the results, it could be concluded that the angiogenic behaviors of endothelial cells at both cellular and molecular level could be significantly stimulated or suppressed by the degradation products of CS and the influences are quite time-dependent

Matrix metalloproteinase activity assays: Importance of zymography.

Science.gov (United States)

Kupai, K; Szucs, G; Cseh, S; Hajdu, I; Csonka, C; Csont, T; Ferdinandy, P

2010-01-01

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways. Copyright 2010 Elsevier Inc. All rights reserved.

Lignin-degrading enzyme activities.

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Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan

2012-01-01

Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays.

Degradation of Akt using protein-catalyzed capture agents.

Science.gov (United States)

Henning, Ryan K; Varghese, Joseph O; Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M; Heath, James R

2016-04-01

Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein-catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein-based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.

Science.gov (United States)

Watzlawick, Hildegard; Morabbi Heravi, Kambiz; Altenbuchner, Josef

2016-10-15

Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is

Bioremediation of lignosulphonates by lignin-degrading basidiomycetous fungi.

Science.gov (United States)

Eugenio, M E; Carbajo, J M; Terrón, M C; González, A E; Villar, J C

2008-07-01

The capability of some ligninolytic fungi to degrade lignosulphonates has been studied. Three lignosulphonates concentrations, three culture media and seven different basidiomycetes in solid-cultures have been assayed to select the conditions for further experiments on submerged cultures. The best results of growth and lignosulphonate decolourization in solid-cultures were obtained with Pycnoporus sanguineus, Coriolus pubescens and Trametes sp. I-62 on Kirk's medium and 1% and 2% of lignosulphonate concentrations. In submerged cultures the lignosulphonate decolourization rate was generally higher when it was added on the 6th day, rather than when it was added from the beginning of the incubation and C. pubescens and P. sanguineus showed again the optimum results of decolourization. Extracellular laccase activity increased with lignosulphonate concentration in all assayed fungi, suggesting that lignosulphonate act as inductors of laccase activity.

Enhanced degradation activity by endophytic bacteria of plants growing in hydrocarbon contaminated soil

Energy Technology Data Exchange (ETDEWEB)

Phillips, L.; Germida, J.J. [Saskatchewan Univ., Saskatoon, SK (Canada); Greer, C.W. [National Research Council of Canada, Montreal, PQ (Canada). Biotechnology Research Inst.

2006-07-01

The feasibility of using phytoremediation for cleaning soils contaminated with petroleum hydrocarbons was discussed. Petroleum hydrocarbons are problematic because of their toxicity, mobility and persistence in the environment. Appropriate clean-up methods are needed, given that 60 per cent of Canada's contaminated sites contain these compounds. Phytoremediation is an in situ biotechnology in which plants are used to facilitate contaminant removal. The approach relies on a synergistic relationship between plants and their root-associated microbial communities. Previous studies on phytoremediation have focussed on rhizosphere communities. However, it is believed that endophytic microbes may also play a vital role in organic contaminant degradation. This study investigated the structural and functional dynamics of both rhizosphere and endophytic microbial communities of plants from a phytoremediation field site in south-eastern Saskatchewan. The former flare pit contains up to 10,000 ppm of F3 to F4 hydrocarbon fractions. Root samples were collected from tall wheatgrass, wild rye, saltmeadow grass, perennial ryegrass, and alfalfa. Culture-based and culture-independent methods were used to evaluate the microbial communities associated with these roots. Most probable number assays showed that the rhizosphere communities contained more n-hexadecane, diesel fuel, and PAH degraders. However, mineralization assays with 14C labelled n-hexadecane, naphthalene, and phenanthrene showed that endophytic communities had more degradation activities per standardized initial degrader populations. Total community DNA samples taken from bulk, rhizosphere, and endophytic samples, were analyzed by denaturing gradient gel electrophoresis. It was shown that specific bacteria increased in endophytic communities compared to rhizosphere communities. It was suggested plants may possibly recruit specific bacteria in response to hydrocarbon contamination, thereby increasing degradation

Application of fluorescent antibody and enzyme-linked immunosorbent assays for TCE and PAH degrading bacteria

Energy Technology Data Exchange (ETDEWEB)

Brigmon, R.L.; Franck, M.; Brey, J.; Scott, D.; Lanclos, K.; Fliermans, C.

1996-07-01

Historically, methods used to identify methanotrophic and polyaromatic hydrocarbon-degrading (PAH) bacteria in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate specific bacteria from other environmental microorganisms. Methanotrophic bacteria have been isolated and characterized from TCE-contaminated soils (Bowman et al. 1993; Fliermans et al., 1988). Fliermans et al., (1988) and others demonstrated that cultures enriched with methane and propane could cometabolically degrade a wide variety of chlorinated aliphatic hydrocarbons including ethylene; 1,2-cisdichloroethylene (c-DCE); 1,2-trans-dichloroethylene (t-DCE); vinyl chloride (VC); toluene; phenol and cresol. Characterization of select microorganisms in the natural setting is important for the evaluation of bioremediation potential and its effectiveness. This realization has necessitated techniques that are selective, sensitive and easily applicable to soils, sediments, and groundwater (Fliermans, et al., 1994). Additionally these techniques can identify and quantify microbial types in situ in real time

Optimization of Phenanthrene-degradation by Dietzia Cinnamea AP for Bioremediation Applications

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Zahra Fathi

2016-07-01

Full Text Available Background: This study focused on isolation and identification of a Phenanthrene (Phe degrader bacterium and optimization of environmental conditions for Phe degradation. Materials and Methods: Enrichment technique was used for isolation and the most effective isolate; named AP was selected based on its Phe biodegradation abilities. The isolate was identified using morphological and biochemical tests as well as16S rDNA sequencing. The effects of various factors such as temperature, pH and C/N on bacterial growth and Phe degradation were investigated using protein assay (Bradford and Gas Chromatography (GC, respectively. Results: The selected isolate was identified as Dietzia cinnamea AP. It was able to degrade Phe at pH 6-10 (optimum at 8, temperatures of 25 -45 °C (optimum at 35 °C and NH4Cl concentrations of 0.5-2.5 gL-1 (optimum at 2 g L-1. By optimization of environmental parameters, within 10 days of fermentation, Phe degradation rate increased by more than 1.2 fold (from 60% to 73%. Conclusion: D. cinnamea AP was found to be an appropriate candidate for bioremediation applications. To the best of our knowledge, this is the first report of D. cinnamea species that can degrade Phe.

Isolation and characterization of diuron-degrading bacteria from lotic surface water.

Science.gov (United States)

Batisson, Isabelle; Pesce, Stéphane; Besse-Hoggan, Pascale; Sancelme, Martine; Bohatier, Jacques

2007-11-01

The bacterial community structure of a diuron-degrading enrichment culture from lotic surface water samples was analyzed and the diuron-degrading strains were selected using a series of techniques combining temporal temperature gradient gel electrophoresis (TTGE) of 16 S rDNA gene V1-V3 variable regions, isolation of strains on agar plates, colony hybridization methods, and biodegradation assays. The TTGE fingerprints revealed that diuron had a strong impact on bacterial community structure and highlighted both diuron-sensitive and diuron-adapted bacterial strains. Two bacterial strains, designated IB78 and IB93 and identified as belonging to Pseudomonas sp. and Stenotrophomonas sp., were isolated and shown to degrade diuron in pure resting cells in a first-order kinetic reaction during the first 24 h of incubation with no 3,4-DCA detected. The percentages of degradation varied from 25% to 60% for IB78 and 20% to 65% for IB93 and for a diuron concentration range from 20 mg/L to 2 mg/L, respectively. It is interesting to note that diuron was less degraded by single isolates than by mixed resting cells, thereby underlining a cumulative effect between these two strains. To the best of our knowledge, this is the first report of diuron-degrading strains isolated from lotic surface water.

Characterization of native fungi responsible for degrading crude oil from the coastal area of Yanbu, Saudi Arabia

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Amr Abd-EL-Mooti EL-Hanafy

2017-01-01

Full Text Available A total of 15 fungal isolates were obtained from oil-contaminated sites near the Red Sea in the Yanbu region. Based on the preliminary DCPIP (2,6-dichlorophenolindophenol assay, three isolates showed promising oil degrading ability. The next-generation sequencing of the ITS-I and ITS-II internal transcribed spacer regions assigned the isolates to Aspergillus and Penicillium. Among these three strains, Y2 (Aspergillus oryzae was the most efficient, degrading about 99% of the crude oil. The degradation rates were corroborated using spectrophotometric and gas chromatography–mass spectrometry analyses after two weeks of cultivation in Bushnell–Haas medium. All the three strains proved to be potent oil-degrading strains and, hence, can be utilized to degrade oil contaminants.

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

Energy Technology Data Exchange (ETDEWEB)

Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

2011-09-10

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

International Nuclear Information System (INIS)

Bloch, Donald B.; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

2011-01-01

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: → A two-hybrid assay was developed to study interactions in macromolecular complexes. → The assay was applied to interactions between components of mRNA P-bodies. → The assay effectively and efficiently identified protein interaction domains. → P-body assembly in mammalian cells differs from that in other species.

Stability-Indicating RP-HPLC Method for Assay of Silver Lactate

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V. Srinivasan

2011-01-01

Full Text Available A simple, economic and time-efficient stability-indicating, reverse-phase high-performance liquid chromatographic (RP-HPLC method has been developed for analysis of silver lactate in the presence of degradation products generated by decomposition. When silver lactate was subjected to acid hydrolysis, base hydrolysis, oxidative, photolytic, humidity and thermal stress, degradation was observed during base hydrolysis, oxidation, humidity and thermal stress. The drug was found to be stable to other stress conditions. Successful chromatographic condition of the drug from the degradation products formed under stress conditions was achieved on a phenomenex Gemini column with potassium dihydrogen phosphate buffer, pH adjusted to 2.2 with orthophosphoric acid, as mobile phase. The method was validated for linearity, precision, specificity and robustness and can be used for quality-control during manufacture and assessment of the stability of samples of silver lactate. To the best of our knowledge, a validated stability-indicating LC assay method for silver lactate based on lactic acid is reported for the first time.

A validated stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant.

Science.gov (United States)

Lalitha Devi, M; Chandrasekhar, K B

2009-12-05

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).

Neuroprotective mechanism of Kai Xin San: upregulation of hippocampal insulin-degrading enzyme protein expression and acceleration of amyloid-beta degradation

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Na Wang

2017-01-01

Full Text Available Kai Xin San is a Chinese herbal formula composed of Radix Ginseng , Poria , Radix Polygalae and Acorus Tatarinowii Rhizome . It has been used in China for many years for treating amnesia. Kai Xin San ameliorates amyloid-β (Aβ-induced cognitive dysfunction and is neuroprotective in vivo , but its precise mechanism remains unclear. Expression of insulin-degrading enzyme (IDE, which degrades Aβ, is strongly correlated with cognitive function. Here, we injected rats with exogenous Aβ42 (200 μM, 5 μL into the hippocampus and subsequently administered Kai Xin San (0.54 or 1.08 g/kg/d intragastrically for 21 consecutive days. Hematoxylin-eosin and Nissl staining revealed that Kai Xin San protected neurons against Aβ-induced damage. Furthermore, enzyme-linked immunosorbent assay, western blot and polymerase chain reaction results showed that Kai Xin San decreased Aβ42 protein levels and increased expression of IDE protein, but not mRNA, in the hippocampus. Our findings reveal that Kai Xin San facilitates hippocampal Aβ degradation and increases IDE expression, which leads, at least in part, to the alleviation of hippocampal neuron injury in rats.

ABTS assay of phenol oxidase activity in soil.

Science.gov (United States)

Floch, Carine; Alarcon-Gutiérrez, Enrique; Criquet, Stéven

2007-12-01

Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 degrees C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 microl of a 0.1 M ABTS solution.

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

Science.gov (United States)

Ismail, Heba M; Yamamoto, Kazuhiro; Vincent, Tonia L; Nagase, Hideaki; Troeberg, Linda; Saklatvala, Jeremy

2015-07-01

Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its

Nondestructive evaluation of a new hydrolytically degradable and photo-clickable PEG hydrogel for cartilage tissue engineering.

Science.gov (United States)

Neumann, Alexander J; Quinn, Timothy; Bryant, Stephanie J

2016-07-15

Photopolymerizable and hydrolytically labile poly(ethylene glycol) (PEG) hydrogels formed from photo-clickable reactions were investigated as cell delivery platforms for cartilage tissue engineering (TE). PEG hydrogels were formed from thiol-norbornene PEG macromers whereby the crosslinks contained caprolactone segments with hydrolytically labile ester linkages. Juvenile bovine chondrocytes encapsulated in the hydrogels were cultured for up to four weeks and assessed biochemically and histologically, using standard destructive assays, and for mechanical and ultrasound properties, as nondestructive assays. Bulk degradation of acellular hydrogels was confirmed by a decrease in compressive modulus and an increase in mass swelling ratio over time. Chondrocytes deposited increasing amounts of sulfated glycosaminoglycans and collagens in the hydrogels with time. Spatially, collagen type II and aggrecan were present in the neotissue with formation of a territorial matrix beginning at day 21. Nondestructive measurements revealed an 8-fold increase in compressive modulus from days 7 to 28, which correlated with total collagen content. Ultrasound measurements revealed changes in the constructs over time, which differed from the mechanical properties, and appeared to correlate with ECM structure and organization shown by immunohistochemical analysis. Overall, non-destructive and destructive measurements show that this new hydrolytically degradable PEG hydrogel is promising for cartilage TE. Designing synthetic hydrogels whose degradation matches tissue growth is critical to maintaining mechanical integrity as the hydrogel degrades and new tissue forms, but is challenging due to the nature of the hydrogel crosslinks that inhibit diffusion of tissue matrix molecules. This study details a promising, new, photo-clickable and synthetic hydrogel whose degradation supports cartilaginous tissue matrix growth leading to the formation of a territorial matrix, concomitant with an

EurEAs_Gplex--A new SNaPshot assay for continental population discrimination and gender identification.

Science.gov (United States)

Daca-Roszak, P; Pfeifer, A; Żebracka-Gala, J; Jarząb, B; Witt, M; Ziętkiewicz, E

2016-01-01

Assays that allow analysis of the biogeographic origin of biological samples in a standard forensic laboratory have to target a small number of highly differentiating markers. Such markers should be easy to multiplex and the assay must perform well in the degraded and scarce biological material. SNPs localized in the genome regions, which in the past were subjected to differential selective pressure in various populations, are the most widely used markers in the studies of biogeographic affiliation. SNPs reflecting biogeographic differences not related to any phenotypic traits are not sufficiently explored. The goal of our study was to identify a small set of SNPs not related to any known pigmentation/phenotype-specific genes, which would allow efficient discrimination between populations of Europe and East Asia. The selection of SNPs was based on the comparative analysis of representative European and Chinese/Japanese samples (B-lymphocyte cell lines), genotyped using the Infinium HumanOmniExpressExome microarray (Illumina). The classifier, consisting of 24 unlinked SNPs (24-SNP classifier), was selected. The performance of a 14-SNP subset of this classifier (14-SNP subclassifier) was tested using genotype data from several populations. The 14-SNP subclassifier differentiated East Asians, Europeans and Africans with ∼100% accuracy; Palestinians, representative of the Middle East, clustered with Europeans, while Amerindians and Pakistani were placed between East Asian and European populations. Based on these results, we have developed a SNaPshot assay (EurEAs_Gplex) for genotyping SNPs from the 14-SNP subclassifier, combined with an additional marker for gender identification. Forensic utility of the EurEAs_Gplex was verified using degraded and low quantity DNA samples. The performance of the EurEAs_Gplex was satisfactory when using degraded DNA; tests using low quantity DNA samples revealed a previously not described source of genotyping errors, potentially

Bovine intestinal bacteria inactivate and degrade ceftiofur and ceftriaxone with multiple beta-lactamases.

Science.gov (United States)

Wagner, R Doug; Johnson, Shemedia J; Cerniglia, Carl E; Erickson, Bruce D

2011-11-01

The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinder-plate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatography-mass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific β-lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum β-lactamases and metallo-β-lactamases. Seven isolates of Bacteroides spp. produced multiple β-lactamases, including possibly CepA, and metallo-β-lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading β-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that β-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple β-lactamases from bacteria in the large intestines of cattle.

Bovine Intestinal Bacteria Inactivate and Degrade Ceftiofur and Ceftriaxone with Multiple β-Lactamases▿

Science.gov (United States)

Wagner, R. Doug; Johnson, Shemedia J.; Cerniglia, Carl E.; Erickson, Bruce D.

2011-01-01

The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinder-plate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatography-mass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific β-lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum β-lactamases and metallo-β-lactamases. Seven isolates of Bacteroides spp. produced multiple β-lactamases, including possibly CepA, and metallo-β-lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading β-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that β-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple β-lactamases from bacteria in the large intestines of cattle. PMID:21876048

Assaying the reporter gene chloramphenicol acetyltransferase

International Nuclear Information System (INIS)

Crabb, D.W.; Minth, C.D.; Dixon, J.E.

1989-01-01

These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product

Evaluation of the influence of fluoroquinolone chemical structure on stability: forced degradation and in silico studies

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André Valle de Bairros

2018-05-01

Full Text Available ABSTRACT Fluoroquinolones are a known antibacterial class commonly used around the world. These compounds present relative stability and they may show some adverse effects according their distinct chemical structures. The chemical hydrolysis of five fluoroquinolones was studied using alkaline and photolytic degradation aiming to observe the differences in molecular reactivity. DFT/B3LYP-6.31G* was used to assist with understanding the chemical structure degradation. Gemifloxacin underwent degradation in alkaline medium. Gemifloxacin and danofloxacin showed more degradation perceptual indices in comparison with ciprofloxacin, enrofloxacin and norfloxacin in photolytic conditions. Some structural features were observed which may influence degradation, such as the presence of five member rings attached to the quinolone ring and the electrostatic positive charges, showed in maps of potential electrostatic charges. These measurements may be used in the design of effective and more stable fluoroquinolones as well as the investigation of degradation products from stress stability assays.

Quantification of hydroxyl radical produced during phacoemulsification.

Science.gov (United States)

Gardner, Jonathan M; Aust, Steven D

2009-12-01

To quantitate hydroxyl radicals produced during phacoemulsification with various irrigating solutions and conditions used in cataract surgery. Chemistry and Biochemistry Department, Utah State University, Logan, Utah, USA. All experiments were performed using an Infiniti Vision System phacoemulsifier with irrigation and aspiration. Hydroxyl radicals were quantitated using electron spin resonance spectroscopy and a spectrophotometric assay for malondialdehyde, which is formed by the oxidation of deoxyribose by the hydroxyl radical. Hydroxyl radical production increased during longitudinal-stroking phacoemulsification as power levels were increased in a nonlinear, nonexponential fashion. The detection of hydroxyl radical was reduced in irrigating solutions containing organic molecules (eg, citrate, acetate, glutathione, dextrose) and further reduced in Navstel, an irrigating solution containing a viscosity-modifying agent, hydroxypropyl methylcellulose. Hydroxyl radicals produced in settings representative of those used in phacoemulsification cataract surgery were quantitated using the deoxyribose method. Hydroxyl radical production was dependent on the level of ultrasound power applied and the irrigating solution used. Oxidative stress on the eye during phacoemulsification may be minimized by using irrigating solutions that contain organic molecules, including the viscosity-modifying agent hydroxypropyl methylcellulose, that can compete for reaction with hydroxyl radicals.

Novel types of DNA-sugar damage in neocarzinostatin cytotoxicity and mutagenesis

International Nuclear Information System (INIS)

Goldberg, I.H.

1986-01-01

Although a number of antitumor antibiotics interact with DNA to form covalent adducts with the bases, relatively few damage DNA by interacting with the deoxyribose moiety. Neocarzinostatin (NCS), a member of a family of macromolecular antibiotics obtained from filtrates of Streptomyces, is such an agent. Many of the biochemical and cellular effects of NCS resemble those of ionizing radiation. Most, possibly all, of the DNA lesions caused by NCS appear to result from the direct attack of an activated form of the drug on the deoxyribose of DNA. This is to be contrasted with ionizing radiation or the antibiotic bleomycin, that damage DNA deoxyribose through the intervention of a reduced form of oxygen. This paper describes the nature of the interaction between the active component of NCS and DNA, on the mechanism of the ensuing deoxyribose damage, and on some of the biological consequences of these actions. 24 refs., 7 figs

Metagenome enrichment approach used for selection of oil-degrading bacteria consortia for drill cutting residue bioremediation.

Science.gov (United States)

Guerra, Alaine B; Oliveira, Jorge S; Silva-Portela, Rita C B; Araújo, Wydemberg; Carlos, Aline C; Vasconcelos, Ana Tereza R; Freitas, Ana Teresa; Domingos, Yldeney Silva; de Farias, Mirna Ferreira; Fernandes, Glauber José Turolla; Agnez-Lima, Lucymara F

2018-04-01

Drill cuttings leave behind thousands of tons of residues without adequate treatment, generating a large environmental liability. Therefore knowledge about the microbial community of drilling residue may be useful for developing bioremediation strategies. In this work, samples of drilling residue were enriched in different culture media in the presence of petroleum, aiming to select potentially oil-degrading bacteria and biosurfactant producers. Total DNA was extracted directly from the drill cutting samples and from two enriched consortia and sequenced using the Ion Torrent platform. Taxonomic analysis revealed the predominance of Proteobacteria in the metagenome from the drill cuttings, while Firmicutes was enriched in consortia samples. Functional analysis using the Biosurfactants and Biodegradation Database (BioSurfDB) revealed a similar pattern among the three samples regarding hydrocarbon degradation and biosurfactants production pathways. However, some statistical differences were observed between samples. Namely, the pathways related to the degradation of fatty acids, chloroalkanes, and chloroalkanes were enriched in consortia samples. The degradation colorimetric assay using dichlorophenolindophenol as an indicator was positive for several hydrocarbon substrates. The consortia were also able to produce biosurfactants, with biosynthesis of iturin, lichnysin, and surfactin among the more abundant pathways. A microcosms assay followed by gas chromatography analysis showed the efficacy of the consortia in degrading alkanes, as we observed a reduction of around 66% and 30% for each consortium in total alkanes. These data suggest the potential use of these consortia in the bioremediation of drilling residue based on autochthonous bioaugmentation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Forced degradation studies of lansoprazole using LC-ESI HRMS and 1 H-NMR experiments: in vitro toxicity evaluation of major degradation products.

Science.gov (United States)

Shankar, G; Borkar, R M; Suresh, U; Guntuku, L; Naidu, V G M; Nagesh, N; Srinivas, R

2017-07-01

Regulatory agencies from all over the world have set up stringent guidelines with regard to drug degradation products due to their toxic effects or carcinogenicity. Lansoprazole, a proton-pump inhibitor, was subjected to forced degradation studies as per ICH guidelines Q1A (R2). The drug was found to degrade under acidic, basic, neutral hydrolysis and oxidative stress conditions, whereas it was found to be stable under thermal and photolytic conditions. The chromatographic separation of the drug and its degradation products were achieved on a Hiber Purospher, C18 (250 × 4.6 mm, 5 μ) column using 10 mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0 ml/min. The eight degradation products (DP1-8) were identified and characterized by UPLC/ESI/HRMS with in-source CID experiments combined with accurate mass measurements. DP-1, DP-2 and DP-3 were formed in acidic, DP-4 in basic, DP-5 in neutral and DP-1, DP-6, DP-7 and DP-8 were in oxidation stress condition Among eight degradation products, five were hitherto unknown degradation products. In addition, one of the major degradation products, DP-2, was isolated by using semi preparative HPLC and other two, DP-6 and DP-7 were synthesized. The cytotoxic effect of these degradation products (DP-2, DP-6 and DP-7) were tested on normal human cells such as HEK 293 (embryonic kidney cells) and RWPE-1(normal prostate epithelial cells) by MTT assay. From the results of cytotoxicity, it was found that lansoprazole as well as its degradation products (DP-2, DP-6 and DP-7) were nontoxic up to 50-μM concentrations, and the latter showed slightly higher cytotoxicity when compared with that of lansoprazole. DNA binding studies using spectroscopic techniques indicate that DP-2, DP-6 and DP-7 molecules interact with ctDNA and may bind to its surface. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Radiation degradation and hemolytic toxicity evaluation of mono azo reactive dyes

International Nuclear Information System (INIS)

Saeed, Q.U.; Bhatti, I.A.; Ashraf, A.

2017-01-01

Monoazo reactive dyes have been synthesized and subjected to degradation before their application. Advanced oxidation process has been recognized as a promising radiation technology for the remediation of hazardous organic compounds. Radiation induced degradation of two mono azo reactive dyes have been tried at different absorbed dose, 5 kGy,10 kGy and 15 kGy. Aqueous solutions of these dyes were treated with gamma radiation using Cs 137 radiation source at Nuclear Institute of Agriculture and Biology (NIAB) Faisalabad. Dyes were evaluated spectrophotometrically by UV-visible and fourier transform infra red (FT-IR) spectroscopic techniques before and after irradiation to analyse their percentage decolorization and degradation. Maximum percentage decolorization of 93% and 63% was achieved for mono azo dyes D1 and D2 at 15 kGy absorbed dose. Toxicity study of these dyes was also tested by haemolytic activity assay. Percentage haemolytic activity of untreated dyes was found within permissible limit showing non toxicity of dye solutions. (author)

Sida tuberculata (Malvaceae): a study based on development of extractive system and in silico and in vitro properties.

Science.gov (United States)

da Rosa, H S; Salgueiro, A C F; Colpo, A Z C; Paula, F R; Mendez, A S L; Folmer, V

2016-07-11

Sida tuberculata (Malvaceae) is a medicinal plant traditionally used in Brazil as an antimicrobial and anti-inflammatory agent. Here, we aimed to investigate the different extractive techniques on phytochemical parameters, as well as to evaluate the toxicity and antioxidant capacity of S. tuberculata extracts using in silico and in vitro models. Therefore, in order to determine the dry residue content and the main compound 20-hydroxyecdysone (20E) concentration, extracts from leaves and roots were prepared testing ethanol and water in different proportions. Extracts were then assessed by Artemia salina lethality test, and toxicity prediction of 20E was estimated. Antioxidant activity was performed by DPPH and ABTS radical scavenger assays, ferric reducing power assay, nitrogen derivative scavenger, deoxyribose degradation, and TBARS assays. HPLC evaluation detected 20E as main compound in leaves and roots. Percolation method showed the highest concentrations of 20E (0.134 and 0.096 mg/mL of extract for leaves and roots, respectively). All crude extracts presented low toxic potential on A. salina (LD50 >1000 µg/mL). The computational evaluation of 20E showed a low toxicity prediction. For in vitro antioxidant tests, hydroethanolic extracts of leaves were most effective compared to roots. In addition, hydroethanolic extracts presented a higher IC50 antioxidant than aqueous extracts. TBARS formation was prevented by leaves hydroethanolic extract from 0.015 and 0.03 mg/mL and for roots from 0.03 and 0.3 mg/mL on egg yolk and rat tissue, respectively (P<0.05). These findings suggest that S. tuberculata extracts are a considerable source of ecdysteroids and possesses a significant antioxidant property with low toxic potential.

Cytotoxic and radioprotective effects of Podophyllum hexandrum.

Science.gov (United States)

Shukla, Sandeep Kumar; Chaudhary, Pankaj; Prem Kumar, Indracanti; Afrin, Farhat; Puri, Satish Chandra; Qazi, Ghulam Nabi; Sharma, Rakesh Kumar

2006-07-01

Podophyllum hexandrum, a herb thriving in Himalayas has already been reported to exhibit antitumor and radioprotective properties. Present study was undertaken to unravel the possible mechanism responsible for the cytotoxic and radioprotective properties of REC-2001, a fraction isolated from the rhizome of P. hexandrum using murine peritoneal macrophages and plasmid DNA as model systems. Cell death, levels of intracellular reactive oxygen species (ROS) and apoptosis were studied employing trypan blue exclusion assay, dichlorofluorescein diacetate and DNA fragmentation assay, respectively. Superoxide anions, hydroxyl radicals and DNA damage were estimated following nitroblue tetrazolium, 2-deoxyribose degradation and plasmid DNA relaxation assays, respectively. Pre-irradiation administration of REC-2001 to peritoneal macrophages in the concentration range of 25-200μg/ml significantly reduced radiation induced ROS generation, DNA damage, apoptosis and cell killing in comparison to radiation control group indicating radioprotective potential. Studies with plasmid DNA indicated the ability of REC-2001 to inhibit 20Gy induced single and double strand breaks further supporting the antioxidative potential. However, REC-2001 in a dose-dependent fashion induced cell death, ROS and DNA fragmentation indicating the cytotoxic nature. REC-2001, in presence of 100μM copper sulfate, generated significant amount of hydroxyl radicals and superoxide anions indicating ability to act as a pro-oxidant in presence of metal ions. The superoxide anion generation was found to be sensitive to metal chelators like EDTA and deferoxamine mesylate (DFR). These results suggest that the ability of REC-2001 to act as a pro-oxidant in presence of metal ions and antioxidant in presence of free radicals might be responsible for cytotoxic and radioprotective properties.

Insight in the PCB-degrading functional community in long-term contaminated soil under bioremediation

Energy Technology Data Exchange (ETDEWEB)

Petric, Ines; Hrsak, Dubravka; Udikovic-Kolic, Nikolina [Ruder Boskovic Inst., Division for Marine and Environmental Research, Zagreb (Croatia); Fingler, Sanja [Inst. for Medical Research and Occupational Health, Zagreb (Croatia); Bru, David; Martin-Laurent, Fabrice [INRA, Univ. der Bourgogne, Soil and Environmental Microbiology, Dijon (France)

2011-02-15

A small-scale bioremediation assay was developed in order to get insight into the functioning of a polychlorinated biphenyl (PCB) degrading community during the time course of bioremediation treatment of a contaminated soil. The study was conducted with the aim to better understand the key mechanisms involved in PCB-removal from soils. Materials and methods Two bioremediation strategies were applied in the assay: (a) biostimulation (addition of carvone as inducer of biphenyl pathway, soya lecithin for improving PCB bioavailability, and xylose as supplemental carbon source) and (b) bioaugmentation with selected seed cultures TSZ7 or Rhodococcus sp. Z6 originating from the transformer station soil and showing substantial PCB-degrading activity. Functional PCB-degrading community was investigated by using molecular-based approaches (sequencing, qPCR) targeting bphA and bphC genes, coding key enzymes of the upper biphenyl pathway, in soil DNA extracts. In addition, kinetics of PCBs removal during the bioremediation treatment was determined using gas chromatography mass spectrometry analyses. Results and discussion bphA-based phylogeny revealed that bioremediation affected the structure of the PCB-degrading community in soils, with Rhodococcus-like bacterial populations developing as dominant members. Tracking of this population further indicated that applied bioremediation treatments led to its enrichment within the PCB-degrading community. The abundance of the PCB-degrading community, estimated by quantifying the copy number of bphA and bphC genes, revealed that it represented up to 0.3% of the total bacterial community. All bioremediation treatments were shown to enhance PCB reduction in soils, with approximately 40% of total PCBs being removed during a 1-year period. The faster PCB reduction achieved in bioaugmented soils suggested an important role of the seed cultures in bioremediation processes. Conclusions The PCBs degrading community was modified in response to

Degradation of 14C-parathion 'in vitro' by microorganisms isolated from a gley humic soil

International Nuclear Information System (INIS)

Andrea, M.M. de; Ruegg, E.F.

1982-01-01

It was determined 'in vitro' the degradation of Parathion by a bacterium and a fungus isolated from a sample of Gley Humic soil previously treated with repeated applications of the insecticide. In a qualitative colorimetric assay hydrolisis of parathion to p-nitrophenol just the bacterium gave a positive answer. In quantitative assays of 14 C-parathion degradation in culture media containing both microorganisms, organic solvents extractions resulted in organic and aqueous phases, which were analysed by liquid scintillation counting and thin-layer chromatography. In a mineral salts medium plus buffer, the bacterium and the fungus behaved differently from the control, because part of the 14 C-insecticide was metabolized to, at least, one metabolite and besides, the microorganisms presented smaller percentages of total recovery. The largest percentage of the radio carbon recovery from the extracts of the medium containing the fungus plus extract of yeast, was obtained from the aqueous phase and the existence of other metabolite was demonstrated by chromatograms of the organic phase. (Author) [pt

Aktivitas Oligoresveratrol dari Kulit Batang Hopea mengarawan (Dipterocarpaceae sebagai Penangkap Radikal Hidroksil

Directory of Open Access Journals (Sweden)

SRI ATUN

2006-06-01

Full Text Available Four oligoresveratrols ranging from dimer to tetramer, isolated from stem bark of Hopea mengarawan (Dipterocarpaceae plants were tested for their activity as hydroxyl radical scavenger. The activity of these compounds was evaluated against the 2-deoxyribose degradation induced by the hydroxyl radical generated via a Fenton-type reaction. Result showed that balanocarpol, heimiol A, vaticanol G, and vaticanol B had IC50 3.83; 15.44; 2.01; and 4.71 μM, respectively. These results suggest that oligoresveratrols from stem bark of H. mengarawan maybe useful as potential sources of natural anti-oxidants.

Activity of Oligoresveratrols from Stem Bark of Hopea mengarawan (Dipterocarpaceae as Hydroxyl Radical Scavenger

Directory of Open Access Journals (Sweden)

SRI ATUN

2006-06-01

Full Text Available Four oligoresveratrols ranging from dimer to tetramer, isolated from stem bark of Hopea mengarawan (Dipterocarpaceae plants were tested for their activity as hydroxyl radical scavenger. The activity of these compounds was evaluated against the 2-deoxyribose degradation induced by the hydroxyl radical generated via a Fenton-type reaction. Result showed that balanocarpol, heimiol A, vaticanol G, and vaticanol B had IC50 3.83; 15.44; 2.01; and 4.71 µM, respectively. These results suggest that oligoresveratrols from stem bark of H. mengarawan maybe useful as potential sources of natural antioxidants.

Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases

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Juliane Schmidt

2017-02-01

Full Text Available Polyurethanes (PU are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC, TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 °C, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

Quantification of ion-induced molecular fragmentation of isolated 2-deoxy-D-ribose molecules

NARCIS (Netherlands)

Alvarado Chacon, F.; Bari, S.; Hoekstra, R.A.; Schlathölter, T.A.

2006-01-01

Recent experiments on low energy ion-induced damage to DNA building blocks indicate that ion induced DNA damage is dominated by deoxyribose disintegration (Phys. Rev. Lett., 2005, 95, 153201). We have studied interactions of keV H+ and Heq+ with isolated deoxyribose molecules by means of high

Enzymatic Degradation of Dynasan 114 SLN - Effect of Surfactants and Particle Size

International Nuclear Information System (INIS)

Olbrich, Carsten; Kayser, Oliver; Mueller, Rainer Helmut

2002-01-01

The degradation velocity of solid lipid nanoparticles (SLN) is - apart from drug diffusion - an important parameter determining drug release in vivo. To assess the effect of stabilizers systematically, Dynasan 114 SLN were produced with ionic surfactants (e.g. cholic acid sodium salt (NaCh), sodium dodecyl sulfate (SDS), cetylpyridiniumchloride (CPC)) and steric stabilizers (Tween 80, Poloxamer 188, 407 and Poloxamine 908) including a mixture of cholic acid sodium salt and Poloxamer 407. In addition, the size effects were investigated. The degradation velocity was measured using an in vitro lipase assay. SLN stabilized with lecithin and NaCh showed the fastest, Tween 80 the intermediate and the high molecular weight Poloxamer 407 the slowest degradation. Size effects were less pronounced for fast degrading particles (e.g. those stabilized with NaCh). No difference in the size range of 180-300-nm was observed, but a distinctly slower degradation of 800-nm SLN could be detected. For slowly degrading particles, more pronounced size effects were found. Size effects are more difficult to assess when the PCS diameters are similar, but small fractions of micrometer particles are present, besides the nanometer bulk population. The measured FFA formation is then a superposition of particles degrading at different speeds due to differences in the shape of the size distribution. Admixing of Poloxamer to NaCh had no delaying effect on the degradation of the Dynasan 114 SLN, indicating an influence of the nature of the lipid matrix that is affecting the stabilizers affinity to and anchoring onto the SLN surface

Marine microbe with potential to adhere and degrade plastic structures

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Alka Kumari

2017-10-01

Full Text Available Extensive usages of plastics have led to their accumulation as a contaminant in natural environment worldwide. Plastic is an inert and non-biodegradable material, due to its complex structure and hydrophobic backbone [1]. Conventional methods for reduction of plastic waste such as burning, land-filling release unwanted toxic chemicals to the environment and harming living organism of land as well as the ocean. There is growing interest in development of strategies for the degradation of plastic wastes to clean the environment [2]. Marine bacteria have evolved with the capability to adapt and grow in the diverse environmental conditions [3]. We studied the ability of marine bacteria for destabilization and utilization of different plastic films (LDPE, HDPE, PVC and PET as a sole source of carbon. An active bacterial strain AIIW2 was selected based on the triphenyl tetrazolium chloride reduction assay, and it was identified as Bacillus species based on 16S rRNA gene sequence. The viability of the strain over the plastic surface was studied and confirmed by bacLight assay with fluorescent probes. Scanning Electron Microscope and Atomic Force Microscope images suggested that bacterial interaction over the plastic surface is causing deterioration and roughness with increasing bacterial incubation time. In Fourier transform infrared spectra of treated plastic film evidenced stretching of the (-CH alkane rock chain and (-CO carbonyl region, suggested the oxidative activities of the bacteria. The results revealed that ability of bacterial strain for instigating their colonization over plastic films and deteriorating the polymeric structure in the absence of other carbon sources [4]. Moreover, production of extracellular enzymes such as esterase, laccase, and dehalogenase which are reported to support utilization of plastics was confirmed by plate assays. In concise, our results suggested that the marine bacterial strain AIIW2 have the ability to utilize

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

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Cécile Feuillie

Full Text Available In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR. In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

Glutaraldehyde degradation in hospital wastewater by photoozonation.

Science.gov (United States)

Kist, Lourdes Teresinha; Rosa, Ellen Caroline; Machado, Enio Leandro; Camargo, Maria Emilia; Moro, Celso Camilo

2013-01-01

In this paper, we assessed aqueous solutions of glutaraldehyde (GA), a chemical used for the disinfection of hospital materials, using advanced oxidative processes, O3, and UV, and the combination of the latter two. Assays with different ozone concentrations at distinct pH levels were conducted to determine the best treatment process. GA concentrations before and after each treatment were measured by spectrophotometry. The best treatment was that which combined O3 and UV, yielding a degradation of 72.0-75.0% in relation to the initial concentration with pH between 4 and 9. Kinetics demonstrated that GA degradation is not dependent on pH, as there was a first-order reaction with a rate constant of k = 0.0180 min(-1) for initial pH 9 and of k = 0.0179 min(-1) for initial pH 7, that is, the values are virtually the same. Secondary wastewater samples were also analysed using the septic tank/filter system of a regional hospital in Vale do Rio Pardo, state of Rio Grande do Sul, southern Brazil. In this case, the characteristics of the wastewater were described and, after treatment, a GA degradation rate of 23.3% was noted, with reductions of 75% for chemical oxygen demand, 81% for biochemical oxygen demand, 68% for turbidity, 70% for surfactants and total disinfection in terms of thermotolerant coliforms.

Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay.

Science.gov (United States)

Rotherham, D; Harbison, S A

2011-04-15

Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for the differentiation of "drug" and "non-drug"Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between "drug" and "non-drug"Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Micropollutant degradation via extracted native enzymes from activated sludge.

Science.gov (United States)

Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

2016-05-15

A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

Parasite-Mediated Degradation of Synthetic Ozonide Antimalarials Impacts In Vitro Antimalarial Activity.

Science.gov (United States)

Giannangelo, Carlo; Stingelin, Lukas; Yang, Tuo; Tilley, Leann; Charman, Susan A; Creek, Darren J

2018-03-01

The peroxide bond of the artemisinins inspired the development of a class of fully synthetic 1,2,4-trioxolane-based antimalarials, collectively known as the ozonides. Similar to the artemisinins, heme-mediated degradation of the ozonides generates highly reactive radical species that are thought to mediate parasite killing by damaging critical parasite biomolecules. We examined the relationship between parasite dependent degradation and antimalarial activity for two ozonides, OZ277 (arterolane) and OZ439 (artefenomel), using a combination of in vitro drug stability and pulsed-exposure activity assays. Our results showed that drug degradation is parasite stage dependent and positively correlates with parasite load. Increasing trophozoite-stage parasitemia leads to substantially higher rates of degradation for both OZ277 and OZ439, and this is associated with a reduction in in vitro antimalarial activity. Under conditions of very high parasitemia (∼90%), OZ277 and OZ439 were rapidly degraded and completely devoid of activity in trophozoite-stage parasite cultures exposed to a 3-h drug pulse. This study highlights the impact of increasing parasite load on ozonide stability and in vitro antimalarial activity and should be considered when investigating the antimalarial mode of action of the ozonide antimalarials under conditions of high parasitemia. Copyright © 2018 American Society for Microbiology.

Macroalgal herbivory on recovering versus degrading coral reefs

Science.gov (United States)

Chong-Seng, K. M.; Nash, K. L.; Bellwood, D. R.; Graham, N. A. J.

2014-06-01

Macroalgal-feeding fishes are considered to be a key functional group on coral reefs due to their role in preventing phase shifts from coral to macroalgal dominance, and potentially reversing the shift should it occur. However, assessments of macroalgal herbivory using bioassay experiments are primarily from systems with relatively high coral cover. This raises the question of whether continued functionality can be ensured in degraded systems. It is clearly important to determine whether the species that remove macroalgae on coral-dominated reefs will still be present and performing significant algal removal on macroalgal-dominated reefs. We compared the identity and effectiveness of macroalgal-feeding fishes on reefs in two conditions post-disturbance—those regenerating with high live coral cover (20-46 %) and those degrading with high macroalgal cover (57-82 %). Using filmed Sargassum bioassays, we found significantly different Sargassum biomass loss between the two conditions; mean assay weight loss due to herbivory was 27.9 ± 4.9 % on coral-dominated reefs and 2.2 ± 1.1 % on reefs with high macroalgal cover. However, once standardised for the availability of macroalgae on the reefs, the rates of removal were similar between the two reef conditions (4.8 ± 4.1 g m-2 h-1 on coral-dominated and 5.3 ± 2.1 g m-2 h-1 on macroalgal-dominated reefs). Interestingly, the Sargassum-assay consumer assemblages differed between reef conditions; nominally grazing herbivores, Siganus puelloides and Chlorurus sordidus, and the browser , Siganus sutor, dominated feeding on high coral cover reefs, whereas browsing herbivores, Naso elegans, Naso unicornis, and Leptoscarus vaigiensis, prevailed on macroalgal-dominated reefs. It appeared that macroalgal density in the surrounding habitat had a strong influence on the species driving the process of macroalgal removal. This suggests that although the function of macroalgal removal may continue, the species responsible may change

RP-HPLC assay method development for Paracetamol and Lornoxicam in combination and characterization of oxidative degradation products of Lornoxicam

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Jain Pritam S.

2013-01-01

Full Text Available A simple, specific, accurate and precise reverse phase high pressure liquid chromatographic method has been developed for the simultaneous determination of Paracetamol and Lornoxicam from tablets and to characterize degradation products of Lornoxicam by reverse phase C18 column (Inertsil ODS 3V C-18, 250 x 4.6 mm, 5 μ. The sample was analyzed using Buffer (0.02504 Molar: Methanol in the ratio of 45:55, as a mobile phase at a flow rate of 1.5 mL/min and detection at 290 nm. The retention time for Paracetamol and Lornoxicam was found to be 2.45 and 9.40 min respectively. The method can be used for estimation of combination of these drugs in tablets. The method was validated as per ICH guidelines. The linearity of developed method was achieved in the range of 249.09 - 747.29 μg/mL (r2=0.9999 for Paracetamol and 4.0125 - 12.0375 μg/mL (r2=0.9999 for Lornoxicam. Recoveries from tablets were between 98 and 102%. The method was validated with respect to linearity, accuracy, precision, robustness and forced degradation studies which further proved the stability-indicating power. During the forced degradation studies lornoxicam was observed to be labile to alkaline hydrolytic stress and oxidative stress (in the solution form. However, it was stable to the acid hydrolytic, photolytic and thermal stress (in both solid and solution form. The degraded products formed were investigated by electrospray ionization (ESI time-of-flight mass spectrometry, NMR and IR spectroscopy. A possible degradation pathway was outlined based on the results. The method was found to be sensitive with a detection limit of 0.193 μg/ml, 2.768 μg/ml and a quantitation limit of 0.638 μg/ml, 9.137 μg/ml for lornoxicam and paracetamol, respectively. Due to these attributes, the proposed method could be used for routine quality control analysis of these drugs in combined dosage forms.

A nuclear DNA-based species determination and DNA quantification assay for common poultry species.

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Ng, J; Satkoski, J; Premasuthan, A; Kanthaswamy, S

2014-12-01

DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50 pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.

Degradable polyphosphazene/poly(alpha-hydroxyester) blends: degradation studies.

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Ambrosio, Archel M A; Allcock, Harry R; Katti, Dhirendra S; Laurencin, Cato T

2002-04-01

Biomaterials based on the polymers of lactic acid and glycolic acid and their copolymers are used or studied extensively as implantable devices for drug delivery, tissue engineering and other biomedical applications. Although these polymers have shown good biocompatibility, concerns have been raised regarding their acidic degradation products, which have important implications for long-term implantable systems. Therefore, we have designed a novel biodegradable polyphosphazene/poly(alpha-hydroxyester) blend whose degradation products are less acidic than those of the poly(alpha-hydroxyester) alone. In this study, the degradation characteristics of a blend of poly(lactide-co-glycolide) (50:50 PLAGA) and poly[(50% ethyl glycinato)(50% p-methylphenoxy) phosphazene] (PPHOS-EG50) were qualitatively and quantitatively determined with comparisons made to the parent polymers. Circular matrices (14mm diameter) of the PLAGA, PPHOS-EG50 and PLAGA-PPHOS-EG50 blend were degraded in non-buffered solutions (pH 7.4). The degraded polymers were characterized for percentage mass loss and molecular weight and the degradation medium was characterized for acid released in non-buffered solutions. The amounts of neutralizing base necessary to bring about neutral pH were measured for each polymer or polymer blend during degradation. The poly(phosphazene)/poly(lactide-co-glycolide) blend required significantly less neutralizing base in order to bring about neutral solution pH during the degradation period studied. The results indicated that the blend degraded at a rate intermediate to that of the parent polymers and that the degradation products of the polyphosphazene neutralized the acidic degradation products of PLAGA. Thus, results from these in vitro degradation studies suggest that the PLAGA-PPHOS-EG50 blend may provide a viable improvement to biomaterials based on acid-releasing organic polymers.

Plasma transthyretin. Tissue sites of degradation and turnover in the rat

International Nuclear Information System (INIS)

Makover, A.; Moriwaki, H.; Ramakrishnan, R.; Saraiva, M.J.; Blaner, W.S.; Goodman, D.S.

1988-01-01

Transthyretin (TTR) is involved in the plasma transport of both retinol and thyroid hormones. TTR is synthesized in the liver and choroid plexus, and in small amounts in several other tissues. A study was conducted to determine the tissue sites of degradation and turnover of TTR in the rat. The study employed TTR labeled with tyramine cellobiose (TC) and the trapped ligand method. Samples of purified rat TTR were labeled either with 125I-TC or directly with 131I. A mixture of the two labeled TTRs was injected intravenously into six rats. Blood samples were collected via a venous catheter for kinetic (turnover) analysis. After 24 or 48 h, the rats were killed, and 23 different tissues/organs were assayed as possible sites of TTR degradation. Derivatization of TTR with TC did not appreciably alter TTR plasma kinetics. Plasma turnover data were best fit by a three-pool model. The mean fractional turnover of plasma TTR was 0.15/h, and of total body TTR 0.04/h. The major sites of TTR degradation were the liver (36-38% of total body TTR degradation, almost all in hepatocytes), muscle (12-15%), and skin (8-10%). Tissues that were sites of 1-8% of body TTR degradation included kidneys, adipose tissue, testes, and the gastrointestinal tract. Less than 1% of total TTR degradation occurred in the other tissues examined. A second study was conducted in which labeled TTR was injected intraventricularly into the cerebrospinal fluid in order to explore the degradation of TTR of choroid plexus origin. The kinetics of the appearance and disappearance of such labeled TTR in plasma were physiologically reasonable, with an estimated turnover of cerebrospinal fluid TTR of the order of 0.33/h. The major tissue sites of degradation of labeled TTR injected into cerebrospinal fluid and into plasma were approximately the same

Guidelines for the use and interpretation of assays for monitoring autophagy.

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Klionsky, Daniel J; Abdalla, Fabio C; Abeliovich, Hagai; Abraham, Robert T; Acevedo-Arozena, Abraham; Adeli, Khosrow; Agholme, Lotta; Agnello, Maria; Agostinis, Patrizia; Aguirre-Ghiso, Julio A; Ahn, Hyung Jun; Ait-Mohamed, Ouardia; Ait-Si-Ali, Slimane; Akematsu, Takahiko; Akira, Shizuo; Al-Younes, Hesham M; Al-Zeer, Munir A; Albert, Matthew L; Albin, Roger L; Alegre-Abarrategui, Javier; Aleo, Maria Francesca; Alirezaei, Mehrdad; Almasan, Alexandru; Almonte-Becerril, Maylin; Amano, Atsuo; Amaravadi, Ravi; Amarnath, Shoba; Amer, Amal O; Andrieu-Abadie, Nathalie; Anantharam, Vellareddy; Ann, David K; Anoopkumar-Dukie, Shailendra; Aoki, Hiroshi; Apostolova, Nadezda; Arancia, Giuseppe; Aris, John P; Asanuma, Katsuhiko; Asare, Nana Y O; Ashida, Hisashi; Askanas, Valerie; Askew, David S; Auberger, Patrick; Baba, Misuzu; Backues, Steven K; Baehrecke, Eric H; Bahr, Ben A; Bai, Xue-Yuan; Bailly, Yannick; Baiocchi, Robert; Baldini, Giulia; Balduini, Walter; Ballabio, Andrea; Bamber, Bruce A; Bampton, Edward T W; Bánhegyi, Gábor; Bartholomew, Clinton R; Bassham, Diane C; Bast, Robert C; Batoko, Henri; Bay, Boon-Huat; Beau, Isabelle; Béchet, Daniel M; Begley, Thomas J; Behl, Christian; Behrends, Christian; Bekri, Soumeya; Bellaire, Bryan; Bendall, Linda J; Benetti, Luca; Berliocchi, Laura; Bernardi, Henri; Bernassola, Francesca; Besteiro, Sébastien; Bhatia-Kissova, Ingrid; Bi, Xiaoning; Biard-Piechaczyk, Martine; Blum, Janice S; Boise, Lawrence H; Bonaldo, Paolo; Boone, David L; Bornhauser, Beat C; Bortoluci, Karina R; Bossis, Ioannis; Bost, Frédéric; Bourquin, Jean-Pierre; Boya, Patricia; Boyer-Guittaut, Michaël; Bozhkov, Peter V; Brady, Nathan R; Brancolini, Claudio; Brech, Andreas; Brenman, Jay E; Brennand, Ana; Bresnick, Emery H; Brest, Patrick; Bridges, Dave; Bristol, Molly L; Brookes, Paul S; Brown, Eric J; Brumell, John H; Brunetti-Pierri, Nicola; Brunk, Ulf T; Bulman, Dennis E; Bultman, Scott J; Bultynck, Geert; Burbulla, Lena F; Bursch, Wilfried; Butchar, Jonathan P; Buzgariu, Wanda; Bydlowski, Sergio P; Cadwell, Ken; Cahová, Monika; Cai, Dongsheng; Cai, Jiyang; Cai, Qian; Calabretta, Bruno; Calvo-Garrido, Javier; Camougrand, Nadine; Campanella, Michelangelo; Campos-Salinas, Jenny; Candi, Eleonora; Cao, Lizhi; Caplan, Allan B; Carding, Simon R; Cardoso, Sandra M; Carew, Jennifer S; Carlin, Cathleen R; Carmignac, Virginie; Carneiro, Leticia A M; Carra, Serena; Caruso, Rosario A; Casari, Giorgio; Casas, Caty; Castino, Roberta; Cebollero, Eduardo; Cecconi, Francesco; Celli, Jean; Chaachouay, Hassan; Chae, Han-Jung; Chai, Chee-Yin; Chan, David C; Chan, Edmond Y; Chang, Raymond Chuen-Chung; Che, Chi-Ming; Chen, Ching-Chow; Chen, Guang-Chao; Chen, Guo-Qiang; Chen, Min; Chen, Quan; Chen, Steve S-L; Chen, WenLi; Chen, Xi; Chen, Xiangmei; Chen, Xiequn; Chen, Ye-Guang; Chen, Yingyu; Chen, Yongqiang; Chen, Yu-Jen; Chen, Zhixiang; Cheng, Alan; Cheng, Christopher H K; Cheng, Yan; Cheong, Heesun; Cheong, Jae-Ho; Cherry, Sara; Chess-Williams, Russ; Cheung, Zelda H; Chevet, Eric; Chiang, Hui-Ling; Chiarelli, Roberto; Chiba, Tomoki; Chin, Lih-Shen; Chiou, Shih-Hwa; Chisari, Francis V; Cho, Chi Hin; Cho, Dong-Hyung; Choi, Augustine M K; Choi, DooSeok; Choi, Kyeong Sook; Choi, Mary E; Chouaib, Salem; Choubey, Divaker; Choubey, Vinay; Chu, Charleen T; Chuang, Tsung-Hsien; Chueh, Sheau-Huei; Chun, Taehoon; Chwae, Yong-Joon; Chye, Mee-Len; Ciarcia, Roberto; Ciriolo, Maria R; Clague, Michael J; Clark, Robert S B; Clarke, Peter G H; Clarke, Robert; Codogno, Patrice; Coller, Hilary A; Colombo, María I; Comincini, Sergio; Condello, Maria; Condorelli, Fabrizio; Cookson, Mark R; Coombs, Graham H; Coppens, Isabelle; Corbalan, Ramon; Cossart, Pascale; Costelli, Paola; Costes, Safia; Coto-Montes, Ana; Couve, Eduardo; Coxon, Fraser P; Cregg, James M; Crespo, José L; Cronjé, Marianne J; Cuervo, Ana Maria; Cullen, Joseph J; Czaja, Mark J; D'Amelio, Marcello; Darfeuille-Michaud, Arlette; Davids, Lester M; Davies, Faith E; De Felici, Massimo; de Groot, John F; de Haan, Cornelis A M; De Martino, Luisa; De Milito, Angelo; De Tata, Vincenzo; Debnath, Jayanta; Degterev, Alexei; Dehay, Benjamin; Delbridge, Lea M D; Demarchi, Francesca; Deng, Yi Zhen; Dengjel, Jörn; Dent, Paul; Denton, Donna; Deretic, Vojo; Desai, Shyamal D; Devenish, Rodney J; Di Gioacchino, Mario; Di Paolo, Gilbert; Di Pietro, Chiara; Díaz-Araya, Guillermo; Díaz-Laviada, Inés; Diaz-Meco, Maria T; Diaz-Nido, Javier; Dikic, Ivan; Dinesh-Kumar, Savithramma P; Ding, Wen-Xing; Distelhorst, Clark W; Diwan, Abhinav; Djavaheri-Mergny, Mojgan; Dokudovskaya, Svetlana; Dong, Zheng; Dorsey, Frank C; Dosenko, Victor; Dowling, James J; Doxsey, Stephen; Dreux, Marlène; Drew, Mark E; Duan, Qiuhong; Duchosal, Michel A; Duff, Karen; Dugail, Isabelle; Durbeej, Madeleine; Duszenko, Michael; Edelstein, Charles L; Edinger, Aimee L; Egea, Gustavo; Eichinger, Ludwig; Eissa, N Tony; Ekmekcioglu, Suhendan; El-Deiry, Wafik S; Elazar, Zvulun; Elgendy, Mohamed; Ellerby, Lisa M; Eng, Kai Er; Engelbrecht, Anna-Mart; Engelender, Simone; Erenpreisa, Jekaterina; Escalante, Ricardo; Esclatine, Audrey; Eskelinen, Eeva-Liisa; Espert, Lucile; Espina, Virginia; Fan, Huizhou; Fan, Jia; Fan, Qi-Wen; Fan, Zhen; Fang, Shengyun; Fang, Yongqi; Fanto, Manolis; Fanzani, Alessandro; Farkas, Thomas; Farré, Jean-Claude; Faure, Mathias; Fechheimer, Marcus; Feng, Carl G; Feng, Jian; Feng, Qili; Feng, Youji; Fésüs, László; Feuer, Ralph; Figueiredo-Pereira, Maria E; Fimia, Gian Maria; Fingar, Diane C; Finkbeiner, Steven; Finkel, Toren; Finley, Kim D; Fiorito, Filomena; Fisher, Edward A; Fisher, Paul B; Flajolet, Marc; Florez-McClure, Maria L; Florio, Salvatore; Fon, Edward A; Fornai, Francesco; Fortunato, Franco; Fotedar, Rati; Fowler, Daniel H; Fox, Howard S; Franco, Rodrigo; Frankel, Lisa B; Fransen, Marc; Fuentes, José M; Fueyo, Juan; Fujii, Jun; Fujisaki, Kozo; Fujita, Eriko; Fukuda, Mitsunori; Furukawa, Ruth H; Gaestel, Matthias; Gailly, Philippe; Gajewska, Malgorzata; Galliot, Brigitte; Galy, Vincent; Ganesh, Subramaniam; Ganetzky, Barry; Ganley, Ian G; Gao, Fen-Biao; Gao, George F; Gao, Jinming; Garcia, Lorena; Garcia-Manero, Guillermo; Garcia-Marcos, Mikel; Garmyn, Marjan; Gartel, Andrei L; Gatti, Evelina; Gautel, Mathias; Gawriluk, Thomas R; Gegg, Matthew E; Geng, Jiefei; Germain, Marc; Gestwicki, Jason E; Gewirtz, David A; Ghavami, Saeid; Ghosh, Pradipta; Giammarioli, Anna M; Giatromanolaki, Alexandra N; Gibson, Spencer B; Gilkerson, Robert W; Ginger, Michael L; Ginsberg, Henry N; Golab, Jakub; Goligorsky, Michael S; Golstein, Pierre; Gomez-Manzano, Candelaria; Goncu, Ebru; Gongora, Céline; Gonzalez, Claudio D; Gonzalez, Ramon; González-Estévez, Cristina; González-Polo, Rosa Ana; Gonzalez-Rey, Elena; Gorbunov, Nikolai V; Gorski, Sharon; Goruppi, Sandro; Gottlieb, Roberta A; Gozuacik, Devrim; Granato, Giovanna Elvira; Grant, Gary D; Green, Kim N; Gregorc, Aleš; Gros, Frédéric; Grose, Charles; Grunt, Thomas W; Gual, Philippe; Guan, Jun-Lin; Guan, Kun-Liang; Guichard, Sylvie M; Gukovskaya, Anna S; Gukovsky, Ilya; Gunst, Jan; Gustafsson, Asa B; Halayko, Andrew J; Hale, Amber N; Halonen, Sandra K; Hamasaki, Maho; Han, Feng; Han, Ting; Hancock, Michael K; Hansen, Malene; Harada, Hisashi; Harada, Masaru; Hardt, Stefan E; Harper, J Wade; Harris, Adrian L; Harris, James; Harris, Steven D; Hashimoto, Makoto; Haspel, Jeffrey A; Hayashi, Shin-ichiro; Hazelhurst, Lori A; He, Congcong; He, You-Wen; Hébert, Marie-Joseé; Heidenreich, Kim A; Helfrich, Miep H; Helgason, Gudmundur V; Henske, Elizabeth P; Herman, Brian; Herman, Paul K; Hetz, Claudio; Hilfiker, Sabine; Hill, Joseph A; Hocking, Lynne J; Hofman, Paul; Hofmann, Thomas G; Höhfeld, Jörg; Holyoake, Tessa L; Hong, Ming-Huang; Hood, David A; Hotamisligil, Gökhan S; Houwerzijl, Ewout J; Høyer-Hansen, Maria; Hu, Bingren; Hu, Chien-An A; Hu, Hong-Ming; Hua, Ya; Huang, Canhua; Huang, Ju; Huang, Shengbing; Huang, Wei-Pang; Huber, Tobias B; Huh, Won-Ki; Hung, Tai-Ho; Hupp, Ted R; Hur, Gang Min; Hurley, James B; Hussain, Sabah N A; Hussey, Patrick J; Hwang, Jung Jin; Hwang, Seungmin; Ichihara, Atsuhiro; Ilkhanizadeh, Shirin; Inoki, Ken; Into, Takeshi; Iovane, Valentina; Iovanna, Juan L; Ip, Nancy Y; Isaka, Yoshitaka; Ishida, Hiroyuki; Isidoro, Ciro; Isobe, Ken-ichi; Iwasaki, Akiko; Izquierdo, Marta; Izumi, Yotaro; Jaakkola, Panu M; Jäättelä, Marja; Jackson, George R; Jackson, William T; Janji, Bassam; Jendrach, Marina; Jeon, Ju-Hong; Jeung, Eui-Bae; Jiang, Hong; Jiang, Hongchi; Jiang, Jean X; Jiang, Ming; Jiang, Qing; Jiang, Xuejun; Jiang, Xuejun; Jiménez, Alberto; Jin, Meiyan; Jin, Shengkan; Joe, Cheol O; Johansen, Terje; Johnson, Daniel E; Johnson, Gail V W; Jones, Nicola L; Joseph, Bertrand; Joseph, Suresh K; Joubert, Annie M; Juhász, Gábor; Juillerat-Jeanneret, Lucienne; Jung, Chang Hwa; Jung, Yong-Keun; Kaarniranta, Kai; Kaasik, Allen; Kabuta, Tomohiro; Kadowaki, Motoni; Kagedal, Katarina; Kamada, Yoshiaki; Kaminskyy, Vitaliy O; Kampinga, Harm H; Kanamori, Hiromitsu; Kang, Chanhee; Kang, Khong Bee; Kang, Kwang Il; Kang, Rui; Kang, Yoon-A; Kanki, Tomotake; Kanneganti, Thirumala-Devi; Kanno, Haruo; Kanthasamy, Anumantha G; Kanthasamy, Arthi; Karantza, Vassiliki; Kaushal, Gur P; Kaushik, Susmita; Kawazoe, Yoshinori; Ke, Po-Yuan; Kehrl, John H; Kelekar, Ameeta; Kerkhoff, Claus; Kessel, David H; Khalil, Hany; Kiel, Jan A K W; Kiger, Amy A; Kihara, Akio; Kim, Deok Ryong; Kim, Do-Hyung; Kim, Dong-Hou; Kim, Eun-Kyoung; Kim, Hyung-Ryong; Kim, Jae-Sung; Kim, Jeong Hun; Kim, Jin Cheon; Kim, John K; Kim, Peter K; Kim, Seong Who; Kim, Yong-Sun; Kim, Yonghyun; Kimchi, Adi; Kimmelman, Alec C; King, Jason S; Kinsella, Timothy J; Kirkin, Vladimir; Kirshenbaum, Lorrie A; Kitamoto, Katsuhiko; Kitazato, Kaio; Klein, Ludger; Klimecki, Walter T; Klucken, Jochen; Knecht, Erwin; Ko, Ben C B; Koch, Jan C; Koga, Hiroshi; Koh, Jae-Young; Koh, Young Ho; Koike, Masato; Komatsu, Masaaki; Kominami, Eiki; Kong, Hee Jeong; Kong, Wei-Jia; Korolchuk, Viktor I; Kotake, Yaichiro; Koukourakis, Michael I; Kouri Flores, Juan B; Kovács, Attila L; Kraft, Claudine; Krainc, Dimitri; Krämer, Helmut; Kretz-Remy, Carole; Krichevsky, Anna M; Kroemer, Guido; Krüger, Rejko; Krut, Oleg; Ktistakis, Nicholas T; Kuan, Chia-Yi; Kucharczyk, Roza; Kumar, Ashok; Kumar, Raj; Kumar, Sharad; Kundu, Mondira; Kung, Hsing-Jien; Kurz, Tino; Kwon, Ho Jeong; La Spada, Albert R; Lafont, Frank; Lamark, Trond; Landry, Jacques; Lane, Jon D; Lapaquette, Pierre; Laporte, Jocelyn F; László, Lajos; Lavandero, Sergio; Lavoie, Josée N; Layfield, Robert; Lazo, Pedro A; Le, Weidong; Le Cam, Laurent; Ledbetter, Daniel J; Lee, Alvin J X; Lee, Byung-Wan; Lee, Gyun Min; Lee, Jongdae; Lee, Ju-Hyun; Lee, Michael; Lee, Myung-Shik; Lee, Sug Hyung; Leeuwenburgh, Christiaan; Legembre, Patrick; Legouis, Renaud; Lehmann, Michael; Lei, Huan-Yao; Lei, Qun-Ying; Leib, David A; Leiro, José; Lemasters, John J; Lemoine, Antoinette; Lesniak, Maciej S; Lev, Dina; Levenson, Victor V; Levine, Beth; Levy, Efrat; Li, Faqiang; Li, Jun-Lin; Li, Lian; Li, Sheng; Li, Weijie; Li, Xue-Jun; Li, Yan-bo; Li, Yi-Ping; Liang, Chengyu; Liang, Qiangrong; Liao, Yung-Feng; Liberski, Pawel P; Lieberman, Andrew; Lim, Hyunjung J; Lim, Kah-Leong; Lim, Kyu; Lin, Chiou-Feng; Lin, Fu-Cheng; Lin, Jian; Lin, Jiandie D; Lin, Kui; Lin, Wan-Wan; Lin, Weei-Chin; Lin, Yi-Ling; Linden, Rafael; Lingor, Paul; Lippincott-Schwartz, Jennifer; Lisanti, Michael P; Liton, Paloma B; Liu, Bo; Liu, Chun-Feng; Liu, Kaiyu; Liu, Leyuan; Liu, Qiong A; Liu, Wei; Liu, Young-Chau; Liu, Yule; Lockshin, Richard A; Lok, Chun-Nam; Lonial, Sagar; Loos, Benjamin; Lopez-Berestein, Gabriel; López-Otín, Carlos; Lossi, Laura; Lotze, Michael T; Lőw, Peter; Lu, Binfeng; Lu, Bingwei; Lu, Bo; Lu, Zhen; Luciano, Frédéric; Lukacs, Nicholas W; Lund, Anders H; Lynch-Day, Melinda A; Ma, Yong; Macian, Fernando; MacKeigan, Jeff P; Macleod, Kay F; Madeo, Frank; Maiuri, Luigi; Maiuri, Maria Chiara; Malagoli, Davide; Malicdan, May Christine V; Malorni, Walter; Man, Na; Mandelkow, Eva-Maria; Manon, Stéphen; Manov, Irena; Mao, Kai; Mao, Xiang; Mao, Zixu; Marambaud, Philippe; Marazziti, Daniela; Marcel, Yves L; Marchbank, Katie; Marchetti, Piero; Marciniak, Stefan J; Marcondes, Mateus; Mardi, Mohsen; Marfe, Gabriella; Mariño, Guillermo; Markaki, Maria; Marten, Mark R; Martin, Seamus J; Martinand-Mari, Camille; Martinet, Wim; Martinez-Vicente, Marta; Masini, Matilde; Matarrese, Paola; Matsuo, Saburo; Matteoni, Raffaele; Mayer, Andreas; Mazure, Nathalie M; McConkey, David J; McConnell, Melanie J; McDermott, Catherine; McDonald, Christine; McInerney, Gerald M; McKenna, Sharon L; McLaughlin, BethAnn; McLean, Pamela J; McMaster, Christopher R; McQuibban, G Angus; Meijer, Alfred J; Meisler, Miriam H; Meléndez, Alicia; Melia, Thomas J; Melino, Gerry; Mena, Maria A; Menendez, Javier A; Menna-Barreto, Rubem F S; Menon, Manoj B; Menzies, Fiona M; Mercer, Carol A; Merighi, Adalberto; Merry, Diane E; Meschini, Stefania; Meyer, Christian G; Meyer, Thomas F; Miao, Chao-Yu; Miao, Jun-Ying; Michels, Paul A M; Michiels, Carine; Mijaljica, Dalibor; Milojkovic, Ana; Minucci, Saverio; Miracco, Clelia; Miranti, Cindy K; Mitroulis, Ioannis; Miyazawa, Keisuke; Mizushima, Noboru; Mograbi, Baharia; Mohseni, Simin; Molero, Xavier; Mollereau, Bertrand; Mollinedo, Faustino; Momoi, Takashi; Monastyrska, Iryna; Monick, Martha M; Monteiro, Mervyn J; Moore, Michael N; Mora, Rodrigo; Moreau, Kevin; Moreira, Paula I; Moriyasu, Yuji; Moscat, Jorge; Mostowy, Serge; Mottram, Jeremy C; Motyl, Tomasz; Moussa, Charbel E-H; Müller, Sylke; Muller, Sylviane; Münger, Karl; Münz, Christian; Murphy, Leon O; Murphy, Maureen E; Musarò, Antonio; Mysorekar, Indira; Nagata, Eiichiro; Nagata, Kazuhiro; Nahimana, Aimable; Nair, Usha; Nakagawa, Toshiyuki; Nakahira, Kiichi; Nakano, Hiroyasu; Nakatogawa, Hitoshi; Nanjundan, Meera; Naqvi, Naweed I; Narendra, Derek P; Narita, Masashi; Navarro, Miguel; Nawrocki, Steffan T; Nazarko, Taras Y; Nemchenko, Andriy; Netea, Mihai G; Neufeld, Thomas P; Ney, Paul A; Nezis, Ioannis P; Nguyen, Huu Phuc; Nie, Daotai; Nishino, Ichizo; Nislow, Corey; Nixon, Ralph A; Noda, Takeshi; Noegel, Angelika A; Nogalska, Anna; Noguchi, Satoru; Notterpek, Lucia; Novak, Ivana; 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2012-04-01

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused

Guidelines for the use and interpretation of assays for monitoring autophagy

Science.gov (United States)

Klionsky, Daniel J.; Abdalla, Fabio C.; Abeliovich, Hagai; Abraham, Robert T.; Acevedo-Arozena, Abraham; Adeli, Khosrow; Agholme, Lotta; Agnello, Maria; Agostinis, Patrizia; Aguirre-Ghiso, Julio A.; Ahn, Hyung Jun; Ait-Mohamed, Ouardia; Ait-Si-Ali, Slimane; Akematsu, Takahiko; Akira, Shizuo; Al-Younes, Hesham M.; Al-Zeer, Munir A.; Albert, Matthew L.; Albin, Roger L.; Alegre-Abarrategui, Javier; Aleo, Maria Francesca; Alirezaei, Mehrdad; Almasan, Alexandru; Almonte-Becerril, Maylin; Amano, Atsuo; Amaravadi, Ravi K.; Amarnath, Shoba; Amer, Amal O.; Andrieu-Abadie, Nathalie; Anantharam, Vellareddy; Ann, David K.; Anoopkumar-Dukie, Shailendra; Aoki, Hiroshi; Apostolova, Nadezda; Arancia, Giuseppe; Aris, John P.; Asanuma, Katsuhiko; Asare, Nana Y.O.; Ashida, Hisashi; Askanas, Valerie; Askew, David S.; Auberger, Patrick; Baba, Misuzu; Backues, Steven K.; Baehrecke, Eric H.; Bahr, Ben A.; Bai, Xue-Yuan; Bailly, Yannick; Baiocchi, Robert; Baldini, Giulia; Balduini, Walter; Ballabio, Andrea; Bamber, Bruce A.; Bampton, Edward T.W.; Juhász, Gábor; Bartholomew, Clinton R.; Bassham, Diane C.; Bast, Robert C.; Batoko, Henri; Bay, Boon-Huat; Beau, Isabelle; Béchet, Daniel M.; Begley, Thomas J.; Behl, Christian; Behrends, Christian; Bekri, Soumeya; Bellaire, Bryan; Bendall, Linda J.; Benetti, Luca; Berliocchi, Laura; Bernardi, Henri; Bernassola, Francesca; Besteiro, Sébastien; Bhatia-Kissova, Ingrid; Bi, Xiaoning; Biard-Piechaczyk, Martine; Blum, Janice S.; Boise, Lawrence H.; Bonaldo, Paolo; Boone, David L.; Bornhauser, Beat C.; Bortoluci, Karina R.; Bossis, Ioannis; Bost, Frédéric; Bourquin, Jean-Pierre; Boya, Patricia; Boyer-Guittaut, Michaël; Bozhkov, Peter V.; Brady, Nathan R; Brancolini, Claudio; Brech, Andreas; Brenman, Jay E.; Brennand, Ana; Bresnick, Emery H.; Brest, Patrick; Bridges, Dave; Bristol, Molly L.; Brookes, Paul S.; Brown, Eric J.; Brumell, John H.; Brunetti-Pierri, Nicola; Brunk, Ulf T.; Bulman, Dennis E.; Bultman, Scott J.; Bultynck, Geert; Burbulla, Lena F.; Bursch, Wilfried; Butchar, Jonathan P.; Buzgariu, Wanda; Bydlowski, Sergio P.; Cadwell, Ken; Cahová, Monika; Cai, Dongsheng; Cai, Jiyang; Cai, Qian; Calabretta, Bruno; Calvo-Garrido, Javier; Camougrand, Nadine; Campanella, Michelangelo; Campos-Salinas, Jenny; Candi, Eleonora; Cao, Lizhi; Caplan, Allan B.; Carding, Simon R.; Cardoso, Sandra M.; Carew, Jennifer S.; Carlin, Cathleen R.; Carmignac, Virginie; Carneiro, Leticia A.M.; Carra, Serena; Caruso, Rosario A.; Casari, Giorgio; Casas, Caty; Castino, Roberta; Cebollero, Eduardo; Cecconi, Francesco; Celli, Jean; Chaachouay, Hassan; Chae, Han-Jung; Chai, Chee-Yin; Chan, David C.; Chan, Edmond Y.; Chang, Raymond Chuen-Chung; Che, Chi-Ming; Chen, Ching-Chow; Chen, Guang-Chao; Chen, Guo-Qiang; Chen, Min; Chen, Quan; Chen, Steve S.-L.; Chen, WenLi; Chen, Xi; Chen, Xiangmei; Chen, Xiequn; Chen, Ye-Guang; Chen, Yingyu; Chen, Yongqiang; Chen, Yu-Jen; Chen, Zhixiang; Cheng, Alan; Cheng, Christopher H.K.; Cheng, Yan; Cheong, Heesun; Cheong, Jae-Ho; Cherry, Sara; Chess-Williams, Russ; Cheung, Zelda H.; Chevet, Eric; Chiang, Hui-Ling; Chiarelli, Roberto; Chiba, Tomoki; Chin, Lih-Shen; Chiou, Shih-Hwa; Chisari, Francis V.; Cho, Chi Hin; Cho, Dong-Hyung; Choi, Augustine M.K.; Choi, DooSeok; Choi, Kyeong Sook; Choi, Mary E.; Chouaib, Salem; Choubey, Divaker; Choubey, Vinay; Chu, Charleen T.; Chuang, Tsung-Hsien; Chueh, Sheau-Huei; Chun, Taehoon; Chwae, Yong-Joon; Chye, Mee-Len; Ciarcia, Roberto; Ciriolo, Maria R.; Clague, Michael J.; Clark, Robert S.B.; Clarke, Peter G.H.; Clarke, Robert; Codogno, Patrice; Coller, Hilary A.; Colombo, María I.; Comincini, Sergio; Condello, Maria; Condorelli, Fabrizio; Cookson, Mark R.; Coombs, Graham H.; Coppens, Isabelle; Corbalan, Ramon; Cossart, Pascale; Costelli, Paola; Costes, Safia; Coto-Montes, Ana; Couve, Eduardo; Coxon, Fraser P.; Cregg, James M.; Crespo, José L.; Cronjé, Marianne J.; Cuervo, Ana Maria; Cullen, Joseph J.; Czaja, Mark J.; D'Amelio, Marcello; Darfeuille-Michaud, Arlette; Davids, Lester M.; Davies, Faith E.; De Felici, Massimo; de Groot, John F.; de Haan, Cornelis A.M.; De Martino, Luisa; De Milito, Angelo; De Tata, Vincenzo; Debnath, Jayanta; Degterev, Alexei; Dehay, Benjamin; Delbridge, Lea M.D.; Demarchi, Francesca; Deng, Yi Zhen; Dengjel, Jörn; Dent, Paul; Denton, Donna; Deretic, Vojo; Desai, Shyamal D.; Devenish, Rodney J.; Di Gioacchino, Mario; Di Paolo, Gilbert; Di Pietro, Chiara; Díaz-Araya, Guillermo; Díaz-Laviada, Inés; Diaz-Meco, Maria T.; Diaz-Nido, Javier; Dikic, Ivan; Dinesh-Kumar, Savithramma P.; Ding, Wen-Xing; Distelhorst, Clark W.; Diwan, Abhinav; Djavaheri-Mergny, Mojgan; Dokudovskaya, Svetlana; Dong, Zheng; Dorsey, Frank C.; Dosenko, Victor; Dowling, James J.; Doxsey, Stephen; Dreux, Marlène; Drew, Mark E.; Duan, Qiuhong; Duchosal, Michel A.; Duff, Karen E.; Dugail, Isabelle; Durbeej, Madeleine; Duszenko, Michael; Edelstein, Charles L.; Edinger, Aimee L.; Egea, Gustavo; Eichinger, Ludwig; Eissa, N. Tony; Ekmekcioglu, Suhendan; El-Deiry, Wafik S.; Elazar, Zvulun; Elgendy, Mohamed; Ellerby, Lisa M.; Eng, Kai Er; Engelbrecht, Anna-Mart; Engelender, Simone; Erenpreisa, Jekaterina; Escalante, Ricardo; Esclatine, Audrey; Eskelinen, Eeva-Liisa; Espert, Lucile; Espina, Virginia; Fan, Huizhou; Fan, Jia; Fan, Qi-Wen; Fan, Zhen; Fang, Shengyun; Fang, Yongqi; Fanto, Manolis; Fanzani, Alessandro; Farkas, Thomas; Farre, Jean-Claude; Faure, Mathias; Fechheimer, Marcus; Feng, Carl G.; Feng, Jian; Feng, Qili; Feng, Youji; Fésüs, László; Feuer, Ralph; Figueiredo-Pereira, Maria E.; Fimia, Gian Maria; Fingar, Diane C.; Finkbeiner, Steven; Finkel, Toren; Finley, Kim D.; Fiorito, Filomena; Fisher, Edward A.; Fisher, Paul B.; Flajolet, Marc; Florez-McClure, Maria L.; Florio, Salvatore; Fon, Edward A.; Fornai, Francesco; Fortunato, Franco; Fotedar, Rati; Fowler, Daniel H.; Fox, Howard S.; Franco, Rodrigo; Frankel, Lisa B.; Fransen, Marc; Fuentes, José M.; Fueyo, Juan; Fujii, Jun; Fujisaki, Kozo; Fujita, Eriko; Fukuda, Mitsunori; Furukawa, Ruth H.; Gaestel, Matthias; Gailly, Philippe; Gajewska, Malgorzata; Galliot, Brigitte; Galy, Vincent; Ganesh, Subramaniam; Ganetzky, Barry; Ganley, Ian G.; Gao, Fen-Biao; Gao, George F.; Gao, Jinming; Garcia, Lorena; Garcia-Manero, Guillermo; Garcia-Marcos, Mikel; Garmyn, Marjan; Gartel, Andrei L.; Gatti, Evelina; Gautel, Mathias; Gawriluk, Thomas R.; Gegg, Matthew E.; Geng, Jiefei; Germain, Marc; Gestwicki, Jason E.; Gewirtz, David A.; Ghavami, Saeid; Ghosh, Pradipta; Giammarioli, Anna M.; Giatromanolaki, Alexandra N.; Gibson, Spencer B.; Gilkerson, Robert W.; Ginger, Michael L.; Ginsberg, Henry N.; Golab, Jakub; Goligorsky, Michael S.; Golstein, Pierre; Gomez-Manzano, Candelaria; Goncu, Ebru; Gongora, Céline; Gonzalez, Claudio D.; Gonzalez, Ramon; González-Estévez, Cristina; González-Polo, Rosa Ana; Gonzalez-Rey, Elena; Gorbunov, Nikolai V.; Gorski, Sharon; Goruppi, Sandro; Gottlieb, Roberta A.; Gozuacik, Devrim; Granato, Giovanna Elvira; Grant, Gary D.; Green, Kim N.; Gregorc, Ales; Gros, Frédéric; Grose, Charles; Grunt, Thomas W.; Gual, Philippe; Guan, Jun-Lin; Guan, Kun-Liang; Guichard, Sylvie M.; Gukovskaya, Anna S.; Gukovsky, Ilya; Gunst, Jan; Gustafsson, Åsa B.; Halayko, Andrew J.; Hale, Amber N.; Halonen, Sandra K.; Hamasaki, Maho; Han, Feng; Han, Ting; Hancock, Michael K.; Hansen, Malene; Harada, Hisashi; Harada, Masaru; Hardt, Stefan E.; Harper, J. Wade; Harris, Adrian L.; Harris, James; Harris, Steven D.; Hashimoto, Makoto; Haspel, Jeffrey A.; Hayashi, Shin-ichiro; Hazelhurst, Lori A.; He, Congcong; He, You-Wen; Hébert, Marie-Josée; Heidenreich, Kim A.; Helfrich, Miep H.; Helgason, Gudmundur V.; Henske, Elizabeth P.; Herman, Brian; Herman, Paul K.; Hetz, Claudio; Hilfiker, Sabine; Hill, Joseph A.; Hocking, Lynne J.; Hofman, Paul; Hofmann, Thomas G.; Höhfeld, Jörg; Holyoake, Tessa L.; Hong, Ming-Huang; Hood, David A.; Hotamisligil, Gökhan S.; Houwerzijl, Ewout J.; Høyer-Hansen, Maria; Hu, Bingren; Hu, Chien-an A.; Hu, Hong-Ming; Hua, Ya; Huang, Canhua; Huang, Ju; Huang, Shengbing; Huang, Wei-Pang; Huber, Tobias B.; Huh, Won-Ki; Hung, Tai-Ho; Hupp, Ted R.; Hur, Gang Min; Hurley, James B.; Hussain, Sabah N.A.; Hussey, Patrick J.; Hwang, Jung Jin; Hwang, Seungmin; Ichihara, Atsuhiro; Ilkhanizadeh, Shirin; Inoki, Ken; Into, Takeshi; Iovane, Valentina; Iovanna, Juan L.; Ip, Nancy Y.; Isaka, Yoshitaka; Ishida, Hiroyuki; Isidoro, Ciro; Isobe, Ken-ichi; Iwasaki, Akiko; Izquierdo, Marta; Izumi, Yotaro; Jaakkola, Panu M.; Jäättelä, Marja; Jackson, George R.; Jackson, William T.; Janji, Bassam; Jendrach, Marina; Jeon, Ju-Hong; Jeung, Eui-Bae; Jiang, Hong; Jiang, Hongchi; Jiang, Jean X.; Jiang, Ming; Jiang, Qing; Jiang, Xuejun; Jiang, Xuejun; Jiménez, Alberto; Jin, Meiyan; Jin, Shengkan V.; Joe, Cheol O.; Johansen, Terje; Johnson, Daniel E.; Johnson, Gail V.W.; Jones, Nicola L.; Joseph, Bertrand; Joseph, Suresh K.; Joubert, Annie M.; Juhász, Gábor; Juillerat-Jeanneret, Lucienne; Jung, Chang Hwa; Jung, Yong-Keun; Kaarniranta, Kai; Kaasik, Allen; Kabuta, Tomohiro; Kadowaki, Motoni; Kågedal, Katarina; Kamada, Yoshiaki; Kaminskyy, Vitaliy O.; Kampinga, Harm H.; Kanamori, Hiromitsu; Kang, Chanhee; Kang, Khong Bee; Kang, Kwang Il; Kang, Rui; Kang, Yoon-A; Kanki, Tomotake; Kanneganti, Thirumala-Devi; Kanno, Haruo; Kanthasamy, Anumantha G.; Kanthasamy, Arthi; Karantza, Vassiliki; Kaushal, Gur P.; Kaushik, Susmita; Kawazoe, Yoshinori; Ke, Po-Yuan; Kehrl, John H.; Kelekar, Ameeta; Kerkhoff, Claus; Kessel, David H.; Khalil, Hany; Kiel, Jan A.K.W.; Kiger, Amy A.; Kihara, Akio; Kim, Deok Ryong; Kim, Do-Hyung; Kim, Dong-Hou; Kim, Eun-Kyoung; Kim, Hyung-Ryong; Kim, Jae-Sung; Kim, Jeong Hun; Kim, Jin Cheon; Kim, John K.; Kim, Peter K.; Kim, Seong Who; Kim, Yong-Sun; Kim, Yonghyun; Kimchi, Adi; Kimmelman, Alec C.; King, Jason S.; Kinsella, Timothy J.; Kirkin, Vladimir; Kirshenbaum, Lorrie A.; Kitamoto, Katsuhiko; Kitazato, Kaio; Klein, Ludger; Klimecki, Walter T.; Klucken, Jochen; Knecht, Erwin; Ko, Ben C.B.; Koch, Jan C.; Koga, Hiroshi; Koh, Jae-Young; Koh, Young Ho; Koike, Masato; Komatsu, Masaaki; Kominami, Eiki; Kong, Hee Jeong; Kong, Wei-Jia; Korolchuk, Viktor I.; Kotake, Yaichiro; Koukourakis, Michael I.; Flores, Juan B. Kouri; Kovács, Attila L.; Kraft, Claudine; Krainc, Dimitri; Krämer, Helmut; Kretz-Remy, Carole; Krichevsky, Anna M.; Kroemer, Guido; Krüger, Rejko; Krut, Oleg; Ktistakis, Nicholas T.; Kuan, Chia-Yi; Kucharczyk, Roza; Kumar, Ashok; Kumar, Raj; Kumar, Sharad; Kundu, Mondira; Kung, Hsing-Jien; Kurz, Tino; Kwon, Ho Jeong; La Spada, Albert R.; Lafont, Frank; Lamark, Trond; Landry, Jacques; Lane, Jon D.; Lapaquette, Pierre; Laporte, Jocelyn F.; László, Lajos; Lavandero, Sergio; Lavoie, Josée N.; Layfield, Robert; Lazo, Pedro A.; Le, Weidong; Le Cam, Laurent; Ledbetter, Daniel J.; Lee, Alvin J.X.; Lee, Byung-Wan; Lee, Gyun Min; Lee, Jongdae; lee, Ju-hyun; Lee, Michael; Lee, Myung-Shik; Lee, Sug Hyung; Leeuwenburgh, Christiaan; Legembre, Patrick; Legouis, Renaud; Lehmann, Michael; Lei, Huan-Yao; Lei, Qun-Ying; Leib, David A.; Leiro, José; Lemasters, John J.; Lemoine, Antoinette; Lesniak, Maciej S.; Lev, Dina; Levenson, Victor V.; Levine, Beth; Levy, Efrat; Li, Faqiang; Li, Jun-Lin; Li, Lian; Li, Sheng; Li, Weijie; Li, Xue-Jun; Li, Yan-Bo; Li, Yi-Ping; Liang, Chengyu; Liang, Qiangrong; Liao, Yung-Feng; Liberski, Pawel P.; Lieberman, Andrew; Lim, Hyunjung J.; Lim, Kah-Leong; Lim, Kyu; Lin, Chiou-Feng; Lin, Fu-Cheng; Lin, Jian; Lin, Jiandie D.; Lin, Kui; Lin, Wan-Wan; Lin, Weei-Chin; Lin, Yi-Ling; Linden, Rafael; Lingor, Paul; Lippincott-Schwartz, Jennifer; Lisanti, Michael P.; Liton, Paloma B.; Liu, Bo; Liu, Chun-Feng; Liu, Kaiyu; Liu, Leyuan; Liu, Qiong A.; Liu, Wei; Liu, Young-Chau; Liu, Yule; Lockshin, Richard A.; Lok, Chun-Nam; Lonial, Sagar; Loos, Benjamin; Lopez-Berestein, Gabriel; López-Otín, Carlos; Lossi, Laura; Lotze, Michael T.; Low, Peter; Lu, Binfeng; Lu, Bingwei; Lu, Bo; Lu, Zhen; Luciano, Fréderic; Lukacs, Nicholas W.; Lund, Anders H.; Lynch-Day, Melinda A.; Ma, Yong; Macian, Fernando; MacKeigan, Jeff P.; Macleod, Kay F.; Madeo, Frank; Maiuri, Luigi; Maiuri, Maria Chiara; Malagoli, Davide; Malicdan, May Christine V.; Malorni, Walter; Man, Na; Mandelkow, Eva-Maria; Manon, Stephen; Manov, Irena; Mao, Kai; Mao, Xiang; Mao, Zixu; Marambaud, Philippe; Marazziti, Daniela; Marcel, Yves L.; Marchbank, Katie; Marchetti, Piero; Marciniak, Stefan J.; Marcondes, Mateus; Mardi, Mohsen; Marfe, Gabriella; Mariño, Guillermo; Markaki, Maria; Marten, Mark R.; Martin, Seamus J.; Martinand-Mari, Camille; Martinet, Wim; Martinez-Vicente, Marta; Masini, Matilde; Matarrese, Paola; Matsuo, Saburo; Matteoni, Raffaele; Mayer, Andreas; Mazure, Nathalie M.; McConkey, David J.; McConnell, Melanie J.; McDermott, Catherine; McDonald, Christine; McInerney, Gerald M.; McKenna, Sharon L.; McLaughlin, BethAnn; McLean, Pamela J.; McMaster, Christopher R.; McQuibban, G. Angus; Meijer, Alfred J.; Meisler, Miriam H.; Meléndez, Alicia; Melia, Thomas J.; Melino, Gerry; Mena, Maria A.; Menendez, Javier A.; Menna-Barreto, Rubem F. S.; Menon, Manoj B.; Menzies, Fiona M.; Mercer, Carol A.; Merighi, Adalberto; Merry, Diane E.; Meschini, Stefania; Meyer, Christian G.; Meyer, Thomas F.; Miao, Chao-Yu; Miao, Jun-Ying; Michels, Paul A.M.; Michiels, Carine; Mijaljica, Dalibor; Milojkovic, Ana; Minucci, Saverio; Miracco, Clelia; Miranti, Cindy K.; Mitroulis, Ioannis; Miyazawa, Keisuke; Mizushima, Noboru; Mograbi, Baharia; Mohseni, Simin; Molero, Xavier; Mollereau, Bertrand; Mollinedo, Faustino; Momoi, Takashi; Monastyrska, Iryna; Monick, Martha M.; Monteiro, Mervyn J.; Moore, Michael N.; Mora, Rodrigo; Moreau, Kevin; Moreira, Paula I.; Moriyasu, Yuji; Moscat, Jorge; Mostowy, Serge; Mottram, Jeremy C.; Motyl, Tomasz; Moussa, Charbel E.-H.; Müller, Sylke; Muller, Sylviane; Münger, Karl; Münz, Christian; Murphy, Leon O.; Murphy, Maureen E.; Musarò, Antonio; Mysorekar, Indira; Nagata, Eiichiro; Nagata, Kazuhiro; Nahimana, Aimable; Nair, Usha; Nakagawa, Toshiyuki; Nakahira, Kiichi; Nakano, Hiroyasu; Nakatogawa, Hitoshi; Nanjundan, Meera; Naqvi, Naweed I.; Narendra, Derek P.; Narita, Masashi; Navarro, Miguel; Nawrocki, Steffan T.; Nazarko, Taras Y.; Nemchenko, Andriy; Netea, Mihai G.; Neufeld, Thomas P.; Ney, Paul A.; Nezis, Ioannis P.; Nguyen, Huu Phuc; Nie, Daotai; Nishino, Ichizo; Nislow, Corey; Nixon, Ralph A.; Noda, Takeshi; Noegel, Angelika A.; Nogalska, Anna; Noguchi, Satoru; Notterpek, Lucia; Novak, Ivana; Nozaki, Tomoyoshi; Nukina, Nobuyuki; Nürnberger, Thorsten; Nyfeler, Beat; Obara, Keisuke; Oberley, Terry D.; Oddo, Salvatore; Ogawa, Michinaga; Ohashi, Toya; Okamoto, Koji; Oleinick, Nancy L.; Oliver, F. Javier; Olsen, Laura J.; Olsson, Stefan; Opota, Onya; Osborne, Timothy F.; Ostrander, Gary K.; Otsu, Kinya; Ou, Jing-hsiung James; Ouimet, Mireille; Overholtzer, Michael; Ozpolat, Bulent; Paganetti, Paolo; Pagnini, Ugo; Pallet, Nicolas; Palmer, Glen E.; Palumbo, Camilla; Pan, Tianhong; Panaretakis, Theocharis; Pandey, Udai Bhan; Papackova, Zuzana; Papassideri, Issidora; Paris, Irmgard; Park, Junsoo; Park, Ohkmae K.; Parys, Jan B.; Parzych, Katherine R.; Patschan, Susann; Patterson, Cam; Pattingre, Sophie; Pawelek, John M.; Peng, Jianxin; Perlmutter, David H.; Perrotta, Ida; Perry, George; Pervaiz, Shazib; Peter, Matthias; Peters, Godefridus J.; Petersen, Morten; Petrovski, Goran; Phang, James M.; Piacentini, Mauro; Pierre, Philippe; Pierrefite-Carle, Valérie; Pierron, Gérard; Pinkas-Kramarski, Ronit; Piras, Antonio; Piri, Natik; Platanias, Leonidas C.; Pöggeler, Stefanie; Poirot, Marc; Poletti, Angelo; Poüs, Christian; Pozuelo-Rubio, Mercedes; Prætorius-Ibba, Mette; Prasad, Anil; Prescott, Mark; Priault, Muriel; Produit-Zengaffinen, Nathalie; Progulske-Fox, Ann; Proikas-Cezanne, Tassula; Przedborski, Serge; Przyklenk, Karin; Puertollano, Rosa; Puyal, Julien; Qian, Shu-Bing; Qin, Liang; Qin, Zheng-Hong; Quaggin, Susan E.; Raben, Nina; Rabinowich, Hannah; Rabkin, Simon W.; Rahman, Irfan; Rami, Abdelhaq; Ramm, Georg; Randall, Glenn; Randow, Felix; Rao, V. Ashutosh; Rathmell, Jeffrey C.; Ravikumar, Brinda; Ray, Swapan K.; Reed, Bruce H.; Reed, John C.; Reggiori, Fulvio; Régnier-Vigouroux, Anne; Reichert, Andreas S.; Reiners, John J.; Reiter, Russel J.; Ren, Jun; Revuelta, José L.; Rhodes, Christopher J.; Ritis, Konstantinos; Rizzo, Elizete; Robbins, Jeffrey; Roberge, Michel; Roca, Hernan; Roccheri, Maria C.; Rocchi, Stephane; Rodemann, H. Peter; Rodríguez de Córdoba, Santiago; Rohrer, Bärbel; Roninson, Igor B.; Rosen, Kirill; Rost-Roszkowska, Magdalena M.; Rouis, Mustapha; Rouschop, Kasper M.A.; Rovetta, Francesca; Rubin, Brian P.; Rubinsztein, David C.; Ruckdeschel, Klaus; Rucker, Edmund B.; Rudich, Assaf; Rudolf, Emil; Ruiz-Opazo, Nelson; Russo, Rossella; Rusten, Tor Erik; Ryan, Kevin M.; Ryter, Stefan W.; Sabatini, David M.; Sadoshima, Junichi; Saha, Tapas; Saitoh, Tatsuya; Sakagami, Hiroshi; Sakai, Yasuyoshi; Salekdeh, Ghasem Hoseini; Salomoni, Paolo; Salvaterra, Paul M.; Salvesen, Guy; Salvioli, Rosa; Sanchez, Anthony M.J.; Sánchez-Alcázar, José A.; Sánchez-Prieto, Ricardo; Sandri, Marco; Sankar, Uma; Sansanwal, Poonam; Santambrogio, Laura; Saran, Shweta; Sarkar, Sovan; Sarwal, Minnie; Sasakawa, Chihiro; Sasnauskiene, Ausra; Sass, Miklós; Sato, Ken; Sato, Miyuki; Schapira, Anthony H.V.; Scharl, Michael; Schätzl, Hermann M.; Scheper, Wiep; Schiaffino, Stefano; Schneider, Claudio; Schneider, Marion E.; Schneider-Stock, Regine; Schoenlein, Patricia V.; Schorderet, Daniel F.; Schüller, Christoph; Schwartz, Gary K.; Scorrano, Luca; Sealy, Linda; Seglen, Per O.; Segura-Aguilar, Juan; Seiliez, Iban; Seleverstov, Oleksandr; Sell, Christian; Seo, Jong Bok; Separovic, Duska; Setaluri, Vijayasaradhi; Setoguchi, Takao; Settembre, Carmine; Shacka, John J.; Shanmugam, Mala; Shapiro, Irving M.; Shaulian, Eitan; Shaw, Reuben J.; Shelhamer, James H.; Shen, Han-Ming; Shen, Wei-Chiang; Sheng, Zu-Hang; Shi, Yang; Shibuya, Kenichi; Shidoji, Yoshihiro; Shieh, Jeng-Jer; Shih, Chwen-Ming; Shimada, Yohta; Shimizu, Shigeomi; Shintani, Takahiro; Shirihai, Orian S.; Shore, Gordon C.; Sibirny, Andriy A.; Sidhu, Stan B.; Sikorska, Beata; Silva-Zacarin, Elaine C.M.; Simmons, Alison; Simon, Anna Katharina; Simon, Hans-Uwe; Simone, Cristiano; Simonsen, Anne; Sinclair, David A.; Singh, Rajat; Sinha, Debasish; Sinicrope, Frank A.; Sirko, Agnieszka; Siu, Parco M.; Sivridis, Efthimios; Skop, Vojtech; Skulachev, Vladimir P.; Slack, Ruth S.; Smaili, Soraya S.; Smith, Duncan R.; Soengas, Maria S.; Soldati, Thierry; Song, Xueqin; Sood, Anil K.; Soong, Tuck Wah; Sotgia, Federica; Spector, Stephen A.; Spies, Claudia D.; Springer, Wolfdieter; Srinivasula, Srinivasa M.; Stefanis, Leonidas; Steffan, Joan S.; Stendel, Ruediger; Stenmark, Harald; Stephanou, Anastasis; Stern, Stephan T.; Sternberg, Cinthya; Stork, Björn; Strålfors, Peter; Subauste, Carlos S.; Sui, Xinbing; Sulzer, David; Sun, Jiaren; Sun, Shi-Yong; Sun, Zhi-Jun; Sung, Joseph J.Y.; Suzuki, Kuninori; Suzuki, Toshihiko; Swanson, Michele S.; Swanton, Charles; Sweeney, Sean T.; Sy, Lai-King; Szabadkai, György; Tabas, Ira; Taegtmeyer, Heinrich; Tafani, Marco; Takács-Vellai, Krisztina; Takano, Yoshitaka; Takegawa, Kaoru; Takemura, Genzou; Takeshita, Fumihiko; Talbot, Nicholas J.; Tan, Kevin S.W.; Tanaka, Keiji; Tanaka, Kozo; Tang, Daolin; Tang, Dingzhong; Tanida, Isei; Tannous, Bakhos A.; Tavernarakis, Nektarios; Taylor, Graham S.; Taylor, Gregory A.; Taylor, J. Paul; Terada, Lance S.; Terman, Alexei; Tettamanti, Gianluca; Thevissen, Karin; Thompson, Craig B.; Thorburn, Andrew; Thumm, Michael; Tian, FengFeng; Tian, Yuan; Tocchini-Valentini, Glauco; Tolkovsky, Aviva M.; Tomino, Yasuhiko; Tönges, Lars; Tooze, Sharon A.; Tournier, Cathy; Tower, John; Towns, Roberto; Trajkovic, Vladimir; Travassos, Leonardo H.; Tsai, Ting-Fen; Tschan, Mario P.; Tsubata, Takeshi; Tsung, Allan; Turk, Boris; Turner, Lorianne S.; Tyagi, Suresh C.; Uchiyama, Yasuo; Ueno, Takashi; Umekawa, Midori; Umemiya-Shirafuji, Rika; Unni, Vivek K.; Vaccaro, Maria I.; Valente, Enza Maria; Van den Berghe, Greet; van der Klei, Ida J.; van Doorn, Wouter G.; van Dyk, Linda F.; van Egmond, Marjolein; van Grunsven, Leo A.; Vandenabeele, Peter; Vandenberghe, Wim P.; Vanhorebeek, Ilse; Vaquero, Eva C.; Velasco, Guillermo; Vellai, Tibor; Vicencio, José Miguel; Vierstra, Richard D.; Vila, Miquel; Vindis, Cécile; Viola, Giampietro; Viscomi, Maria Teresa; Voitsekhovskaja, Olga V.; von Haefen, Clarissa; Votruba, Marcela; Wada, Keiji; Wade-Martins, Richard; Walker, Cheryl L.; Walsh, Craig M.; Walter, Jochen; Wan, Xiang-Bo; Wang, Aimin; Wang, Chenguang; Wang, Dawei; Wang, Fan; Wang, Fen; Wang, Guanghui; Wang, Haichao; Wang, Hong-Gang; Wang, Horng-Dar; Wang, Jin; Wang, Ke; Wang, Mei; Wang, Richard C.; Wang, Xinglong; Wang, Xiujie J.; Wang, Ying-Jan; Wang, Yipeng; Wang, Zhen-Bo; Wang, Zhigang Charles; Wang, Zhinong; Wansink, Derick G.; Ward, Diane M.; Watada, Hirotaka; Waters, Sarah L.; Webster, Paul; Wei, Lixin; Weihl, Conrad C.; Weiss, William A.; Welford, Scott M.; Wen, Long-Ping; Whitehouse, Caroline A.; Whitton, J. Lindsay; Whitworth, Alexander J.; Wileman, Tom; Wiley, John W.; Wilkinson, Simon; Willbold, Dieter; Williams, Roger L.; Williamson, Peter R.; Wouters, Bradly G.; Wu, Chenghan; Wu, Dao-Cheng; Wu, William K.K.; Wyttenbach, Andreas; Xavier, Ramnik J.; Xi, Zhijun; Xia, Pu; Xiao, Gengfu; Xie, Zhiping; Xie, Zhonglin; Xu, Da-zhi; Xu, Jianzhen; Xu, Liang; Xu, Xiaolei; Yamamoto, Ai; Yamamoto, Akitsugu; Yamashina, Shunhei; Yamashita, Michiaki; Yan, Xianghua; Yanagida, Mitsuhiro; Yang, Dun-Sheng; Yang, Elizabeth; Yang, Jin-Ming; Yang, Shi Yu; Yang, Wannian; Yang, Wei Yuan; Yang, Zhifen; Yao, Meng-Chao; Yao, Tso-Pang; Yeganeh, Behzad; Yen, Wei-Lien; Yin, Jia-Jing; Yin, Xiao-Ming; Yoo, Ook-Joon; Yoon, Gyesoon; Yoon, Seung-Yong; Yorimitsu, Tomohiro; Yoshikawa, Yuko; Yoshimori, Tamotsu; Yoshimoto, Kohki; You, Ho Jin; Youle, Richard J.; Younes, Anas; Yu, Li; Yu, Long; Yu, Seong-Woon; Yu, Wai Haung; Yuan, Zhi-Min; Yue, Zhenyu; Yun, Cheol-Heui; Yuzaki, Michisuke; Zabirnyk, Olga; Silva-Zacarin, Elaine; Zacks, David; Zacksenhaus, Eldad; Zaffaroni, Nadia; Zakeri, Zahra; Zeh, III, Herbert J.; Zeitlin, Scott O.; Zhang, Hong; Zhang, Hui-Ling; Zhang, Jianhua; Zhang, Jing-Pu; Zhang, Lin; Zhang, Long; Zhang, Ming-Yong; Zhang, Xu Dong; Zhao, Mantong; Zhao, Yi-Fang; Zhao, Ying; Zhao, Zhizhuang J.; Zheng, Xiaoxiang; Zhivotovsky, Boris; Zhong, Qing; Zhou, Cong-Zhao; Zhu, Changlian; Zhu, Wei-Guo; Zhu, Xiao-Feng; Zhu, Xiongwei; Zhu, Yuangang; Zoladek, Teresa; Zong, Wei-Xing; Zorzano, Antonio; Zschocke, Jürgen; Zuckerbraun, Brian

2012-01-01

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused

Chemical composition and antioxidant/antimicrobial activities in supercritical carbon dioxide fluid extract of Gloiopeltis tenax.

Science.gov (United States)

Zheng, Jiaojiao; Chen, Yicun; Yao, Fen; Chen, Weizhou; Shi, Ganggang

2012-12-01

Gloiopeltis tenax (G. tenax) is widely distributed along the Chinese coastal areas and is commonly used in the treatment of diarrhea and colitis. This study aimed at investigating the bioactivities of the volatile constituents in G. tenax. We extracted the essential constituents of G. tenax by supercritical carbon dioxide extraction (CO₂-SFE), then identified and analyzed the constituents by gas chromatography-mass spectrometry (GC-MS). In total, 30 components were identified in the G. tenax extract. The components showed remarkable antioxidant activity (radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH)), lipid peroxidation inhibition capacity (in a β-carotene/linoleic acid-coupled oxidation reaction), and hydroxyl radical-scavenging activity (by deoxyribose degradation by iron-dependent hydroxyl radical), compared to butylated hydroxytoluene. In microdilution assays, G. tenax extracts showed a moderate inhibitory effects on Staphyloccocus aureus (minimum inhibitory concentration (MIC) = 3.9 mg/mL), Enterococcus faecalis (7.8 mg/mL), Pseudomonas aeruginosa (15.6 mg/mL), and Escherichia coli (3.9 mg/mL). Antioxidant and antimicrobial activities of G. tenax were related to the active chemical composition. These results suggest that the CO₂-SFE extract from G. tenax has potential to be used as a natural antioxidant and antimicrobial agent in food processing.

Chemical Composition and Antioxidant/Antimicrobial Activities in Supercritical Carbon Dioxide Fluid Extract of Gloiopeltis tenax

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Jiaojiao Zheng

2012-11-01

Full Text Available Gloiopeltis tenax (G. tenax is widely distributed along the Chinese coastal areas and is commonly used in the treatment of diarrhea and colitis. This study aimed at investigating the bioactivities of the volatile constituents in G. tenax. We extracted the essential constituents of G. tenax by supercritical carbon dioxide extraction (CO2-SFE, then identified and analyzed the constituents by gas chromatography-mass spectrometry (GC-MS. In total, 30 components were identified in the G. tenax extract. The components showed remarkable antioxidant activity (radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH, lipid peroxidation inhibition capacity (in a β-carotene/linoleic acid-coupled oxidation reaction, and hydroxyl radical-scavenging activity (by deoxyribose degradation by iron-dependent hydroxyl radical, compared to butylated hydroxytoluene. In microdilution assays, G. tenax extracts showed a moderate inhibitory effects on Staphyloccocus aureus (minimum inhibitory concentration (MIC = 3.9 mg/mL, Enterococcus faecalis (7.8 mg/mL, Pseudomonas aeruginosa (15.6 mg/mL, and Escherichia coli (3.9 mg/mL. Antioxidant and antimicrobial activities of G. tenax were related to the active chemical composition. These results suggest that the CO2-SFE extract from G. tenax has potential to be used as a natural antioxidant and antimicrobial agent in food processing.

Influence of exogenous fibrolytic enzymes on in vitro and in sacco degradation of forages for ruminants

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Lorenzo Carreón

2010-02-01

Full Text Available An in vitro assay was carried out to evaluate the effects of exogenous fibrolytic enzymes (1, 2, 3 and 4 g/kg DM powder preparation containing xylanase and cellulase from Aspergillus niger and Trichoderma viride on DM, NDF and ADF degradation of alfalfa hay, corn silage, corn stover, elephant grass, Guinea grass and oat straw. Kinetics data of in vitro degradations were analyzed. The potentially degradable fraction and degradation rate of NDF and ADF of alfalfa increased quadratically (P<0.05 as the inclusion level of enzyme increased up to 3 g. The others forages were not affected by the enzyme. An in sacco trail was performed using four Holstein steers fitted with ruminal cannulas to evaluate the effects of the exogenous fibrolytic enzymes (3 g/kg DM on DM, NDF and ADF degradation of alfalfa hay and corn stover. Kinetics data were also analyzed. The potentially degradable fraction degradation of NDF (62.0 vs 65.7% and ADF (52.8 vs 56.9%, of alfalfa hay were increased (P<0.05 by the exogenous fibrolytic enzymes, but no differences were found for corn stover. These results suggest that the enzymes increased in vitro and in sacco fibre degradation only for alfalfa hay.

Identification and in vitro cytotoxicity of ochratoxin A degradation products formed during coffee roasting.

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Cramer, Benedikt; Königs, Maika; Humpf, Hans-Ulrich

2008-07-23

The mycotoxin ochratoxin A is degraded by up to 90% during coffee roasting. In order to investigate this degradation, model heating experiments with ochratoxin A were carried out, and the reaction products were analyzed by HPLC-DAD and HPLC-MS/MS. Two ochratoxin A degradation products were identified, and their structure and absolute configuration were determined. As degradation reactions, the isomerization to 14-(R)-ochratoxin A and the decarboxylation to 14-decarboxy-ochratoxin A were identified. Subsequently, an analytical method for the determination of these compounds in roasted coffee was developed. Quantification was carried out by HPLC-MS/MS and the use of stable isotope dilution analysis. By using this method for the analysis of 15 coffee samples from the German market, it could be shown that, during coffee roasting, the ochratoxin A diastereomer 14-(R)-ochratoxin A was formed in amounts of up to 25.6% relative to ochratoxin A. The decarboxylation product was formed only in traces. For toxicity evaluations, first preliminary cell culture assays were performed with the two new substances. Both degradation products exhibited higher IC50 values and caused apoptotic effects with higher concentrations than ochratoxin A in cultured human kidney epithelial cells. Thus, these cell culture data suggest that the degradation products are less cytotoxic than ochratoxin A.

γ-radiolytical degradation of levofloxacin lactate and the activity of the byproducts

International Nuclear Information System (INIS)

Cao Dongmei; Zhang Xiaohong; Guan Yu; Zhu Wei; Zhang HaiQian

2010-01-01

Recently antibiotics wastewater in environment has induced the increment of bacterial resistance. This paper is to investigate the gamma radiolysis of wastewater containing 10 μg/mL levofloxacin lactate (LVF). It has been found that the antibiotic was removed more than 99% with 1 kGy under air while the G-value decreased with the dose increment.Five main degraded products (m/z 346, 330, 318, 302, 274) and the most probable radiolysis pathway were identified by liquid chromatography-mass spectrometry (LC-MS). Compared with the degradation under N 2 , the radiolytical mechanism was suggested.In the active assay, 2 μg/mL was the inhibitory concentration.Compared with the concentration of 4 μg/mL, the minimal inhibitory concentration (MIC), degraded products analysis can be suggested that the byproduct m/z 346 might have the anti-E. coli activity. It has been shown that 3 kGy is the appropriate dose for the radiolytical treatment of LVF. To sum up, the gamma radiation technique is an effective method for decomposing antibiotics, and it is necessary to take the activity of degraded products into consideration. (authors)

Metagenomic Functional Potential Predicts Degradation Rates of a Model Organophosphorus Xenobiotic in Pesticide Contaminated Soils

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Thomas C. Jeffries

2018-02-01

Full Text Available Chemical contamination of natural and agricultural habitats is an increasing global problem and a major threat to sustainability and human health. Organophosphorus (OP compounds are one major class of contaminant and can undergo microbial degradation, however, no studies have applied system-wide ecogenomic tools to investigate OP degradation or use metagenomics to understand the underlying mechanisms of biodegradation in situ and predict degradation potential. Thus, there is a lack of knowledge regarding the functional genes and genomic potential underpinning degradation and community responses to contamination. Here we address this knowledge gap by performing shotgun sequencing of community DNA from agricultural soils with a history of pesticide usage and profiling shifts in functional genes and microbial taxa abundance. Our results showed two distinct groups of soils defined by differing functional and taxonomic profiles. Degradation assays suggested that these groups corresponded to the organophosphorus degradation potential of soils, with the fastest degrading community being defined by increases in transport and nutrient cycling pathways and enzymes potentially involved in phosphorus metabolism. This was against a backdrop of taxonomic community shifts potentially related to contamination adaptation and reflecting the legacy of exposure. Overall our results highlight the value of using holistic system-wide metagenomic approaches as a tool to predict microbial degradation in the context of the ecology of contaminated habitats.

Antioxidant Generation during Coffee Roasting: A Comparison and Interpretation from Three Complementary Assays

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Sebastian E. W. Opitz

2014-11-01

Full Text Available Coffee is a major source of dietary antioxidants; some are present in the green bean, whereas others are generated during roasting. However, there is no single accepted analytical method for their routine determination. This paper describes the adaption of three complementary assays (Folin-Ciocalteu (FC, ABTS and ORAC for the routine assessment of antioxidant capacity of beverages, their validation, and use for determining the antioxidant capacities of extracts from coffee beans at different stages in the roasting process. All assays showed a progressive increase in antioxidant capacity during roasting to a light roast state, consistent with the production of melanoidins having a higher antioxidant effect than the degradation of CGAs. However, the three assays gave different numbers for the total antioxidant capacity of green beans relative to gallic acid (GA, although the range of values was much smaller when chlorogenic acid (CGA was used as reference. Therefore, although all three assays indicated that there was an increase in antioxidant activity during coffee roasting, and the large differences in responses to GA and CGA illustrate their different sensitivities to different types of antioxidant molecule.

Phytate degradation by fungi and bacteria that inhabit sawdust and coffee residue composts.

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Fathallh Eida, Mohamed; Nagaoka, Toshinori; Wasaki, Jun; Kouno, Kenji

2013-01-01

Phytate is the primary source of organic phosphorus, but it cannot be directly utilized by plants and is strongly adsorbed by the soil, reducing bioavailability. Composting is a process used to improve the bioavailability of phytate in organic wastes through degradation by microorganisms. In this study, we aimed to investigate the phytate-degrading ability of fungi and bacteria that inhabit sawdust compost and coffee residue compost, and their contribution to the composting process. In the plate assay, the fungi that formed clear zones around their colonies belonged to the genera Mucor, Penicillium, Galactomyces, Coniochaeta, Aspergillus, and Fusarium, while the bacteria belonged to the genera Pseudomonas, Enterobacter, Chitinophaga, and Rahnella. Eight fungal isolates (genera Mucor, Penicillium, Galactomyces, and Coniochaeta) and four bacterial isolates (genera Pseudomonas, Enterobacter, and Rahnella) were selected to evaluate phytase activity in their liquid culture and their ability to degrade phytate in organic materials composed of mushroom media residue and rice bran. The selected fungi degraded phytate in organic materials to varying degrees. Penicillium isolates showed the highest degradation ability and Coniochaeta isolate exhibited relatively high degradation ability. The clear zone diameters of these fungal isolates displayed significantly positive and negative correlations with inorganic and phytate phosphorus contents in the organic materials after incubation, respectively; however, none of the selected bacteria reduced phytate phosphorus in organic materials. It is therefore possible that fungi are major contributors to phytate degradation during composting.

Molecular Structure of Nucleic Acids

Indian Academy of Sciences (India)

Molecular Structure of Nucleic Acids. A Structure for Deoxyribose Nucleic Acid. J. D. Watson and F. H. C. Crick. Medical Research Council Unit for the Study of the Molecular Structure of Biological. Systems, Cavendish Laboratory, Cambridge. April 2. We wish to suggest a structure for the salt of deoxyribose nucleic acid ...

Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

Science.gov (United States)

Leatham, Gary F.

1985-01-01

Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22°C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-β-d-glucosidase, β-N-acetyl-d-glucosaminidase, α-d-galactosidase, β-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting. PMID:16346918

Branched multifunctional polyether polyketals: variation of ketal group structure enables unprecedented control over polymer degradation in solution and within cells.

Science.gov (United States)

Shenoi, Rajesh A; Narayanannair, Jayaprakash K; Hamilton, Jasmine L; Lai, Benjamin F L; Horte, Sonja; Kainthan, Rajesh K; Varghese, Jos P; Rajeev, Kallanthottathil G; Manoharan, Muthiah; Kizhakkedathu, Jayachandran N

2012-09-12

Multifunctional biocompatible and biodegradable nanomaterials incorporating specific degradable linkages that respond to various stimuli and with defined degradation profiles are critical to the advancement of targeted nanomedicine. Herein we report, for the first time, a new class of multifunctional dendritic polyether polyketals containing different ketal linkages in their backbone that exhibit unprecedented control over degradation in solution and within the cells. High-molecular-weight and highly compact poly(ketal hydroxyethers) (PKHEs) were synthesized from newly designed α-epoxy-ω-hydroxyl-functionalized AB(2)-type ketal monomers carrying structurally different ketal groups (both cyclic and acyclic) with good control over polymer properties by anionic ring-opening multibranching polymerization. Polymer functionalization with multiple azide and amine groups was achieved without degradation of the ketal group. The polymer degradation was controlled primarily by the differences in the structure and torsional strain of the substituted ketal groups in the main chain, while for polymers with linear (acyclic) ketal groups, the hydrophobicity of the polymer may play an additional role. This was supported by the log P values of the monomers and the hydrophobicity of the polymers determined by fluorescence spectroscopy using pyrene as the probe. A range of hydrolysis half-lives of the polymers at mild acidic pH values was achieved, from a few minutes to a few hundred days, directly correlating with the differences in ketal group structures. Confocal microscopy analyses demonstrated similar degradation profiles for PKHEs within live cells, as seen in solution and the delivery of fluorescent marker to the cytosol. The cell viability measured by MTS assay and blood compatibility determined by complement activation, platelet activation, and coagulation assays demonstrate that PKHEs and their degradation products are highly biocompatible. Taken together, these data

Isolation and identification of bacterial consortia responsible for degrading oil spills from the coastal area of Yanbu, Saudi Arabia

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Amr Abd-EL Mooti EL Hanafy

2016-01-01

Full Text Available Twenty-three crude-oil-degrading bacteria were isolated from oil-contaminated sites near the Red Sea. Based on a high growth rate on crude oil and on hydrocarbon degradation ability, four strains were selected from the 23 isolated strains for further study. These four strains were selected on the basis of dichlorophenolindophenol assay. The nucleotide sequences of the 16S rRNA gene showed that these isolated strains belonged to genus Pseudomonas and Nitratireductor. Among the four isolates, strains S5 (Pseudomonas sp., 95% and 4b (Nitratireductor sp., 70% were the most effective ones in degrading crude oil. Using a spectrophotometer and gas chromatography–mass spectrometry, degradation of more than 90% of the crude oil was observed after two weeks of cultivation in Bushnell–Haas medium. The results showed that these strains have the ability to degrade crude oil and may be used for environmental remediation.

Investigation of the profile and kinetics of degradation of rivaroxaban using HPLC, TLC-densitometry and LC/MS/MS: Application to pre-formulation studies

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Mohamed A. Abdallah

2015-06-01

Full Text Available Rivaroxaban (RIVA, an amide group-containing oral anticoagulant was subjected to stress conditions commonly required for the registration of pharmaceuticals: base and acid-catalyzed hydrolysis (0.1 M, 60 °C, 3–6 h, oxidation (10% H2O2, 24 h, photodegradation (300–800 nm, 24 h and thermal decomposition (50 °C, 6 h. Two major degradation products were separated and identified using TLC and LC/MS/MS, respectively. An orthogonal stability-indicating testing protocol (RP-HPLC and NP-TLC-densitometry was developed and validated according to ICH guidelines. Both assays enabled the determination of RIVA in the presence of its degradation products as well as the kinetics of degradation. Determination was carried out over a concentration range of (5.00–50.00 μg/mL and (0.40–12.00 μg/band with an accuracy of (100.81% ± 1.03 and (100.29% ± 1.08 for HPLC and TLC-densitometry, respectively. Results indicated that RIVA was stable towards oxidation, photodegradation and thermal decomposition but extremely sensitive to hydrolysis. Two major degradation products were detected in the case of base-catalyzed hydrolysis while only one degradation product was detected upon acid-catalyzed hydrolysis. This could be attributed to the presence of amide groups in RIVA structure of different stability profiles. The kinetics of hydrolysis was investigated in more detail and the reaction was found to follow the pseudo first order kinetics, as confirmed by the results of both HPLC and TLC-densitometric assays. The applicability of the assay for the determination of RIVA content and dissolution pattern of the innovator product as well as three generic formulations was demonstrated.

Microbial degradation and impact of Bracken toxin ptaquiloside on microbial communities in soil

DEFF Research Database (Denmark)

Engel, Pernille; Brandt, Kristian Koefoed; Rasmussen, Lars Holm

2007-01-01

), but not in the NZ soil (weak acid loamy Entisol). In the DK soil PTA turnover was predominantly due to microbial degradation (biodegradation); chemical hydrolysis was occurring mainly in the uppermost A horizon where pH was very low (3.4). Microbial activity (basal respiration) and growth ([3H]leucine incorporation...... assay) increased after PTA exposure, indicating that the Bracken toxin served as a C substrate for the organotrophic microorganisms. On the other hand, there was no apparent impact of PTA on community size as measured by substrate-induced respiration or composition as indicated by community......-level physiological profiles. Our results demonstrate that PTA stimulates microbial activity and that microorganisms play a predominant role for rapid PTA degradation in Bracken-impacted soils....

Use of mycelia as paths for the isolation of contaminant‐degrading bacteria from soil

Science.gov (United States)

Furuno, Shoko; Remer, Rita; Chatzinotas, Antonis; Harms, Hauke; Wick, Lukas Y.

2012-01-01

Summary Mycelia of fungi and soil oomycetes have recently been found to act as effective paths boosting bacterial mobility and bioaccessibility of contaminants in vadose environments. In this study, we demonstrate that mycelia can be used for targeted separation and isolation of contaminant‐degrading bacteria from soil. In a ‘proof of concept’ study we developed a novel approach to isolate bacteria from contaminated soil using mycelia of the soil oomycete Pythium ultimum as translocation networks for bacteria and the polycyclic aromatic hydrocarbon naphthalene (NAPH) as selective carbon source. NAPH‐degrading bacterial isolates were affiliated with the genera Xanthomonas, Rhodococcus and Pseudomonas. Except for Rhodococcus the NAPH‐degrading isolates exhibited significant motility as observed in standard swarming and swimming motility assays. All steps of the isolation procedures were followed by cultivation‐independent terminal 16S rRNA gene terminal fragment length polymorphism (T‐RFLP) analysis. Interestingly, a high similarity (63%) between both the cultivable NAPH‐degrading migrant and the cultivable parent soil bacterial community profiles was observed. This suggests that mycelial networks generally confer mobility to native, contaminant‐degrading soil bacteria. Targeted, mycelia‐based dispersal hence may have high potential for the isolation of bacteria with biotechnologically useful properties. PMID:22014110

Glycerol-plasticised silk membranes made using formic acid are ductile, transparent and degradation-resistant.

Science.gov (United States)

Allardyce, Benjamin J; Rajkhowa, Rangam; Dilley, Rodney J; Redmond, Sharon L; Atlas, Marcus D; Wang, Xungai

2017-11-01

Regenerated silk fibroin membranes tend to be brittle when dry. The use of plasticisers such as glycerol improve membrane ductility, but, when combined with aqueous processing, can lead to a higher degradation rate than solvent-annealed membranes. This study investigated the use of formic acid as the solvent with glycerol to make deformable yet degradation-resistant silk membranes. Here we show that membranes cast using formic acid had low light scattering, with a diffuse transmittance of less than 5% over the visible wavelengths, significantly lower than the 20% transmittance of aqueous derived silk/glycerol membranes. They had 64% β-sheet content and lost just 30% of the initial silk weight over 6h when tested with an accelerated enzymatic degradation assay, in comparison the aqueous membranes completely degraded within this timeframe. The addition of glycerol also improved the maximum elongation of formic acid derived membranes from under 3% to over 100%. They also showed good cytocompatibility and supported the adhesion and migration of human tympanic membrane keratinocytes. Formic acid based, silk/glycerol membranes may be of great use in medical applications such as repair of tympanic membrane perforation or ocular applications where transparency and resistance to enzymatic degradation are important. Copyright © 2017 Elsevier B.V. All rights reserved.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

Science.gov (United States)

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

A Cancer Cell-Activatable Aptamer-Reporter System for One-Step Assay of Circulating Tumor Cells

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Zihua Zeng

2014-01-01

Full Text Available The current antibody-mediated numeration assays of circulating tumor cells (CTCs require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

Flow cytofluorometric assay of human whole blood leukocyte DNA degradation in response to Yersinia pestis and Staphylococcus aureus

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Kravtsov, Alexander L.; Grebenyukova, Tatyana P.; Bobyleva, Elena V.; Golovko, Elena M.; Malyukova, Tatyana A.; Lyapin, Mikhail N.; Kostyukova, Tatyana A.; Yezhov, Igor N.; Kuznetsov, Oleg S.

2001-05-01

Human leukocytes containing less than 2C DNA per cell (damaged or dead cells) were detected and quantified by flow cytometry and DNA-specific staining with ethidium bromide and mithramycin in whole blood infected with Staphylococcus aureus or Yersinia pestis. Addition of live S. aureus to the blood (100 microbe cells per one leukocyte) resulted in rapid degradation of leukocyte DNA within 3 to 6 hours of incubation at 37 degree(s)C. However, only about 50 percent cells were damaged and the leukocytes with the intact genetic apparatus could be found in the blood for a period up to 24 hours. The leukocyte injury was preceded by an increase of DNA per cell content (as compared to the normal one) that was likely to be connected with the active phagocytosis of S. aureus by granulocytes (2C DNA of diploid phagocytes plus the all bacterial DNA absorbed). In response to the same dose of actively growing (at 37 degree(s)C) virulent Y. pestis cells, no increase in DNA content per cell could be observed in the human blood leukocytes. The process of the leukocyte DNA degradation started after a 6-hour incubation, and between 18 to 24 hours of incubation about 90 percent leukocytes (phagocytes and lymphocytes) lost their specific DNA fluorescence. These results demonstrated a high potential of flow cytometry in comparative analysis in vitro of the leukocyte DNA degradation process in human blood in response to bacteria with various pathogenic properties. They agree with the modern idea of an apoptotic mechanism of immunosuppression in plague.

Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

International Nuclear Information System (INIS)

Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

2008-01-01

Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis

Ecofriendly degradation of sulfonated diazo dye C.I. Reactive Green 19A using Micrococcus glutamicus NCIM-2168.

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Saratale, R G; Saratale, G D; Chang, J S; Govindwar, S P

2009-09-01

Micrococcus glutamicus NCIM-2168 exhibited complete decolorization and degradation of C.I. Reactive Green 19A (an initial concentration of 50 mg l(-1)) within 42 h at temperature 37 degrees C and pH 8, under static condition. Extent of mineralization was determined with total organic carbon (TOC) and chemical oxygen demand (COD) measurement, showing a satisfactory reduction of TOC (72%) and COD (66%) within 42 h. Enzyme studies shows involvement of oxidoreductive enzymes in decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Reactive Green 19A into various metabolites. The microbial toxicity and phytotoxicity assay revealed that the degradation of Reactive Green 19A produced nontoxic metabolites. In addition, the M. glutamicus strain was applied to decolorize a mixture of ten reactive dyes showing a 63% decolorization (in terms of decrease in ADMI value) within 72 h, along with 48% and 42% reduction in TOC and COD under static condition.

A LDR-PCR approach for multiplex polymorphisms genotyping of severely degraded DNA with fragment sizes <100 bp.

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Zhang, Zhen; Wang, Bao-Jie; Guan, Hong-Yu; Pang, Hao; Xuan, Jin-Feng

2009-11-01

Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini-short tandem repeat-polymerase chain reaction (PCR) and mini-sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small-scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.

Preparation of supramolecular hydrogel-enzyme hybrids exhibiting biomolecule-responsive gel degradation.

Science.gov (United States)

Shigemitsu, Hajime; Fujisaku, Takahiro; Onogi, Shoji; Yoshii, Tatsuyuki; Ikeda, Masato; Hamachi, Itaru

2016-09-01

Hydrogelators are small, self-assembling molecules that form supramolecular nanofiber networks that exhibit unique dynamic properties. Development of supramolecular hydrogels that degrade in response to various biomolecules could potentially be used for applications in areas such as drug delivery and diagnostics. Here we provide a synthetic procedure for preparing redox-responsive supramolecular hydrogelators that are used to create hydrogels that degrade in response to oxidizing or reducing conditions. The synthesis takes ∼2-4 d, and it can potentially be carried out in parallel to prepare multiple hydrogelator candidates. This described solid-phase peptide synthesis protocol can be used to produce previously described hydrogelators or to construct a focused molecular library to efficiently discover and optimize new hydrogelators. In addition, we describe the preparation of redox-responsive supramolecular hydrogel-enzyme hybrids that are created by mixing aqueous solutions of hydrogelators and enzymes, which requires 2 h for completion. The resultant supramolecular hydrogel-enzyme hybrids exhibit gel degradation in response to various biomolecules, and can be rationally designed by connecting the chemical reactions of the hydrogelators with enzymatic reactions. Gel degradation in response to biomolecules as triggers occurs within a few hours. We also describe the preparation of hydrogel-enzyme hybrids arrayed on flat glass slides, enabling high-throughput analysis of biomolecules such as glucose, uric acid, lactate and so on by gel degradation, which is detectable by the naked eye. The protocol requires ∼6 h to prepare the hydrogel-enzyme hybrid array and to complete the biomolecule assay.

Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

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Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H.; Burk, Robert D.

2015-01-01

In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution. PMID:26086730

Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

Directory of Open Access Journals (Sweden)

Koenraad Van Doorslaer

2015-06-01

Full Text Available In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

Science.gov (United States)

Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

2015-06-01

In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

Progress of organic matter degradation and maturity of compost produced in a large-scale composting facility.

Science.gov (United States)

Nakasaki, Kiyohiko; Marui, Taketoshi

2011-06-01

To monitor the progress of organic matter degradation in a large-scale composting facility, the percentage of organic matter degradation was determined by measuring CO(2) evolution during recomposting of compost samples withdrawn from the facility. The percentage of organic matter degradation was calculated as the ratio of the amount of CO(2) evolved from compost raw material to that evolved from each sample during recomposting in the laboratory composting apparatus. It was assumed that the difference in the cumulative emission of CO(2) between the compost raw material and a sample corresponds to the amount of CO( 2) evolved from the sample in the composting facility. Using this method, the changes in organic matter degradation during composting in practical large-scale composting facilities were estimated and it was found that the percentage of organic matter degradation increased more vigorously in the earlier stages than in the later stages of composting. The percentage of organic matter degradation finally reached 78 and 55% for the compost produced from garbage-animal manure mixture and distillery waste (shochu residue), respectively. It was thus ascertained that organic matter degradation progressed well in both composting facilities. Furthermore, by performing a plant growth assay, it was observed that the compost products of both the facilities did not inhibit seed germination and thus were useful in promoting plant growth.

Isolation of Thermophilic Lignin Degrading Bacteria from Oil-Palm Empty Fruit Bunch (EFB) Compost

Science.gov (United States)

Lai, C. M. T.; Chua, H. B.; Danquah, M. K.; Saptoro, A.

2017-06-01

Empty Fruit Bunch (EFB) is a potential and sustainable feedstock for bioethanol production due to its high cellulosic content and availability in Malaysia. Due to high lignin content of EFB and the lack of effective delignification process, commercial bioethanol production from EFB is presently not viable. Enzymatic delignification has been identified as one of the key steps in utilising EFB as a feedstock for bioethanol conversion. To date, limited work has been reported on the isolation of lignin degrading bacteria. Hence, there is a growing interest to search for new lignin degrading bacteria with greater tolerance to temperature and high level of ligninolytic enzymes for more effective lignin degradation. This study aimed to isolate and screen thermophilic ligninolytic microorganisms from EFB compost. Ten isolates were successfully isolated from EFB compost. Although they are not capable of decolorizing Methylene Blue (MB) dye under agar plate assay method, they are able to utilize lignin mimicked compound - guaiacol as a sole carbon on the agar plate assay. This infers that there is no correlation of ligninolytic enzymes with dye decolourization for all the isolates that have been isolated. However, they are able to produce ligninolytic enzymes (Lignin peroxidase, Manganese peroxidase, Laccase) in Minimal Salt Medium with Kraft Lignin (MSM-KL) with Lignin Peroxidase (LiP) as the predominant enzyme followed by Manganese Peroxidase (MnP) and Laccase (Lac). Among all the tested isolates, CLMT 29 has the highest LiP production up to 8.7673 U/mL following 24 h of growth.

Random assay in radioimmunoassay: Feasibility and application compared with batch assay

Energy Technology Data Exchange (ETDEWEB)

Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

2016-12-15

The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

Kinetic measurements of the hydrolytic degradation of cefixime: effect of Captisol complexation and water-soluble polymers.

Science.gov (United States)

Mallick, Subrata; Mondal, Arijit; Sannigrahi, Santanu

2008-07-01

We have taken kinetic measurements of the hydrolytic degradation of cefixime, and have studied the effect of Captisol complexation and water-soluble polymers on that degradation. The phase solubility of cefixime in Captisol was determined. Kinetic measurements were carried out as a function of pH and temperature. High-performance liquid chromatography (HPLC) was performed to assay all the samples of phase-solubility analysis and kinetic measurements. Chromatographic separation of the degradation products was also performed by HPLC. FT-IR spectroscopy was used to investigate the presence of any interaction between cefixime and Captisol and soluble polymer. The phase-solubility study showed A(L)-type behaviour. The pH-rate profile of cefixime exhibited a U-shaped profile whilst the degradation of cefixime alone was markedly accelerated with elevated temperature. A strong stabilizing influence of the cefixime-Captisol complexation and hypromellose was observed against aqueous mediated degradation, as compared with povidone and macrogol. The unfavourable effect of povidone and macrogol may have been due to the steric hindrance, which prevented the guest molecule from entering the cyclodextrin cavity, whereas hypromellose did not produce any steric hindrance.

Toxicological Assessment and UV/TiO2-Based Induced Degradation Profile of Reactive Black 5 Dye

Science.gov (United States)

Bilal, Muhammad; Rasheed, Tahir; Iqbal, Hafiz M. N.; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

2018-01-01

In this study, the toxicological and degradation profile of Reactive Black 5 (RB5) dye was evaluated using a UV/TiO2-based degradation system. Fourier transform infrared spectroscopy (FT-IR), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) techniques were used to evaluate the degradation level of RB5. The UV-Vis spectral analysis revealed the disappearance of peak intensity at 599 nm (λmax). The FT-IR spectrum of UV/TiO2 treated dye sample manifest appearance of new peaks mainly because of the degraded product and/or disappearance of some characteristics peaks which were present in the untreated spectrum. The HPLC profile verified the RB5 degradation subject to the formation of metabolites at different retention times. A stable color removal higher than 96% with COD removal in the range of 74-82.3% was noted at all evaluated dye concentrations. The tentative degradation pathway of RB5 is proposed following a careful analysis of the intermediates identified by UPLC-MS. Toxicity profile of untreated and degraded dye samples was monitored using three types of human cell lines via MTT assay and acute toxicity testing with Artemia salina. In conclusion, the UV/TiO2-based degradation system could be effectively employed for the remediation of textile wastewater comprising a high concentration of reactive dyes.

Intrinsic immunogenicity of rapidly-degradable polymers evolves during degradation.

Science.gov (United States)

Andorko, James I; Hess, Krystina L; Pineault, Kevin G; Jewell, Christopher M

2016-03-01

Recent studies reveal many biomaterial vaccine carriers are able to activate immunostimulatory pathways, even in the absence of other immune signals. How the changing properties of polymers during biodegradation impact this intrinsic immunogenicity is not well studied, yet this information could contribute to rational design of degradable vaccine carriers that help direct immune response. We use degradable poly(beta-amino esters) (PBAEs) to explore intrinsic immunogenicity as a function of the degree of polymer degradation and polymer form (e.g., soluble, particles). PBAE particles condensed by electrostatic interaction to mimic a common vaccine approach strongly activate dendritic cells, drive antigen presentation, and enhance T cell proliferation in the presence of antigen. Polymer molecular weight strongly influences these effects, with maximum stimulation at short degradation times--corresponding to high molecular weight--and waning levels as degradation continues. In contrast, free polymer is immunologically inert. In mice, PBAE particles increase the numbers and activation state of cells in lymph nodes. Mechanistic studies reveal that this evolving immunogenicity occurs as the physicochemical properties and concentration of particles change during polymer degradation. This work confirms the immunological profile of degradable, synthetic polymers can evolve over time and creates an opportunity to leverage this feature in new vaccines. Degradable polymers are increasingly important in vaccination, but how the inherent immunogenicity of polymers changes during degradation is poorly understood. Using common rapidly-degradable vaccine carriers, we show that the activation of immune cells--even in the absence of other adjuvants--depends on polymer form (e.g., free, particulate) and the extent of degradation. These changing characteristics alter the physicochemical properties (e.g., charge, size, molecular weight) of polymer particles, driving changes in

Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

International Nuclear Information System (INIS)

Walia, S.; Khan, A.; Rosenthal, N.

1990-01-01

Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community

Degradation and Stabilization of Peptide Hormones in Human Blood Specimens.

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Jizu Yi

Full Text Available Plasma hormone peptides, including GLP-1, GIP, Glucagon, and OXM, possess multiple physiological roles and potential therapeutic and diagnostic utility as biomarkers in the research of metabolic disorders. These peptides are subject to proteolytic degradation causing preanalytical variations. Stabilization for accurate quantitation of these active peptides in ex vivo blood specimens is essential for drug and biomarker development. We investigated the protease-driven instability of these peptides in conventional serum, plasma, anticoagulated whole blood, as well as whole blood and plasma stabilized with protease inhibitors. The peptide was monitored by both time-course Matrix-Assisted Laser Desorption Ionization Time-to-Flight Mass Spectrometry (MALDI -TOF MS and Ab-based assay (ELISA or RIA. MS enabled the identification of proteolytic fragments. In non-stabilized blood samples, the results clearly indicated that dipeptidyl peptidase-IV (DPP-IV removed the N-terminal two amino acid residues from GLP-1, GIP and OXM(1-37 and not-yet identified peptidase(s cleave(s the full-length OXM(1-37 and its fragments. DPP-IV also continued to remove two additional N-terminal residues of processed OXM(3-37 to yield OXM(5-37. Importantly, both DPP-IV and other peptidase(s activities were inhibited efficiently by the protease inhibitors included in the BD P800* tube. There was preservation of GLP-1, GIP, OXM and glucagon in the P800 plasma samples with half-lives > 96, 96, 72, and 45 hours at room temperature (RT, respectively. In the BD P700* plasma samples, the stabilization of GLP-1 was also achieved with half-life > 96 hours at RT. The stabilization of these variable peptides increased their utility in drug and/or biomarker development. While stability results of GLP-1 obtained with Ab-based assay were consistent with those obtained by MS analysis, the Ab-based results of GIP, Glucagon, and OXM did not reflect the time-dependent degradations revealed by MS

In vitro study on the degradation of lithium-doped hydroxyapatite for bone tissue engineering scaffold

Energy Technology Data Exchange (ETDEWEB)

Wang, Yaping; Yang, Xu; Gu, Zhipeng; Qin, Huanhuan [College of Polymer Science and Engineering, Sichuan University, Chengdu 610065 (China); Li, Li [Department of Oncology, The 452 Hospital of Chinese PLA, Chengdu, Sichuan Province 610021 (China); Liu, Jingwang [College of Polymer Science and Engineering, Sichuan University, Chengdu 610065 (China); Yu, Xixun, E-mail: yuxixun@163.com [College of Polymer Science and Engineering, Sichuan University, Chengdu 610065 (China)

2016-09-01

Li-doped hydroxyapatite (LiHA) which is prepared through introducing low dose of Li into hydroxyapatite (HA) has been increasingly studied as a bone tissue-engineered scaffold. The degradation properties play a crucial role in the success of long-term implantation of a bone tissue-engineered construct. Herein, the in vitro degradation behaviors of LiHA scaffolds via two approaches were investigated in this study: solution-mediated degradation and osteoblast-mediated degradation. In solution-mediated degradation, after being immersed in simulated body fluid (SBF) for some time, some characteristics of these scaffolds (such as release of ionized lithium and phosphate, pH change, mechanical properties, cytocompatibility and SEM surface characterization) were systematically tested. A similar procedure was also employed to research the degradation behaviors of LiHA scaffolds in osteoblast-mediated degradation. The results suggested that the degradation in SBF and degradation in culture medium with cell existed distinguishing mechanisms. LiHA scaffolds were degraded via a hydrolytic mechanism when they were soaked in SBF. Upon degradation, an apatite precipitation (layer) was formed on the surfaces of scaffolds. While a biological mechanism was presented for the degradation of scaffolds in cell-mediated degradation. Compared with pure HA, LiHA scaffolds had a better effect on the growth of osteoblast cells, meanwhile, the release amount of PO{sub 4}{sup 3−} in a degradation medium indicated that osteoblasts could accelerate the degradation of LiHA due to the more physiological activities of osteoblast. According to the results from compressive strength test, doping Li into HA could enhance the strength of HA. Moreover, the results from MTT assay and SEM observation showed that the degradation products of LiHA scaffolds were beneficial to the proliferation of osteoblasts. The results of this research can provide the theoretical basis for the clinical application of Li

Removal of antibiotics in wastewater by enzymatic treatment with fungal laccase - Degradation of compounds does not always eliminate toxicity.

Science.gov (United States)

Becker, Dennis; Varela Della Giustina, Saulo; Rodriguez-Mozaz, Sara; Schoevaart, Rob; Barceló, Damià; de Cazes, Matthias; Belleville, Marie-Pierre; Sanchez-Marcano, José; de Gunzburg, Jean; Couillerot, Olivier; Völker, Johannes; Oehlmann, Jörg; Wagner, Martin

2016-11-01

In this study, the performance of immobilised laccase (Trametes versicolor) was investigated in combination with the mediator syringaldehyde (SYR) in removing a mixture of 38 antibiotics in an enzymatic membrane reactor (EMR). Antibiotics were spiked in osmosed water at concentrations of 10μg·L(-1) each. Laccase without mediator did not reduce the load of antibiotics significantly. The addition of SYR enhanced the removal: out of the 38 antibiotics, 32 were degraded by >50% after 24h. In addition to chemical analysis, the samples' toxicity was evaluated in two bioassays (a growth inhibition assay and the Microtox assay). Here, the addition of SYR resulted in a time-dependent increase of toxicity in both bioassays. In cooperation with SYR, laccase effectively removes a broad range of antibiotics. However, this enhanced degradation induces unspecific toxicity. If this issue is resolved, enzymatic treatment may be a valuable addition to existing water treatment technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Matrix effects of TRU [transuranic] assays using the SWEPP PAN assay system

International Nuclear Information System (INIS)

Smith, J.R.

1990-08-01

The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of 239 Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs

Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

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Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul [Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul (Korea, Republic of); Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo [Seoul National University college of Medicine, Seoul (Korea, Republic of)

2009-08-15

Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2+-1.7%, 3.9+-2.1%, 7.1+-6.2%, 11.2+-7.2%. The CVs by random assay were 2.1+-1.7%, 4.8+-3.1%, 3.6+-4.8%, and 7.4+-6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

International Nuclear Information System (INIS)

Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul; Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo

2009-01-01

Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2±1.7%, 3.9±2.1%, 7.1±6.2%, 11.2±7.2%. The CVs by random assay were 2.1±1.7%, 4.8±3.1%, 3.6±4.8%, and 7.4±6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

Characterization of the biodegradation, bioremediation and detoxification capacity of a bacterial consortium able to degrade the fungicide thiabendazole.

Science.gov (United States)

Perruchon, Chiara; Pantoleon, Anastasios; Veroutis, Dimitrios; Gallego-Blanco, Sara; Martin-Laurent, F; Liadaki, Kalliopi; Karpouzas, Dimitrios G

2017-12-01

Thiabendazole (TBZ) is a persistent fungicide used in the post-harvest treatment of fruits. Its application results in the production of contaminated effluents which should be treated before their environmental discharge. In the absence of efficient treatment methods in place, biological systems based on microbial inocula with specialized degrading capacities against TBZ could be a feasible treatment approach. Only recently the first bacterial consortium able to rapidly transform TBZ was isolated. This study aimed to characterize its biodegradation, bioremediation and detoxification potential. The capacity of the consortium to mineralize 14 C-benzyl-ring labelled TBZ was initially assessed. Subsequent tests evaluated its degradation capacity under various conditions (range of pH, temperatures and TBZ concentration levels) and relevant practical scenarios (simultaneous presence of other postharvest compounds) and its bioaugmentation potential in soils contaminated with increasing TBZ levels. Finally cytotoxicity assays explored its detoxification potential. The consortium effectively mineralized the benzoyl ring of the benzimidazole moiety of TBZ and degraded spillage level concentrations of the fungicide in aqueous cultures (750 mg L -1 ) and in soil (500 mg kg -1 ). It maintained its high degradation capacity in a wide range of pH (4.5-7.5) and temperatures (15-37 °C) and in the presence of other pesticides (ortho-phenylphenol and diphenylamine). Toxicity assays using the human liver cancer cell line HepG2 showed a progressive decrease in cytotoxicity, concomitantly with the biodegradation of TBZ, pointing to a detoxification process. Overall, the bacterial consortium showed high potential for future implementation in bioremediation and biodepuration applications.

Radioreceptor opioid assay

International Nuclear Information System (INIS)

Miller, R.J.; Chang, K.-J.

1981-01-01

A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

Isolation and Characterization of Phenanthrene Degrading Bacteria from Diesel Fuel-Contaminated Antarctic Soils

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Alejandro Gran-Scheuch

2017-08-01

Full Text Available Antarctica is an attractive target for human exploration and scientific investigation, however the negative effects of human activity on this continent are long lasting and can have serious consequences on the native ecosystem. Various areas of Antarctica have been contaminated with diesel fuel, which contains harmful compounds such as heavy metals and polycyclic aromatic hydrocarbons (PAH. Bioremediation of PAHs by the activity of microorganisms is an ecological, economical, and safe decontamination approach. Since the introduction of foreign organisms into the Antarctica is prohibited, it is key to discover native bacteria that can be used for diesel bioremediation. By following the degradation of the PAH phenanthrene, we isolated 53 PAH metabolizing bacteria from diesel contaminated Antarctic soil samples, with three of these isolates exhibiting a high phenanthrene degrading capacity. In particular, the Sphingobium xenophagum D43FB isolate showed the highest phenanthrene degradation ability, generating up to 95% degradation of initial phenanthrene. D43FB can also degrade phenanthrene in the presence of its usual co-pollutant, the heavy metal cadmium, and showed the ability to grow using diesel-fuel as a sole carbon source. Microtiter plate assays and SEM analysis revealed that S. xenophagum D43FB exhibits the ability to form biofilms and can directly adhere to phenanthrene crystals. Genome sequencing analysis also revealed the presence of several genes involved in PAH degradation and heavy metal resistance in the D43FB genome. Altogether, these results demonstrate that S. xenophagum D43FB shows promising potential for its application in the bioremediation of diesel fuel contaminated-Antarctic ecosystems.

Efficient degradation of Azo dyes by a newly isolated fungus Trichoderma tomentosum under non-sterile conditions.

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He, Xiao-Ling; Song, Chao; Li, Yuan-Yuan; Wang, Ning; Xu, Lei; Han, Xin; Wei, Dong-Sheng

2018-04-15

A fast-growing fungus with remarkable ability to degrade several azo dyes under non-sterile conditions was isolated and identified. This fungus was identified as Trichoderma tomentosum. Textile effluent of ten-fold dilution could be decolorized by 94.9% within 72h before optimization. Acid Red 3R model wastewater with a concentration of 85.5mgL -1 could be decolorized by 99.2% within the same time after optimization. High-level of manganese peroxidase and low-level of lignin peroxidase activities were detected during the process of decolorization from the culture supernatant, indicating the possible involvement of two enzymes in azo dye decolorization. No aromatic amine products were detected from the degradation products of Acid Red 3R by gas chromatography-mass spectrometry (GC/MS) analysis, indicating the possible involvement of a special symmetrical oxidative degradation pathway. Phytotoxicity assay confirmed the lower toxicity toward the test plant seeds of the degradation products when compared to the original dye. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of Aliphatic and Aromatic Compounds Degradation by Indigenous Bacteria Isolated from Soil Contaminated with Petroleum

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Farhad Gilavand

2015-12-01

Full Text Available Background:  The major of this study was to isolate oil-degrading bacteria from soil contaminated with petroleum and examining the removal of hydrocarbons by these bacteria. Methods: Oil-degrading colonies were purified from the samples obtained of around Ahvaz oil wells. Organic matter degradation was investigated with 1 g of crude oil in basal salt medium (BSM as sole carbon source. The growth rate was determined through total protein assay and hydrocarbon consuming was measured through organic carbon oxidation and titration by dichromate as oxidizing agent. Results: Two potential isolates named S1 and S2 strains were screened and identified as Planococcus and Pseudomonas aeruginosa. As results for S1 and S2 could degrade 80.86 and 65.6% of olive oil, 59.6 and 35.33 of crude oil, while 32 and 26.15 % of coal tar were consumed during 14 days incubation. Conclusion: The results of this investigation showed these indigenous strains high capability to biodegradation at short time and are desirable alternatives for treatment of oil pollutants.

The Effect of Static Stretch on Elastin Degradation in Arteries

Science.gov (United States)

Chow, Ming-Jay; Choi, Myunghwan; Yun, Seok Hyun; Zhang, Yanhang

2013-01-01

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h−1) is almost twice that of digestion without stretch (0.069±0.028 h−1). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation. PMID:24358135

Photoactive TiO2 Films Formation by Drain Coating for Endosulfan Degradation

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Natalia Tapia-Orozco

2013-01-01

Full Text Available Heterogeneous photocatalysis is an advanced oxidation process in which a photoactive catalyst, such as TiO2, is attached to a support to produce free radical species known as reactive oxygen species (ROS that can be used to break down toxic organic compounds. In this study, the draining time, annealing temperature, and draining/annealing cycles for TiO2 films grown by the drain coating method were evaluated using a 23 factorial experimental design to determine the photoactivity of the films via endosulfan degradation. The TiO2 films prepared with a large number of draining/annealing cycles at high temperatures enhanced (P>0.05 endosulfan degradation and superoxide radical generation after 30 minutes of illumination with UV light. We demonstrated a negative correlation (R2=0.69; P>0.01 between endosulfan degradation and superoxide radical generation. The endosulfan degradation rates were the highest at 30 minutes with the F6 film. In addition, films prepared using conditions F1, F4, and F8 underwent an adsorption/desorption process. The kinetic reaction constants, Kapp (min−1, were 0.0101, 0.0080, 0.0055, 0.0048, and 0.0035 for F6, F2, F5, F3, and F1, respectively. The endosulfan metabolites alcohol, ether, and lactone were detected and quantified at varying levels in all photocatalytic assays.

Silver nanoparticles: a potential nanocatalyst for the rapid degradation of starch hydrolysis by α-amylase.

Science.gov (United States)

Ernest, Vinita; Shiny, P J; Mukherjee, Amitava; Chandrasekaran, N

2012-05-01

Silver nanoparticles (AgNPs) are proven to be an effective catalytic material for various applications due to their excellent optical and electronic properties. In this paper, we describe a novel approach for the degradation of starch using the catalytic behaviour of AgNPs in an enzyme catalysed reaction of starch hydrolysis by α-amylase. AgNPs were synthesized by soluble starch reducing silver nitrate to silver atoms. An increase of 4.7-fold in reducing sugar formation and 1.5 times faster enzyme activity confirmed the catalytic activity of AgNPs as a nanocatalyst. Surprisingly, starch degradation tests revealed that 9.9 mg of starch was hydrolysed within 5 min, which corroborates with the reducing sugar assay. In short, the present study paves way for the faster degradation of starch by immobilizing the enzyme onto the surface of the AgNP, which could be a promising application in the food industry. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enhanced photocatalytic degradation of dyes under sunlight using biocompatible TiO2 nanoparticles

Science.gov (United States)

Bharati, B.; Sonkar, A. K.; Singh, N.; Dash, D.; Rath, Chandana

2017-08-01

As TiO2 is one of the most popular photocatalysts, we have studied here the photocatalytic degradation of the most common dyestuffs like rhodamine B (RhB), congo red (CR) and methylene blue (MB), which mainly come from the textile and photographic industries using nanoparticles of TiO2. Nanoparticles of TiO2 synthesized through a simple and cost effective sol-gel technique crystallizes in the anatase phase, showing a band gap less than that of bulk value. Particles consisting of coherently scattered domains of size 33 nm are found to be agglomerated and polycrystalline in nature. While the degradation rates of MB, CR and RhB after irradiating with a renewable source of energy, i.e. sunlight, show 100% degradation, TiO2 irradiated with UV light of 4.8 eV shows a much slower degradation rate. To use the waste water after photocatalysis, we examine further the biocompatibile nature of the TiO2 nanoparticles by platelet interaction activity, hemolysis effect and MTT assay. It is worth mentioning here that TiO2 nanoparticles are found to be highly hemocompatible, show no platelet aggregation, and the level of intracellular ROS in human platelets does not show significant change in ROS level. We conclude that TiO2 nanoparticles constitute an excellent photocatalyst and biocompatible material, and that after photocatalytic degradation of dye effluents obtained from textile industries, purified water can be used in agriculture and domestic sectors.

In Silico Discovery of Novel Potent Antioxidants on the Basis of Pulvinic Acid and Coumarine Derivatives and Their Experimental Evaluation.

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Rok Martinčič

Full Text Available A pigment from the edible mushroom Xerocomus badius norbadione A, which is a natural derivative of pulvinic acid, was found to possess antioxidant properties. Since the pulvinic acid represents a novel antioxidant scaffold, several other derivatives were recently synthetized and evaluated experimentally, along with some structurally related coumarine derivatives. The obtained data formed the basis for the construction of several quantitative structure-activity and pharmacophore models, which were employed in the virtual screening experiments of compound libraries and for the prediction of their antioxidant activity, with the goal of discovering novel compounds possessing antioxidant properties. A final prioritization list of 21 novel compounds alongside 8 established antioxidant compounds was created for their experimental evaluation, consisting of the DPPH assay, 2-deoxyribose assay, β-carotene bleaching assay and the cellular antioxidant activity assay. Ten novel compounds from the tetronic acid and barbituric acid chemical classes displayed promising antioxidant activity in at least one of the used assays, that is comparable to or even better than some standard antioxidants. Compounds 5, 7 and 9 displayed good activity in all the assays, and were furthermore effective preventers of oxidative stress in human peripheral blood mononuclear cells, which are promising features for the potential therapeutic use of such compounds.

Validation, optimisation, and application data in support of the development of a targeted selected ion monitoring assay for degraded cardiac troponin T

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Alexander S. Streng

2016-06-01

Full Text Available Cardiac troponin T (cTnT fragmentation in human serum was investigated using a newly developed targeted selected ion monitoring assay, as described in the accompanying article: “Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T” [1]. This article presents data describing aspects of the validation and optimisation of this assay. The data consists of several figures, an excel file containing the results of a sequence identity search, and a description of the raw mass spectrometry (MS data files, deposited in the ProteomeXchange repository with id PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003187.

Toxicity assessment of pesticide triclosan by aquatic organisms and degradation studies.

Science.gov (United States)

Taştan, Burcu Ertit; Tekinay, Turgay; Çelik, Hatice Sena; Özdemir, Caner; Cakir, Dilara Nur

2017-12-01

Triclosan is considered as an important contaminant and is widely used in personal care products as an antimicrobial agent. This study demonstrates the biodegradation of triclosan by two freshwater microalgae and the acute toxicity of triclosan and 2,4-dichlorophenol. The effects of culture media and light on biodegradation of triclosan and the changing morphology of microalgae were systematically studied. Geitlerinema sp. and Chlorella sp. degraded 82.10% and 92.83% of 3.99 mg/L of triclosan at 10 days, respectively. The microalgal growth inhibition assay confirmed absence of toxic effects of triclosan on Chlorella sp., even at higher concentration (50 mg/L) after 72 h exposure. HPLC analysis showed that 2,4-dichlorophenol was produced as degradation product of triclosan by Geitlerinema sp. and Chlorella sp. This study proved to be beneficial to understand biodegradation and acute toxicity of triclosan by microalgae in order to provide aquatic environmental protection. Copyright © 2017 Elsevier Inc. All rights reserved.

Thermal Degradation Kinetics Modeling of Benzophenones and Xanthones during High-Temperature Oxidation of Cyclopia genistoides (L.) Vent. Plant Material.

Science.gov (United States)

Beelders, Theresa; de Beer, Dalene; Joubert, Elizabeth

2015-06-10

Degradation of the major benzophenones, iriflophenone-3-C-glucoside-4-O-glucoside and iriflophenone-3-C-glucoside, and the major xanthones, mangiferin and isomangiferin, of Cyclopia genistoides followed first-order reaction kinetics during high-temperature oxidation of the plant material at 80 and 90 °C. Iriflophenone-3-C-glucoside-4-O-glucoside was shown to be the most thermally stable compound. Isomangiferin was the second most stable compound at 80 °C, while its degradation rate constant was influenced the most by increased temperature. Mangiferin and iriflophenone-3-C-glucoside had comparable degradation rate constants at 80 °C. The thermal degradation kinetic model was subsequently evaluated by subjecting different batches of plant material to oxidative conditions (90 °C/16 h). The model accurately predicted the individual contents of three of the compounds in aqueous extracts prepared from oxidized plant material. The impact of benzophenone and xanthone degradation was reflected in the decreased total antioxidant capacity of the aqueous extracts, as determined using the oxygen radical absorbance capacity and DPPH(•) scavenging assays.

Degradation of /sup 14/C-parathion 'in vitro' by microorganisms isolated from a gley humic soil

Energy Technology Data Exchange (ETDEWEB)

Andrea, M.M. de; Ruegg, E.F. (Instituto Biologico, Sao Paulo (Brazil). Centro de Radioisotopos)

It was determined 'in vitro' the degradation of Parathion by a bacterium and a fungus isolated from a sample of Gley Humic soil previously treated with repeated applications of the insecticide. In a qualitative colorimetric assay hydrolysis of parathion to p-nitrophenol just the bacterium gave a positive answer. In quantitative assays of /sup 14/C-parathion degradation in culture media containing both microorganisms, organic solvents extractions resulted in organic and aqueous phases, which were analysed by liquid scintillation counting and thin-layer chromatography. In a mineral salts medium plus buffer, the bacterium and the fungus behaved differently from the control, because part of the /sup 14/C-insecticide was metabolized to, at least, one metabolite and besides, the microorganisms presented smaller percentages of total recovery. The largest percentage of the radio carbon recovery from the extracts of the medium containing the fungus plus extract of yeast, was obtained from the aqueous phase and the existence of other metabolite was demonstrated by chromatograms of the organic phase.

Aerobic degradation of N-methyl-4-nitroaniline (MNA by Pseudomonas sp. strain FK357 isolated from soil.

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Fazlurrahman Khan

Full Text Available N-Methyl-4-nitroaniline (MNA is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA, 4-aminophenol (4-AP, and 1, 2, 4-benzenetriol (BT as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH2 substituent reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.

Selection of non-destructive assay methods: Neutron counting or calorimetric assay?

International Nuclear Information System (INIS)

Cremers, T.L.; Wachter, J.R.

1994-01-01

The transition of DOE facilities from production to D ampersand D has lead to more measurements of product, waste, scrap, and other less attractive materials. Some of these materials are difficult to analyze by either neutron counting or calorimetric assay. To determine the most efficacious analysis method, variety of materials, impure salts and hydrofluorination residues have been assayed by both calorimetric assay and neutron counting. New data will be presented together with a review of published data. The precision and accuracy of these measurements are compared to chemistry values and are reported. The contribution of the gamma ray isotopic determination measurement to the overall error of the calorimetric assay or neutron assay is examined and discussed. Other factors affecting selection of the most appropriate non-destructive assay method are listed and considered

Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

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Carina Ladeira

2015-06-01

The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

Microbial Activity and Silica Degradation in Rice Straw

Science.gov (United States)

Kim, Esther Jin-kyung

Abundantly available agricultural residues like rice straw have the potential to be feedstocks for bioethanol production. Developing optimized conditions for rice straw deconstruction is a key step toward utilizing the biomass to its full potential. One challenge associated with conversion of rice straw to bioenergy is its high silica content as high silica erodes machinery. Another obstacle is the availability of enzymes that hydrolyze polymers in rice straw under industrially relevant conditions. Microbial communities that colonize compost may be a source of enzymes for bioconversion of lignocellulose to products because composting systems operate under thermophilic and high solids conditions that have been shown to be commercially relevant. Compost microbial communities enriched on rice straw could provide insight into a more targeted source of enzymes for the breakdown of rice straw polysaccharides and silica. Because rice straw is low in nitrogen it is important to understand the impact of nitrogen concentrations on the production of enzyme activity by the microbial community. This study aims to address this issue by developing a method to measure microbial silica-degrading activity and measure the effect of nitrogen amendment to rice straw on microbial activity and extracted enzyme activity during a high-solids, thermophilic incubation. An assay was developed to measure silica-degrading enzyme or silicase activity. This process included identifying methods of enzyme extraction from rice straw, identifying a model substrate for the assay, and optimizing measurement techniques. Rice straw incubations were conducted with five different levels of nitrogen added to the biomass. Microbial activity was measured by respiration and enzyme activity. A microbial community analysis was performed to understand the shift in community structure with different treatments. With increased levels of nitrogen, respiration and cellulose and hemicellulose degrading activity

In vitro assays for predicting tumor cell response to radiation by apoptotic pathways

International Nuclear Information System (INIS)

Algan, Oe.; Hanks, G.E.; Biade, S.; Chapman, J.D.

1995-01-01

Purpose: We had previously shown that the rate of spontaneous and radiation-induced apoptosis was significantly greater in well-differentiated compared to anaplastic Dunning prostate carcinomas. The goal of this study was to define the most useful assay for quantifying radiation-induced apoptotic cell death and to determine if measured rates of radiation-induced apoptosis in tumor cell populations can predict treatment outcome. Materials and Methods: The time course and extent of radiation-induced apoptosis after single doses of Cesium-137 gamma-rays were measured by five different assays. These included gross DNA degradation, nucleosome ladder formation, labeling of 3'-OH ends in DNA with an immunofluorescence probe, immunofluorescence vital stains (LIVE/DEAD[reg] EUKOLIGHT TM ) and trypan blue. The majority of these studies were performed with DU-145 human prostate cells. Data was analyzed to determine the component of cell inactivation resulting from apoptosis with the modified linear quadratic equation, -1n (SF) = (α a + α p ) D + β p D 2 , were α a represents cell inactivation by radiation-induced apoptosis, α p and β p represent cell death by proliferative mechanisms and D represents radiation dose. Results: These studies indicated that DU-145 cell death after radiation occurs over two distinct time periods. The first phase of death begins shortly after irradiation and plateaus within 16-24 hr. This process of cell death has properties consistent with apoptosis as determined by 3'-OH DNA end-labeling and nucleosome ladder assays. The second phase of cell death (determined by viability staining) begins approximately 48 hr after irradiation and continues until the remainder of inactivated cells express their death. This longer phase of cell inactivation probably represents proliferative cell death and other non-apoptotic mechanisms. The five different assays were performed on DU-145 cells 24 hr after irradiation with 10 Gy. Significant nucleosome ladders

ENHANCEMENT OF RESISTANCE TO OXIDATIVE DEGRADATION OF NATURAL RUBBER THROUGH LATEX DEGRADATION

Institute of Scientific and Technical Information of China (English)

无

1998-01-01

A fully characterised natural rubber latex was subjected to mechanical degradation by stirring at intervals. The resistance to oxidative degradation of the different samples were studied by measuring the Plasticity retention indices (PRI).The results show that there is an enhancement of the PRI from 57% for the undegraded rubber to 79% for the one-hour degraded sample. Further degradation resulted in decrease of PRI as time of degradation increased. Therefore, the one-hour degraded sample is a special rubber with high oxidation resistance which is of great importance in engineering.

Efficient plant biomass degradation by thermophilic fungus Myceliophthora heterothallica.

Science.gov (United States)

van den Brink, Joost; van Muiswinkel, Gonny C J; Theelen, Bart; Hinz, Sandra W A; de Vries, Ronald P

2013-02-01

Rapid and efficient enzymatic degradation of plant biomass into fermentable sugars is a major challenge for the sustainable production of biochemicals and biofuels. Enzymes that are more thermostable (up to 70°C) use shorter reaction times for the complete saccharification of plant polysaccharides compared to hydrolytic enzymes of mesophilic fungi such as Trichoderma and Aspergillus species. The genus Myceliophthora contains four thermophilic fungi producing industrially relevant thermostable enzymes. Within this genus, isolates belonging to M. heterothallica were recently separated from the well-described species M. thermophila. We evaluate here the potential of M. heterothallica isolates to produce efficient enzyme mixtures for biomass degradation. Compared to the other thermophilic Myceliophthora species, isolates belonging to M. heterothallica and M. thermophila grew faster on pretreated spruce, wheat straw, and giant reed. According to their protein profiles and in vitro assays after growth on wheat straw, (hemi-)cellulolytic activities differed strongly between M. thermophila and M. heterothallica isolates. Compared to M. thermophila, M. heterothallica isolates were better in releasing sugars from mildly pretreated wheat straw (with 5% HCl) with a high content of xylan. The high levels of residual xylobiose revealed that enzyme mixtures of Myceliophthora species lack sufficient β-xylosidase activity. Sexual crossing of two M. heterothallica showed that progenies had a large genetic and physiological diversity. In the future, this will allow further improvement of the plant biomass-degrading enzyme mixtures of M. heterothallica.

Optimization of a colorimetric assay for glycosylated human serum albumin

International Nuclear Information System (INIS)

Bohney, J.P.; Feldhoff, R.C.

1986-01-01

The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100 0 C. A NaBH 4 reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with [ 3 H]glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation

A validated specific stability-indicating RP-HPLC assay method for Ambrisentan and its related substances.

Science.gov (United States)

Narayana, M B V; Chandrasekhar, K B; Rao, B M

2014-09-01

A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability). © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Isolation and characterization of mesotrione-degrading Bacillus sp. from soil

International Nuclear Information System (INIS)

Batisson, Isabelle; Crouzet, Olivier; Besse-Hoggan, Pascale; Sancelme, Martine; Mangot, Jean-Francois; Mallet, Clarisse; Bohatier, Jacques

2009-01-01

Dissipation kinetics of mesotrione, a new triketone herbicide, sprayed on soil from Limagne (Puy-de-Dome, France) showed that the soil microflora were able to biotransform it. Bacteria from this soil were cultured in mineral salt solution supplemented with mesotrione as sole source of carbon for the isolation of mesotrione-degrading bacteria. The bacterial community structure of the enrichment cultures was analyzed by temporal temperature gradient gel electrophoresis (TTGE). The TTGE fingerprints revealed that mesotrione had an impact on bacterial community structure only at its highest concentrations and showed mesotrione-sensitive and mesotrione-adapted strains. Two adapted strains, identified as Bacillus sp. and Arthrobacter sp., were isolated by colony hybridization methods. Biodegradation assays showed that only the Bacillus sp. strain was able to completely and rapidly biotransform mesotrione. Among several metabolites formed, 2-amino-4-methylsulfonylbenzoic acid (AMBA) accumulated in the medium. Although sulcotrione has a chemical structure closely resembling that of mesotrione, the isolates were unable to degrade it. - A Bacillus sp. strain isolated from soil was able to completely and rapidly biotransform the triketone herbicide mesotrione

PI3K/AKT signaling inhibits NOTCH1 lysosome-mediated degradation.

Science.gov (United States)

Platonova, Natalia; Manzo, Teresa; Mirandola, Leonardo; Colombo, Michela; Calzavara, Elisabetta; Vigolo, Emilia; Cermisoni, Greta Chiara; De Simone, Daria; Garavelli, Silvia; Cecchinato, Valentina; Lazzari, Elisa; Neri, Antonino; Chiaramonte, Raffaella

2015-06-06

The pathways of NOTCH and PI3K/AKT are dysregulated in about 60% and 48% of T-cell acute lymphoblastic leukemia (T-ALL) patients, respectively. In this context, they interact and cooperate in controlling tumor cell biology. Here, we propose a novel mechanism by which the PI3K/AKT pathway regulates NOTCH1 in T-ALL, starting from the evidence that the inhibition of PI3K/AKT signaling induced by treatment with LY294002 or transient transfection with a dominant negative AKT mutant downregulates NOTCH1 protein levels and activity, without affecting NOTCH1 transcription. We showed that the withdrawal of PI3K/AKT signaling was associated to NOTCH1 phosphorylation in tyrosine residues and monoubiquitination of NOTCH1 detected by Ubiquitin capture assay. Co-immunoprecipitation assay and colocalization analysis further showed that the E3 ubiquitin ligase c-Cbl interacts and monoubiquitinates NOTCH1, activating its lysosomal degradation. These results suggest that the degradation of NOTCH1 could represent a mechanism of control by which NOTCH1 receptors are actively removed from the cell surface. This mechanism is finely regulated by the PI3K/AKT pathway in physiological conditions. In pathological conditions characterized by PI3K/AKT hyperactivation, such as T-ALL, the excessive AKT signaling could lead to NOTCH1 signaling dysregulation. Therefore, a therapeutic strategy directed to PI3K/AKT in T-ALL could contemporaneously inhibit the dysregulated NOTCH1 signaling. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.

Science.gov (United States)

Mang, Samuel; Bucher, Hannes; Nickolaus, Peter

2016-01-01

The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.

Microbead agglutination based assays

KAUST Repository

Kodzius, Rimantas

2013-01-21

We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures.

Science.gov (United States)

Tinh, Nguyen Thi Ngoc; Asanka Gunasekara, R A Y S; Boon, Nico; Dierckens, Kristof; Sorgeloos, Patrick; Bossier, Peter

2007-10-01

Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.

The Anti-Inflammatory Effects of Lipoxygenase and Cyclo-Oxygenase Inhibitors in Inflammation-Induced Human Fetal Glia Cells and the Aβ Degradation Capacity of Human Fetal Astrocytes in an Ex vivo Assay

Directory of Open Access Journals (Sweden)

Rea Pihlaja

2017-05-01

Full Text Available Chronic inflammation is a common phenomenon present in the background of multiple neurodegenerative diseases, including Alzheimer's disease (AD. The arachidonic acid pathway overproduces proinflammatory eicosanoids during these states and glial cells in the brain gradually lose their vital functions of protecting and supporting neurons. In this study, the role of different key enzymes of the eicosanoid pathway mediating inflammatory responses was examined in vitro and ex vivo using human fetal glial cells. Astrocytes and microglia were exposed to proinflammatory agents i.e., cytokines interleukin 1-β (IL-1β and tumor necrosis factor (TNF-α. ELISA assays were used to examine the effects of inhibitors of key enzymes in the eicosanoid pathway. Inhibitors for 5-lipoxygenase (5-LOX and cyclo-oxygenase 2 (COX-2 in both cell types and 5-, 12-, and 15-LOX-inhibitor in astrocytes reduced significantly IL-6 secretion, compared to exposed glial cells without inhibitors. The cytokine antibody array showed that especially treatments with 5, -12, and -15 LOX inhibitor in astrocytes, 5-LOX inhibitor in microglia and COX-2 inhibitor in both glial cell types significantly reduced the expression of multiple proinflammatory cytokines. Furthermore, human fetal astrocytes and microglia were cultured on top of AD-affected and control human brain sections for 30 h. According to the immunochemical evaluation of the level of total Aβ, astrocytes were very efficient at degrading Aβ from AD-affected brain sections ex vivo; simultaneously added enzyme inhibitors did not increase their Aβ degradation capabilities. Microglia were not able to reduce the level of total Aβ during the 30 h incubation time.

Persistent activation of NF-kappaB related to IkappaB's degradation profiles during early chemical hepatocarcinogenesis

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García-Román Rebeca

2007-04-01

Full Text Available Abstract Background To define the NF-kappaB activation in early stages of hepatocarcinogenesis and its IkappaB's degradation profiles in comparison to sole liver regeneration. Methods Western-blot and EMSA analyses were performed for the NF-kappaB activation. The transcriptional activity of NF-kappaB was determined by RT-PCR of the IkappaB-α mRNA. The IkappaB's degradation proteins were determined by Western-blot assay. Results We demonstrated the persistent activation of NF-kappaB during early stages of hepatocarcinogenesis, which reached maximal level 30 min after partial hepatectomy. The DNA binding and transcriptional activity of NF-kappaB, were sustained during early steps of hepatocarcinogenesis in comparison to only partial hepatectomy, which displayed a transitory NF-kappaB activation. In early stages of hepatocarconogenesis, the IkappaB-α degradation turned out to be acute and transitory, but the low levels of IkappaB-β persisted even 15 days after partial hepatectomy. Interestingly, IkappaB-β degradation is not induced after sole partial hepatectomy. Conclusion We propose that during liver regeneration, the transitory stimulation of the transcription factor response, assures blockade of NF-kappaB until recovery of the total mass of the liver and the persistent NF-kappaB activation in early hepatocarcinogenesis may be due to IkappaB-β and IkappaB-α degradation, mainly IkappaB-β degradation, which contributes to gene transcription related to proliferation required for neoplasic progression.

A thermo-degradable hydrogel with light-tunable degradation and drug release.

Science.gov (United States)

Hu, Jingjing; Chen, Yihua; Li, Yunqi; Zhou, Zhengjie; Cheng, Yiyun

2017-01-01

The development of thermo-degradable hydrogels is of great importance in drug delivery. However, it still remains a huge challenge to prepare thermo-degradable hydrogels with inherent degradation, reproducible, repeated and tunable dosing. Here, we reported a thermo-degradable hydrogel that is rapidly degraded above 44 °C by a facile chemistry. Besides thermo-degradability, the hydrogel also undergoes rapid photolysis with ultraviolet light. By embedding photothermal nanoparticles or upconversion nanoparticles into the gel, it can release the entrapped cargoes such as dyes, enzymes and anticancer drugs in an on-demand and dose-tunable fashion upon near-infrared light exposure. The smart hydrogel works well both in vitro and in vivo without involving sophisticated syntheses, and is well suited for clinical cancer therapy due to the high transparency and non-invasiveness features of near-infrared light. Copyright © 2016 Elsevier Ltd. All rights reserved.

Organic matter degradation in Chilean sediments - following nature's own degradation experiment

DEFF Research Database (Denmark)

Langerhuus, Alice Thoft; Niggemann, Jutta; Lomstein, Bente Aagaard

ORGANIC MATTER DEGRADATION IN CHILEAN SEDIMENTS – FOLLOWING NATURE’S OWN DEGRADATION EXPERIMENT Degradation of sedimentary organic matter was studied at two stations from the shelf of the Chilean upwelling region. Sediment cores were taken at 1200 m and 800 m water depth and were 4.5 m and 7.5 m...... in length, respectively. The objective of this study was to assess the degradability of the organic matter from the sediment surface to the deep sediments. This was done by analysing amino acids (both L- and D-isomers) and amino sugars in the sediment cores, covering a timescale of 15.000 years. Diagenetic...... indicators (percentage of carbon and nitrogen present as amino acid carbon and nitrogen, the ratio between a protein precursor and its non-protein degradation product and the percentage of D-amino acids) revealed ongoing degradation in these sediments, indicating that microorganisms were still active in 15...

Evidence for catabolite degradation in the glucose-dependent inactivation of yeast cytoplasmic malate dehydrogenase

International Nuclear Information System (INIS)

Neeff, J.; Haegele, E.; Nauhaus, J.; Heer, U.; Mecke, D.

1978-01-01

The cytoplasmic malate dehydrogenase of Saccharomyces cerevisiae was radioactively labeled during its synthesis on a glucose-free derepression medium. After purification a sensitive radioimmunoassay for this enzyme could be developed. The assay showed that after the physiological, glucose-dependent 'catabolite inactivation' of cytoplasmic malate dehydrogenase an inactive enzyme protein is immunologically not detectable. Together with the irreversibility of this reaction in vivo this finding strongly suggests a proteolytic mechanism of enzyme inactivation. For this process the term 'catabolite degradation' is used. (orig.) [de

The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus.

Science.gov (United States)

Wang, Yafei; Peng, Wei; Zhou, Xu; Huang, Fei; Shao, Lingyun; Luo, Meizhong

2014-09-01

Agrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process. Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins. We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system. Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS). © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1

Science.gov (United States)

Song, Jinlong; Shi, Yanhua; Li, Kang; Zhao, Bin; Yan, Yanchun

2013-01-01

A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L−1 and 56.33 nmol min−1, respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments. PMID:24155944

Radiation cross-linked collagen/dextran dermal scaffolds: effects of dextran on cross-linking and degradation.

Science.gov (United States)

Zhang, Yaqing; Zhang, Xiangmei; Xu, Ling; Wei, Shicheng; Zhai, Maolin

2015-01-01

Ionizing radiation effectively cross-links collagen into network with enhanced anti-degradability and biocompatibility, while radiation-cross-linked collagen scaffold lacks flexibility, satisfactory surface appearance, and performs poor in cell penetration and ingrowth. To make the radiation-cross-linked collagen scaffold to serve as an ideal artificial dermis, dextran was incorporated into collagen. Scaffolds with the collagen/dextran (Col/Dex) ratios of 10/0, 7/3, and 5/5 were fabricated via (60)Co γ-irradiation cross-linking, followed by lyophilization. The morphology, microstructure, physicochemical, and biological properties were investigated. Compared with pure collagen, scaffolds with dextran demonstrated more porous appearance, enhanced hydrophilicity while the cross-linking density was lower with the consequence of larger pore size, higher water uptake, as well as reduced stiffness. Accelerated degradation was observed when dextran was incorporated in both the in vitro and in vivo assays, which led to earlier integration with cell and host tissue. The effect of dextran on degradation was ascribed to the decreased cross-linking density, looser microstructure, more porous and hydrophilic surface. Considering the better appearance, softness, moderate degradation rate due to controllable cross-linking degree and good biocompatibility as well, radiation-cross-linked collagen/dextran scaffolds are expected to serve as promising artificial dermal substitutes.

Curcumin Quantum Dots Mediated Degradation of Bacterial Biofilms

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Ashish K. Singh

2017-08-01

Full Text Available Bacterial biofilm has been reported to be associated with more than 80% of bacterial infections. Curcumin, a hydrophobic polyphenol compound, has anti-quorum sensing activity apart from having antimicrobial action. However, its use is limited by its poor aqueous solubility and rapid degradation. In this study, we attempted to prepare quantum dots of the drug curcumin in order to achieve enhanced solubility and stability and investigated for its antimicrobial and antibiofilm activity. We utilized a newer two-step bottom up wet milling approach to prepare Curcumin Quantum Dots (CurQDs using acetone as a primary solvent. Minimum inhibitory concentration against select Gram-positive and Gram-negative bacteria was performed. The antibiofilm assay was performed at first using 96-well tissue culture plate and subsequently validated by Confocal Laser Scanning Microscopy. Further, biofilm matrix protein was isolated using formaldehyde sludge and TCA/Acetone precipitation method. Protein extracted was incubated with varying concentration of CurQDs for 4 h and was subjected to SDS–PAGE. Molecular docking study was performed to observe interaction between curcumin and phenol soluble modulins as well as curli proteins. The biophysical evidences obtained from TEM, SEM, UV-VIS, fluorescence, Raman spectroscopy, and zeta potential analysis confirmed the formation of curcumin quantum dots with increased stability and solubility. The MICs of curcumin quantum dots, as observed against both select gram positive and negative bacterial isolates, was observed to be significantly lower than native curcumin particles. On TCP assay, Curcumin observed to be having antibiofilm as well as biofilm degrading activity. Results of SDS–PAGE and molecular docking have shown interaction between biofilm matrix proteins and curcumin. The results indicate that aqueous solubility and stability of Curcumin can be achieved by preparing its quantum dots. The study also demonstrates

S100A4 interacts with p53 in the nucleus and promotes p53 degradation.

Science.gov (United States)

Orre, L M; Panizza, E; Kaminskyy, V O; Vernet, E; Gräslund, T; Zhivotovsky, B; Lehtiö, J

2013-12-05

S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor.

Science.gov (United States)

Shoji, Atsushi; Suenaga, Yumiko; Hosaka, Atsushi; Ishida, Yuuki; Yanagida, Akio; Sugawara, Masao

2017-10-25

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly. Copyright © 2017 Elsevier B.V. All rights reserved.

Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays.

Science.gov (United States)

Souberbielle, Jean-Claude; Fayol, Véronique; Sault, Corinne; Lawson-Body, Ethel; Kahan, André; Cormier, Catherine

2005-02-01

The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P 50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.

Mechanisms of strand break formation in DNA due to the direct effect of ionizing radiation: the dependency of free base release on the length of alternating CG oligodeoxynucleotides.

Science.gov (United States)

Sharma, Kiran K; Razskazovskiy, Yuriy; Purkayastha, Shubhadeep; Bernhard, William A

2009-06-11

The question of how NA base sequence influences the yield of DNA strand breaks produced by the direct effect of ionizing radiation was investigated in a series of oligodeoxynucleotides of the form (d(CG)(n))(2) and (d(GC)(n))(2). The yields of free base release from X-irradiated DNA films containing 2.5 waters/nucleotide were measured by HPLC as a function of oligomer length. For (d(CG)(n))(2), the ratio of the Gua yield to Cyt yield, R, was relatively constant at 2.4-2.5 for n = 2-4 and it decreased to 1.2 as n increased from 5 to 10. When Gua was moved to the 5' end, for example going from d(CG)(5) to d(GC)(5), R dropped from 1.9 +/- 0.1 to 1.1 +/- 0.1. These effects are poorly described if the chemistry at the oligomer ends is assumed to be independent of the remainder of the oligomer. A mathematical model incorporating charge transfer through the base stack was derived to explain these effects. In addition, EPR was used to measure the yield of trapped-deoxyribose radicals at 4 K following X-irradiation at 4 K. The yield of free base release was substantially greater, by 50-100 nmol/J, than the yield of trapped-deoxyribose radicals. Therefore, a large fraction of free base release stems from a nonradical intermediate. For this intermediate, a deoxyribose carbocation formed by two one-electron oxidations is proposed. This reaction pathway requires that the hole (electron loss site) transfers through the base stack and, upon encountering a deoxyribose hole, oxidizes that site to form a deoxyribose carbocation. This reaction mechanism provides a consistent way of explaining both the absence of trapped radical intermediates and the unusual dependence of free base release on oligomer length.

Optimization of a colorimetric assay for glycosylated human serum albumin

Energy Technology Data Exchange (ETDEWEB)

Bohney, J.P.; Feldhoff, R.C.

1986-05-01

The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

Mechanistic Insights into Elastin Degradation by Pseudolysin, the Major Virulence Factor of the Opportunistic Pathogen Pseudomonas aeruginosa

Science.gov (United States)

Yang, Jie; Zhao, Hui-Lin; Ran, Li-Yuan; Li, Chun-Yang; Zhang, Xi-Ying; Su, Hai-Nan; Shi, Mei; Zhou, Bai-Cheng; Chen, Xiu-Lan; Zhang, Yu-Zhong

2015-01-01

Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1’ positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1’ sub-sites played key roles in the binding of pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection. PMID:25905792

Degradation of microbial polyesters.

Science.gov (United States)

Tokiwa, Yutaka; Calabia, Buenaventurada P

2004-08-01

Microbial polyhydroxyalkanoates (PHAs), one of the largest groups of thermoplastic polyesters are receiving much attention as biodegradable substitutes for non-degradable plastics. Poly(D-3-hydroxybutyrate) (PHB) is the most ubiquitous and most intensively studied PHA. Microorganisms degrading these polyesters are widely distributed in various environments. Although various PHB-degrading microorganisms and PHB depolymerases have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. Distributions of PHB-degrading microorganisms, factors affecting the biodegradability of PHB, and microbial and enzymatic degradation of PHB are discussed in this review. We also propose an application of a new isolated, thermophilic PHB-degrading microorganism, Streptomyces strain MG, for producing pure monomers of PHA and useful chemicals, including D-3-hydroxycarboxylic acids such as D-3-hydroxybutyric acid, by enzymatic degradation of PHB.

Inhibition of Prenylation Promotes Caspase 3 Activation, Lamin B Degradation and Loss in Metabolic Cell Viability in Pancreatic β-Cells

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Khadija G. Syeda

2017-10-01

Full Text Available Background/Aims: Lamins are intermediate filament proteins that constitute the main components of the lamina underlying the inner-nuclear membrane and serve to organize chromatin. Lamins (e.g., lamin B undergo posttranslational modifications (e.g., isoprenylation at their C-terminal cysteine residues. Such modifications are thought to render optimal association of lamins with the nuclear envelop. Using human islets, rodent islets, and INS-1 832/13 cells, we recently reported significant metabolic defects under glucotoxic and endoplasmic reticulum (ER stress conditions, including caspase 3 activation and lamin B degradation. The current study is aimed at further understanding the regulatory roles of protein prenylation in the induction of the aforestated metabolic defects. Methods: Subcellular phase partitioning assay was done using Triton X-114. Cell morphology and metabolic cell viability assays were carried out using standard methodologies. Results: We report that exposure of pancreatic β-cells to Simvastatin, an inhibitor of mevalonic acid (MVA biosynthesis, and its downstream isoprenoid derivatives, or FTI-277, an inhibitor of farnesyltransferase that mediates farnesylation of lamins, leads to activation of caspase 3 and lamin B degradation. Furthermore, Simvastatin-treatment increased activation of p38MAPK (a stress kinase and inhibited ERK1/2 (regulator of cell proliferation. Inhibition of farnesylation also resulted in the release of degraded lamin B into the cytosolic fraction and promoted loss in metabolic cell viability. Conclusion: Based on these findings we conclude that protein prenylation plays key roles in islet β-cell function. These findings affirm further support to the hypothesis that defects in prenylation pathway induce caspase-3 activation and nuclear lamin degradation in pancreatic β-cells under the duress of metabolic stress (e.g., glucotoxicity.

Establishment of an Arabidopsis callus system to study the interrelations of biosynthesis, degradation and accumulation of carotenoids

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Schaub, Patrick; Rodriguez-Franco, Marta; Cazzonelli, Christopher Ian; Álvarez, Daniel; Wüst, Florian

2018-01-01

The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high

Technological Potential of Lactobacillus Strains Isolated from Fermented Green Olives: In Vitro Studies with Emphasis on Oleuropein-Degrading Capability

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Massimo Iorizzo

2016-01-01

Full Text Available Technological properties of two strains of Lactobacillus plantarum (B3 and B11 and one of Lactobacillus pentosus (B4, previously isolated from natural fermented green olives, have been studied in vitro. Acidifying ability, salt, temperature, and pH tolerances of all strains were found in the range reported for similar strains produced in Italy and optimal growth conditions were found to be 6.0–8.0 pH, 15–30°C temperature, and less than 6% NaCl. Moreover, all strains showed very good tolerance to common olive phenol content (0.3% total phenol and high oleuropein-degrading capability. It was found that medium composition affected the bacterial oleuropein degradation. B11 strain grown in a nutrient-rich medium showed a lower oleuropein-degrading action than when it was cultivated in nutrient-poor medium. Furthermore, enzymatic activity assays revealed that oleuropein depletion did not correspond to an increase of hydroxytyrosol, evidencing that bacterial strains could efficiently degrade oleuropein via a mechanism different from hydrolysis.

Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

International Nuclear Information System (INIS)

Garaj-Vrhovac, V.; Kopjar, N.

2003-01-01

Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

Analysis of the impact of random summing on passive assay of pebble bed reactor fuel using gamma-ray spectrometry

Science.gov (United States)

Chen, J.; Hawari, A. I.

2007-08-01

Pebble bed reactors (PBR) are characterized by multi-pass fuel systems in which spherical fuel pebbles are circulated through the core until they reach a proposed burnup limit. The fuel is assayed on-line to ensure that the burnup limit is not breached. However, random summing effects can impact the response of the burnup measurement system and result in distortions that degrade the accuracy of the assay results. Monte Carlo analysis was performed to estimate the magnitude and effect of random summing on the absolute and relative indicators that have been identified as usable in on-line assay. For a throughput rate of 10 5 counts/s and trapezoidal pulse shaping of the signals, the results show that absolute indicators suffer from severe distortions due to this effect. Relative indicators are found to be resistant to random summing with the deviation in the ratio of peak areas remaining less than 5-15% depending on pulse width.

p-Nitrophenol degradation by electro-Fenton process: Pathway, kinetic model and optimization using central composite design.

Science.gov (United States)

Meijide, J; Rosales, E; Pazos, M; Sanromán, M A

2017-10-01

The chemical process scale-up, from lab studies to industrial production, is challenging and requires deep knowledge of the kinetic model and the reactions that take place in the system. This knowledge is also useful in order to be employed for the reactor design and the determination of the optimal operational conditions. In this study, a model substituted phenol such as p-nitrophenol was degraded by electro-Fenton process and the reaction products yielded along the treatment were recorded. The kinetic model was developed using Matlab software and was based on main reactions that occurred until total mineralization which allowed predicting the degradation pathway under this advanced oxidation process. The predicted concentration profiles of p-nitrophenol, their intermediates and by-products in electro-Fenton process were validated with experimental assays and the results were consistent. Finally, based on the developed kinetic model the degradation process was optimized using central composite design taking as key parameters the ferrous ion concentration and current density. Copyright © 2017 Elsevier Ltd. All rights reserved.

Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity.

Science.gov (United States)

Shahbazi, Razieh; Kasra-Kermanshahi, Roha; Gharavi, Sara; Moosavi-Nejad, Zahra; Borzooee, Faezeh

2013-06-01

Sodium dodecyl sulfate (SDS) is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS-degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene. Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS). Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp) and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg. The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation.

Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil.

Science.gov (United States)

Zuo, Zhenqiang; Gong, Ting; Che, You; Liu, Ruihua; Xu, Ping; Jiang, Hong; Qiao, Chuanling; Song, Cunjiang; Yang, Chao

2015-06-01

Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (10(6) cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.

Assay strategies and methods for phospholipases

International Nuclear Information System (INIS)

Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

1991-01-01

Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

Degradation and protection of DNAzymes on human skin.

Science.gov (United States)

Marquardt, Kay; Eicher, Anna-Carola; Dobler, Dorota; Höfer, Frank; Schmidts, Thomas; Schäfer, Jens; Renz, Harald; Runkel, Frank

2016-10-01

DNAzymes are catalytic nucleic acid based molecules that have become a new class of active pharmaceutical ingredients (API). Until now, five DNAzymes have entered clinical trials. Two of them were tested for topical application, whereby dermally applied DNAzymes had been prone to enzymatic degradation. To protect the DNAzymes the enzymatic activity of human skin has to be examined. Therefore, the enzymatic activity of human skin was qualitatively and quantitatively analyzed. Activity similar to that of DNase II could be identified and the specific activity was determined to be 0.59Units/mg. These results were used to develop an in vitro degradation assay to screen different kinds of protective systems on human skin. The chosen protective systems consisted of biodegradable chitosans or polyethylenimine, which forms polyplexes when combined with DNAzymes. The polyplexes were characterized in terms of particle size, zeta potential, stability and degree of complexation. The screening revealed that the protective efficiency of the polyplexes depended on the polycation and the charge ratio (ξ). At a critical ξ ratio between 1.0 and 4.1 and at a maximal zeta potential, sufficient protection of the DNAzyme was achieved. The results of this study will be helpful for the development of a protective dermal drug delivery systems using polyplexes. Copyright © 2016 Elsevier B.V. All rights reserved.

Degradation Capability of n-hexadecane Degrading Bacteria from Petroleum Contaminated Soils

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PENG Huai-li

2017-05-01

Full Text Available Samplings were performed in the petroleum contaminated soils of Dongying, Shandong Province of China. Degrading bacteria was isolated through enrichment in a Bushnel-Hass medium, with n-hexadecane as the sole source of carbon and energy. Then the isolated strains were identified by amplification of 16S rDNA gene and sequencing. The strain TZSX2 was selected as the powerful bacteria with stronger degradation ability, which was then identified as Rhodococcus hoagii genera based on the constructing results of the phylogenetic tree. The optimum temperature that allowed both high growth and efficient degradation ratio was in the scope of 28~36 ℃, and gas chromatography results showed that approximately more than 30% of n-hexadecane could be degraded in one week of incubation within the temperature range. Moreover, the strain TZSX2 was able to grow in high concentrations of n-hexadecane. The degradation rate reached 79% when the initial n-hexadecane concentration was 2 mL·L-1,while it still achieved 12% with n-hexadecane concentration of 20 mL·L-1. The optimal pH was 9 that allowed the highest growth and the greatest degradation rate of 91%. Above all, the screened strain TZSX2 showed high capabilities of alkali tolerance with excellent degradation efficiency for even high concentration of n-hexadecane, and thus it would be quite suitable for the remediation of petroleum contaminated soils especially in the extreme environment.

Abiotic Degradation and Toxicological Impacts of Pharmaceuticals and Personal Care Products (PPCPs) in Surface Waters: Roles of Mineral Sediments and Solar Radiation

Science.gov (United States)

Rubasinghege, G. R. S.; Rijal, H.; Maldonado-Torres, S.; Gurung, R.; Rogelj, S.; Piyasena, M.

2017-12-01

The growing medical and personal needs of human populations have escalated release of pharmaceuticals and personal care products into surface waters. This work investigates abiotic degradation pathways of a particular PPCP, ibuprofen, in the presence of a major mineral component of sedimentation (kaolinite clay), as well as the health effects of the primary compound and its degradation products. Results from these studies showed that the rate and extent of ibuprofen degradation is greatly influenced by the presence of sedimentation particles and solar radiation. In the absence of solar radiation, the dominant reaction mechanism was observed to be the adsorption of ibuprofen onto sedimentation particle surface where surface silanol groups play a key role. In contrast, under solar radiation and in the presence of clay particles, ibuprofen breaks down to several fractions. The decay rates were at least 6-fold higher for irradiated samples compared to those of dark conditions. Toxicity of primary ibuprofen and its secondary residues were tested on three microorganisms: Bacillus megaterium, Pseudoaltermonas atlantica; and algae from the Chlorella genus. The results from the biological assays show that primary PPCP is more toxic than the mixture of secondary products. Overall, however, biological assays carried out using only 4-acetylbenzoic acid, the most abundant secondary product, show a higher toxic effect on algae compared to its parent compound.

Solid phase assays

International Nuclear Information System (INIS)

Reese, M.G.; Johnson, L.R.; Ransom, D.K.

1980-01-01

In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells

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Xiao-hong Kang

2017-04-01

Full Text Available Background/Aims: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC. Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. Methods: Cell proliferation was analyzed by cell counting kit-8 (CCK-8 assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. Results: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3β. GSK-3 inhibitor and GSK-3β siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. Conclusion: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3β activation was involved in bufalin-induced apoptosis.

Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

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Surang Chankhamhaengdecha

2013-01-01

Full Text Available Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL quorum sensing (QS system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9% and 68 (51.5% of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30±3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

Energy Technology Data Exchange (ETDEWEB)

Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

2006-06-25

Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

International Nuclear Information System (INIS)

Singer, Timothy M.; Lambert, Iain B.; Williams, Andrew; Douglas, George R.; Yauk, Carole L.

2006-01-01

Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

DNA comet assay to identify different freezing temperatures of irradiated liver chicken

International Nuclear Information System (INIS)

Duarte, Renato C.; Mozeika, Michel A.; Fanaro, Gustavo B.; Villavicencio, Anna L.C.H.; Marchioni, Eric

2009-01-01

The cold chain is a succession of steps which maintain the food at low temperature. The thawed food never be frozen again and the best solution being to consume it quickly to avoid the microorganism growth which causes decay and nutrients damage. One of most important point is that freezing process, unlike irradiation, do not destroy microorganisms, only inactive them as long as they remain in a frozen state. The Comet Assay is an original test used to detect irradiated foods that's recognize the DNA damage and can then be used to control the overall degradation of the food and in a certain extend to evaluate the damage caused by irradiation, different forms of freeze and storage time on liver chicken cells. Different freezing temperatures were used, deep freeze -196 deg C and slow freeze -10 deg C. Samples were irradiated in a 60 Co irradiator with 1.5, 3.0 and 4.5 kGy radiation doses. Fast freezing technique induces a low percent of DNA degradation comparing to slow freezing technique. This procedure could be a good choose to chicken freezing processing. (author)

DNA comet assay to identify different freezing temperatures of irradiated liver chicken

Energy Technology Data Exchange (ETDEWEB)

Duarte, Renato C.; Mozeika, Michel A.; Fanaro, Gustavo B.; Villavicencio, Anna L.C.H. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: renatocduarte@yahoo.com.br; Marchioni, Eric [Universite de Strasbourg, Illkirch (France). Faculte de Pharmacie. Lab. de Chimie Analytique et Sciences de l' Aliment

2009-07-01

The cold chain is a succession of steps which maintain the food at low temperature. The thawed food never be frozen again and the best solution being to consume it quickly to avoid the microorganism growth which causes decay and nutrients damage. One of most important point is that freezing process, unlike irradiation, do not destroy microorganisms, only inactive them as long as they remain in a frozen state. The Comet Assay is an original test used to detect irradiated foods that's recognize the DNA damage and can then be used to control the overall degradation of the food and in a certain extend to evaluate the damage caused by irradiation, different forms of freeze and storage time on liver chicken cells. Different freezing temperatures were used, deep freeze -196 deg C and slow freeze -10 deg C. Samples were irradiated in a {sup 60}Co irradiator with 1.5, 3.0 and 4.5 kGy radiation doses. Fast freezing technique induces a low percent of DNA degradation comparing to slow freezing technique. This procedure could be a good choose to chicken freezing processing. (author)

Drift Degradation Analysis

International Nuclear Information System (INIS)

D. Kicker

2004-01-01

Degradation of underground openings as a function of time is a natural and expected occurrence for any subsurface excavation. Over time, changes occur to both the stress condition and the strength of the rock mass due to several interacting factors. Once the factors contributing to degradation are characterized, the effects of drift degradation can typically be mitigated through appropriate design and maintenance of the ground support system. However, for the emplacement drifts of the geologic repository at Yucca Mountain, it is necessary to characterize drift degradation over a 10,000-year period, which is well beyond the functional period of the ground support system. This document provides an analysis of the amount of drift degradation anticipated in repository emplacement drifts for discrete events and time increments extending throughout the 10,000-year regulatory period for postclosure performance. This revision of the drift degradation analysis was developed to support the license application and fulfill specific agreement items between the U.S. Nuclear Regulatory Commission (NRC) and the U.S. Department of Energy (DOE). The earlier versions of ''Drift Degradation Analysis'' (BSC 2001 [DIRS 156304]) relied primarily on the DRKBA numerical code, which provides for a probabilistic key-block assessment based on realistic fracture patterns determined from field mapping in the Exploratory Studies Facility (ESF) at Yucca Mountain. A key block is defined as a critical block in the surrounding rock mass of an excavation, which is removable and oriented in an unsafe manner such that it is likely to move into an opening unless support is provided. However, the use of the DRKBA code to determine potential rockfall data at the repository horizon during the postclosure period has several limitations: (1) The DRKBA code cannot explicitly apply dynamic loads due to seismic ground motion. (2) The DRKBA code cannot explicitly apply loads due to thermal stress. (3) The DRKBA

Drift Degradation Analysis

Energy Technology Data Exchange (ETDEWEB)

D. Kicker

2004-09-16

Degradation of underground openings as a function of time is a natural and expected occurrence for any subsurface excavation. Over time, changes occur to both the stress condition and the strength of the rock mass due to several interacting factors. Once the factors contributing to degradation are characterized, the effects of drift degradation can typically be mitigated through appropriate design and maintenance of the ground support system. However, for the emplacement drifts of the geologic repository at Yucca Mountain, it is necessary to characterize drift degradation over a 10,000-year period, which is well beyond the functional period of the ground support system. This document provides an analysis of the amount of drift degradation anticipated in repository emplacement drifts for discrete events and time increments extending throughout the 10,000-year regulatory period for postclosure performance. This revision of the drift degradation analysis was developed to support the license application and fulfill specific agreement items between the U.S. Nuclear Regulatory Commission (NRC) and the U.S. Department of Energy (DOE). The earlier versions of ''Drift Degradation Analysis'' (BSC 2001 [DIRS 156304]) relied primarily on the DRKBA numerical code, which provides for a probabilistic key-block assessment based on realistic fracture patterns determined from field mapping in the Exploratory Studies Facility (ESF) at Yucca Mountain. A key block is defined as a critical block in the surrounding rock mass of an excavation, which is removable and oriented in an unsafe manner such that it is likely to move into an opening unless support is provided. However, the use of the DRKBA code to determine potential rockfall data at the repository horizon during the postclosure period has several limitations: (1) The DRKBA code cannot explicitly apply dynamic loads due to seismic ground motion. (2) The DRKBA code cannot explicitly apply loads due to thermal

Cometabolic Degradation of Dibenzofuran and Dibenzothiophene by a Naphthalene-Degrading Comamonas sp. JB.

Science.gov (United States)

Ji, Xiangyu; Xu, Jing; Ning, Shuxiang; Li, Nan; Tan, Liang; Shi, Shengnan

2017-12-01

Comamonas sp. JB was used to investigate the cometabolic degradation of dibenzofuran (DBF) and dibenzothiophene (DBT) with naphthalene as the primary substrate. Dehydrogenase and ATPase activity of the growing system with the presence of DBF and DBT were decreased when compared to only naphthalene in the growing system, indicating that the presence of DBF and DBT inhibited the metabolic activity of strain JB. The pathways and enzymes involved in the cometabolic degradation were tested. Examination of metabolites elucidated that strain JB cometabolically degraded DBF to 1,2-dihydroxydibenzofuran, subsequently to 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid, and finally to catechol. Meanwhile, strain JB cometabolically degraded DBT to 1,2-dihydroxydibenzothiophene and subsequently to the ring cleavage product. A series of naphthalene-degrading enzymes including naphthalene dioxygenase, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase, salicylate hydroxylase, and catechol 2,3-oxygenase have been detected, confirming that naphthalene was the real inducer of expression the degradation enzymes and metabolic pathways were controlled by naphthalene-degrading enzymes.

Evaluation of total PSA assay on vitros ECi and correlation with Kryptor-PSA assay.

Science.gov (United States)

Cassinat, B; Wacquet, M; Toubert, M E; Rain, J D; Schlageter, M H

2001-01-01

An increasing number of multiparametric immuno-analysers for PSA assays are available. As different immuno-assays may vary in their analytical quality and their accuracy for the follow-up of patients, expertise is necessary for each new assay. The PSA assay on the Vitros-ECi analyser has been evaluated and compared with the PSA assay from the Kryptor analyser. Variation coefficients were 0.91 to 1.98% for within-run assays, and 4.2% to 5.4% for interassay (PSA levels = 0.8 microgram/L to 33.6 micrograms/L). Dilution tests showed 93 to 136% recovery until 70 micrograms/L PSA. Functional sensitivity was estimated at 0.03 microgram/L. Equimolarity of the test was confirmed. Correlation of PSA levels measured with Vitros-ECi and Kryptor analysers displayed a correlation coefficient r2 of 0.9716. The half-lives and doubling times of PSA were similar using both methods. Vitros-ECi PSA assay meets the major criteria for the management of prostate cancer patients.

Hormone assay

International Nuclear Information System (INIS)

Eisentraut, A.M.

1977-01-01

An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

Intermittent degradation and schizotypy

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Matthew W. Roché

2015-06-01

Full Text Available Intermittent degradation refers to transient detrimental disruptions in task performance. This phenomenon has been repeatedly observed in the performance data of patients with schizophrenia. Whether intermittent degradation is a feature of the liability for schizophrenia (i.e., schizotypy is an open question. Further, the specificity of intermittent degradation to schizotypy has yet to be investigated. To address these questions, 92 undergraduate participants completed a battery of self-report questionnaires assessing schizotypy and psychological state variables (e.g., anxiety, depression, and their reaction times were recorded as they did so. Intermittent degradation was defined as the number of times a subject’s reaction time for questionnaire items met or exceeded three standard deviations from his or her mean reaction time after controlling for each item’s information processing load. Intermittent degradation scores were correlated with questionnaire scores. Our results indicate that intermittent degradation is associated with total scores on measures of positive and disorganized schizotypy, but unrelated to total scores on measures of negative schizotypy and psychological state variables. Intermittent degradation is interpreted as potentially derivative of schizotypy and a candidate endophenotypic marker worthy of continued research.

Prediction of phospholipidosis-inducing potential of drugs by in vitro biochemical and physicochemical assays followed by multivariate analysis.

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Kuroda, Yukihiro; Saito, Madoka

2010-03-01

An in vitro method to predict phospholipidosis-inducing potential of cationic amphiphilic drugs (CADs) was developed using biochemical and physicochemical assays. The following parameters were applied to principal component analysis, as well as physicochemical parameters: pK(a) and clogP; dissociation constant of CADs from phospholipid, inhibition of enzymatic phospholipid degradation, and metabolic stability of CADs. In the score plot, phospholipidosis-inducing drugs (amiodarone, propranolol, imipramine, chloroquine) were plotted locally forming the subspace for positive CADs; while non-inducing drugs (chlorpromazine, chloramphenicol, disopyramide, lidocaine) were placed scattering out of the subspace, allowing a clear discrimination between both classes of CADs. CADs that often produce false results by conventional physicochemical or cell-based assay methods were accurately determined by our method. Basic and lipophilic disopyramide could be accurately predicted as a nonphospholipidogenic drug. Moreover, chlorpromazine, which is often falsely predicted as a phospholipidosis-inducing drug by in vitro methods, could be accurately determined. Because this method uses the pharmacokinetic parameters pK(a), clogP, and metabolic stability, which are usually obtained in the early stages of drug development, the method newly requires only the two parameters, binding to phospholipid, and inhibition of lipid degradation enzyme. Therefore, this method provides a cost-effective approach to predict phospholipidosis-inducing potential of a drug. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Optimizing Polychlorinated Biphenyl Degradation by Flavonoid-Induced Cells of the Rhizobacterium Rhodococcus erythropolis U23A.

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Thi Thanh My Pham

Full Text Available There is evidence that many plant secondary metabolites may act as signal molecules to trigger the bacterial ability to metabolize polychlorinated biphenyls (PCBs during the rhizoremediation process. However, the bases for the PCB rhizoremediation process are still largely unknown. The rhizobacterium Rhodococcus erythropolis U23A is unable to use flavanone as a growth substrate. However, on the basis of an assay that monitors the amount of 4-chlorobenzoate produced from 4-chlorobiphenyl by cells grown co-metabolically on flavanone plus sodium acetate, this flavonoid was previously found to be a potential inducer of the U23A biphenyl catabolic pathway. In this work, and using the same assay, we identified ten other flavonoids that did not support growth, but that acted as inducers of the U23A biphenyl pathway, and we confirmed flavonoid induction of the biphenyl catabolic pathway using quantitative real-time polymerase chain reaction (RT-qPCR on the bphA gene. We also examined the effect of the growth co-substrate on flavonoid induction. Sodium acetate was replaced by glucose, mannose, sucrose, or mannitol, which are sugars found in plant root exudates. The data showed that the level of induction of strain U23A biphenyl-degrading enzymes was significantly influenced by the nature and concentration of the flavonoid in the growth medium, as well as by the substrate used for growth. Sucrose allowed for an optimal induction response for most flavonoids. Some flavonoids, such as flavone and isoflavone, were better inducers of the biphenyl catabolic enzymes than biphenyl itself. We also found that all flavonoids tested in this work were metabolized by strain U23A during co-metabolic growth, but that the metabolite profiles, as well as the level of efficiency of degradation, differed for each flavonoid. To obtain insight into how flavonoids interact with strain U23A to promote polychlorinated biphenyl (PCB degradation, we determined the concentration of

Chemical composition and biological activity of the essential oil from Thymus lanceolatus.

Science.gov (United States)

Khadir, Abdelmounaim; Sobeh, Mansour; Gad, Haidy A; Benbelaid, Fethi; Bendahou, Mourad; Peixoto, Herbenya; Sporer, Frank; Ashour, Mohamed L; Wink, Michael

2016-01-01

Thymus lanceolatus is a rare species, which grows wild in Algeria and Tunis. It is used traditionally as a drink and to flavor and preserve meat and poultry. The composition of the essential oil was determined by GLC/FID and GLC/MS. Forty-nine components were identified and quantified, accounting for 96.75% of the total detected components in the oil. The oxygenated monoterpenes (74.85%) constitute the major class of volatile secondary metabolites in the oil. Thymol was the most abundant constituent (69.61%) followed by γ-terpinene (8.38%). The antioxidant activity was evaluated using both diphenylpicrylhydrazyl (DPPH˙) reduction and 2-deoxyribose (2-DR) degradation prevention methods. The oil showed a very potent antioxidant activity with IC(50) values of 0.20 ± 0.07 and 4.96 ± 0.39 μg/mL for the DPPH˙ and 2-DR methods, respectively. The antimicrobial activity of the oil was assessed using the agar diffusion method, and the in vitro cytotoxicity on five different cancer cells was examined using the MTT assay. The oil revealed promising inhibitory activity against Gram positive bacteria, especially Bacillus subtilis and Streptococcus pyogenes with an MIC value of 62.5 μg/mL. Additionally, the highest cytotoxic activity was observed against the HL-60 cells with an IC(50) of 113.5 μg/mL. These results validate some of their traditional uses in food preservation.

The Study of the Microbes Degraded Polystyrene

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Zhi-Long Tang

2017-01-01

Full Text Available Under the observation that Tenebrio molitor and Zophobas morio could eat polystyrene (PS, we setup the platform to screen the gut microbes of these two worms. To take advantage of that Tenebrio molitor and Zophobas morio can eat and digest polystyrene as its diet, we analyzed these special microbes with PS plate and PS turbidity system with time courses. There were two strains TM1 and ZM1 which isolated from Tenebrio molitor and Zophobas morio, and were identified by 16S rDNA sequencing. The results showed that TM1 and ZM1 were cocci-like and short rod shape Gram-negative bacteria under microscope. The PS plate and turbidity assay showed that TM1 and ZM1 could utilize polystyrene as their carbon sources. The further study of PS degraded enzyme and cloning warrants our attention that this platform will be an excellent tools to explore and solve this problem.

Characterization and degradation potential of diesel-degrading bacterial strains for application in bioremediation.

Science.gov (United States)

Balseiro-Romero, María; Gkorezis, Panagiotis; Kidd, Petra S; Van Hamme, Jonathan; Weyens, Nele; Monterroso, Carmen; Vangronsveld, Jaco

2017-10-03

Bioremediation of polluted soils is a promising technique with low environmental impact, which uses soil organisms to degrade soil contaminants. In this study, 19 bacterial strains isolated from a diesel-contaminated soil were screened for their diesel-degrading potential, biosurfactant (BS) production, and biofilm formation abilities, all desirable characteristics when selecting strains for re-inoculation into hydrocarbon-contaminated soils. Diesel-degradation rates were determined in vitro in minimal medium with diesel as the sole carbon source. The capacity to degrade diesel range organics (DROs) of strains SPG23 (Arthobacter sp.) and PF1 (Acinetobacter oleivorans) reached 17-26% of total DROs after 10 days, and 90% for strain GK2 (Acinetobacter calcoaceticus). The amount and rate of alkane degradation decreased significantly with increasing carbon number for strains SPG23 and PF1. Strain GK2, which produced BSs and biofilms, exhibited a greater extent, and faster rate of alkane degradation compared to SPG23 and PF1. Based on the outcomes of degradation experiments, in addition to BS production, biofilm formation capacities, and previous genome characterizations, strain GK2 is a promising candidate for microbial-assisted phytoremediation of diesel-contaminated soils. These results are of particular interest to select suitable strains for bioremediation, not only presenting high diesel-degradation rates, but also other characteristics which could improve rhizosphere colonization.

A multiplex PCR mini-barcode assay to identify processed shark products in the global trade.

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Diego Cardeñosa

Full Text Available Protecting sharks from overexploitation has become global priority after widespread population declines have occurred. Tracking catches and trade on a species-specific basis has proven challenging, in part due to difficulties in identifying processed shark products such as fins, meat, and liver oil. This has hindered efforts to implement regulations aimed at promoting sustainable use of commercially important species and protection of imperiled species. Genetic approaches to identify shark products exist but are typically based on sequencing or amplifying large DNA regions and may fail to work on heavily processed products in which DNA is degraded. Here, we describe a novel multiplex PCR mini-barcode assay based on two short fragments of the cytochrome oxidase I (COI gene. This assay can identify to species all sharks currently listed on the Convention of International Trade of Endangered Species (CITES and most shark species present in the international trade. It achieves species diagnosis based on a single PCR and one to two downstream DNA sequencing reactions. The assay is capable of identifying highly processed shark products including fins, cooked shark fin soup, and skin-care products containing liver oil. This is a straightforward and reliable identification method for data collection and enforcement of regulations implemented for certain species at all governance levels.

Stability-indicating UPLC method for determination of Valsartan and their degradation products in active pharmaceutical ingredient and pharmaceutical dosage forms.

Science.gov (United States)

Krishnaiah, Ch; Reddy, A Raghupathi; Kumar, Ramesh; Mukkanti, K

2010-11-02

A simple, precise, accurate stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the quantitative determination of purity of Valsartan drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. The method was developed using Waters Aquity BEH C18 (100 mm x 2.1 mm, 1.7 microm) column with mobile phase containing a gradient mixture of solvents A and B. The eluted compounds were monitored at 225 nm, the run time was within 9.5 min, which Valsartan and its seven impurities were well separated. Valsartan was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Valsartan was found to degrade significantly in acid and oxidative stress conditions and stable in base, hydrolytic and photolytic degradation conditions. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per international conference on harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of Valsartan in pharmaceutical dosage forms.

Evaluation of fungal- and photo-degradation as potential treatments for the removal of sunscreens BP3 and BP1

International Nuclear Information System (INIS)

Gago-Ferrero, Pablo; Badia-Fabregat, Marina; Olivares, Alba; Piña, Benjamin; Blánquez, Paqui; Vicent, Teresa; Caminal, Gloria; Díaz-Cruz, M. Silvia

2012-01-01

Photodecomposition might be regarded as one of the most important abiotic factors affecting the fate of UV absorbing compounds in the environment and photocatalysis has been suggested as an effective method to degrade organic pollutants. However, UV filters transformation appears to be a complex process, barely addressed to date. The white rot fungus Trametes versicolor is considered as a promising alternative to conventional aerobic bacterial degradation, as it is able to metabolise a wide range of xenobiotics. This study focused on both degradation processes of two widely used UV filters, benzophenone-3 (BP3) and benzophenone-1 (BP1). Fungal treatment resulted in the degradation of more than 99% for both sunscreens in less than 24 h, whereas photodegradation was very inefficient, especially for BP3, which remained unaltered upon 24 h of simulated sunlight irradiation. Analysis of metabolic compounds generated showed BP1 as a minor by-product of BP3 degradation by T. versicolor while the main intermediate metabolites were glycoconjugate derivatives. BP1 and BP3 showed a weak, but significant estrogenic activity (EC50 values of 0.058 mg/L and 12.5 mg/L, respectively) when tested by recombinant yeast assay (RYA), being BP1 200-folds more estrogenic than BP3. Estrogenic activity was eliminated during T. versicolor degradation of both compounds, showing that none of the resulting metabolites possessed significant estrogenic activity at the concentrations produced. These results demonstrate the suitability of this method to degrade both sunscreen agents and to eliminate estrogenic activity. - Highlights: ► Fungus T. versicolor is able to degrade totally BP3 and BP1 in few hours in a fluidised bed bioreactor. ► BP3 is not degraded under simulated sunlight. ► Glycoconjugates have been identified as the main intermediate metabolites. ► Decrease in endocrine activity was found in both photodegradation and biodegradation.

Developments in polymer degradation - 7

International Nuclear Information System (INIS)

Grassie, N.

1987-01-01

A selection of topics which are representative of the continually expanding area of polymer degradation is presented. The aspects emphasised include the products of degradation of specific polymers, degradation by high energy radiation and mechanical forces, fire retardant studies and the special role of small radicals in degradation processes. (author)

Constitutively internalized dopamine transporter is targeted to late endosomes and lysosomal degradation in heterologous cell lines and dopaminergic neurons

DEFF Research Database (Denmark)

Eriksen, Jacob; Madsen, Kenneth; Vægter, Christian Bjerggaard

and amphetamine, a substrate of the DAT. In antibody feeding experiments we observed that Tac-DAT was constitutively internalized faster than Tac alone and using an ELISA based assay we could quantify time-dependent intracellular accumulation of the transporter. Incubation with inhibitors of lysosomal degradation...... (leupeptin, chloroquine, or ammonium chloride) increased the amount of transporter accumulated intracellularly over time, suggesting that constitutively endocytosed transporter was targeted to lysosomal degradation. This was further supported by expression of Tac-DAT in the immortalized dopaminergic cell...... dopaminergic neurons and visualized the DAT directly in the neurons using the fluorescent cocaine analog JHC 1-064. These data showed pronounced colocalization upon constitutive internalization with Lysotracker, a late endosomal/lysosomal marker; however only little co-lolization was observed with Alexa488...

The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

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Bas-jan Van Der Leede

2015-08-01

Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

Apparent Contradiction: Psychrotolerant Bacteria from Hydrocarbon-Contaminated Arctic Tundra Soils That Degrade Diterpenoids Synthesized by Trees

Science.gov (United States)

Yu, Zhongtang; Stewart, Gordon R.; Mohn, William W.

2000-01-01

Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82°N, 62°W). According to most-probable-number assays, resin acid degraders were abundant (103 to 104 propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (106 to 107 propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genus Pseudomonas. Strain DhA-95 is a member of the genus Sphingomonas. All four strains are psychrotolerant, with growth temperature ranges of 4°C to 30°C (DhA-91 and DhA-95) or 4°C to 22°C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22°C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two Mycobacterium sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents. PMID:11097882

Purex diluent degradation

International Nuclear Information System (INIS)

Tallent, O.K.; Mailen, J.C.; Pannell, K.D.

1984-02-01

The chemical degradation of normal paraffin hydrocarbon (NPH) diluents both in the pure state and mixed with 30% tributyl phosphate (TBP) was investigated in a series of experiments. The results show that degradation of NPH in the TBP-NPH-HNO 3 system is consistent with the active chemical agent being a radical-like nitrogen dioxide (NO 2 ) molecule, not HNO 3 as such. Spectrophotometric, gas chromatographic, mass spectrographic, and titrimetric methods were used to identify the degradation products, which included alkane nitro and nitrate compounds, alcohols, unsaturated alcohols, nitro alcohols, nitro alkenes, ketones, and carboxylic acids. The degradation rate was found to increase with increases in the HNO 3 concentration and the temperature. The rate was decreased by argon sparging to remove NO 2 and by the addition of butanol, which probably acts as a NO 2 scavenger. 13 references, 11 figures

Iodinated contrast media electro-degradation: process performance and degradation pathways.

Science.gov (United States)

Del Moro, Guido; Pastore, Carlo; Di Iaconi, Claudio; Mascolo, Giuseppe

2015-02-15

The electrochemical degradation of six of the most widely used iodinated contrast media was investigated. Batch experiments were performed under constant current conditions using two DSA® electrodes (titanium coated with a proprietary and patented mixed metal oxide solution of precious metals such as iridium, ruthenium, platinum, rhodium and tantalum). The degradation removal never fell below 85% (at a current density of 64 mA/cm(2) with a reaction time of 150 min) when perchlorate was used as the supporting electrolyte; however, when sulphate was used, the degradation performance was above 80% (at a current density of 64 mA/cm(2) with a reaction time of 150 min) for all of the compounds studied. Three main degradation pathways were identified, namely, the reductive de-iodination of the aromatic ring, the reduction of alkyl aromatic amides to simple amides and the de-acylation of N-aromatic amides to produce aromatic amines. However, as amidotrizoate is an aromatic carboxylate, this is added via the decarboxylation reaction. The investigation did not reveal toxicity except for the lower current density used, which has shown a modest toxicity, most likely for some reaction intermediates that are not further degraded. In order to obtain total removal of the contrast media, it was necessary to employ a current intensity between 118 and 182 mA/cm(2) with energy consumption higher than 370 kWh/m(3). Overall, the electrochemical degradation was revealed to be a reliable process for the treatment of iodinated contrast media that can be found in contaminated waters such as hospital wastewater or pharmaceutical waste-contaminated streams. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of SUMO in RNF4-mediated promyelocytic leukemia protein (PML) degradation: sumoylation of PML and phospho-switch control of its SUMO binding domain dissected in living cells.

Science.gov (United States)

Percherancier, Yann; Germain-Desprez, Delphine; Galisson, Frédéric; Mascle, Xavier H; Dianoux, Laurent; Estephan, Patricia; Chelbi-Alix, Mounira K; Aubry, Muriel

2009-06-12

Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.

Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones

International Nuclear Information System (INIS)

Schulster, D.

1977-01-01

Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique. (author)

Relationship between the radioisotopic footpad assay and other immunological assays in tumor bearing rats

International Nuclear Information System (INIS)

Mizushima, Yutaka; Takeichi, Noritoshi; Minami, Akio; Kasai, Masaharu; Itaya, Toshiyuki

1981-01-01

KMT-17, a fibrosarcoma induced by 3-methylcholanthrene in a WKA rat, is a sensitive tumor to various kinds of immunological assays and is a suitable model tumor for the study of the immune status in tumor bearing hosts. The antitumor immune response of KMT-17 bearing rats was studied by a radioisotopic footpad assay (FPA) in comparison with other in vivo and in vitro assays. Delayed hypersensitivity to tumor antigens measured by the FPA was observed from the 8th day after transplantation of KMT-17 cells, reached a peak on the 12 - 15th day, and then declined in the late stage on the 17th day. The kinetics of the FPA correlated well with those of an in vivo Winn assay and of an in vitro lymphocyte cytotoxicity assay ( 51 Cr-release assay). The appearance of an antitumor antibody detected by a complement dependent cytotoxicity test also correlated well with the kinetics of the FPA. A growth inhibition assay (GIA) for non-specific cell-mediated immunity also showed similar kinetics to that of the FPA. The delayed hypersensitivity footpad reaction to tumor cell extracts measured by this FPA was tumor-specific. These results suggest that the FPA is a simple and reliable in vivo assay for evaluating antitumor immunity in tumor bearing hosts. (author)

Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

Directory of Open Access Journals (Sweden)

Angela Filomena

2017-10-01

Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling

DEFF Research Database (Denmark)

Barascuk, Natasha; Vassiliadis, Efstathios; Larsen, Lise

2011-01-01

Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine.......Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine....

Liquor oligoclonal bands assay: interpretation, correlation with other laboratory assays and importance for diagnostics of neurological disorders

OpenAIRE

Bagdonas, Dovydas

2017-01-01

Aim: to analyse the possible relationship between liquor IgG oligoclonal bands assay and other laboratory assays in neurological patients. Objectives: to determine the frequency of oligoclonal bands in neurological patients; to compare the results between serum and liquor laboratory assays in dependence of oligoclonal bands assay results; to evaluate the relationships between oligoclonal bands assay and serological-immunological assays for infectious diseases, gender, age and neurological ...

A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.

Directory of Open Access Journals (Sweden)

Anna eGasperl

2015-12-01

Full Text Available Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP by fructan exohydrolases (FEHs to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding.

Anthocyanins degradation during storage of Hibiscus sabdariffa extract and evolution of its degradation products.

Science.gov (United States)

Sinela, André; Rawat, Nadirah; Mertz, Christian; Achir, Nawel; Fulcrand, Hélène; Dornier, Manuel

2017-01-01

Degradation parameters of two main anthocyanins from roselle extract (Hibiscus sabdariffa L.) stored at different temperatures (4-37°C) over 60days were determined. Anthocyanins and some of their degradation products were monitored and quantified using HPLC-MS and DAD. Degradation of anthocyanins followed first-order kinetics and reaction rate constants (k values), which were obtained by non-linear regression, showed that the degradation rate of delphinidin 3-O-sambubioside was higher than that of cyanidin 3-O-sambubioside with k values of 9.2·10(-7)s(-1) and 8.4·10(-7)s(-1) at 37°C respectively. The temperature dependence of the rate of anthocyanin degradation was modeled by the Arrhenius equation. Degradation of delphinidin 3-O-sambubioside (Ea=90kJmol(-1)) tended to be significantly more sensitive to an increase in temperature than cyanidin 3-O-sambubioside (Ea=80kJmol(-1)). Degradation of these anthocyanins formed scission products (gallic and protocatechuic acids respectively) and was accompanied by an increase in polymeric color index. Copyright © 2016 Elsevier Ltd. All rights reserved.

Scopadulciol, Isolated from Scoparia dulcis, Induces β-Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells.

Science.gov (United States)

Fuentes, Rolly G; Toume, Kazufumi; Arai, Midori A; Sadhu, Samir K; Ahmed, Firoj; Ishibashi, Masami

2015-04-24

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased β-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of β-catenin induced by 1. The 1-induced degradation of β-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of β-catenin, which resulted in the inhibition of TCF/β-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation.

Directory of Open Access Journals (Sweden)

Beatriz Orosa

2017-01-01

Full Text Available Hypersensitive response programmed cell death (HR-PCD is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.

Endogenous Locus Reporter Assays.

Science.gov (United States)

Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

2018-01-01

Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

Isolation, Identification and Phenotypic Characterization of Microcystin-Degrading Bacteria from Lake Erie

Science.gov (United States)

Krishnan, A.; Mou, X. J.

2015-12-01

Lake Erie, the smallest and warmest lake among the Laurentian Great Lakes, is known for its problem of eutrophication and frequent occurrence of harmful cyanobacterial blooms (CyanoHABs). One major harmful effect of CyanoHABs is the production of cyanotoxins, especially microcystins. Microcystins (MC) are a group of hepatotoxins and the predominant variant of them is MC-LR. Field measurements and lab experiments indicate that MC degradation in Lake Erie is mainly carried out by indigenous bacteria. However, our knowledge on taxa involved in this process is very limited. This study aimed to fill this knowledge gap using a culture-dependent approach. Water and surface sediment samples were collected from Lake Erie in 2014 and 2015 and enriched with MC-LR. Cells were plated on a number of culturing media. The obtained pure bacterial cultures were screened for MC degrading abilities by MT2 BIO-LOG assays and by growing cells in liquid media containing MC-LR as the sole carbon source. In the latter experiment, MC concentrations were measured using HPLC. Isolates showing positive MC degradation activities in the screening steps were designated MC+ bacteria and characterized based on their phenotypic properties, including colony pigmentation, elevation, opacity, margin, gram nature and motility. The taxonomic identity of MC+ bacteria was determined by 16S rRNA gene full-length DNA sequencing. The presence of mlrA, a gene encoding MC cleavage pathway, was detected by PCR. Our culturing efforts obtained 520 pure cultures; 44 of them were identified as MC+. These MC+ isolates showed diversity in taxonomic identities and differed in their morphology, gram nature, colony characteristics and motility. PCR amplification of mlrA gene yield negative results for all MC+ isolates, indicating that the primers that were used may not be ubiquitous enough to cover the heterogeneity of mlrA genes or, more likely, alternative degradative genes/pathways were employed by Lake Erie bacteria

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

Science.gov (United States)

Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

2018-01-01

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

Particle passage kinetics and neutral detergent fiber degradability of silage of pineapple waste (aerial parts under different packing densities

Directory of Open Access Journals (Sweden)

Graciele Araújo de Oliveira Caetano

2014-01-01

Full Text Available The objective of this study was to determine the kinetics of in situ degradability parameters of the dry matter (DM and neutral detergent fiber (NDF and the passage of materials originating from the ensilage of the waste from pineapple cultivation (aerial parts. The four treatments utilized were silage of pineapple waste compacted at 600, 700, 900 and 1000 kg/m³. After ensiling the material from the pineapple cultivation, the particle-transit and rumen-degradation kinetics were analyzed. For the analysis of particle transit, chromium was utilized as a marker to mark the fiber. Passage rates were determined by retrieving the markers in the feces of the animals. In the degradation assay, samples were incubated in nylon bags for 0, 6, 18, 48 and 96 hours. The behavior observed in the regression curves of the variables analyzed describes high correlation between them, i.e., the time during which the silage is retained in the rumen influences its digestibility and its degradation rate. Although the silage compacted at 900 kg/m³ shows a larger potentially digestible fraction, it is recommended that it be ensiled at a compaction density of approximately 750 kg/m³ due to the lower cost and shorter mean retention time in the rumen-reticulum and rumen fill, thereby increasing the ruminal degradation and passage dynamics.

Individually and Synergistic Degradation of Hydrocarbons by Biosurfactant Producing Bacteria

Directory of Open Access Journals (Sweden)

Amirarsalan Kavyanifard

2016-02-01

Full Text Available Background: Increasing worldwide contamination with hydrocarbons has urged environmental remediation using biological agents such as bacteria. Our goal here was to study the phylogenetic relationship of two crude oil degrader bacteria and investigation of their ability to degrade hydrocarbons. Materials and Methods: Phylogenetic relationship of isolates was determined using morphological and biochemical characteristics and 16S rDNA gene sequencing. Optimum conditions of each isolate for crude oil degradation were investigated using one factor in time method. The rate of crude oil degradation by individual and consortium bacteria was assayed via Gas chromatography–mass spectrometry (GC-MS analysis. Biosurfactant production was measured by Du Noüy ring method using Krüss-K6 tensiometer. Results: The isolates were identified as Dietzia cinnamea KA1 and Dietzia cinnamea AP and clustered separately, while both are closely related to each other and with other isolates of Dietzia cinnamea. The optimal conditions for D. cinnamea KA1 were 35°C, pH9.0, 510 mM NaCl, and minimal requirement of 46.5 mM NH4Cl and 2.10 mM NaH2PO4. In the case of D. cinnamea AP, the values were 30°C, pH8.0, 170 mM NaCl, and minimal requirement of 55.8 mM NH4Cl and 2.10 mM NaH2PO4, respectively. Gas chromatography – Mass Spectroscopy (GC-MS analysis showed that both isolates were able to utilize various crude oil compounds, but D. cinnamea KA1 was more efficient individually and consortium of isolates was the most. The isolates were able to grow and produce biosurfactant when cultured in MSM supplemented with crude oil and optimization of MSM conditions lead to increase in biosurfactant production. Conclusion: To the best of our knowledge this is the first report of synergistic relationship between two strains of D. cinnamea in biodegradation of crude oil components, including poisonous and carcinogenic compound in a short time.

High-density polyethylene (HDPE)-degrading potential bacteria from marine ecosystem of Gulf of Mannar, India.

Science.gov (United States)

Balasubramanian, V; Natarajan, K; Hemambika, B; Ramesh, N; Sumathi, C S; Kottaimuthu, R; Rajesh Kannan, V

2010-08-01

Assessment of high-density polyethylene (HDPE)-degrading bacteria isolated from plastic waste dumpsites of Gulf of Mannar. Rationally, 15 bacteria (GMB1-GMB15) were isolated by enrichment technique. GMB5 and GMB7 were selected for further studies based on their efficiency to degrade the HDPE and identified as Arthrobacter sp. and Pseudomonas sp., respectively. Assessed weight loss of HDPE after 30 days of incubation was nearly 12% for Arthrobacter sp. and 15% for Pseudomonas sp. The bacterial adhesion to hydrocarbon (BATH) assay showed that the cell surface hydrophobicity of Pseudomonas sp. was higher than Arthrobacter sp. Both fluorescein diacetate hydrolysis and protein content of the biofilm were used to test the viability and protein density of the biomass. Acute peak elevation was observed between 2 and 5 days of inoculation for both bacteria. Fourier transform infrared (FT-IR) spectrum showed that keto carbonyl bond index (KCBI), Ester carbonyl bond index (ECBI) and Vinyl bond index (VBI) were increased indicating changes in functional group(s) and/or side chain modification confirming the biodegradation. The results pose us to suggest that both Pseudomonas sp. and Arthrobacter sp. were proven efficient to degrade HDPE, albeit the former was more efficacious, yet the ability of latter cannot be neglected. Recent alarm on ecological threats to marine system is dumping plastic waste in the marine ecosystem and coastal arena by anthropogenic activity. In maintenance phase of the plastic-derived polyethylene waste, the microbial degradation plays a major role; the information accomplished in this work will be the initiating point for the degradation of polyethylene by indigenous bacterial population in the marine ecosystem and provides a novel eco-friendly solution in eco-management.

RP-HPLC assay method development for Paracetamol and Lornoxicam in combination and characterization of oxidative degradation products of Lornoxicam

OpenAIRE

Jain Pritam S.; Patel Miketa A.; Chaudhari Amar J.; Surana Sanjay J.

2013-01-01

A simple, specific, accurate and precise reverse phase high pressure liquid chromatographic method has been developed for the simultaneous determination of Paracetamol and Lornoxicam from tablets and to characterize degradation products of Lornoxicam by reverse phase C18 column (Inertsil ODS 3V C-18, 250 x 4.6 mm, 5 μ). The sample was analyzed using Buffer (0.02504 Molar): Methanol in the ratio of 45:55, as a mobile phase at a flow rate of 1.5 mL/min and de...

Drift Degradation Analysis

International Nuclear Information System (INIS)

G.H. Nieder-Westermann

2005-01-01

The outputs from the drift degradation analysis support scientific analyses, models, and design calculations, including the following: (1) Abstraction of Drift Seepage; (2) Seismic Consequence Abstraction; (3) Structural Stability of a Drip Shield Under Quasi-Static Pressure; and (4) Drip Shield Structural Response to Rock Fall. This report has been developed in accordance with ''Technical Work Plan for: Regulatory Integration Modeling of Drift Degradation, Waste Package and Drip Shield Vibratory Motion and Seismic Consequences'' (BSC 2004 [DIRS 171520]). The drift degradation analysis includes the development and validation of rockfall models that approximate phenomenon associated with various components of rock mass behavior anticipated within the repository horizon. Two drift degradation rockfall models have been developed: the rockfall model for nonlithophysal rock and the rockfall model for lithophysal rock. These models reflect the two distinct types of tuffaceous rock at Yucca Mountain. The output of this modeling and analysis activity documents the expected drift deterioration for drifts constructed in accordance with the repository layout configuration (BSC 2004 [DIRS 172801])

Direct 125I-radioligand assays for serum progesterone compared with assays involving extraction of serum

International Nuclear Information System (INIS)

Ratcliffe, W.A.; Corrie, J.E.T.; Dalziel, A.H.; Macpherson, J.S.

1982-01-01

Two direct radioimmunoassays for progesterone in 50 μL of unextracted serum or plasma with assays involving extraction of serum were compared. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11α-hemisuccinyl conjugate and the radioligand 125 I-labeled progesterone 11α-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r > 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum

Absolute nuclear material assay

Science.gov (United States)

Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

2010-07-13

A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Science.gov (United States)

Klionsky, Daniel J.; Abeliovich, Hagai; Agostinis, Patrizia; Agrawal, Devendra K.; Aliev, Gjumrakch; Askew, David S.; Baba, Misuzu; Baehrecke, Eric H.; Bahr, Ben A.; Ballabio, Andrea; Bamber, Bruce A.; Bassham, Diane C.; Bergamini, Ettore; Bi, Xiaoning; Biard-Piechaczyk, Martine; Blum, Janice S.; Bredesen, Dale E.; Brodsky, Jeffrey L.; Brumell, John H.; Brunk, Ulf T.; Bursch, Wilfried; Camougrand, Nadine; Cebollero, Eduardo; Cecconi, Francesco; Chen, Yingyu; Chin, Lih-Shen; Choi, Augustine; Chu, Charleen T.; Chung, Jongkyeong; Clarke, Peter G.H.; Clark, Robert S.B.; Clarke, Steven G.; Clavé, Corinne; Cleveland, John L.; Codogno, Patrice; Colombo, María I.; Coto-Montes, Ana; Cregg, James M.; Cuervo, Ana Maria; Debnath, Jayanta; Demarchi, Francesca; Dennis, Patrick B.; Dennis, Phillip A.; Deretic, Vojo; Devenish, Rodney J.; Di Sano, Federica; Dice, J. Fred; DiFiglia, Marian; Dinesh-Kumar, Savithramma; Distelhorst, Clark W.; Djavaheri-Mergny, Mojgan; Dorsey, Frank C.; Dröge, Wulf; Dron, Michel; Dunn, William A.; Duszenko, Michael; Eissa, N. Tony; Elazar, Zvulun; Esclatine, Audrey; Eskelinen, Eeva-Liisa; Fésüs, László; Finley, Kim D.; Fuentes, José M.; Fueyo, Juan; Fujisaki, Kozo; Galliot, Brigitte; Gao, Fen-Biao; Gewirtz, David A.; Gibson, Spencer B.; Gohla, Antje; Goldberg, Alfred L.; Gonzalez, Ramon; González-Estévez, Cristina; Gorski, Sharon; Gottlieb, Roberta A.; Häussinger, Dieter; He, You-Wen; Heidenreich, Kim; Hill, Joseph A.; Høyer-Hansen, Maria; Hu, Xun; Huang, Wei-Pang; Iwasaki, Akiko; Jäättelä, Marja; Jackson, William T.; Jiang, Xuejun; Jin, Shengkan; Johansen, Terje; Jung, Jae U.; Kadowaki, Motoni; Kang, Chanhee; Kelekar, Ameeta; Kessel, David H.; Kiel, Jan A.K.W.; Kim, Hong Pyo; Kimchi, Adi; Kinsella, Timothy J.; Kiselyov, Kirill; Kitamoto, Katsuhiko; Knecht, Erwin; Komatsu, Masaaki; Kominami, Eiki; Kondo, Seiji; Kovács, Attila L.; Kroemer, Guido; Kuan, Chia-Yi; Kumar, Rakesh; Kundu, Mondira; Landry, Jacques; Laporte, Marianne; Le, Weidong; Lei, Huan-Yao; Lenardo, Michael J.; Levine, Beth; Lieberman, Andrew; Lim, Kah-Leong; Lin, Fu-Cheng; Liou, Willisa; Liu, Leroy F.; Lopez-Berestein, Gabriel; López-Otín, Carlos; Lu, Bo; Macleod, Kay F.; Malorni, Walter; Martinet, Wim; Matsuoka, Ken; Mautner, Josef; Meijer, Alfred J.; Meléndez, Alicia; Michels, Paul; Miotto, Giovanni; Mistiaen, Wilhelm P.; Mizushima, Noboru; Mograbi, Baharia; Monastyrska, Iryna; Moore, Michael N.; Moreira, Paula I.; Moriyasu, Yuji; Motyl, Tomasz; Münz, Christian; Murphy, Leon O.; Naqvi, Naweed I.; Neufeld, Thomas P.; Nishino, Ichizo; Nixon, Ralph A.; Noda, Takeshi; Nürnberg, Bernd; Ogawa, Michinaga; Oleinick, Nancy L.; Olsen, Laura J.; Ozpolat, Bulent; Paglin, Shoshana; Palmer, Glen E.; Papassideri, Issidora; Parkes, Miles; Perlmutter, David H.; Perry, George; Piacentini, Mauro; Pinkas-Kramarski, Ronit; Prescott, Mark; Proikas-Cezanne, Tassula; Raben, Nina; Rami, Abdelhaq; Reggiori, Fulvio; Rohrer, Bärbel; Rubinsztein, David C.; Ryan, Kevin M.; Sadoshima, Junichi; Sakagami, Hiroshi; Sakai, Yasuyoshi; Sandri, Marco; Sasakawa, Chihiro; Sass, Miklós; Schneider, Claudio; Seglen, Per O.; Seleverstov, Oleksandr; Settleman, Jeffrey; Shacka, John J.; Shapiro, Irving M.; Sibirny, Andrei; Silva-Zacarin, Elaine C.M.; Simon, Hans-Uwe; Simone, Cristiano; Simonsen, Anne; Smith, Mark A.; Spanel-Borowski, Katharina; Srinivas, Vickram; Steeves, Meredith; Stenmark, Harald; Stromhaug, Per E.; Subauste, Carlos S.; Sugimoto, Seiichiro; Sulzer, David; Suzuki, Toshihiko; Swanson, Michele S.; Tabas, Ira; Takeshita, Fumihiko; Talbot, Nicholas J.; Tallóczy, Zsolt; Tanaka, Keiji; Tanaka, Kozo; Tanida, Isei; Taylor, Graham S.; Taylor, J. Paul; Terman, Alexei; Tettamanti, Gianluca; Thompson, Craig B.; Thumm, Michael; Tolkovsky, Aviva M.; Tooze, Sharon A.; Truant, Ray; Tumanovska, Lesya V.; Uchiyama, Yasuo; Ueno, Takashi; Uzcátegui, Néstor L.; van der Klei, Ida; Vaquero, Eva C.; Vellai, Tibor; Vogel, Michael W.; Wang, Hong-Gang; Webster, Paul; Wiley, John W.; Xi, Zhijun; Xiao, Gutian; Yahalom, Joachim; Yang, Jin-Ming; Yap, George; Yin, Xiao-Ming; Yoshimori, Tamotsu; Yu, Li; Yue, Zhenyu; Yuzaki, Michisuke; Zabirnyk, Olga; Zheng, Xiaoxiang; Zhu, Xiongwei; Deter, Russell L.

2009-01-01

Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response. PMID:18188003

Degradation of diclofenac by UV-activated persulfate process: Kinetic studies, degradation pathways and toxicity assessments.

Science.gov (United States)

Lu, Xian; Shao, Yisheng; Gao, Naiyun; Chen, Juxiang; Zhang, Yansen; Xiang, Huiming; Guo, Youluo

2017-07-01

Diclofenac (DCF) is the frequently detected non-steroidal pharmaceuticals in the aquatic environment. In this study, the degradation of DCF was evaluated by UV-254nm activated persulfate (UV/PS). The degradation of DCF followed the pseudo first-order kinetics pattern. The degradation rate constant (k obs ) was accelerated by UV/PS compared to UV alone and PS alone. Increasing the initial PS dosage or solution pH significantly enhanced the degradation efficiency. Presence of various natural water constituents had different effects on DCF degradation, with an enhancement or inhibition in the presence of inorganic anions (HCO 3 - or Cl - ) and a significant inhibition in the presence of NOM. In addition, preliminary degradation mechanisms and major products were elucidated using LC-MS/MS. Hydroxylation, decarbonylation, ring-opening and cyclation reaction involving the attack of SO 4 • - or other substances, were the main degradation mechanism. TOC analyzer and Microtox bioassay were employed to evaluate the mineralization and cytotoxicity of solutions treated by UV/PS at different times, respectively. Limited elimination of TOC (32%) was observed during the mineralization of DCF. More toxic degradation products and their related intermediate species were formed, and the UV/PS process was suitable for removing the toxicity. Of note, longer degradation time may be considered for the final toxicity removal. Copyright © 2017. Published by Elsevier Inc.

Assay method and compositions

International Nuclear Information System (INIS)

1977-01-01

Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

α-amylase assay and action pattern determination using radioactive substrate, HPLC, and a radioactive flow detector

International Nuclear Information System (INIS)

Marsili, R.T.; Ostapenko, H.

1987-01-01

A new assay system is presented for the analysis of α-amylase. The disappearance of 14 C-labeled starch substrate and the appearance of its radioactive degradation products were monitored by HPLC and a radioactive flow detector/integrator. The hydrolysis of radioactive substrate was proportional to enzyme concentration when two commercially available α-amylase preparations of Bacillus subtilis origin were studied. The method demonstrated an average recovery of 101.7 +/- 6.5% when modified food starch was spiked with amylase and analyzed. In addition, the method was shown to be useful for predicting detailed action patterns of various types of amylases

Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.

Science.gov (United States)

Mitani, Takakazu; Watanabe, Shun; Yoshioka, Yasukiyo; Katayama, Shigeru; Nakamura, Soichiro; Ashida, Hitoshi

2017-12-01

Theobromine, a methylxanthine derived from cacao beans, reportedly has various health-promoting properties but molecular mechanism by which effects of theobromine on adipocyte differentiation and adipogenesis remains unclear. In this study, we aimed to clarify the molecular mechanisms of the anti-adipogenic effect of theobromine in vitro and in vivo. ICR mice (4week-old) were administered with theobromine (0.1g/kg) for 7days. Theobromine administration attenuated gains in body and epididymal adipose tissue weights in mice and suppressed expression of adipogenic-associated genes in mouse adipose tissue. In 3T3-L1 preadipocytes, theobromine caused degradation of C/EBPβ protein by the ubiquitin-proteasome pathway. Pull down assay showed that theobromine selectively interacts with adenosine receptor A1 (AR1), and AR1 knockdown inhibited theobromine-induced C/EBPβ degradation. Theobromine increased sumoylation of C/EBPβ at Lys133. Expression of the small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) gene, coding for a desumoylation enzyme, was suppressed by theobromine. In vivo knockdown studies showed that AR1 knockdown in mice attenuated the anti-adipogenic effects of theobromine in younger mice. Theobromine suppresses adipocyte differentiation and induced C/EBPβ degradation by increasing its sumoylation. Furthermore, the inhibition of AR1 signaling is important for theobromine-induced C/EBPβ degradation. Copyright © 2017 Elsevier B.V. All rights reserved.

Photo-fenton degradation of diclofenac: identification of main intermediates and degradation pathway.

Science.gov (United States)

Pérez-Estrada, Leónidas A; Malato, Sixto; Gernjak, Wolfgang; Agüera, Ana; Thurman, E Michael; Ferrer, Imma; Fernández-Alba, Amadeo R

2005-11-01

In recent years, the presence of pharmaceuticals in the aquatic environment has been of growing interest. These new contaminants are important because many of them are not degraded under the typical biological treatments applied in the wastewater treatment plants and represent a continuous input into the environment. Thus, compounds such as diclofenac are present in surface waters in all Europe and a crucial need for more enhanced technologies that can reduce its presence in the environment has become evident. In this sense, advanced oxidation processes (AOPs) represent a good choice for the treatment of hazardous nonbiodegradable pollutants. This work deals with the solar photodegradation of diclofenac, an antiinflammatory drug, in aqueous solutions by photo-Fenton reaction. A pilot-scale facility using a compound parabolic collector (CPC) reactor was used for this study. Results obtained show rapid and complete oxidation of diclofenac after 60 min, and total mineralization (disappearance of dissolved organic carbon, DOC) after 100 min of exposure to sunlight. Although diclofenac precipitates during the process at low pH, its degradation takes place in the homogeneous phase governed by a precipitation-redissolution-degradation process. Establishment of the reaction pathway was made possible by a thorough analysis of the reaction mixture identifying the main intermediate products generated. Gas chromatography-mass spectrometry (GC/ MS) and liquid chromatography coupled with time-of-flight mass spectrometry (LC/TOF-MS) were used to identify 18 intermediates, in two tentative degradation routes. The main one was based on the initial hydroxylation of the phenylacetic acid moiety in the C-4 position and subsequent formation of a quinone imine derivative that was the starting point for further multistep degradation involving hydroxylation, decarboxylation, and oxidation reactions. An alternative route was based on the transient preservation of the biphenyl amino moiety

Characterization of Methane Degradation and Methane-Degrading Microbes in Alaska Coastal Water

Energy Technology Data Exchange (ETDEWEB)

Kirchman, David L. [Univ. of Delaware, Lewes, DE (United States)

2012-03-29

The net flux of methane from methane hydrates and other sources to the atmosphere depends on methane degradation as well as methane production and release from geological sources. The goal of this project was to examine methane-degrading archaea and organic carbon oxidizing bacteria in methane-rich and methane-poor sediments of the Beaufort Sea, Alaska. The Beaufort Sea system was sampled as part of a multi-disciplinary expedition (Methane in the Arctic Shelf or MIDAS) in September 2009. Microbial communities were examined by quantitative PCR analyses of 16S rRNA genes and key methane degradation genes (pmoA and mcrA involved in aerobic and anaerobic methane degradation, respectively), tag pyrosequencing of 16S rRNA genes to determine the taxonomic make up of microbes in these sediments, and sequencing of all microbial genes (metagenomes ). The taxonomic and functional make-up of the microbial communities varied with methane concentrations, with some data suggesting higher abundances of potential methane-oxidizing archaea in methane-rich sediments. Sequence analysis of PCR amplicons revealed that most of the mcrA genes were from the ANME-2 group of methane oxidizers. According to metagenomic data, genes involved in methane degradation and other degradation pathways changed with sediment depth along with sulfate and methane concentrations. Most importantly, sulfate reduction genes decreased with depth while the anaerobic methane degradation gene (mcrA) increased along with methane concentrations. The number of potential methane degradation genes (mcrA) was low and inconsistent with other data indicating the large impact of methane on these sediments. The data can be reconciled if a small number of potential methane-oxidizing archaea mediates a large flux of carbon in these sediments. Our study is the first to report metagenomic data from sediments dominated by ANME-2 archaea and is one of the few to examine the entire microbial assemblage potentially involved in

Isolation and characterization of bacteria capable of degrading polycyclic aromatic hydrocarbons (PAHs) and organophosphorus pesticides from PAH-contaminated soil in Hilo, Hawaii.

Science.gov (United States)

Seo, Jong-Su; Keum, Young-Soo; Harada, Renee M; Li, Qing X

2007-07-11

Nineteen bacterial strains were isolated from petroleum-contaminated soil in Hilo, HI, and characterized by two different spray-plated methods, turbidity test in liquid medium, and 16S rRNA gene sequence analysis. Analysis of the soil showed 13 polycyclic aromatic hydrocarbons (PAHs) in a range from 0.6 to 30 mg/kg of dry weight each and 12 PAH metabolites. Five distinct bacterial strains (C3, C4, P1-1, JS14, and JS19b1) selected from preliminary plating and turbidity tests were further tested for PAH degradation through single PAH degradation assay. Strains C3, C4, and P1-1 degraded phenanthrene (40 mg/L) completely during 7 days of incubation. Strain JS14 degraded fluoranthene (40 mg/L) completely during 10 days of incubation. Strain JS19b1 degraded 100% of phenanthrene (40 mg/L) in 7 days, 77% of fluorene (40 mg/L) in 14 days, 97% of fluoranthene (40 mg/L) in 10 days, and 100% of pyrene (40 mg/L) in 14 days. Turbidity tests showed that strains P1-1, JS14, and JS19b1 utilized several organophosphorus pesticides as growth substrate. P1-1 can degrade carbofenothion, chlorfenvinphos, diazinon, fonofos, and pirimiphos-methyl. JS14 can transform chlorfenvinphos and diazinon. JS19b1 can break down diazinon, pirimiphos-methyl, and temephos.

Development and validation of stability indicating UPLC assay method for ziprasidone active pharma ingredient

Directory of Open Access Journals (Sweden)

Sonam Mittal

2012-01-01

Full Text Available Background: Ziprasidone, a novel antipsychotic, exhibits a potent highly selective antagonistic activity on D2 and 5HT2A receptors. Literature survey for ziprasidone revealed several analytical methods based on different techniques but no UPLC method has been reported so far. Aim: Aim of this research paper is to present a simple and rapid stability indicating isocratic, ultra performance liquid chromatographic (UPLC method which was developed and validated for the determination of ziprasidone active pharmaceutical ingredient. Forced degradation studies of ziprasidone were studied under acid, base, oxidative hydrolysis, thermal stress and photo stress conditions. Materials and Methods: The quantitative determination of ziprasidone drug was performed on a Supelco analytical column (100×2.1 mm i.d., 2.7 ΅m with 10 mM ammonium acetate buffer (pH: 6.7 and acetonitrile (ACN as mobile phase with the ratio (55:45-Buffer:ACN at a flow rate of 0.35 ml/ min. For UPLC method, UV detection was made at 318 nm and the run time was 3 min. Developed UPLC method was validated as per ICH guidelines. Results and Conclusion: Mild degradation of the drug substance was observed during oxidative hydrolysis and considerable degradation observed during basic hydrolysis. During method validation, parameters such as precision, linearity, ruggedness, stability, robustness, and specificity were evaluated, which remained within acceptable limits. Developed UPLC method was successfully applied for evaluating assay of Ziprasidone active Pharma ingredient.

Statistical modeling for degradation data

CERN Document Server

Lio, Yuhlong; Ng, Hon; Tsai, Tzong-Ru

2017-01-01

This book focuses on the statistical aspects of the analysis of degradation data. In recent years, degradation data analysis has come to play an increasingly important role in different disciplines such as reliability, public health sciences, and finance. For example, information on products’ reliability can be obtained by analyzing degradation data. In addition, statistical modeling and inference techniques have been developed on the basis of different degradation measures. The book brings together experts engaged in statistical modeling and inference, presenting and discussing important recent advances in degradation data analysis and related applications. The topics covered are timely and have considerable potential to impact both statistics and reliability engineering.

Extensions and applications of degradation modeling

International Nuclear Information System (INIS)

Hsu, F.; Subudhi, M.; Samanta, P.K.; Vesely, W.E.

1991-01-01

Component degradation modeling being developed to understand the aging process can have many applications with potential advantages. Previous work has focused on developing the basic concepts and mathematical development of a simple degradation model. Using this simple model, times of degradations and failures occurrences were analyzed for standby components to detect indications of aging and to infer the effectiveness of maintenance in preventing age-related degradations from transforming to failures. Degradation modeling approaches can have broader applications in aging studies and in this paper, the authors discuss some of the extensions and applications of degradation modeling. The extensions and applications of the degradation modeling approaches discussed are: (a) theoretical developments to study reliability effects of different maintenance strategies and policies, (b) relating aging-failure rate to degradation rate, and (c) application to a continuously operating component

The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA

International Nuclear Information System (INIS)

Hanif, Sarmad; Shamim, Uzma; Ullah, M.F.; Azmi, Asfar S.; Bhat, Showket H.; Hadi, S.M.

2008-01-01

Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence of transition metal ions such as copper. In this paper we show that similar to various other classes of polyphenols, delphinidin is also capable of causing oxidative degradation of cellular DNA. Lymphocytes were exposed to various concentrations of delphinidin (10, 20, 50 μM) for 1 h and the DNA breakage was assessed using single cell alkaline gel electrophoresis (Comet assay). Inhibition of DNA breakage by several scavengers of reactive oxygen species (ROS) indicated that it is caused by the formation of ROS. Incubation of lymphocytes with neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation in intact lymphocytes in a dose dependent manner. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. We have further shown that delphinidin is able to degrade DNA in cell nuclei and that such DNA degradation is also inhibited by neocuproine suggesting that nuclear copper is mobilized in this reaction. These results indicate that the generation of ROS possibly occurs through mobilization of endogenous copper ions. The results are in support of our hypothesis that the prooxidant activity of plant polyphenols may be an important mechanism for their anticancer properties

Degradation of 2,4-D in soils by Fe₃O₄ nanoparticles combined with stimulating indigenous microbes.

Science.gov (United States)

Fang, Guodong; Si, Youbin; Tian, Chao; Zhang, Gangya; Zhou, Dongmei

2012-03-01

Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) in soils by Fe₃O₄ nanoparticles combined with soil indigenous microbes was investigated, and the effects of Fe₃O₄ nanoparticles on soil microbial populations and enzyme activities were also studied. The soils contaminated with 2,4-D were treated with Fe₃O₄ nanoparticles. The microbial populations and enzyme activities were analyzed by dilution plate method and chemical assay, respectively, and the concentration of 2,4-D in soil was determined by high-performance liquid chromatography (HPLC). The results indicated that Fe₃O₄ nanoparticles combined with soil indigenous microbes led to a higher degradation efficiency of 2,4-D than the treatments with Fe₃O₄ nanoparticles or indigenous microbes alone. The degradation of 2,4-D in soils followed the pseudo first-order kinetic. The half-lives of 2,4-D degradation (DT₅₀) of the combined treatments were 0.9, 1.9 and 3.1 days in a Red soil, Vertisol and Alfisol, respectively, which implied that the DT₅₀ of the combination treatments were significantly shorter than that of the treatments Fe₃O₄ nanoparticles or indigenous microbes alone. The effects of Fe₃O₄ nanoparticles on soil microbial populations and enzyme activities were also investigated and compared with the α-Fe₂O₃ nanoparticles. The results suggested that the α-Fe₂O₃ nanoparticles had only comparatively small effects on degradation of 2,4-D in soils, while the Fe₃O₄ nanoparticles not only degraded 2,4-D in soils but also increased the soil microbial populations and enzyme activities; the maximum increase in enzyme activities were 67.8% (amylase), 53.8% (acid phosphatase), 26.5% (catalase) and 38.0% (urease), compared with the untreated soil. Moreover, the introduction of Fe₃O₄ nanoparticles at the different dosage resulted in a variable degradation efficiency of 2,4-D in soil. The method of combining Fe₃O₄ nanoparticles with indigenous soil microbes may

Mangrove microniches determine the structural and functional diversity of enriched petroleum hydrocarbon-degrading consortia.

Science.gov (United States)

Gomes, Newton C M; Flocco, Cecilia G; Costa, Rodrigo; Junca, Howard; Vilchez, Ramiro; Pieper, Dietmar H; Krögerrecklenfort, Ellen; Paranhos, Rodolfo; Mendonça-Hagler, Leda C S; Smalla, Kornelia

2010-11-01

In this study, the combination of culture enrichments and molecular tools was used to identify bacterial guilds, plasmids and functional genes potentially important in the process of petroleum hydrocarbon (PH) decontamination in mangrove microniches (rhizospheres and bulk sediment). In addition, we aimed to recover PH-degrading consortia (PHDC) for future use in remediation strategies. The PHDC were enriched with petroleum from rhizosphere and bulk sediment samples taken from a mangrove chronically polluted with oil hydrocarbons. Southern blot hybridization (SBH) assays of PCR amplicons from environmental DNA before enrichments resulted in weak positive signals for the functional gene types targeted, suggesting that PH-degrading genotypes and plasmids were in low abundance in the rhizosphere and bulk sediments. However, after enrichment, these genes were detected and strong microniche-dependent differences in the abundance and composition of hydrocarbonoclastic bacterial populations, plasmids (IncP-1α, IncP-1β, IncP-7 and IncP-9) and functional genes (naphthalene, extradiol and intradiol dioxygenases) were revealed by in-depth molecular analyses [PCR-denaturing gradient gel electrophoresis and hybridization (SBH and microarray)]. Our results suggest that, despite the low abundance of PH-degrading genes and plasmids in the environmental samples, the original bacterial composition of the mangrove microniches determined the structural and functional diversity of the PHDC enriched. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

DENTINE CARIES: ACID-TOLERANT MICROORGANISMS AND ASPECTS ON COLLAGEN DEGRADATION.

Science.gov (United States)

Lager, Anders Hedenbjörk

2014-01-01

. Key findings: Each investigated lesion harbored a unique microbiota in terms of both species composition and numbers of microorganisms. This indicates that various combinations of aciduric microorganisms can colonize, survive in and probably also propagate dentine carious lesions. We also found that solid pH-selective agars can be used successfully to select acid-tolerant microorganisms in caries lesions. This would preserve their phenotypic traits for further study. In Paper III, the relation between salivary levels of matrix metalloproteinase-8 (MMP-8), salivary levels of tissue inhibitor of MMP (TIMP-1), and the presence of manifest caries lesions in a large number of subjects was investigated. Saliva samples were collected and analyzed for concentrations of MMP-8, TIMP-1 and total protein using immunofluorometric assays, enzyme linked immunosorbent assays and Bradford assays, respectively. Key findings: Subjects with manifest caries lesions had significantly elevated levels of salivary MMP-8 compared to subjects without caries lesions. TIMP-1 was not significant in any case. In Paper IV, a new method for generating bioactive demineralized dentine matrix substrate (DDM) was developed using a dialysis system and two different demineralization approaches (acetic acid or EDTA). The generated DDM was subsequently analyzed for the presence of type 1 collagen, active MMP-8 and hydroxyproline (HYP) levels using SDS-PAGE, ELISA or immunofluorescence assay. Key findings: Both demineralization methods produced a substrate rich in collagen and with preserved MMP-8 activity. This report presents new knowledge on the composition of the acid tolerant dentine caries microbiota from three levels in dentine carious lesions and on the efficacy of operative caries removal on the numbers of viable microorganisms in the caries free cavity using two operative methods. Moreover, the basic mechanisms behind collagen degradation in the dentine caries process are studied from both a

Applications and extensions of degradation modeling

International Nuclear Information System (INIS)

Hsu, F.; Subudhi, M.; Samanta, P.K.; Vesely, W.E.

1991-01-01

Component degradation modeling being developed to understand the aging process can have many applications with potential advantages. Previous work has focused on developing the basic concepts and mathematical development of a simple degradation model. Using this simple model, times of degradations and failures occurrences were analyzed for standby components to detect indications of aging and to infer the effectiveness of maintenance in preventing age-related degradations from transforming to failures. Degradation modeling approaches can have broader applications in aging studies and in this paper, we discuss some of the extensions and applications of degradation modeling. The application and extension of degradation modeling approaches, presented in this paper, cover two aspects: (1) application to a continuously operating component, and (2) extension of the approach to analyze degradation-failure rate relationship. The application of the modeling approach to a continuously operating component (namely, air compressors) shows the usefulness of this approach in studying aging effects and the role of maintenance in this type component. In this case, aging effects in air compressors are demonstrated by the increase in both the degradation and failure rate and the faster increase in the failure rate compared to the degradation rate shows the ineffectiveness of the existing maintenance practices. Degradation-failure rate relationship was analyzed using data from residual heat removal system pumps. A simple linear model with a time-lag between these two parameters was studied. The application in this case showed a time-lag of 2 years for degradations to affect failure occurrences. 2 refs

Applications and extensions of degradation modeling

Energy Technology Data Exchange (ETDEWEB)

Hsu, F.; Subudhi, M.; Samanta, P.K. [Brookhaven National Lab., Upton, NY (United States); Vesely, W.E. [Science Applications International Corp., Columbus, OH (United States)

1991-12-31

Component degradation modeling being developed to understand the aging process can have many applications with potential advantages. Previous work has focused on developing the basic concepts and mathematical development of a simple degradation model. Using this simple model, times of degradations and failures occurrences were analyzed for standby components to detect indications of aging and to infer the effectiveness of maintenance in preventing age-related degradations from transforming to failures. Degradation modeling approaches can have broader applications in aging studies and in this paper, we discuss some of the extensions and applications of degradation modeling. The application and extension of degradation modeling approaches, presented in this paper, cover two aspects: (1) application to a continuously operating component, and (2) extension of the approach to analyze degradation-failure rate relationship. The application of the modeling approach to a continuously operating component (namely, air compressors) shows the usefulness of this approach in studying aging effects and the role of maintenance in this type component. In this case, aging effects in air compressors are demonstrated by the increase in both the degradation and failure rate and the faster increase in the failure rate compared to the degradation rate shows the ineffectiveness of the existing maintenance practices. Degradation-failure rate relationship was analyzed using data from residual heat removal system pumps. A simple linear model with a time-lag between these two parameters was studied. The application in this case showed a time-lag of 2 years for degradations to affect failure occurrences. 2 refs.

Applications and extensions of degradation modeling

Energy Technology Data Exchange (ETDEWEB)

Hsu, F.; Subudhi, M.; Samanta, P.K. (Brookhaven National Lab., Upton, NY (United States)); Vesely, W.E. (Science Applications International Corp., Columbus, OH (United States))

1991-01-01

Component degradation modeling being developed to understand the aging process can have many applications with potential advantages. Previous work has focused on developing the basic concepts and mathematical development of a simple degradation model. Using this simple model, times of degradations and failures occurrences were analyzed for standby components to detect indications of aging and to infer the effectiveness of maintenance in preventing age-related degradations from transforming to failures. Degradation modeling approaches can have broader applications in aging studies and in this paper, we discuss some of the extensions and applications of degradation modeling. The application and extension of degradation modeling approaches, presented in this paper, cover two aspects: (1) application to a continuously operating component, and (2) extension of the approach to analyze degradation-failure rate relationship. The application of the modeling approach to a continuously operating component (namely, air compressors) shows the usefulness of this approach in studying aging effects and the role of maintenance in this type component. In this case, aging effects in air compressors are demonstrated by the increase in both the degradation and failure rate and the faster increase in the failure rate compared to the degradation rate shows the ineffectiveness of the existing maintenance practices. Degradation-failure rate relationship was analyzed using data from residual heat removal system pumps. A simple linear model with a time-lag between these two parameters was studied. The application in this case showed a time-lag of 2 years for degradations to affect failure occurrences. 2 refs.

Expert system for transuranic waste assay

Energy Technology Data Exchange (ETDEWEB)

Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

1989-01-01

Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

Expert system for transuranic waste assay

International Nuclear Information System (INIS)

Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

1989-01-01

Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Energy Technology Data Exchange (ETDEWEB)

Boucas, Rodrigo Ippolito [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Trindade, Edvaldo S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Departamento de Biologia Celular, Universidade Federal do Parana, Curitiba, Parana (Brazil); Tersariol, Ivarne L.S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP (Brazil); Dietrich, Carl P. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Nader, Helena B. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil)], E-mail: hbnader.bioq@epm.br

2008-06-23

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

International Nuclear Information System (INIS)

Boucas, Rodrigo Ippolito; Trindade, Edvaldo S.; Tersariol, Ivarne L.S.; Dietrich, Carl P.; Nader, Helena B.

2008-01-01

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture

Calibration and validation of the 14C-labelled polyethylene glycol-binding assay for tannins in tropical browse

International Nuclear Information System (INIS)

Mlambo, V.; Makkar, H.P.S.

2005-01-01

This study evaluates the radiolabelled polyethylene glycol (PEG)-binding procedure [Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996. Polyethylene glycol-binding to plant samples as an assay for the biological effects of tannins: predicting the negative effects of tannins in Mediterranean browse on rumen degradation. J. Agric. Food Chem. 44, 3230-3234] for tannin analysis, using 27 tropical browse plants. In this method, the amount of PEG bound to a plant sample is assumed to be a reflection of its tannin content. The method was modified to exclude the use of non-tanniniferous substrate for estimating non-specific binding (NSB) in tannin-containing substrates. Non-specific binding values varied widely (0.4-2.8 mg PEG/100 mg DM tannin-free substrate) when the tannin-free substrate was changed from wheat straw to either rye grass or maize shoots. We therefore propose a modified radiolabelled PEG-binding method to estimate the level of PEG-binding (PEGb) to tannin-containing foliage without using tannin-free substrate to correct for non-specific binding. In this approach, incremental levels of each tanniniferous substrate were used to generate PEGb values. The resultant linear response was analysed and tannin activity was expressed as the slope of the response curve (PEGbSlope) observed for each substrate. The slope takes into account the non-specific binding in each substrate, thus PEGbSlope does not require correction for NSB using tannin-free samples. This approach improved the correlation between PEGb and the 125 I-labelled bovine serum albumin precipitation assay. Relationships between the modified PEG-binding assay and radiolabelled bovine serum albumin assay, in vitro tannin bioassay and colorimetric assays are presented. (author)

Resistance irrelevant CYP417A2v2 was found degrading insecticide in Laodelphax striatellus.

Science.gov (United States)

Miah, Mohammad Asaduzzaman; Elzaki, Mohammed Esmail Abdalla; Han, Zhaojun

2017-07-01

Cytochrome P450 monooxygenases (CYPs) usually overexpressed in resistant strain were found involved in oxidative detoxification of insecticides. In this study, an investigation was conducted to confirm if resistance irrelevant CYPs which were not overexpressed in resistant strain before, were capable of degrading insecticides. Three resistance irrelevant CYPs viz. CYP417A2v2, CYP425A1v2, and CYP4DJ1 from CYP4 family of Laodelphax striatellus were randomly selected for experiments. CYP417A2v2 and CYP425A1v2 were found expressed successfully in Sf9 cell line while CYP4DJ1 was not expressed successfully and out of two expressed CYPs, only CYP417A2v2 showed its efficient catalytic activity. For catalytic activity, three traditional model probe substrates and five insecticides were assayed. For the probe substrates screened, p -nitroanisole and ethoxycoumarin were preferentially metabolized by CYP417A2v2 (specific activity 3.76 ± 1.22 and 1.63 ± 0.37 nmol min -1  mg protein -1 , respectively) and they may be potential diagnostic probes for this enzyme. Among insecticides, only imidacloprid was efficiently degraded by CYP417A2v2. Incubation of imidacloprid with CYP417A2v2 of L. striatellus and subsequent HPLC, LC-MS, and MS/MS analysis revealed the formation of imidacloprid metabolites, that is, 4' or 5'hydroxy-imidacloprid by hydroxylation. This result implies the exemption of CYPs character that it is not always, all the CYPs degrading insecticides being selected and overexpressed in resistant strains and the degrading CYPs without mutations to upregulate could be candidates during insecticide resistance evolution. This characterization of individual insect CYPs in insecticide degradation can provide insight for better understand of insecticide resistance development.

Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

DEFF Research Database (Denmark)

Hendriksen, Niels Bohse; Winding, Anne

2012-01-01

Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soils provide numerous essential ecosystem services such as carbon cycling...... of soil microbial functions is still needed. In soil, enzymes originate from a variety of organisms, notably fungi and bacteria and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity will reflect...... the functional diversity and activity of the microorganisms involved in decomposition processes. Their activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysacharides as cellulose, hemicellulose...

Bacteria-mediated bisphenol A degradation.

Science.gov (United States)

Zhang, Weiwei; Yin, Kun; Chen, Lingxin

2013-07-01

Bisphenol A (BPA) is an important monomer in the manufacture of polycarbonate plastics, food cans, and other daily used chemicals. Daily and worldwide usage of BPA and BPA-contained products led to its ubiquitous distribution in water, sediment/soil, and atmosphere. Moreover, BPA has been identified as an environmental endocrine disruptor for its estrogenic and genotoxic activity. Thus, BPA contamination in the environment is an increasingly worldwide concern, and methods to efficiently remove BPA from the environment are urgently recommended. Although many factors affect the fate of BPA in the environment, BPA degradation is mainly depended on the metabolism of bacteria. Many BPA-degrading bacteria have been identified from water, sediment/soil, and wastewater treatment plants. Metabolic pathways of BPA degradation in specific bacterial strains were proposed, based on the metabolic intermediates detected during the degradation process. In this review, the BPA-degrading bacteria were summarized, and the (proposed) BPA degradation pathway mediated by bacteria were referred.

First results with a radioreceptor-assay (TRAK-Assay) for TSH-receptor-autoantibodies

International Nuclear Information System (INIS)

Becker, W.; Reiners, C.; Boerner, W.

1983-01-01

A new radioreceptor-assay (TRAK-assay) for autoantibodies against TSH-receptors was tested in 48 untreated thyrotoxic patients (26 regional autonomies, 22 toxic diffuse goiters). None of the 26 patients with regional autonomy showed positive autoantibody-titers. 4 patients with toxic diffuse goiter and thyrotoxic exophthalmos were TRAK-positive. Positive titers of microsomal and thyreoglobulin autoantibodies could be seen in 8 of 9 patients with positive TRAK-titers. In accordance with the conventional methods for detecting thyroid-stimulating immunoglobulins the new TRAK-assay seems to be suited for differentiating between immunogenic toxic diffuse goiter (Graves' disease) and goiter with disseminated autonomy as well as for prediction of relapse. (orig.) [de

Anaerobic degradation of dairy wastewater in intermittent UASB reactors: influence of effluent recirculation.

Science.gov (United States)

Couras, C S; Louros, V L; Gameiro, T; Alves, N; Silva, A; Capela, M I; Arroja, L M; Nadais, H

2015-01-01

This work studied the influence of effluent recirculation upon the kinetics of anaerobic degradation of dairy wastewater in the feedless phase of intermittent upflow anaerobic sludge bed (UASB) reactors. Several laboratory-scale tests were performed with different organic loads in closed circuit UASB reactors inoculated with adapted flocculent sludge. The data obtained were used for determination of specific substrate removal rates and specific methane production rates, and adjusted to kinetic models. A high initial substrate removal was observed in all tests due to adsorption of organic matter onto the anaerobic biomass which was not accompanied by biological substrate degradation as measured by methane production. Initial methane production rate was about 45% of initial soluble and colloidal substrate removal rate. This discrepancy between methane production rate and substrate removal rate was observed mainly on the first day of all experiments and was attenuated on the second day, suggesting that the feedless period of intermittent UASB reactors treating dairy wastewater should be longer than one day. Effluent recirculation expressively raised the rate of removal of soluble and colloidal substrate and methane productivity, as compared with results for similar assays in batch reactors without recirculation. The observed bed expansion was due to the biogas production and the application of effluent recirculation led to a sludge bed contraction after all the substrates were degraded. The settleability of the anaerobic sludge improved by the introduction of effluent recirculation this effect being more pronounced for the higher loads.

Evaluation of fungal- and photo-degradation as potential treatments for the removal of sunscreens BP3 and BP1

Energy Technology Data Exchange (ETDEWEB)

Gago-Ferrero, Pablo, E-mail: pablo.gago@idaea.csic.es [Departament de Quimica Ambiental, IDAEA-CSIC, C/ Jordi Girona 18-26, 08034 Barcelona (Spain); Badia-Fabregat, Marina, E-mail: marina.badia@uab.cat [Departament d' Enginyeria Quimica, Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain); Olivares, Alba, E-mail: esalba.olivares@idaea.csic.es [Departament de Quimica Ambiental, IDAEA-CSIC, C/ Jordi Girona 18-26, 08034 Barcelona (Spain); Pina, Benjamin, E-mail: benjami.pina@idaea.csic.es [Departament de Quimica Ambiental, IDAEA-CSIC, C/ Jordi Girona 18-26, 08034 Barcelona (Spain); Blanquez, Paqui, E-mail: paqui.blanquez@uab.cat [Departament d' Enginyeria Quimica, Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain); Vicent, Teresa, E-mail: teresa.vicent@uab.cat [Departament d' Enginyeria Quimica, Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain); Caminal, Gloria, E-mail: gloria.caminal@uab.cat [Unitat de Biocatalisi Aplicada associada al IQAC (CSIC-UAB). Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain); Diaz-Cruz, M. Silvia, E-mail: silvia.diaz@idaea.csic.es [Departament de Quimica Ambiental, IDAEA-CSIC, C/ Jordi Girona 18-26, 08034 Barcelona (Spain); and others

2012-06-15

Photodecomposition might be regarded as one of the most important abiotic factors affecting the fate of UV absorbing compounds in the environment and photocatalysis has been suggested as an effective method to degrade organic pollutants. However, UV filters transformation appears to be a complex process, barely addressed to date. The white rot fungus Trametes versicolor is considered as a promising alternative to conventional aerobic bacterial degradation, as it is able to metabolise a wide range of xenobiotics. This study focused on both degradation processes of two widely used UV filters, benzophenone-3 (BP3) and benzophenone-1 (BP1). Fungal treatment resulted in the degradation of more than 99% for both sunscreens in less than 24 h, whereas photodegradation was very inefficient, especially for BP3, which remained unaltered upon 24 h of simulated sunlight irradiation. Analysis of metabolic compounds generated showed BP1 as a minor by-product of BP3 degradation by T. versicolor while the main intermediate metabolites were glycoconjugate derivatives. BP1 and BP3 showed a weak, but significant estrogenic activity (EC50 values of 0.058 mg/L and 12.5 mg/L, respectively) when tested by recombinant yeast assay (RYA), being BP1 200-folds more estrogenic than BP3. Estrogenic activity was eliminated during T. versicolor degradation of both compounds, showing that none of the resulting metabolites possessed significant estrogenic activity at the concentrations produced. These results demonstrate the suitability of this method to degrade both sunscreen agents and to eliminate estrogenic activity. - Highlights: Black-Right-Pointing-Pointer Fungus T. versicolor is able to degrade totally BP3 and BP1 in few hours in a fluidised bed bioreactor. Black-Right-Pointing-Pointer BP3 is not degraded under simulated sunlight. Black-Right-Pointing-Pointer Glycoconjugates have been identified as the main intermediate metabolites. Black-Right-Pointing-Pointer Decrease in endocrine activity

Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

Science.gov (United States)

Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

2013-09-01

Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men. © 2013 American Society of Andrology and European Academy of Andrology.

Study of PP/montmorillonite composite degradation

International Nuclear Information System (INIS)

Baer, Marcia; Granado, Carlos J.F.

2009-01-01

The objective of this work was to produce composites of PP/sodium bentonite and PP/ organophilic bentonite through melt intercalation and analyze the degradation produced by ultraviolet irradiation. The XRD results showed that the samples of nature bentonite had better interaction with de polymer and produced intercalated nanocomposite. The effect of UV irradiation on degradation was observed after 24 hours of exposition. The samples showed the same photoproducts and at the same proportion until 240 hours of UV exposition; with 480 hours the organophilize bentonite composite showed higher degradation than other ones. The superficial cracks increased with degradation time. The degradation occurs due chromophores impurities presented in the samples, thus samples with sodium clay show higher degradation, and organophilic clay contains ammonium salt that contribute to increase the degradation. (author)

Solar photocatalytic degradation and detoxification of EU priority substances

Energy Technology Data Exchange (ETDEWEB)

Hincapie, M. [Facultad de Ingeniera Ambiental, Universidad de Medellin, Carrera 87 No. 30-65, P.O. Box 1983, Medellin (Colombia); Maldonado, M.I.; Oller, I.; Gernjak, W.; Malato, S. [Plataforma Solar de Almeria-CIEMAT, Carretera Senes km4, 04200 Tabernas (Almeria) (Spain); Sanchez-Perez, J.A.; Ballesteros, M.M. [Departamento de Ingenieria Quimica, Universidad de Almeria Crta de Sacramento s/n, 04120 Almeria (Spain)

2005-04-15

Several different pesticides (alachlor, atrazine, chlorfenvinphos, diuron, isoproturon and pentachlorophenol) considered PS (priority substances) by the European Commission and dissolved in water at 50mg/L (or at maximum water solubility) have been degraded at pilot-plant scale using photo-Fenton and TiO{sub 2} photocatalysis driven by solar energy. Two different iron concentrations (2 and 55mg/L) and TiO{sub 2} at 200mg/L have been tested and discussed, using mainly TOC mineralisation for comparison of treatment effectiveness. Vibrio fischeri (Microtox{sup (}R)) toxicity assays were also employed for evaluating the photocatalytic treatments, and comparison between these results and parent compound disappearance, TOC evolution and anion (or ammonia) release were discussed. Almost complete mineralisation and total detoxification were always attained. It has been demonstrated that evolution of chloride could be a key-parameter for predicting toxicity of chlorinated compounds.

Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine

International Nuclear Information System (INIS)

Lim, Sue Ping; Callen, David F; Kumar, Raman; Akkamsetty, Yamini; Wang, Wen; Ho, Kristen; Neilsen, Paul M; Walther, Diego J; Suetani, Rachel J; Prestidge, Clive

2013-01-01

Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression

Ordered bulk degradation via autophagy

DEFF Research Database (Denmark)

Dengjel, Jörn; Kristensen, Anders Riis; Andersen, Jens S

2008-01-01

During amino acid starvation, cells undergo macroautophagy which is regarded as an unspecific bulk degradation process. Lately, more and more organelle-specific autophagy subtypes such as reticulophagy, mitophagy and ribophagy have been described and it could be shown, depending on the experimental...... at proteasomal and lysosomal degradation ample cross-talk between the two degradation pathways became evident. Degradation via autophagy appeared to be ordered and regulated at the protein complex/organelle level. This raises several important questions such as: can macroautophagy itself be specific and what...

In vitro degradation of ribosomes.

Science.gov (United States)

Mora, G; Rivas, A

1976-12-01

The cytoplasmic ribosomes from Euglena gracilis var. bacillaris are found to be of two types taking into consideration their stability "in vitro". In the group of unstable ribosomes the large subunit is degraded. The other group apparently does not suffer any degradation under the conditions described. However the RNAs extracted from both types of ribosomes are degraded during sucrose density gradients. The degradation of the largest RNA species has been reported previously, but no comment has been made about the stability of the ribosome itself.

Lysosomal degradation of membrane lipids.

Science.gov (United States)

Kolter, Thomas; Sandhoff, Konrad

2010-05-03

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

How do polymers degrade?

Science.gov (United States)

Lyu, Suping

2011-03-01

Materials derived from agricultural products such as cellulose, starch, polylactide, etc. are more sustainable and environmentally benign than those derived from petroleum. However, applications of these polymers are limited by their processing properties, chemical and thermal stabilities. For example, polyethylene terephthalate fabrics last for many years under normal use conditions, but polylactide fabrics cannot due to chemical degradation. There are two primary mechanisms through which these polymers degrade: via hydrolysis and via oxidation. Both of these two mechanisms are related to combined factors such as monomer chemistry, chain configuration, chain mobility, crystallinity, and permeation to water and oxygen, and product geometry. In this talk, we will discuss how these materials degrade and how the degradation depends on these factors under application conditions. Both experimental studies and mathematical modeling will be presented.

Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

Science.gov (United States)

Yoshizaki, Yuki; Mori, Yutaro; Tsuzaki, Yoshihito; Mori, Takayasu; Nomura, Naohiro; Wakabayashi, Mai; Takahashi, Daiei; Zeniya, Moko; Kikuchi, Eriko; Araki, Yuya; Ando, Fumiaki; Isobe, Kiyoshi; Nishida, Hidenori; Ohta, Akihito; Susa, Koichiro; Inoue, Yuichi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi; Sohara, Eisei

2015-11-13

Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal. Copyright © 2015 Elsevier Inc. All rights reserved.

Fully glutathione degradable waterborne polyurethane nanocarriers: Preparation, redox-sensitivity, and triggered intracellular drug release

Energy Technology Data Exchange (ETDEWEB)

Omrani, Ismail; Babanejad, Niloofar; Shendi, Hasan Kashef; Nabid, Mohammad Reza, E-mail: m-nabid@sbu.ac.ir

2017-01-01

Polyurethanes are important class of biomaterials that are extensively used in medical devices. In spite of their easy synthesis, polyurethanes that are fully degradable in response to the intracellular reducing environment are less explored for controlled drug delivery. Herein, a novel glutathione degradable waterborne polyurethane (WPU) nanocarrier for redox triggered intracellular delivery of a model lipophilic anticancer drug, doxorubicin (DOX) is reported. The WPU was prepared from polyaddition reaction of isophorone diisocyanate (IPDI) and a novel linear polyester polyol involving disulfide linkage, disulfide labeled chain extender, dimethylolpropionic acid (DMPA) using dibutyltin dilaurate (DBTDL) as a catalyst. The resulting polyurethane self-assembles into nanocarrier in water. The dynamic light scattering (DLS) measurements and scanning electron microscope (SEM) revealed fast swelling and disruption of nanocarriers under an intracellular reduction-mimicking environment. The in vitro release studies showed that DOX was released in a controlled and redox-dependent manner. MTT assays showed that DOX-loaded WPU had a high in vitro antitumor activity in both HDF noncancer cells and MCF- 7 cancer cells. In addition, it is found that the blank WPU nanocarriers are nontoxic to HDF and MCF-7 cells even at a high concentration of 2 mg/mL. Hence, nanocarriers based on disulfide labeled WPU have appeared as a new class of biocompatible and redox-degradable nanovehicle for efficient intracellular drug delivery. - Highlights: • A novel fully glutathione degradable waterborne polyurethane was developed. • The waterborne nanocarrier with disulfide bonds in both hard and soft segment were developed for redox-triggered intracellular delivery of DOX. • The polyester diol bearing disulfide bonds in the backbone was prepared by a polycondensation polymerization reaction.

Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster

International Nuclear Information System (INIS)

Garcia, J.V.; Fenton, B.W.; Rosner, M.R.

1988-01-01

An insulin-degrading enzyme (IDE) from the cytoplasm of Drosophila Kc cells has been purified and characterized. The purified enzyme is a monomer with an s value of 7.2 S, an apparent K/sub m/ for porcine insulin of 3 μM, and a specific activity of 3.3 nmol of porcine insulin degraded/(min x mg). N-Terminal sequence analysis of the gel-purified enzyme gave a single, serine-rich sequence. The Drosophila IDE shares a number of properties in common with its mammalian counterpart. The enzyme could be specifically affinity-labeled with [ 125 I]insulin, has a molecular weight of 110K, and has a pI of 5.3. Although Drosophila Kc cells grow at room temperature, the optimal enzyme activity assay conditions parallel those of the mammalian IDE: 37 0 C and a pH range of 7-8. The Drosophila IDE activity, like the mammalian enzymes, is inhibited by bacitracin and sulfhydryl-specific reagents. Similarly, the Drosophila IDE activity is insensitive to glutathione as well as protease inhibitors such as aprotinin and leupeptin. Insulin-like growth factor II, equine insulin, and porcine insulin compete for degradation of [ 125 I]insulin at comparable concentrations (approximately 10 -6 M), whereas insulin-like growth factor I and the individual A and B chains of insulin are less effective. The high degree of evolutionary conservation between the Drosophila and mammalian IDE suggest an important role for this enzyme in the metabolism of insulin and also provides further evidence for the existence of a complete insulin-like system in invertebrate organisms such as Drosophila

Enzymatic degradation of polycaprolactone–gelatin blend

International Nuclear Information System (INIS)

Banerjee, Aditi; Chatterjee, Kaushik; Madras, Giridhar

2015-01-01

Blends of polycaprolactone (PCL), a synthetic polymer and gelatin, natural polymer offer a optimal combination of strength, water wettability and cytocompatibility for use as a resorbable biomaterial. The enzymatic degradation of PCL, gelatin and PCL–gelatin blended films was studied in the presence of lipase (Novozym 435, immobilized) and lysozyme. Novozym 435 degraded the PCL films whereas lysozyme degraded the gelatin. Though Novozym 435 and lysozyme individually could degrade PCL–gelatin blended films, the combination of these enzymes showed the highest degradation of these blended films. Moreover, the enzymatic degradation was much faster when fresh enzymes were added at regular intervals. The changes in physico-chemical properties of polymer films due to degradation were studied by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. These results have important implications for designing resorbable biomedical implants. (paper)

The role of autophagy in chloroplast degradation and chlorophagy in immune defenses during Pst DC3000 (AvrRps4 infection.

Directory of Open Access Journals (Sweden)

Junjian Dong

Full Text Available BACKGROUND: Chlorosis of leaf tissue normally observed during pathogen infection may result from the degradation of chloroplasts. There is a growing evidence to suggest that the chloroplast plays a significant role during pathogen infection. Although most degradation of the organelles and cellular structures in plants is mediated by autophagy, its role in chloroplast catabolism during pathogen infection is largely unknown. RESULTS: In this study, we investigated the function of autophagy in chloroplast degradation during avirulent Pst DC3000 (AvrRps4 infection. We examined the expression of defensive marker genes and suppression of bacterial growth using the electrolyte leakage assay in normal light (N and low light (L growing environments of wild-type and atg5-1 plants during pathogen treatment. Stroma-targeted GFP proteins (CT-GFP were observed with LysoTracker Red (LTR staining of autophagosome-like structures in the vacuole. The results showed that Arabidopsis expressed a significant number of small GFP-labeled bodies when infected with avirulent Pst DC3000 (AvrRps4. While barely detectable, there were small GFP-labeled bodies in plants with the CT-GFP expressing atg5-1 mutation. The results showed that chloroplast degradation depends on autophagy and this may play an important role in inhibiting pathogen growth. CONCLUSION: Autophagy plays a role in chloroplast degradation in Arabidopsis during avirulent Pst DC3000 (AvrRps4 infection. Autophagy dependent chloroplast degradation may be the primary source of reactive oxygen species (ROS as well as the pathogen-response signaling molecules that induce the defense response.

Safeguards and Non-destructive Assay

International Nuclear Information System (INIS)

Carchon, R.; Bruggeman, M.

2001-01-01

SCK-CEN's programme on safeguards and non-destructive assay includes: (1) various activities to assure nuclear materials accountancy; (2) contributes to the implementation of Integrated Safeguards measures in Belgium and to assist the IAEA through the Belgian Support Programme; (3) renders services to internal and external customers in the field of safeguards; (4) improves passive neutron coincidence counting techniques for waste assay and safeguards verification measurements by R and D on correlation algorithms implemented via software or dedicated hardware; (5) improves gamma assay techniques for waste assay by implementing advanced scanning techniques and different correlation algorithms; and (6) develops numerical calibration techniques. Major achievements in these areas in 2000 are reported

Microbial Enzymatic Degradation of Biodegradable Plastics.

Science.gov (United States)

Roohi; Bano, Kulsoom; Kuddus, Mohammed; Zaheer, Mohammed R; Zia, Qamar; Khan, Mohammed F; Ashraf, Ghulam Md; Gupta, Anamika; Aliev, Gjumrakch

2017-01-01

The renewable feedstock derived biodegradable plastics are important in various industries such as packaging, agricultural, paper coating, garbage bags and biomedical implants. The increasing water and waste pollution due to the available decomposition methods of plastic degradation have led to the emergence of biodegradable plastics and biological degradation with microbial (bacteria and fungi) extracellular enzymes. The microbes utilize biodegradable polymers as the substrate under starvation and in unavailability of microbial nutrients. Microbial enzymatic degradation is suitable from bioremediation point of view as no waste accumulation occurs. It is important to understand the microbial interaction and mechanism involved in the enzymatic degradation of biodegradable plastics under the influence of several environmental factors such as applied pH, thermo-stability, substrate molecular weight and/or complexity. To study the surface erosion of polymer film is another approach for hydrolytic degradation characteristion. The degradation of biopolymer is associated with the production of low molecular weight monomer and generation of carbon dioxide, methane and water molecule. This review reported the degradation study of various existing biodegradable plastics along with the potent degrading microbes (bacteria and fungi). Patents available on plastic biodegradation with biotechnological significance is also summarized in this paper. This paper assesses that new disposal technique should be adopted for the degradation of polymers and further research is required for the economical production of biodegradable plastics along with their enzymatic degradation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The use of Taka-diastase in a [3H]poly(A) hybridization assay of oligo(U) sequences in RNA

International Nuclear Information System (INIS)

De Herdt, E.; Kondo, M.; Slegers, H.

1981-01-01

A reliable assay for uridylate sequences longer than 10 is described. The procedure is based on the hybridization of [ 3 H]poly(A) with poly(U) or oligo(U) sequences in high ionic conditions and a subsequent degradation of single stranded polynucleotides with purified Taka-diastase. A 1:2 complex between poly(A) and poly(U) is formed on which one poly(U) strand is digested by Taka-diastase. The procedure is especially suitable for the detection and quantitation of Usub(n) (n > 10) in RNA preparations. (Auth.)

Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

DEFF Research Database (Denmark)

Shetty, Radhakrishna; Vestergaard, Mike; Jessen, Flemming

2017-01-01

processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase......Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production...... was estimated to be 77 kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation...

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation.

Science.gov (United States)

Goel, Garima; Pandey, Piyush; Sood, Anchal; Bisht, Sandeep; Maheshwari, D K; Sharma, G D

2012-06-01

Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT(T)) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10(-6). DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.

Novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s bearing functionalizable carbonate building blocks: II. Enzymatic biodegradation and in vitro biocompatibility assay.

Science.gov (United States)

Yang, Jing; Tian, Weisheng; Li, Qiaobo; Li, Yang; Cao, Amin

2004-01-01

In a previous study, we have reported chemical synthesis of novel aliphatic poly(butylene succinate-co-cyclic carbonate) P(BS-co-CC)s bearing various functionalizable carbonate building blocks, and this work will continue to present our new studies on their enzymatic degradation and in vitro cell biocompatibility assay. First, enzymatic degradation of the novel P(BS-co-CC) film samples was investigated with two enzymes of lipase B Candida Antartic (Novozyme 435) and lipase Porcine Pancreas PPL, and it was revealed that copolymerizing linear poly(butylene succinate) PBS with a functionalizable carbonate building block could remarkably accelerate the enzymatic degradation of a synthesized product P(BS-co-CC), and its biodegradation behavior was found to strongly depend on the overall impacts of several important factors as the cyclic carbonate (CC) comonomer structure and molar content, molar mass, thermal characteristics, morphology, the enzyme-substrate specificity, and so forth. Further, the biodegraded residual film samples and water-soluble enzymatic degradation products were allowed to be analyzed by means of proton nuclear magnetic resonance (1H NMR), gel permeation chromatograph (GPC), differential scanning calorimeter (DSC), attenuated total reflection FTIR (ATR-FTIR), scanning electron microscope (SEM), and liquid chromatograph-mass spectrometry (LC-MS). On the experimental evidences, an exo-type mechanism of enzymatic chain hydrolysis preferentially occurring in the noncrystalline domains was suggested for the synthesized new P(BS-co-CC) film samples. With regard to their cell biocompatibilities, an assay with NIH 3T3 mouse fibroblast cell was conducted using the novel synthesized P(BS-co-CC) films as substrates with respect to the cell adhesion and proliferation, and these new biodegradable P(BS-co-CC) samples were found to exhibit as low cell toxicity as the PLLA control, particularly the two samples of poly(butylene succinate-co-18.7 mol % dimethyl

Effect of benzene and ethylbenzene on the transcription of methyl-tert-butyl ether degradation genes of Methylibium petroleiphilum PM1.

Science.gov (United States)

Joshi, Geetika; Schmidt, Radomir; Scow, Kate M; Denison, Michael S; Hristova, Krassimira R

2016-09-01

Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds' impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1.

Degradation of volatile hydrocarbons from steam-classified solid waste by a mixture of aromatic hydrocarbon-degrading bacteria.

Science.gov (United States)

Leahy, Joseph G; Tracy, Karen D; Eley, Michael H

2003-03-01

Steam classification is a process for treatment of solid waste that allows recovery of volatile organic compounds from the waste via steam condensate and off-gases. A mixed culture of aromatic hydrocarbon-degrading bacteria was used to degrade the contaminants in the condensate, which contained approx. 60 hydrocarbons, of which 38 were degraded within 4 d. Many of the hydrocarbons, including styrene, 1,2,4-trimethylbenzene, naphthalene, ethylbenzene, m-/p-xylene, chloroform, 1,3-dichloropropene, were completely or nearly completely degraded within one day, while trichloroethylene and 1,2,3-trichloropropane were degraded more slowly.

Radioactive wastes assay technique and equipment

International Nuclear Information System (INIS)

Lee, K. M.; Hong, D. S; Kim, T. K.; Bae, S. M.; Shon, J. S.; Hong, K. P.

2004-12-01

The waste inventory records such as the activities and radio- nuclides contained in the waste packages are to be submitted with the radioactive wastes packages for the final disposal. The nearly around 10,000 drums of waste stocked in KAERI now should be assayed for the preparation of the waste inventory records too. For the successive execution of the waste assay, the investigation into the present waste assay techniques and equipment are to be taken first. Also the installation of the waste assay equipment through the comprehensive design, manufacturing and procurement should be proceeded timely. As the characteristics of the KAERI-stocked wastes are very different from that of the nuclear power plant and those have no regular waste streams, the application of the in-direct waste assay method using the scaling factors are not effective for the KAERI-generated wastes. Considering for the versal conveniency including the accuracy over the wide range of waste forms and the combination of assay time and sensitivity, the TGS(Tomographic Gamma Scanner) is appropriate as for the KAERI -generated radioactive waste assay equipment

Calibration and validation of the {sup 14}C-labelled polyethylene glycol-binding assay for tannins in tropical browse

Energy Technology Data Exchange (ETDEWEB)

Mlambo, V. [Animal Production Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf (Austria)]. E-mail: vmlambo@agric.uniswa.sz; Makkar, H.P.S. [Animal Production and Health Section, Joint FAO/IAEA Division of Nuclear Techniques in Agriculture and Food, International Atomic Energy Agency, Vienna (Austria)

2005-08-19

This study evaluates the radiolabelled polyethylene glycol (PEG)-binding procedure [Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996. Polyethylene glycol-binding to plant samples as an assay for the biological effects of tannins: predicting the negative effects of tannins in Mediterranean browse on rumen degradation. J. Agric. Food Chem. 44, 3230-3234] for tannin analysis, using 27 tropical browse plants. In this method, the amount of PEG bound to a plant sample is assumed to be a reflection of its tannin content. The method was modified to exclude the use of non-tanniniferous substrate for estimating non-specific binding (NSB) in tannin-containing substrates. Non-specific binding values varied widely (0.4-2.8 mg PEG/100 mg DM tannin-free substrate) when the tannin-free substrate was changed from wheat straw to either rye grass or maize shoots. We therefore propose a modified radiolabelled PEG-binding method to estimate the level of PEG-binding (PEGb) to tannin-containing foliage without using tannin-free substrate to correct for non-specific binding. In this approach, incremental levels of each tanniniferous substrate were used to generate PEGb values. The resultant linear response was analysed and tannin activity was expressed as the slope of the response curve (PEGbSlope) observed for each substrate. The slope takes into account the non-specific binding in each substrate, thus PEGbSlope does not require correction for NSB using tannin-free samples. This approach improved the correlation between PEGb and the {sup 125}I-labelled bovine serum albumin precipitation assay. Relationships between the modified PEG-binding assay and radiolabelled bovine serum albumin assay, in vitro tannin bioassay and colorimetric assays are presented. (author)

Isolation and identification of aromatic hydrocarbon degrading yeasts present in gasoline tanks of urbans vehicles

Directory of Open Access Journals (Sweden)

Nathalia Catalina Delgadillo-Ordoñez

2017-07-01

Full Text Available Yeast isolates were obtained from fuel tanks of vehicles in order to assess their potential use in the degradation of aromatic hydrocarbons. Growth assays were performed in minimum mineral medium using different aromatic hydrocarbons (benzene, toluene, naphthalene, phenanthrene, and pyrene as the sole carbon source. Isolates that showed growth in any of the tested polycyclic aromatic hydrocarbons were identified by Sanger sequencing of the ITS1 and ITS2 rDNA molecular markers. A total of 16 yeasts strains were isolated, and three showed remarkable growth in media with aromatic hydrocarbons as the sole carbon source. These strains belong to the genus Rhodotorula, and correspond to the species Rhodotorula calyptogenae (99,8% identity and Rhodotorula dairenensis (99,8% identity.  These strains grew in benzene, toluene, naphthalene, phenanthrene and pyrene. This study demonstrates for the first time that yeasts of the genus Rhodotorula inhabit pipelines and fuel tanks of vehicles and that remove   aromatic hydrocarbons that are environmental pollutants. Our results suggest that these yeasts are potential candidates for aromatic hydrocarbon degradation as part of bioremediation strategies.

Direct measurement of human plasma corticotropin-releasing hormone by two-site immunoradiometric assay

International Nuclear Information System (INIS)

Linton, E.A.; McLean, C.; Nieuwenhuyzen Kruseman, A.C.; Tilders, F.J.; Van der Veen, E.A.; Lowry, P.J.

1987-01-01

A ''two-site'' immunoradiometric assay (IRMA) which allows the direct estimation of human CRH (hCRH) in plasma is described. Using this IRMA, basal levels of CRH in normal subjects ranged from 2-28 pg/mL [mean, 15 +/- 7 (+/- SD) pg/mL; n = 58]. Values in men and women were similar. Plasma CRH values within this range were also found in patients with Cushing's syndrome, Addison's disease, and Nelson's syndrome, with no correlation between plasma CRH and ACTH levels in these patients. Elevated plasma CRH levels were found in pregnant women near term [1462 +/- 752 (+/- SD) pg/mL; n = 55], and the dilution curve of this CRH-like immunoreactivity paralleled the IRMA standard curve. After its immunoadsorption from maternal plasma, this CRH-like material eluted on reverse phase high performance liquid chromatography with a retention time identical to that of synthetic CRH and had equipotent bioactivity with the synthetic peptide in the perfused anterior pituitary cell bioassay. Circulating CRH was not detected in Wistar rats, even after adrenalectomy and subsequent ether stress. Synthetic hCRH was degraded by fresh human plasma relatively slowly; 65% of added CRH remained after 1 h of incubation at 37 C. Degradation was inhibited by heat treatment (54 C; 1 h), cold treatment (4 C; 4 h), or freezing and thawing. Loss of synthetic rat CRH occurred more rapidly when fresh rat plasma was used; only 20% of added CRH remained under the same conditions. The inability to measure CRH in peripheral rat plasma may be due to the presence of active CRH-degrading enzymes which fragment the CRH molecule into forms not recognized by the CRH IRMA

Degradation analysis of thin film photovoltaic modules

International Nuclear Information System (INIS)

Radue, C.; Dyk, E.E. van

2009-01-01

Five thin film photovoltaic modules were deployed outdoors under open circuit conditions after a thorough indoor evaluation. Two technology types were investigated: amorphous silicon (a-Si:H) and copper indium gallium diselenide (CIGS). Two 14 W a-Si:H modules, labelled Si-1 and Si-2, were investigated. Both exhibited degradation, initially due to the well-known light-induced degradation described by Staebler and Wronski [Applied Physics Letters 31 (4) (1977) 292], and thereafter due to other degradation modes such as cell degradation. The various degradation modes contributing to the degradation of the a-Si:H modules will be discussed. The initial maximum power output (P MAX ) of Si-1 was 9.92 W, with the initial light-induced degradation for Si-1 ∼30% and a total degradation of ∼42%. For Si-2 the initial P MAX was 7.93 W, with initial light-induced degradation of ∼10% and a total degradation of ∼17%. Three CIGS modules were investigated: two 20 W modules labelled CIGS-1 and CIGS-2, and a 40 W module labelled CIGS-3. CIGS-2 exhibited stable performance while CIGS-1 and CIGS-3 exhibited degradation. CIGS is known to be stable over long periods of time, and thus the possible reasons for the degradation of the two modules are discussed.

Degradation analysis of thin film photovoltaic modules

Energy Technology Data Exchange (ETDEWEB)

Radue, C., E-mail: chantelle.radue@nmmu.ac.z [Department of Physics, PO Box 77000, Nelson Mandela Metropolitan University, Port Elizabeth 6031 (South Africa); Dyk, E.E. van [Department of Physics, PO Box 77000, Nelson Mandela Metropolitan University, Port Elizabeth 6031 (South Africa)

2009-12-01

Five thin film photovoltaic modules were deployed outdoors under open circuit conditions after a thorough indoor evaluation. Two technology types were investigated: amorphous silicon (a-Si:H) and copper indium gallium diselenide (CIGS). Two 14 W a-Si:H modules, labelled Si-1 and Si-2, were investigated. Both exhibited degradation, initially due to the well-known light-induced degradation described by Staebler and Wronski [Applied Physics Letters 31 (4) (1977) 292], and thereafter due to other degradation modes such as cell degradation. The various degradation modes contributing to the degradation of the a-Si:H modules will be discussed. The initial maximum power output (P{sub MAX}) of Si-1 was 9.92 W, with the initial light-induced degradation for Si-1 approx30% and a total degradation of approx42%. For Si-2 the initial P{sub MAX} was 7.93 W, with initial light-induced degradation of approx10% and a total degradation of approx17%. Three CIGS modules were investigated: two 20 W modules labelled CIGS-1 and CIGS-2, and a 40 W module labelled CIGS-3. CIGS-2 exhibited stable performance while CIGS-1 and CIGS-3 exhibited degradation. CIGS is known to be stable over long periods of time, and thus the possible reasons for the degradation of the two modules are discussed.

Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation.

Science.gov (United States)

Yuan, Y; Zhang, G Q; Chai, W; Ni, M; Xu, C; Chen, J Y

2016-10-01

Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1.Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J

Radioactive waste package assay facility. Volume 1. Application of assay technology

International Nuclear Information System (INIS)

Findlay, D.J.S.; Green, T.H.; Molesworth, T.V.; Staniforth, D.; Strachan, N.R.; Rogers, J.D.; Wise, M.O.; Forrest, K.R.

1992-01-01

This report, in three volumes, covers the work carried out by Taylor Woodrow Construction Ltd., and two major sub-contractors: Harwell Laboratory (AEA Technology) and Siemens Plessey Controls Ltd., on the development of a radioactive waste package assay facility, for cemented 500 litre intermediate level waste drums. In volume 1, the reasons for assay are considered together with the various techniques that can be used, and the information that can be obtained. The practical problems associated with the use of the various techniques in an integrated assay facility are identified, and the key parameters defined. Engineering and operational features are examined and provisional designs proposed for facilities at three throughput levels: 15,000, 750 and 30 drums per year respectively. The capital and operating costs for such facilities have been estimated. A number of recommendations are made for further work. 16 refs., 14 figs., 13 tabs

Robust PV Degradation Methodology and Application

Energy Technology Data Exchange (ETDEWEB)

Jordan, Dirk [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Deline, Christopher A [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Kurtz, Sarah [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Kimball, Greg [SunPower; Anderson, Mike [SunPower

2017-11-15

The degradation rate plays an important role in predicting and assessing the long-term energy generation of PV systems. Many methods have been proposed for extracting the degradation rate from operational data of PV systems, but most of the published approaches are susceptible to bias due to inverter clipping, module soiling, temporary outages, seasonality, and sensor degradation. In this manuscript, we propose a methodology for determining PV degradation leveraging available modeled clear-sky irradiance data rather than site sensor data, and a robust year-over-year (YOY) rate calculation. We show the method to provide reliable degradation rate estimates even in the case of sensor drift, data shifts, and soiling. Compared with alternate methods, we demonstrate that the proposed method delivers the lowest uncertainty in degradation rate estimates for a fleet of 486 PV systems.

Automation of the dicentric chromosome assay and related assays

International Nuclear Information System (INIS)

Balajee, Adayabalam S.; Dainiak, Nicholas

2016-01-01

Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

Toxicological aspects of photocatalytic degradation of selected xenobiotics with nano-sized Mn-doped TiO{sub 2}

Energy Technology Data Exchange (ETDEWEB)

Ozmen, Murat, E-mail: murat.ozmen@inonu.edu.tr [Inonu University, Faculty of Science, Department of Biology, Malatya (Turkey); Güngördü, Abbas [Inonu University, Faculty of Science, Department of Biology, Malatya (Turkey); Erdemoglu, Sema [Inonu University, Faculty of Science, Department of Chemistry, Malatya (Turkey); Ozmen, Nesrin [Inonu University, Faculty of Education, Department of Science Teaching Program, Malatya (Turkey); Asilturk, Meltem [Akdeniz University, Department of Materials Science and Engineering, Antalya (Turkey)

2015-08-15

Highlights: • Undoped and Mn-doped TiO{sub 2} nanoparticles were synthesized and characterized. • The photocatalytic efficiency of the photocatalysts was evaluated for BPA and ATZ. • Toxicity of photocatalysts and photocatalytic by-products were determined. • Mn-doped TiO{sub 2} nanoparticles did not cause significant lethality on X. laevis. • Degradation of BPA caused a significant reduction of lethal effects. - Abstract: The toxic effects of two selected xenobiotics, bisphenol A (BPA) and atrazine (ATZ), were evaluated after photocatalytic degradation using nano-sized, Mn-doped TiO{sub 2}. Undoped and Mn-doped TiO{sub 2} nanoparticles were synthesized. The samples were characterized by X-ray diffractometry (XRD), scanning electron microscopy (SEM), UV–vis-diffuse reflectance spectra (DRS), X-ray fluorescence spectroscopy (XRF), and BET surface area. The photocatalytic efficiency of the undoped and Mn-doped TiO{sub 2} was evaluated for BPA and ATZ. The toxicity of the synthesized photocatalysts and photocatalytic by-products of BPA and ATZ was determined using frog embryos and tadpoles, zebrafish embryos, and bioluminescent bacteria. Possible toxic effects were also evaluated using selected enzyme biomarkers. The results showed that Mn-doped TiO{sub 2} nanoparticles did not cause significant lethality in Xenopus laevis embryos and tadpoles, but nonfiltered samples caused lethality in zebrafish. Furthermore, Mn-doping of TiO{sub 2} increased the photocatalytic degradation capability of nanoparticles, and it successfully degraded BPA and AZT, but degradation of AZT caused an increase of the lethal effects on both tadpoles and fish embryos. Degradation of BPA caused a significant reduction of lethal effects, especially after 2–4 h of degradation. However, biochemical assays showed that both Mn-doped TiO{sub 2} and the degradation by-products caused a significant change of selected biomarkers on X. laevis tadpoles; thus, the ecological risks of Mn

Abiotic degradation of plastic films

Science.gov (United States)

Ángeles-López, Y. G.; Gutiérrez-Mayen, A. M.; Velasco-Pérez, M.; Beltrán-Villavicencio, M.; Vázquez-Morillas, A.; Cano-Blanco, M.

2017-01-01

Degradable plastics have been promoted as an option to mitigate the environmental impacts of plastic waste. However, there is no certainty about its degradability under different environmental conditions. The effect of accelerated weathering (AW), natural weathering (NW) and thermal oxidation (TO) on different plastics (high density polyethylene, HDPE; oxodegradable high density polyethylene, HDPE-oxo; compostable plastic, Ecovio ® metalized polypropylene, PP; and oxodegradable metalized polypropylene, PP-oxo) was studied. Plastics films were exposed to AW per 110 hours; to NW per 90 days; and to TO per 30 days. Plastic films exposed to AW and NW showed a general loss on mechanical properties. The highest reduction in elongation at break on AW occurred to HDPE-oxo (from 400.4% to 20.9%) and was higher than 90% for HDPE, HDPE-oxo, Ecovio ® and PP-oxo in NW. No substantial evidence of degradation was found on plastics exposed to TO. Oxo-plastics showed higher degradation rates than their conventional counterparts, and the compostable plastic was resistant to degradation in the studied abiotic conditions. This study shows that degradation of plastics in real life conditions will vary depending in both, their composition and the environment.

Effect of ultrasonic degradation of hyaluronic acid extracted from rooster comb on antioxidant and antiglycation activities.

Science.gov (United States)

Hafsa, Jawhar; Chaouch, Mohamed Aymen; Charfeddine, Bassem; Rihouey, Christophe; Limem, Khalifa; Le Cerf, Didier; Rouatbi, Sonia; Majdoub, Hatem

2017-12-01

Recently, low-molecular-weight hyaluronic acid (LMWHA) has been reported to have novel features, such as free radical scavenging activities, antioxidant activities and dietary supplements. In this study, hyaluronic acid (HA) was extracted from rooster comb and LMWHA was obtained by ultrasonic degradation in order to assess their antioxidant and antiglycation activities. Molecular weight (Mw) and the content of glucuronic acid (GlcA) were used as the index for comparison of the effect of ultrasonic treatment. The effects on the structure were determined by ultraviolet (UV) spectra and Fourier transform infrared spectra (FTIR). The antioxidant activity was determined by three analytical assays (DPPH, NO and TBARS), and the inhibitory effect against glycated-BSA was also assessed. The GlcA content of HA and LMWHA was estimated at about 48.6% and 47.3%, respectively. The results demonstrate that ultrasonic irradiation decreases the Mw (1090-181 kDa) and intrinsic viscosity (1550-473 mL/g), which indicate the cleavage of the glycosidic bonds. The FTIR and UV spectra did not significantly change before and after degradation. The IC 50 value of HA and LWMHA was 1.43, 0.76 and 0.36 mg/mL and 1.20, 0.89 and 0.17 mg/mL toward DPPH, NO and TBARS, respectively. Likewise LMWHA exhibited significant inhibitory effects on the AGEs formation than HA. The results demonstrated that the ultrasonic irradiation did not damage and change the chemical structure of HA after degradation; furthermore, decreasing Mw and viscosity of LMWHA after degradation may enhance the antioxidant and antiglycation activity.

The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.

Science.gov (United States)

Kee, Nalise Low Ah; Krause, Jason; Blatch, Gregory L; Muramoto, Koji; Sakka, Kazuo; Sakka, Makiko; Naudé, Ryno J; Wagner, Leona; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich; Mielicki, Wojciech P; Frost, Carminita L

2015-10-01

Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice.

Directory of Open Access Journals (Sweden)

Lavanya Tayi

Full Text Available Xanthomonas oryzae pv.oryzae (Xoo causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA, one pectin methyl esterase (pmt and two pectate lyases (pel and pelL. There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43 grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA. Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43 in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice.

Science.gov (United States)

Tayi, Lavanya; Maku, Roshan V; Patel, Hitendra Kumar; Sonti, Ramesh V

2016-01-01

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Impact of temperature and substrate concentration on degradation rates of acetate, propionate and hydrogen and their links to microbial community structure.

Science.gov (United States)

Zhao, Jing; Westerholm, Maria; Qiao, Wei; Yin, Dongmin; Bi, Shaojie; Jiang, Mengmeng; Dong, Renjie

2018-05-01

The present study investigates the conversion of acetate, propionate and hydrogen consumption linked to the microbial community structure and related to temperature and substrate concentration. Biogas reactors were continuously fed with coffee powder (20 g-COD/L) or acetate (20, 40, and 60 g-COD/L) and operated for 193 days at 37 °C or 55 °C conditions. Starting HRT was 23 days which was then reduced to 7 days. The kinetics of acetate and propionate degradation and hydrogen consumption rates were measured in batch assays. At HRT 7 days, the degradation rate of propionate was higher in thermophilic batches, while acetate degradation rate was higher at mesophilic conditions. The gaseous hydrogen consumption in acetate reactors increased proportionally with temperature and substrate concentration, while the dissolved hydrogen was not affected. The relative high abundance of hydrogentrophic methanogens indicated that the methanogenesis was directed towards the syntrophic acetate oxidation pathway at high acetate concentration and high temperature. Copyright © 2018 Elsevier Ltd. All rights reserved.

The radiation degradation of polypropylene

International Nuclear Information System (INIS)

De Hollain, G.

1977-04-01

Polypropylene is used extensively in the manufacture of disposable medical devices because of its superior properties. Unfortunately this polymer does not lend itself well to radiation sterilization, undergoing serious degradation which affects the mechanical properties of the polymer. In this paper the effects of radiation on the mechanical and physical properties of polypropylene are discussed. A programme of research to minimize the radiation degradation of this polymer through the addition of crosslinking agents to counteract the radiation degradation is proposed. It is furthermore proposed that a process of annealing of the irradiated polymer be investigated in order to minimize the post-irradiation degradation of the polypropylene [af

Degradation of thiram in soil

International Nuclear Information System (INIS)

Raghu, K.; Murthy, N.B.K.; Kumarsamy, R.

1975-01-01

Determination of the residual 35 S labelled tetramethylthiuram disulfide showed that the fungicide persisted longer in sterilized than in unsterilized soil, while the chloroform extractable radioactivity decreased, the water extractable radioactivity increased with increase in time. However, in sterilized soil the water extractable radioactivity remained more or less constant. Degradation of the fungicide was further demonstrated by the release of C 35 S 2 from soil treated with labelled thiram. Dimethylamine was found to be one of the degradation products. A bacterium isolated from thiram-enriched soil could degrade the fungicide in shake culture. The degradation pathways of thiram in sterilized and unsterilized soils are discussed. (author)

Radiometric assays for glycerol, glucose, and glycogen

International Nuclear Information System (INIS)

Bradley, D.C.; Kaslow, H.R.

1989-01-01

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays

Harmonization of radiobiological assays: why and how?

International Nuclear Information System (INIS)

Prasanna, Pataje G.

2014-01-01

The International Atomic Energy Agency has made available a technical manual for cytogenetic biodosimetry assays (dicentric chromosome aberration (DCA) and cytokinesis-block micronucleus (CBMN) assays) used for radiation dose assessment in radiation accidents. The International Standardization Organization, which develops standards and guidelines, also provides an avenue for laboratory accreditation, has developed guidelines and recommendations for performing cytogenetic biodosimetry assays. Harmonization of DCA and CBMN assays, has improved their accuracy. Double-blinded inter-laboratory comparison studies involving several networks have further validated DCA and CBMN assays and improved the confidence in their potential use for radiation dose assessment in mass casualties. This kind of international harmonization is lacking for pre-clinical radiobiology assays. The widely used pre-clinical assays that are relatively important to set stage for clinical trials include clonogenic assays, flow-cytometry assays, apoptotic assays, and tumor regression and growth delay assays. However, significant inter-laboratory variations occur with respect to data among laboratories. This raises concerns on the reliability and reproducibility of preclinical data that drives further development and translation. Lack of reproducibility may stem from a variety of factors such as poor scientist training, less than optimal experimental design, inadequate description of methodology, and impulse to publish only the positive data etc. Availability of technical manuals, standard operating procedures, accreditation avenues for laboratories performing such assays, inter-laboratory comparisons, and use of standardized protocols are necessary to enhance reliability and reproducibility. Thus, it is important that radiobiological assays are harmonized for laboratory protocols to ensure successful translation of pre-clinical research on radiation effect modulators to help design clinic trials with

Study of the degradation performance (TOC, BOD, and toxicity) of bisphenol A by the photo-Fenton process.

Science.gov (United States)

Pérez-Moya, M; Kaisto, T; Navarro, M; Del Valle, L J

2017-03-01

Degradation of bisphenol A (BPA, 0.5 L, 30 mg L -1 ) was studied by photo-Fenton treatment, while Fenton reagents were variables. The efficiency of the degradation process was evaluated by the reduction of total organic carbon (TOC), the biochemical oxygen demand (BOD), and toxicity. For toxicity analysis, bacterial methods were found infeasible, but the in vitro assay of VERO cells culture was successfully applied. Experiments according to a 2 2 design of experiments (DOE) with star points and three center points for statistical validity allowed selecting those process conditions (Fe(II) and H 2 O 2 load) that maximized the process performance. Photo-Fenton process effectively eliminated BPA and partly degraded its by-products (residual TOC TOC = 92 %) was attained. Toxicity was also detected to 50 % of cellular mortality even at long reaction times. However, 40.25 mg L -1 of H 2 O 2 decreased residual TOC to 70 % while cell mortality decreased down to 25 %. With more H 2 O 2 , the residual TOC decreased down to 15 % but cell mortality remained within the 20-25 % level. Photo-Fenton increased the biodegradability and reduced the toxicity of the studied sample.

Degradation of Insecticides in Poultry Manure: Determining the Insecticidal Treatment Interval for Managing House Fly (Diptera: Muscidae) Populations in Poultry Farms.

Science.gov (United States)

Ong, Song-Quan; Ab Majid, Abdul Hafiz; Ahmad, Hamdan

2016-04-01

It is crucial to understand the degradation pattern of insecticides when designing a sustainable control program for the house fly, Musca domestica (L.), on poultry farms. The aim of this study was to determine the half-life and degradation rates of cyromazine, chlorpyrifos, and cypermethrin by spiking these insecticides into poultry manure, and then quantitatively analyzing the insecticide residue using ultra-performance liquid chromatography. The insecticides were later tested in the field in order to study the appropriate insecticidal treatment intervals. Bio-assays on manure samples were later tested at 3, 7, 10, and 15 d for bio-efficacy on susceptible house fly larvae. Degradation analysis demonstrated that cyromazine has the shortest half-life (3.01 d) compared with chlorpyrifos (4.36 d) and cypermethrin (3.75 d). Cyromazine also had a significantly greater degradation rate compared with chlorpyrifos and cypermethrin. For the field insecticidal treatment interval study, 10 d was the interval that had been determined for cyromazine due to its significantly lower residue; for ChCy (a mixture of chlorpyrifos and cypermethrin), the suggested interval was 7 d. Future work should focus on the effects of insecticide metabolites on targeted pests and the poultry manure environment.

MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

Science.gov (United States)

Schmitt, Sebastian; Höfner, Georg; Wanner, Klaus T

2014-08-05

Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure.

Science.gov (United States)

Co, Michelle; Zettersten, Camilla; Nyholm, Leif; Sjöberg, Per J R; Turner, Charlotta

2012-02-24

Experiments with birch bark samples have been carried to enable a distinction between extraction and degradation effects during pressurised hot water extraction. Two samples, E80 and E180, contained birch bark extracts obtained after extraction at 80 and 180°C for up to 45 min, respectively. Two other samples, P80 and P180, were only extracted for 5 min at the two temperatures and were thereafter filtered and hydrothermally treated at 80 and 180°C, respectively. During the latter treatment, samples were collected at different times to assess the stability of the extracted compounds. An offline DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as well as a high performance liquid chromatographic separation coupled to an electrochemical detector, were used to determine the antioxidant capacity of the processed samples. The results obtained with the different techniques were compared to assess the yield of the extraction and degradation processes. In addition, an online hyphenated system comprising high performance liquid chromatography coupled to diode-array; electrochemical; and tandem mass spectrometric detection (HPLC-DAD-ECD-MS/MS) was used to study the compositions of the extracts in more detail. The results for the samples processed at 80°C showed that the extraction reached a steady-state already after 5 min, and that the extracted compounds were stable throughout the entire extraction process. Processing at 180°C, on the other hand, gave rise to partly degraded extracts with a multitude of peaks in both the diode array and electrochemical detectors, and a higher antioxidant capacity compared to for the extracts obtained at 80°C. It is concluded that HPLC-DAD-ECD is a more appropriate technique for the determination of antioxidants than the DPPH assay. The mass spectrometric results indicate that one of the extracted antioxidants, catechin, was isomerised to its diastereoisomers; (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin. Copyright �

Development of a stability-indicating high performance liquid chromatography method for assay of erythromycin ethylsuccinate in powder for oral suspension dosage form

Directory of Open Access Journals (Sweden)

Fahimeh Kamarei

2014-12-01

Full Text Available In this study an effective method was developed to assay erythromycin ethylsuccinate for an oral suspension dosage form. The chromatographic separation was achieved on an X-Terra™ C18 analytical column. A mixture of acetonitrile–ammonium dihydrogen phosphate buffer (0.025 mol L-1 (60:40, V/V (pH 7.0 was used as the mobile phase, effluent flow rate monitored at 1.0 mL min−1, and UV detection at 205 nm. In forced degradation studies, the effects of acid, base, oxidation, UV light and temperature were investigated showing no interference in the peak of drug. The proposed method was validated in terms of specificity, linearity, robustness, precision and accuracy. The method was linear at concentrations ranging from 400 to 600 μg mL−1, precise (intra- and inter-day relative standard deviations <0.65, accurate (mean recovery; 99.5%. The impurities and degradation products of erythromycin ethylsuccinate were selectively determined with good resolution in both the raw material and the final suspension forms. The method could be useful for both routine analytical and quality control assays of erythromycin ethylsuccinate in commercial powder for an oral suspension dosage form and it could be a very powerful tool to investigate the chemical stability of erythromycin ethylsuccinate.

Self-degradable Cementitious Sealing Materials

Energy Technology Data Exchange (ETDEWEB)

Sugama, T.; Butcher, T., Lance Brothers, Bour, D.

2010-10-01

A self-degradable alkali-activated cementitious material consisting of a sodium silicate activator, slag, Class C fly ash, and sodium carboxymethyl cellulose (CMC) additive was formulated as one dry mix component, and we evaluated its potential in laboratory for use as a temporary sealing material for Enhanced Geothermal System (EGS) wells. The self-degradation of alkali-activated cementitious material (AACM) occurred, when AACM heated at temperatures of {ge}200 C came in contact with water. We interpreted the mechanism of this water-initiated self-degradation as resulting from the in-situ exothermic reactions between the reactants yielded from the dissolution of the non-reacted or partially reacted sodium silicate activator and the thermal degradation of the CMC. The magnitude of self-degradation depended on the CMC content; its effective content in promoting degradation was {ge}0.7%. In contrast, no self-degradation was observed from CMC-modified Class G well cement. For 200 C-autoclaved AACMs without CMC, followed by heating at temperatures up to 300 C, they had a compressive strength ranging from 5982 to 4945 psi, which is {approx}3.5-fold higher than that of the commercial Class G well cement; the initial- and final-setting times of this AACM slurry at 85 C were {approx}60 and {approx}90 min. Two well-formed crystalline hydration phases, 1.1 nm tobermorite and calcium silicate hydrate (I), were responsible for developing this excellent high compressive strength. Although CMC is an attractive, as a degradation-promoting additive, its addition to both the AACM and the Class G well cement altered some properties of original cementitious materials; among those were an extending their setting times, an increasing their porosity, and lowering their compressive strength. Nevertheless, a 0.7% CMC-modified AACM as self-degradable cementitious material displayed the following properties before its breakdown by water; {approx}120 min initial- and {approx}180 min final

Clonogenic assay: adherent cells.

Science.gov (United States)

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

2011-03-13

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

Science.gov (United States)

Nikolova, Teodora; Marini, Federico; Kaina, Bernd

2017-10-01

Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

Quantifying the degradation of degradable implants and bone formation in the femoral condyle using micro-CT 3D reconstruction.

Science.gov (United States)

Xu, Yichi; Meng, Haoye; Yin, Heyong; Sun, Zhen; Peng, Jiang; Xu, Xiaolong; Guo, Quanyi; Xu, Wenjing; Yu, Xiaoming; Yuan, Zhiguo; Xiao, Bo; Wang, Cheng; Wang, Yu; Liu, Shuyun; Lu, Shibi; Wang, Zhaoxu; Wang, Aiyuan

2018-01-01

Degradation limits the application of magnesium alloys, and evaluation methods for non-traumatic in vivo quantification of implant degradation and bone formation are imperfect. In the present study, a micro-arc-oxidized AZ31 magnesium alloy was used to evaluate the degradation of implants and new bone formation in 60 male New Zealand white rabbits. Degradation was monitored by weighing the implants prior to and following implantation, and by performing micro-computed tomography (CT) scans and histological analysis after 1, 4, 12, 24, 36, and 48 weeks of implantation. The results indicated that the implants underwent slow degradation in the first 4 weeks, with negligible degradation in the first week, followed by significantly increased degradation during weeks 12-24 (Pformation increased as the implant degraded. The findings concluded that micro-CT, which is useful for providing non-traumatic, in vivo , quantitative and precise data, has great value for exploring the degradation of implants and novel bone formation.

Recovering of images degraded by atmosphere

Science.gov (United States)

Lin, Guang; Feng, Huajun; Xu, Zhihai; Li, Qi; Chen, Yueting

2017-08-01

Remote sensing images are seriously degraded by multiple scattering and bad weather. Through the analysis of the radiative transfer procedure in atmosphere, an image atmospheric degradation model considering the influence of atmospheric absorption multiple scattering and non-uniform distribution is proposed in this paper. Based on the proposed model, a novel recovering method is presented to eliminate atmospheric degradation. Mean-shift image segmentation and block-wise deconvolution are used to reduce time cost, retaining a good result. The recovering results indicate that the proposed method can significantly remove atmospheric degradation and effectively improve contrast compared with other removal methods. The results also illustrate that our method is suitable for various degraded remote sensing, including images with large field of view (FOV), images taken in side-glance situations, image degraded by atmospheric non-uniform distribution and images with various forms of clouds.

Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11

Energy Technology Data Exchange (ETDEWEB)

Yuki Kasai; Yumiko Kodama; Yoh Takahata; Toshihiro Hoaki; Kazuya Watanabe [Marine Biotechnology Institute, Kamaishi (Japan)

2007-09-15

Azoarcus sp. strain DN11 is a denitrifying bacterium capable of benzene degradation under anaerobic conditions. The present study evaluated strain DN11 for its application to bioaugmentation of benzene-contaminated underground aquifers. Strain DN11 could grow on benzene, toluene, m-xylene, and benzoate as the sole carbon and energy sources under nitrate-reducing conditions, although o- and p-xylenes were transformed in the presence of toluene. Phenol was not utilized under anaerobic conditions. Kinetic analysis of anaerobic benzene degradation estimated its apparent affinity and inhibition constants to be 0.82 and 11 {mu}M, respectively. Benzene-contaminated groundwater taken from a former coal-distillation plant site in Aichi, Japan was anaerobically incubated in laboratory bottles and supplemented with either inorganic nutrients (nitrogen, phosphorus, and nitrate) alone, or the nutrients plus strain DN11, showing that benzene was significantly degraded only when DN11 was introduced. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments, and quantitative PCR revealed that DN11 decreased after benzene was degraded. Following the decrease in DN11 16S rRNA gene fragments corresponding to bacteria related to Owenweeksia hongkongensis and Pelotomaculum isophthalicum, appeared as strong bands, suggesting possible metabolic interactions in anaerobic benzene degradation. Results suggest that DN11 is potentially useful for degrading benzene that contaminates underground aquifers at relatively low concentrations. 50 refs., 6 figs., 1 tab.

Injectable, degradable, electroactive nanocomposite hydrogels containing conductive polymer nanoparticles for biomedical applications.

Science.gov (United States)

Wang, Qinmei; Wang, Qiong; Teng, Wei

2016-01-01

Injectable electroactive hydrogels (eGels) are promising in regenerative medicine and drug delivery, however, it is still a challenge to obtain such hydrogels simultaneously possessing other properties including uniform structure, degradability, robustness, and biocompatibility. An emerging strategy to endow hydrogels with desirable properties is to incorporate functional nanoparticles in their network. Herein, we report the synthesis and characterization of an injectable hydrogel based on oxidized alginate (OA) crosslinking gelatin reinforced by electroactive tetraaniline-graft-OA nanoparticles (nEOAs), where nEOAs are expected to impart electroactivity besides reinforcement without significantly degrading the other properties of hydrogels. Assays of transmission electron microscopy, (1)H nuclear magnetic resonance, and dynamic light scattering reveal that EOA can spontaneously and quickly self-assemble into robust nanoparticles in water, and this nanoparticle structure can be kept at pH 3~9. Measurement of the gel time by rheometer and the stir bar method confirms the formation of the eGels, and their gel time is dependent on the weight content of nEOAs. As expected, adding nEOAs to hydrogels does not cause the phase separation (scanning electron microscopy observation), but it improves mechanical strength up to ~8 kPa and conductivity up to ~10(-6) S/cm in our studied range. Incubating eGels in phosphate-buffered saline leads to their further swelling with an increase of water content <6% and gradual degradation. When growing mesenchymal stem cells on eGels with nEOA content ≤14%, the growth curves and morphology of cells were found to be similar to that on tissue culture plastic; when implanting these eGels on a chick chorioallantoic membrane for 1 week, mild inflammation response appeared without any other structural changes, indicating their good in vitro and in vivo biocompatibility. With injectability, uniformity, degradability, electroactivity, relative

Stability-indicating UPLC method for determining related substances and degradants in dronedarone.

Science.gov (United States)

Pydimarry, Surya Prakash Rao; Cholleti, Vijay Kumar; Vangala, Ranga Reddy

2014-08-01

A simple, sensitive and reproducible method was developed on ultra-performance liquid chromatography coupled with photodiode array detection for the quantitative determination of dronedarone hydrochloride (DRO) in drug substance and pharmaceutical dosage forms. The method is applicable for the quantification of related substances and assays of drug substances. Chromatographic separation was achieved on Acquity UPLC BEH C8 100 mm, 2.1 mm and 1.7 µm columns, using gradient elution within a short run time of 10.0 min. The eluted compounds were monitored at 288 nm, the flow rate was 0.5 mL/min and the column oven temperature was maintained at 40°C. The resolution of DRO and 11 impurities (potentials and by-products) was greater than 2.0 for all pairs of components. The high correlation coefficient value (>0.9995) indicates the clear correlations between the concentrations of investigated compound and their peak areas within the test ranges. The repeatability and intermediate precision, expressed by the relative standard deviation, were less than 2.5%. The accuracy and validity of the method were further ascertained by performing recovery studies via a spike method. The accuracy of the method, expressed as relative error, was satisfactory. No interference was observed from concomitant substances normally added to the tablets. DRO was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. DRO was found to degrade significantly in acid and base stress conditions and to remain stable in thermal, photolytic degradation, oxidative and hydrolytic conditions. The degradation products were well resolved from primary peak and its impurities, proving that the method is stability indicating. The developed method was validated as per International Conference on Harmonization guidelines with respect to specificity, limit of detection, limit of quantification, linearity, accuracy, precision, solution stability and robustness

Multicentre comparison of a diagnostic assay

DEFF Research Database (Denmark)

Waters, Patrick; Reindl, Markus; Saiz, Albert

2016-01-01

) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

Directory of Open Access Journals (Sweden)

Sarah J Fletcher

Full Text Available Tight junctions (TJs link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes. By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

International Nuclear Information System (INIS)

Yousaf, Sohail; Afzal, Muhammad; Reichenauer, Thomas G.; Brady, Carrie L.; Sessitsch, Angela

2011-01-01

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants. - Highlights: → E. ludwigii strains efficiently colonized plants in a non-sterile soil environment. → E. ludwigii strains efficiently expressed alkane degradation genes in plants. → E. ludwigii efficiently degraded alkane contaminations and promoted plant growth. → E. ludwigii interacted more effectively with Italian ryegrass than with other plants. → Degradation activity varied with plant and microbial genotype as well as with time. - Enterobacter ludwigii strains belonging to the E. cloacae complex are able to efficiently degrade alkanes when associated with plants and to promote plant growth.

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

Energy Technology Data Exchange (ETDEWEB)

Yousaf, Sohail [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria); Afzal, Muhammad [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria); National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad (Pakistan); Reichenauer, Thomas G. [AIT Austrian Institute of Technology GmbH, Environmental Resources and Technologies Unit, A-2444 Seibersdorf (Austria); Brady, Carrie L. [Forestry and Agricultural Biotechnology Institute, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria (South Africa); Sessitsch, Angela, E-mail: angela.sessitsch@ait.ac.at [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria)

2011-10-15

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants. - Highlights: > E. ludwigii strains efficiently colonized plants in a non-sterile soil environment. > E. ludwigii strains efficiently expressed alkane degradation genes in plants. > E. ludwigii efficiently degraded alkane contaminations and promoted plant growth. > E. ludwigii interacted more effectively with Italian ryegrass than with other plants. > Degradation activity varied with plant and microbial genotype as well as with time. - Enterobacter ludwigii strains belonging to the E. cloacae complex are able to efficiently degrade alkanes when associated with plants and to promote plant growth.

An ultrafiltration assay for lysyl oxidase

International Nuclear Information System (INIS)

Shackleton, D.R.; Hulmes, D.J.

1990-01-01

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware

Influence of water solubility, side chain degradability and side chain configuration on the degradation of phthalic acid esters under methanogenic conditions

Energy Technology Data Exchange (ETDEWEB)

Alnervik, M.

1996-12-31

Water solubility and degradability of side chains estrifying phthalic acid are factors possible to influence the degradation of phthalic acid esters (PAEs). To investigate the importance of these factors degradation of butyl 2-ethylhexyl phthalate (BEHP), bis(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), dihexyl phthalate (DHP), dioctyl phthalate (DOP) and didecyl phthalate (DDP) were examined under methanogenic conditions as well as was the degradability of the alcohols estrifying these PAEs. We also investigated if the degradation of resistant PAEs could be stimulated by the addition of a degradable PAE. Synthesis of degradation intermediates and two methods for PAE analyses are presented. The investigation showed that all alcohols were degraded to methane and carbon dioxide and that the degradation of PAE occurred in incubations amended with BBP, BEHP, DHP and DBP, whilst DEHP, DOP and DDP were unaffected throughout the experimental period. BBP added to incubations with DEHP, could not stimulate DEHP degradation. In conclusion, the degradability of alcohols estrifying phthalic acid in this study does not affect the anaerobic degradability of PAEs. Water solubility of a PAE can not be rejected as a factor limiting phthalate degradation under methanogenic conditions. Anaerobic degradation of persistent PAEs can not be stimulated by mixing it with a degradable phthalate. 23 refs, 11 figs, 2 tabs

Direct ubiquitin independent recognition and degradation of a folded protein by the eukaryotic proteasomes-origin of intrinsic degradation signals.

Directory of Open Access Journals (Sweden)

Amit Kumar Singh Gautam

Full Text Available Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a origin and identification of an intrinsic degradation signal in the substrate, b identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably

SCFSLF-mediated cytosolic degradation of S-RNase is required for cross-pollen compatibility in S-RNase-based self-incompatibility in Petunia hybrida

Directory of Open Access Journals (Sweden)

Yongbiao eXue

2014-07-01

Full Text Available Many flowering plants adopt self-incompatibility (SI to maintain their genetic diversity. In species of Solanaceae, Plantaginaceae and Rosaceae, SI is genetically controlled by a single S-locus with multiple haplotypes. The S-locus has been shown to encode S-RNases expressed in pistil and multiple SLF (S-locus F-box proteins in pollen controlling the female and male specificity of SI, respectively. S-RNases appear to function as a cytotoxin to reject self-pollen. In addition, SLFs have been shown to form SCF (SKP1/Cullin1/F-box complexes to serve as putative E3 ubiquitin ligase to interact with S-RNases. Previously, two different mechanisms, the S-RNase degradation and the S-RNase compartmentalization, have been proposed as the restriction mechanisms of S-RNase cytotoxicity allowing compatible pollination. In this study, we have provided several lines of evidence in support of the S-RNase degradation mechanism by a combination of cellular, biochemical and molecular biology approaches. First, both immunogold labeling and subcellular fractionation assays showed that two key pollen SI factors, PhSLF-S3L and PhSSK1 (SLF-interacting SKP1-like1 from Petunia hybrida, a Solanaceous species, are co-localized in cytosols of both pollen grains and tubes. Second, PhS3L-RNases are mainly detected in the cytosols of both self and non-self pollen tubes after pollination. Third, we found that both PhS3-RNases and PhS3L-RNases directly interact with PhSLF-S3L by yeast two-hybrid and co-immunoprecipitation assays. Fourth, S-RNases are specifically degraded in compatible pollen tubes by non-self SLF action. Taken together, our results demonstrate that SCFSLF-mediated non-self S-RNase degradation occurs in the cytosol of pollen tube through the ubiquitin/26S proteasome system serving as the major mechanism to neutralize S-RNase cytotoxicity during compatible pollination in P. hybrida.

Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3.

Science.gov (United States)

Li, Shao-Jia; Yin, Xue-Ren; Wang, Wen-Li; Liu, Xiao-Fen; Zhang, Bo; Chen, Kun-Song

2017-06-15

Citric acid is the predominant organic acid of citrus fruit. Degradation of citric acid occurs during fruit development, influencing fruit acidity. Associations of CitAco3 transcripts and citric acid degradation have been reported for citrus fruit. Here, transient overexpression of CitAco3 significantly reduced the citric acid content of citrus leaves and fruits. Using dual luciferase assays, it was shown that CitNAC62 and CitWRKY1 could transactivate the promoter of CitAco3. Subcellular localization results showed that CitWRKY1 was located in the nucleus and CitNAC62 was not. Yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays indicated that the two differently located transcription factors could interact with each other. Furthermore, BiFC showed that the protein-protein interaction occurred only in the nucleus, indicating the potential mobility of CitNAC62 in plant cells. A synergistic effect on citrate content was observed between CitNAC62 and CitWRKY1. Transient overexpression of CitNAC62 or CitWRKY1 led to significantly lower citrate content in citrus fruit. The combined expression of CitNAC62 and CitWRKY1 resulted in lower citrate content compared with the expression of CitNAC62 or CitWRKY1 alone. The transcript abundance of CitAco3 was consistent with the citrate content. Thus, we propose that a complex of CitWRKY1 and CitNAC62 contributes to citric acid degradation in citrus fruit, potentially via modulation of CitAco3. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Androgen receptor (AR) degradation enhancer ASC-J9® in an FDA-approved formulated solution suppresses castration resistant prostate cancer cell growth.

Science.gov (United States)

Cheng, Max A; Chou, Fu-Ju; Wang, Keliang; Yang, Rachel; Ding, Jie; Zhang, Qiaoxia; Li, Gonghui; Yeh, Shuyuan; Xu, Defeng; Chang, Chawnshang

2018-03-28

ASC-J9 ® is a recently-developed androgen receptor (AR)-degradation enhancer that effectively suppresses castration resistant prostate cancer (PCa) cell proliferation and invasion. The optimal half maximum inhibitory concentrations (IC 50 ) of ASC-J9 ® at various PCa cell confluences (20%, 50%, and 100%) were assessed via both short-term MTT growth assays and long-term clonogenic proliferation assays. Our results indicate that the IC 50 values for ASC-J9 ® increased with increasing cell confluency. The IC 50 values were significantly decreased in PCa AR-positive cells compared to PCa AR-negative cells or in normal prostate cells. This suggests that ASC-J9 ® may function mainly via targeting the AR-positive PCa cells with limited unwanted side-effects to suppress the surrounding normal prostate cells. Mechanism dissection indicated that ASC-J9 ® might function via altering the apoptosis signals to suppress the PCa AR-negative PC-3 cells. Preclinical studies using multiple in vitro PCa cell lines and an in vivo mouse model with xenografted castration-resistant PCa CWR22Rv1 cells demonstrated that ASC-J9 ® has similar AR degradation effects when dissolved in FDA-approved solvents, including DMSO, PEG-400:Tween-80 (95:5), DMA:Labrasol:Tween-80 (10:45:45), and DMA:Labrasol:Tween-20 (10:45:45). Together, results from preclinical studies suggest a potential new therapy with AR-degradation enhancer ASC-J9 ® may potentially be ready to be used in human clinical trials in order to better suppress PCa at later castration resistant stages. Copyright © 2017 Elsevier B.V. All rights reserved.

Short-stack modeling of degradation in solid oxide fuel cells. Part I. Contact degradation

Energy Technology Data Exchange (ETDEWEB)

Gazzarri, J.I. [Department of Mechanical Engineering, University of British Columbia, 2054-6250 Applied Science Lane, Vancouver, BC V6T 1Z4 (Canada); Kesler, O. [Department of Mechanical and Industrial Engineering, University of Toronto, 5 King' s College Road, Toronto, ON M5S 3G8 (Canada)

2008-01-21

As the first part of a two paper series, we present a two-dimensional impedance model of a working solid oxide fuel cell (SOFC) to study the effect of contact degradation on the impedance spectrum for the purpose of non-invasive diagnosis. The two dimensional modeled geometry includes the ribbed interconnect, and is adequate to represent co- and counter-flow configurations. Simulated degradation modes include: cathode delamination, interconnect oxidation, and interconnect-cathode detachment. The simulations show differences in the way each degradation mode impacts the impedance spectrum shape, suggesting that identification is possible. In Part II, we present a sensitivity analysis of the results to input parameter variability that reveals strengths and limitations of the method, as well as describing possible interactions between input parameters and concurrent degradation modes. (author)

Short-stack modeling of degradation in solid oxide fuel cells. Part I. Contact degradation

Science.gov (United States)

Gazzarri, J. I.; Kesler, O.

As the first part of a two paper series, we present a two-dimensional impedance model of a working solid oxide fuel cell (SOFC) to study the effect of contact degradation on the impedance spectrum for the purpose of non-invasive diagnosis. The two dimensional modeled geometry includes the ribbed interconnect, and is adequate to represent co- and counter-flow configurations. Simulated degradation modes include: cathode delamination, interconnect oxidation, and interconnect-cathode detachment. The simulations show differences in the way each degradation mode impacts the impedance spectrum shape, suggesting that identification is possible. In Part II, we present a sensitivity analysis of the results to input parameter variability that reveals strengths and limitations of the method, as well as describing possible interactions between input parameters and concurrent degradation modes.

Working session 1: Tubing degradation

International Nuclear Information System (INIS)

Kharshafdjian, G.; Turluer, G.

1997-01-01

A general introductory overview of the purpose of the group and the general subject area of SG tubing degradation was given by the facilitator. The purpose of the session was described as to open-quotes develop conclusions and proposals on regulatory and technical needs required to deal with the issues of SG tubing degradation.close quotes Types, locations and characteristics of tubing degradation in steam generators were briefly reviewed. The well-known synergistic effects of materials, environment, and stress and strain/strain rate, subsequently referred to by the acronym open-quotes MESSclose quotes by some of the group members, were noted. The element of time (i.e., evolution of these variables with time) was emphasized. It was also suggested that the group might want to consider the related topics of inspection capabilities, operational variables, degradation remedies, and validity of test data, and some background information in these areas was provided. The presentation given by Peter Millet during the Plenary Session was reviewed; Specifically, the chemical aspects and the degradation from the secondary side of the steam generator were noted. The main issues discussed during the October 1995 EPRI meeting on secondary side corrosion were reported, and a listing of the potential SG tube degradations was provided and discussed

Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

Science.gov (United States)

Ballari, Rajashekhar V; Martin, Asha

2013-12-01

DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal. Copyright © 2013 Elsevier Ltd. All rights reserved.

Endocytic collagen degradation

DEFF Research Database (Denmark)

Madsen, Daniel H.; Jürgensen, Henrik J.; Ingvarsen, Signe Ziir

2012-01-01

it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked...... up-regulation of uPARAP/Endo180 in activated fibroblasts and hepatic stellate cells located close to the collagen deposits. In a hepatic stellate cell line, uPARAP/Endo180 was shown to be active in, and required for, the uptake and intracellular degradation of collagen. To evaluate the functional...... groups of mice clearly revealed a fibrosis protective role of uPARAP/Endo180. This effect appeared to directly reflect the activity of the collagen receptor, since no compensatory events were noted when comparing the mRNA expression profiles of the two groups of mice in an array system focused on matrix-degrading...

Kynurenine pathway in psychosis: evidence of increased tryptophan degradation.

LENUS (Irish Health Repository)

Barry, Sandra

2009-05-01

The kynurenine pathway of tryptophan degradation may serve to integrate disparate abnormalities heretofore identified in research aiming to elucidate the complex aetiopathogenesis of psychotic disorders. Post-mortem brain tissue studies have reported elevated kynurenine and kynurenic acid in the frontal cortex and upregulation of the first step of the pathway in the anterior cingulate cortex of individuals with schizophrenia. In this study, we examined kynurenine pathway activity by measuring tryptophan breakdown, a number of pathway metabolites and interferon gamma (IFN-gamma), which is the preferential activator of the first-step enzyme, indoleamine dioxygenase (IDO), in the plasma of patients with major psychotic disorder. Plasma tryptophan, kynurenine pathway metabolites were measured using high-performance liquid chromatography (HPLC) in 34 patients with a diagnosis on the psychotic spectrum (schizophrenia or schizoaffective disorder) and in 36 healthy control subjects. IFN-gamma was measured using enzyme-linked immunosorbent assay (ELISA). The mean tryptophan breakdown index (kynurenine\\/tryptophan) was significantly higher in the patient group compared with controls (P < 0.05). IFN-gamma measures did not differ between groups (P = 0.23). No relationship was found between measures of psychopathology, symptom severity and activity in the first step in the pathway. A modest correlation was established between the tryptophan breakdown index and illness duration. These results provide evidence for kynurenine pathway upregulation, specifically involving the first enzymatic step, in patients with major psychotic disorder. Increased tryptophan degradation in psychoses may have potential consequences for the treatment of these disorders by informing the development of novel therapeutic compounds.

Comparing aye-aye (Daubentonia madagascariensis) presence and distribution between degraded and non-degraded forest within Ranomafana National Park, Madagascar.

Science.gov (United States)

Farris, Zach J; Morelli, Toni Lyn; Sefczek, Timothy; Wright, Patricia C

2011-01-01

The aye-aye is considered the most widely distributed lemur in Madagascar; however, the effect of forest quality on aye-aye abundance is unknown. We compared aye-aye presence across degraded and non-degraded forest at Ranomafana National Park, Madagascar. We used secondary signs (feeding sites, high activity sites) as indirect cues of aye-aye presence and Canarium trees as an indicator of resource availability. All 3 measured variables indicated higher aye-aye abundance within non-degraded forest; however, the differences across forest type were not significant. Both degraded and non-degraded forests showed a positive correlation between feeding sites and high activity sites. We found that Canarium, an important aye-aye food source, was rare and had limited dispersal, particularly across degraded forest. This preliminary study provides baseline data for aye-aye activity and resource utilization across degraded and non-degraded forests. Copyright © 2011 S. Karger AG, Basel.

Fabrication of nanocrystalline hydroxyapatite doped degradable composite hollow fiber for guided and biomimetic bone tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Zhang Ning [Department of Bioengineering, Clemson University, Clemson, SC, 29634 (United States); Nichols, Heather L. [Department of Bioengineering, Clemson University, Clemson, SC, 29634 (United States); Tylor, Shila [Department of Bioengineering, Clemson University, Clemson, SC, 29634 (United States); Wen Xuejun [Department of Bioengineering, Clemson University, Clemson, SC, 29634 (United States)]. E-mail: xjwen@clemson.edu

2007-04-15

Natural bone tissue possesses a nanocomposite structure interwoven in a three-dimensional (3-D) matrix, which plays critical roles in conferring appropriate physical and biological properties to the bone tissue. Single type of material may not be sufficient to mimic the composition, structure and properties of native bone, therefore, composite materials consisting of both polymers, bioceramics, and other inorganic materials have to be designed. Among a variety of candidate materials, polymer-nanoparticle composites appear most promising for bone tissue engineering applications because of superior mechanical properties, improved durability, and surface bioactivity when compared with conventional polymers or composites. The long term objective of this project is to use highly aligned, bioactive, biodegradable scaffold mimicking natural histological structure of human long bone, and to engineer and regenerate human long bone both in vitro and in vivo. In this study, bioactive, degradable, and highly permeable composite hollow fiber membranes (HFMs) were fabricated using a wet phase phase-inversion approach. The structure of the hollow fiber membranes was examined using scanning electron microscopy (SEM); degradation behavior was examined using weigh loss assay, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC); and bioactivity was evaluated with the amount of calcium deposition from the culture media onto HFM surface. Doping PLGA HFMs with nanoHA results in a more bioactive and slower degrading HFM than pure PLGA HFMs.

Self-bioremediation of cork-processing wastewaters by (chloro)phenol-degrading bacteria immobilised onto residual cork particles.

Science.gov (United States)

del Castillo, I; Hernández, P; Lafuente, A; Rodríguez-Llorente, I D; Caviedes, M A; Pajuelo, E

2012-04-15

Cork manufacturing is a traditional industry in Southern Europe, being the main application of this natural product in wine stoppers and insulation. Cork processing begins at boiling the raw material. As a consequence, great volumes of dark wastewaters, with elevated concentrations of chlorophenols, are generated, which must be depurated through costly physicochemical procedures before discarding them into public water courses. This work explores the potential of bacteria, isolated from cork-boiling waters storage ponds, in bioremediation of the same effluent. The bacterial population present in cork-processing wastewaters was analysed by DGGE; low bacterial biodiversity was found. Aerobic bacteria were isolated and investigated for their tolerance against phenol and two chlorophenols. The most tolerant strains were identified by sequencing 16S rDNA. The phenol-degrading capacity was investigated by determining enzyme activities of the phenol-degrading pathway. Moreover, the capacity to form biofilms was analysed in a microtitre plate assay. Finally, the capacity to form biofilms onto the surface of residual small cork particles was evaluated by acridine staining followed by epifluorescence microscopy and by SEM. A low-cost bioremediation system, using phenol-degrading bacteria immobilised onto residual cork particles (a by-product of the industry) is proposed for the remediation of this industrial effluent (self-bioremediation). Copyright © 2011 Elsevier Ltd. All rights reserved.

Radiation degradation of alginate and chitosan

Energy Technology Data Exchange (ETDEWEB)

Nagasawa, Naotsugu; Mitomo, Hiroshi [Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma (Japan); Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

2000-03-01

Alginate and chitosan were irradiated in solid or aqueous solution condition with Co{sup 60} gamma rays in the dose range of 20 to 500 kGy. Degradation was observed both in solid and solution conditions. The degradation in solution was remarkably greater than that in solid. For example, the molecular weight of alginate in 4%(w/v) solution decreased from 2 x 10{sup 5} for 0 kGy to 6 x 10{sup 3} for 50 kGy irradiation while the equivalent degradation by solid irradiation required 500 kGy. The activated species from irradiated water must be responsible for the degradation in solution. The degradation was also accompanied with the color change of alginate: the color became deep brown for highly degraded alginate. UV spectra showed a distinct absorption peak at 265 nm for colored alginates, increasing with dose. The fact that discoloration of colored alginate was caused on exposure to ozone suggests a formation of double bond in pyranose-ring by scission of glycosidic bond. Degradation behavior of chitosan in irradiation was almost the same as that of alginate. (author)

Modified batch anaerobic digestion assay for testing efficiencies of trace metal additives to enhance methane production of energy crops.

Science.gov (United States)

Brulé, Mathieu; Bolduan, Rainer; Seidelt, Stephan; Schlagermann, Pascal; Bott, Armin

2013-01-01

Batch biochemical methane potential (BMP) assays to evaluate the methane yield of biogas substrates such as energy crops are usually carried out with undiluted inoculum. A BMP assay was performed on two energy crops (green cuttings and grass silage). Anaerobic digestion was performed both with and without supplementation of three commercial additives containing trace metals in liquid, solid or adsorbed form (on clay particles). In order to reveal positive effects of trace metal supplementation on the methane yield, besides undiluted inoculum, 3-fold and 10-fold dilutions of the inoculum were applied for substrate digestion. Diluted inoculum variants were supplemented with both mineral nutrients and pH-buffering substances to prevent a collapse of the digestion process. As expected, commercial additives had no effect on the digestion process performed with undiluted inoculum, while significant increases of methane production through trace element supplementation could be observed on the diluted variants. The effect of inoculum dilution may be twofold: (1) decrease in trace metal supplementation from the inoculum and (2) reduction in the initial number of bacterial cells. Bacteria require higher growth rates for substrate degradation and hence have higher trace element consumption. According to common knowledge of the biogas process, periods with volatile fatty acids accumulation and decreased pH may have occurred in the course ofanaerobic digestion. These effects may have led to inhibition, not only ofmethanogenes and acetogenes involved in the final phases of methane production, but also offibre-degrading bacterial strains involved in polymer hydrolysis. Further research is required to confirm this hypothesis.

Nano-immunosafety: issues in assay validation

International Nuclear Information System (INIS)

Boraschi, Diana; Italiani, Paola; Oostingh, Gertie J; Duschl, Albert; Casals, Eudald; Puntes, Victor F; Nelissen, Inge

2011-01-01

Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

Data transformation methods for multiplexed assays

Science.gov (United States)

Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

2013-07-23

Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

Modelling land degradation in IMAGE 2

NARCIS (Netherlands)

Hootsmans RM; Bouwman AF; Leemans R; Kreileman GJJ; MNV

2001-01-01

Food security may be threatened by loss of soil productivity as a result of human-induced land degradation. Water erosion is the most important cause of land degradation, and its effects are irreversible. This report describes the IMAGE land degradation model developed for describing current and

Biosurfactant and Degradative Enzymes Mediated Crude Oil Degradation by Bacterium Bacillus subtilis A1

Science.gov (United States)

Parthipan, Punniyakotti; Preetham, Elumalai; Machuca, Laura L.; Rahman, Pattanathu K. S. M.; Murugan, Kadarkarai; Rajasekar, Aruliah

2017-01-01

In this work, the biodegradation of the crude oil by the potential biosurfactant producing Bacillus subtilis A1 was investigated. The isolate had the ability to synthesize degradative enzymes such as alkane hydroxylase and alcohol dehydrogenase at the time of biodegradation of hydrocarbon. The biosurfactant producing conditions were optimized as pH 7.0, temperature 40°C, 2% sucrose and 3% of yeast extract as best carbon and nitrogen sources for maximum production of biosurfactant (4.85 g l-1). Specifically, the low molecular weight compounds, i.e., C10–C14 were completely degraded, while C15–C19 were degraded up to 97% from the total hydrocarbon pools. Overall crude oil degradation efficiency of the strain A1 was about 87% within a short period of time (7 days). The accumulated biosurfactant from the biodegradation medium was characterized to be lipopeptide in nature. The strain A1 was found to be more robust than other reported biosurfactant producing bacteria in degradation efficiency of crude oil due to their enzyme production capability and therefore can be used to remove the hydrocarbon pollutants from contaminated environment. PMID:28232826

Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake.

Science.gov (United States)

Barnard, Anna; Posocco, Paola; Pricl, Sabrina; Calderon, Marcelo; Haag, Rainer; Hwang, Mark E; Shum, Victor W T; Pack, Daniel W; Smith, David K

2011-12-21

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery. © 2011 American Chemical Society

Spectrophotometric Enzyme Assays for High-Throughput Screening

Directory of Open Access Journals (Sweden)

Jean-Louis Reymond

2004-01-01

Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

Radioenzymatic assay of DOPA (3,4-dihydroxyphenylalanine)

International Nuclear Information System (INIS)

Johnson, G.A.; Gren, J.M.; Kupiecki, R.

1978-01-01

We modified the single-isotope radioenzymatic assay for catecholamines [Life Sci. 21, 625(1977)] to assay 3,4-dihydroxyphenylalanine (DOPA). DOPA decarboxylase is used to convert DOPA to dopamine, which concurrently is converted to [ 3 H]-3-O-methyldopamine in the presence of catechol-O-methyltransferase and [methyl- 3 H]-S-adenosylmethionine and assayed radioenzymatically. For assay of plasma DOPA, 50 μl of untreated plasma is added directly into the incubation mixture. A duplicate mixture containing an internal standard requires a second 50-μl aliquot of plasma. Because the assay measures both DOPA and endogenous dopamine, two additional aliquots of plasma must be assayed for dopamine in the absence of the decarboxylase by the differential assay; DOPA is estimated by difference. The assay is sensitive to 25 pg (500 ng/liter of plasma). Analysis of DOPA (DOPA plus dopamine) and the concurrent differential assay of catecholamines in at least 10 samples can be done in a single working day. Plasma DOPA concentrations for 42 normotensive adults were 1430 +- 19 ng/liter (mean +- SEM). In contrast, dopamine concentrations for these same subjects averaged 23 +- 20 ng/liter. Values for the 24 women subjects (1510 +- 62 ng/liter) significantly (P = 0.04) exceeded those for the men

RAS - Screens & Assays - Drug Discovery

Science.gov (United States)

The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

Comparison of Acute Toxicity of Algal Metabolites Using Bioluminescence Inhibition Assay

Directory of Open Access Journals (Sweden)

Hansa Jeswani

2015-01-01

Full Text Available Microalgae are reported to degrade hazardous compounds. However, algae, especially cyanobacteria are known to produce secondary metabolites which may be toxic to flora, fauna and human beings. The aim of this study was selection of an appropriate algal culture for biological treatment of biomass gasification wastewater based on acute toxicity considerations. The three algae that were selected were Spirulina sp., Scenedesmus abundans and a fresh water algal consortium. Acute toxicity of the metabolites produced by these algal cultures was tested at the end of log phase using the standard bioluminescence inhibition assay based on Vibrio fischeri NRRLB 11174. Scenedesmus abundans and a fresh water algal consortium dominated by cyanobacteria such as Phormidium, Chroococcus and Oscillatoria did not release much toxic metabolites at the end of log phase and caused only about 20% inhibition in bioluminescence. In comparison, Spirulina sp. released toxic metabolites and caused 50% bioluminescence inhibition at 3/5 times dilution of the culture supernatant (EC50.

The fluorometric microculture cytotoxicity assay.

Science.gov (United States)

Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

2008-01-01

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

Solution assay instrument operations manual

International Nuclear Information System (INIS)

Li, T.K.; Marks, T.; Parker, J.L.

1983-09-01

An at-line solution assay instrument (SAI) has been developed and installed in a plutonium purification and americium recovery process area in the Los Alamos Plutonium Processing Facility. The instrument was designed for accurate, timely, and simultaneous nondestructive analysis of plutonium and americium in process solutions that have a wide range of concentrations and americium/plutonium ratios and for routine operation by process technicians who lack instrumentation background. The SAI, based on transmission-corrected, high-resolution gamma-ray spectroscopy, has two measurement stations attached to a single multichannel analyzer/computer system. To ensure the quality of assay results, the SAI has an internal measurement control program, which requires daily and weekly check runs and monitors key aspects of all assay runs. For a 25-ml sample, the assay precision is 5 g/l within a 2000-s count time

Construction of genetically engineered bacteria that degrades organophosphorus pesticide residues and can be easily detected by the fluorescence.

Science.gov (United States)

Li, Qin; Wang, Pan; Chen, Rui; Li, Wei; Wu, Yi-Jun

2014-01-01

Organophosphorus compounds (OPs) are widely used in agriculture and industry and there is increased concern about their toxicological effects in the environment. Bioremediation can offer an efficient and cost-effective option for the removal of OPs. Herein, we describe the construction of a genetically engineered microorganism (GEM) that can degrade OPs and be directly detected and monitored in the environment using an enhanced green fluorescent protein (EGFP) fusion strategy. The coding regions of EGFP, a reporter protein that can fluoresce by itself, and organophosphorus hydrolase (OPH), which has a broad substrate specificity and is able to hydrolyse a number of organophosphorus pesticides, were cloned into the expression vector pET-28b. The fusion protein of EGFP-OPH was expressed in E. coli BL21 (DE3) and the protein expression reached the highest level at 11 h after isopropyl beta-D-thiogalactopyranoside induction. The fluorescence of the GEM was detected by fluorescence spectrophotometry and microscopy, and its ability to degrade OPs was determined by OPH activity assay. Those GEM that express the fusion protein (EGFP and OPH) exhibited strong fluorescence intensity and also potent hydrolase activity, which could be used to degrade organophosphorus pesticide residues in the environment and can also be directly monitored by fluorescence.

Bacterial diversity exploration in hydrocarbon polluted soil: metabolic potential and degrader community evolution revealed by isotope labeling

International Nuclear Information System (INIS)

Martin, F.

2011-01-01

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds produced by incomplete combustion of organic matter. They are a source of environmental pollution, especially associated to oil product exploitation, and represent a threat for living organisms including human beings because of their toxicity. Many bacteria capable of degrading PAHs have been isolated and studied. However, since less than 5% of soil bacteria can be cultivated in the laboratory, bacterial species able to degrade PAHs in situ have been poorly studied. The first goal of this study was to identify bacteria that degrade PAHs in soil using culture-independent molecular methods. To this end, a strategy known a stable isotope probing has been implemented based on the use of phenanthrene, a three rings PAH, in which the natural isotope of carbon was replaced by 13 C. This molecule has been introduced as a tracer in microcosms containing soil from a constructed wetlands collecting contaminated water from highway runoff. Bacteria having incorporated the 13 C were then identified by 16 S rRNA gene sequence analysis after PCR amplification from labeled genomic DNA extracted from soil. The results show that so far little studied Betaproteobacteria, belonging to the genera Acidovorax, Rhodoferax, Hydrogenophaga and Thiobacillus, as well as Rhodocyclaceae, were the key players in phenanthrene degradation. Predominance of Betaproteobacteries was established thanks to quantitative PCR measurements. A dynamic analysis of bacterial diversity also showed that the community structure of degraders depended on phenanthrene bioavailability. In addition, the phylogenetic diversity of ring-hydroxylating di-oxygenases, enzymes involved in the first step of PAH degradation, has been explored. We detected new sequences, mostly related to di-oxygenases from Sphingomonadales and Burkholderiales. For the first time, we were able to associate a catalytic activity for oxidation of PAHs to partial gene sequences

PWR degraded core analysis

International Nuclear Information System (INIS)

Gittus, J.H.

1982-04-01

A review is presented of the various phenomena involved in degraded core accidents and the ensuing transport of fission products from the fuel to the primary circuit and the containment. The dominant accident sequences found in the PWR risk studies published to date are briefly described. Then chapters deal with the following topics: the condition and behaviour of water reactor fuel during normal operation and at the commencement of degraded core accidents; the generation of hydrogen from the Zircaloy-steam and the steel-steam reactions; the way in which the core deforms and finally melts following loss of coolant; debris relocation analysis; containment integrity; fission product behaviour during a degraded core accident. (U.K.)

Making transuranic assay measurements using modern controllers

International Nuclear Information System (INIS)

Kuckertz, T.H.; Caldwell, J.T.; Medvick, P.A.; Kunz, W.E.; Hastings, R.D.

1987-01-01

This paper describes methodology and computer-controlled instrumentation developed at the Los Alamos National Laboratory that accurately performs nondestructive assays of large containers bearing transuranic wastes and nonradioactive matrix materials. These assay systems can measure fissile isotopes with 1-mg sensitivity and spontaneous neutron-emitting isotopes at a 10-mg sensitivity. The assays are performed by neutron interrogation, detection, and counting in a custom assay chamber. An International Business Machines Personal Computer (IBM-PC) is used to control the CAMAC-based instrumentation system that acquires the assay data. 6 refs., 7 figs

Time-resolved immunofluorometric assay of serum ferritin

Energy Technology Data Exchange (ETDEWEB)

Yan, Yao [China Inst. of Atomic Energy, Beijing (China)

2007-06-15

This assay is a solid phase, two-site fluoroimmunometric assay based on the direct sandwish technique. Standards or samples containing ferritin are first reacted with immobilized anti-ferritin antibodies. Then the europium-lablled antibodies are reacted with the bound antigen. The range of this assay is 2-1000 ng/mL. The analytical sentivity is better than 0.05 ng/mL. The intra-assay variation and inter-assay variation are both below 5%; This kit was compared with Wallac DELFIA kit. The correlation is r=0.96. (authors)

Ecodynamics of oil-degrading bacteria and significance of marine mixed populations in the degradation of petroleum compounds

International Nuclear Information System (INIS)

Venkateswaran, Kasthuri; Tanaka, Hiroki; Komukai, Shyoko

1993-01-01

Ecological studies, screening of hydrocarbon-degrading bacteria, and studies of the potentials of various single and mixed bacterial populations in the utilization of petroleum compounds were carried out to understand the microbial hydrocarbon degradation process in marine ecosystems. Populations of hydrocarbon utilizers were larger in coastal regions than in pelagic environments. Ecological observations indicated that oil-degrading bacteria were ubiquitously distributed in both temperate and tropical environments, irrespective of oil-polluted and unpolluted ecosystem. Bacteria were grown with n-tet-radecane, pristane, propylbenzene, phenanthrene, and crude oil as the sole carbon source; and substrate specificities of the purified strains were characterized. Based on the assimilation characteristics of the isolated strains, an artificial mixed-culture system was constructed. Biodegradation of crude oil by the natural mixed population was found to be higher than by the artificial mixed population. However, when some of the substrate-specific degraders were artificially mixed with natural microflora, the degradation of hard-to-degrade aromatic hydrocarbon fractions of crude oil was enhanced

Geodiversity and land degradation in Hungary

Science.gov (United States)

Őrsi, Anna

2014-05-01

Geodiversity represents a variety of natural values, but they are threatened by a series of anthropogenic activities and land degradation processes. Their effect depends on the intensity of the processes and the sensitivity of the area in question. As a consequence of land degradation processes not only biodiversity but also geodiversity can be damaged and deteriorated. The appearance of the natural landscape changes and natural processes may not have a decisive role in landscape development any more. Some of the damages are irreversible because fundamental changes happen in the landscape, or the processes having created the original forms are no longer in operation. Small scale land degradation processes may be reversible if nature is still capable of reproducing the original state. The most important land degradation processes are desertification and soil erosion. Mining, waste disposal, urbanisation and construction activities, agriculture, inaccurate forest and water management, tourism, unsuitable land use can also lead to severe land degradation problems. The objective of the paper is to show Hungarian examples to all land degradation processes that threaten geodiversity. The results will be shown on a series of maps showing land degradation processes endangering geodiversity in Hungary. A detailed analysis of smaller study sites will be provided to show the effects of certain land degradation processes on landform development and on the changes of geodiversity. This research is supported by the Hungarian Scientific Research Fund (OTKA), project Nr. 10875.

Scintillation proximity assay

International Nuclear Information System (INIS)

Hart, H.

1980-01-01

In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

Insights into the processes behind the contamination of degraded human teeth and bone samples with exogenous sources of DNA

DEFF Research Database (Denmark)

Gilbert, M. T. P.; Hansen, Anders J.; Willerslev, E.

2006-01-01

A principal problem facing human DNA studies that use old and degraded remains is contamination from other sources of human DNA. In this study we have attempted to contaminate deliberately bones and teeth sampled from a medieval collection excavated in Trondheim, Norway, in order to investigate......, prior to assaying for the residual presence of the handler's DNA. Surprisingly, although our results suggest that a large proportion of the teeth were contaminated with multiple sources of human DNA prior to our investigation, we were unable to contaminate the samples with further human DNA. One...

Degradations and Rearrangement Reactions

Science.gov (United States)

Zhang, Jianbo

This section deals with recent reports concerning degradation and rearrangement reactions of free sugars as well as some glycosides. The transformations are classified in chemical and enzymatic ways. In addition, the Maillard reaction will be discussed as an example of degradation and rearrangement transformation and its application in current research in the fields of chemistry and biology.

COMPARATIVE ANALYSIS USING DIPIRONA DEGRADATION PROCESS WITH PHOTO-FENTON UV-C LIGHT AND SOLAR RADIATION

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Daniella Carla Napoleão

2015-01-01

Full Text Available The contamination of water bodies is a major concern on the part of scientists from different parts of the world. Domestic and industrial activities are the cause of the daily pouring of various types of pollutants which are in most cases resistant to conventional treatments of waters. Among the contaminants, especially noteworthy are the drugs in which it is found that 50% to 90% are discarded without treatment. The concerns about these substances are the adverse effects to human health and animals, especially in aquatic environments. The advanced oxidation processes (AOP have been studied and applied as an efficient alternative treatment, in order that it can be applied to the degradation of the different pollutants, considering that can generate hydroxyl radicals, highly reactive even somewhat selective. This study evaluated the efficiency of the photo-Fenton process using UV-C radiation and sunlight to degradation of the drug dipyrone in aqueous solution contaminated with the active ingredient of the drug at a concentration of 20 mg.L-1. Assays were performed with 50 mL aliquots of the solution following 23 factorial designs with central point, and the variables studied: addition of H2O2, adding FeSO4.7H2O and time. The detection and quantification of dipyrone before and after the AOP was performed by high performance liquid chromatography (HPLC and verified that about DE100% degradation of the compound was obtained.

Antifoam degradation testing

Energy Technology Data Exchange (ETDEWEB)

Lambert, D. P. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Zamecnik, J. R. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Newell, D. D. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Williams, M. S. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL)

2015-08-20

This report describes the results of testing to quantify the degradation products resulting from the dilution and storage of Antifoam 747. Antifoam degradation is of concern to the Defense Waste Processing Facility (DWPF) due to flammable decomposition products in the vapor phase of the Chemical Process Cell vessels, as well as the collection of flammable and organic species in the offgas condensate. The discovery that hexamethyldisiloxane is formed from the antifoam decomposition was the basis for a Potential Inadequacy in the Safety Analysis declaration by the DWPF.

Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes.

Science.gov (United States)

Davis, Carl K; Webb, Richard I; Sly, Lindsay I; Denman, Stuart E; McSweeney, Chris S

2012-06-01

Microbial dehalogenation of chlorinated compounds in anaerobic environments is well known, but the degradation of fluorinated compounds under similar conditions has rarely been described. Here, we report on the isolation of a bovine rumen bacterium that metabolizes fluoroacetate under anaerobic conditions, the mode of degradation and its presence in gut ecosystems. The bacterium was identified using 16S rRNA gene sequence analysis as belonging to the phylum Synergistetes and was designated strain MFA1. Growth was stimulated by amino acids with greater quantities of amino acids metabolized in the presence of fluoroacetate, but sugars were not fermented. Acetate, formate, propionate, isobutryate, isovalerate, ornithine and H(2) were end products of amino acid metabolism. Acetate was the primary end product of fluoroacetate dehalogenation, and the amount produced correlated with the stoichiometric release of fluoride which was confirmed using fluorine nuclear magnetic resonance ((19) F NMR) spectroscopy. Hydrogen and formate produced in situ were consumed during dehalogenation. The growth characteristics of strain MFA1 indicated that the bacterium may gain energy via reductive dehalogenation. This is the first study to identify a bacterium that can anaerobically dehalogenate fluoroacetate. Nested 16S rRNA gene-specific PCR assays detected the bacterium at low numbers in the gut of several herbivore species. © 2012 Commonwealth of Australia.

Assessing sediment contamination using six toxicity assays

Directory of Open Access Journals (Sweden)

Allen G. BURTON Jr.

2001-08-01

Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

Using microorganisms to aid in hydrocarbon degradation

International Nuclear Information System (INIS)

Black, W.; Zamora, J.

1993-01-01

Aliphatic hydrocarbons are threatening the potable water supply and the aquatic ecosystem. Given the right microbial inhabitant(s), a large portion of these aliphatic hydrocarbons could be biodegraded before reaching the water supply. The authors' purpose is to isolate possible oil-degrading organisms. Soil samples were taken from hydrocarbon-laden soils at petroleum terminals, a petroleum refinery waste-treatment facility, a sewage-treatment plant grease collector, a site of previous bioremediation, and various other places. Some isolates known to be good degraders were obtained from culture collection services. These samples were plated on a 10w-30 multigrade motor oil solid medium to screen for aliphatic hydrocarbon degraders. The degrading organisms were isolated, identified, and tested (CO 2 evolution, BOD, and COD) to determine the most efficient degrader(s). Thirty-seven organisms were tested, and the most efficient degraders were Serratia marcescens, Escherichia coli, and Enterobacter agglomerans

A Bayesian approach to degradation-based burn-in optimization for display products exhibiting two-phase degradation patterns

International Nuclear Information System (INIS)

Yuan, Tao; Bae, Suk Joo; Zhu, Xiaoyan

2016-01-01

Motivated by the two-phase degradation phenomena observed in light displays (e.g., plasma display panels (PDPs), organic light emitting diodes (OLEDs)), this study proposes a new degradation-based burn-in testing plan for display products exhibiting two-phase degradation patterns. The primary focus of the burn-in test in this study is to eliminate the initial rapid degradation phase, while the major purpose of traditional burn-in tests is to detect and eliminate early failures from weak units. A hierarchical Bayesian bi-exponential model is used to capture two-phase degradation patterns of the burn-in population. Mission reliability and total cost are introduced as planning criteria. The proposed burn-in approach accounts for unit-to-unit variability within the burn-in population, and uncertainty concerning the model parameters, mainly in the hierarchical Bayesian framework. Available pre-burn-in data is conveniently incorporated into the burn-in decision-making procedure. A practical example of PDP degradation data is used to illustrate the proposed methodology. The proposed method is compared to other approaches such as the maximum likelihood method or the change-point regression. - Highlights: • We propose a degradation-based burn-in test for products with two-phase degradation. • Mission reliability and total cost are used as planning criteria. • The proposed burn-in approach is built within the hierarchical Bayesian framework. • A practical example was used to illustrate the proposed methodology.

Thermal degradation of deoxynivalenol during maize bread baking.

Science.gov (United States)

Numanoglu, E; Gökmen, V; Uygun, U; Koksel, H

2012-01-01

The thermal degradation of deoxynivalenol (DON) was determined at isothermal baking conditions within the temperature range of 100-250°C, using a crust-like model, which was prepared with naturally contaminated maize flour. No degradation was observed at 100°C. For the temperatures of 150, 200 and 250°C, thermal degradation rate constants (k) were calculated and temperature dependence of DON degradation was observed by using Arrhenius equation. The degradation of DON obeyed Arrhenius law with a regression coefficient of 0.95. A classical bread baking operation was also performed at 250°C for 70 min and the rate of DON degradation in the bread was estimated by using the kinetic data derived from the model study. The crust and crumb temperatures recorded during bread baking were used to calculate the thermal degradation rate constants (k) and partial DON degradations at certain time intervals. Using these data, total degradation at the end of the entire baking process was predicted for both crust and crumb. This DON degradation was consistent with the experimental degradation data, confirming the accuracy of kinetic constants determined by means of the crust-like model.

Operationalizing measurement of forest degradation

DEFF Research Database (Denmark)

Dons, Klaus; Smith-Hall, Carsten; Meilby, Henrik

2015-01-01

. In Tanzania, charcoal production is considered a major cause of forest degradation, but is challenging to quantify due to sub-canopy biomass loss, remote production sites and illegal trade. We studied two charcoal production sites in dry Miombo woodland representing open woodland conditions near human......Quantification of forest degradation in monitoring and reporting as well as in historic baselines is among the most challenging tasks in national REDD+ strategies. However, a recently introduced option is to base monitoring systems on subnational conditions such as prevalent degradation activities...

Photocatalytic Degradation of 4-Nitrophenol by C, N-TiO2: Degradation Efficiency vs. Embryonic Toxicity of the Resulting Compounds

Science.gov (United States)

Osin, Oluwatomiwa A.; Yu, Tianyu; Cai, Xiaoming; Jiang, Yue; Peng, Guotao; Cheng, Xiaomei; Li, Ruibin; Qin, Yao; Lin, Sijie

2018-06-01

The photocatalytic activity of TiO2 based photocatalysts can be improved by structural modification and elemental doping. In this study, through rational design, one type of carbon and nitrogen co-doped TiO2 (C, N-TiO2) photocatalyst with mesoporous structure was synthesized with improved photocatalytic activity in degrading 4-nitrophenol under simulated sunlight irradiation. The photocatalytic degradation efficiency of the C, N-TiO2 was much higher than the anatase TiO2 (A-TiO2) based on absorbance and HPLC analyses. Moreover, using zebrafish embryos, we showed that the intermediate degradation compounds generated by photocatalytic degradation of 4-nitrophenol had higher toxicity than the parent compound. A repeated degradation process was necessary to render complete degradation and non-toxicity to the zebrafish embryos. Our results demonstrated the importance of evaluating the photocatalytic degradation efficiency in conjunction with the toxicity assessment of the degradation compounds.

X-ray induced degradation of DNA in Aspergillus nidulans cells comparative analysis of UV- and X-ray induced DNA degradation

International Nuclear Information System (INIS)

Zinchenko, V.V.; Babykin, M.M.

1980-01-01

Irradiating cells of Aspergillus nidulans of the wild type in the logarythmical growth phase with X-rays leads to a certain retention in DNA synthesis. This period is characterized by an insignificant fermentative DNA degradation connected with a process of its repair. There is no direct dependence between the radiation dose and the level of DNA degradation. The investigation of X-ray induced DNA degradation in a number of UVS-mutants permits to show the existence of two branches of DNA degradation - dependent and independent of the exogenic energy source. The dependence of DNA degradation on albumen synthesis prior to irradiation and after it, is demonstrated. It is supposed that the level of X-ray induced DNA degradation is determined by two albumen systems, one of which initiates degradation and the other terminates it. The comparative analysis of UV and X-ray induced DNA degradation is carried out

Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

Science.gov (United States)

Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

2006-01-01

Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

Science.gov (United States)

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

STRUCTURAL PERFORMANCE OF DEGRADED REINFORCED CONCRETE MEMBERS

International Nuclear Information System (INIS)

Braverman, J.I.; Miller, C.A.; Ellingwood, B.R.; Naus, D.J.; Hofmayer, C.H.; Bezler, P.; Chang, T.Y.

2001-01-01

This paper describes the results of a study to evaluate, in probabilistic terms, the effects of age-related degradation on the structural performance of reinforced concrete members at nuclear power plants. The paper focuses on degradation of reinforced concrete flexural members and shear walls due to the loss of steel reinforcing area and loss of concrete area (cracking/spalling). Loss of steel area is typically caused by corrosion while cracking and spalling can be caused by corrosion of reinforcing steel, freeze-thaw, or aggressive chemical attack. Structural performance in the presence of uncertainties is depicted by a fragility (or conditional probability of failure). The effects of degradation on the fragility of reinforced concrete members are calculated to assess the potential significance of various levels of degradation. The fragility modeling procedures applied to degraded concrete members can be used to assess the effects of degradation on plant risk and can lead to the development of probability-based degradation acceptance limits

Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

DEFF Research Database (Denmark)

Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.

1990-01-01

We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u......, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function......-PA-inhibitor complexes bound to the receptor with a Kd of approximately 100 pM at 4 degrees C. Transferring the cells to 37 degrees C led to degradation to amino acids of up to 50% of the cell-bound u-PA-inhibitor complexes, whereas the degradation of uncomplexed u-PA was 15%; the remaining ligand was recovered...

Assessment of the degradation efficiency of full-scale biogas plants: A comparative study of degradation indicators.

Science.gov (United States)

Li, Chao; Nges, Ivo Achu; Lu, Wenjing; Wang, Haoyu

2017-11-01

Increasing popularity and applications of the anaerobic digestion (AD) process has necessitated the development and identification of tools for obtaining reliable indicators of organic matter degradation rate and hence evaluate the process efficiency especially in full-scale, commercial biogas plants. In this study, four biogas plants (A1, A2, B and C) based on different feedstock, process configuration, scale and operational performance were selected and investigated. Results showed that the biochemical methane potential (BMP) based degradation rate could be use in incisively gauging process efficiency in lieu of the traditional degradation rate indicators. The BMP degradation rates ranged from 70 to 90% wherein plants A2 and C showed the highest throughput. This study, therefore, corroborates the feasibility of using the BMP degradation rate as a practical tool for evaluating process performance in full-scale biogas processes and spots light on the microbial diversity in full-scale biogas processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

Science.gov (United States)

Wang, Xiaofu; Chen, Xiaoyun; Xu, Junfeng; Dai, Chen; Shen, Wenbiao

2015-10-01

This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Degradation of shape memory effect

International Nuclear Information System (INIS)

Vandermeer, R.A.

1983-01-01

An important parameter for deciding whether or not a SME alloy is suitable for practical applications is the magnitude of the strain reversal accompanying martensite reversion. This research is concerned with elucidating metallurgical factors that cause degradation of this heat-activated recovery strain, E/sub R/. After explaining what is meant by degradation, two manifestations of degradation recently identified in near-monotectoid uranium-niobium alloys are described. The first was associated with the onset of plastic deformation of the martensite beyond the reversible strain limit, E/sub L/; a reduction of E/sub R/ from 5.25% at 8% total strain, i.e. E/sub L/, to 2.9% at 12% total strain was observed. A second type of degradation depended strongly on the heating rate during reversion; the E/sub R/ for an imposed strain of 6.95% was reduced from a value of 5.25% to 1.3% when the heating rate was decreased from 40 0 /sec to 0.05 0 /sec. Degradation was attributed to a change in the transformation path and the interjection of time-dependent, low temperature aging reactions

Characterization of the novel dimethyl sulfide-degrading bacterium Alcaligenes sp. SY1 and its biochemical degradation pathway

Energy Technology Data Exchange (ETDEWEB)

Sun, Yiming; Qiu, Jiguo; Chen, Dongzhi; Ye, Jiexu; Chen, Jianmeng, E-mail: jchen@zjut.edu.cn

2016-03-05

Highlights: • A novel efficient DMS-degrading bacterium Alcaligenes sp. SY1 was identified. • A RSM was applied to optimize incubation condition of Alcaligenes sp. SY1. • SIP was applied as C{sup 13} labelled DMS to trace intermediates during DMS degradation. • Kinetics of DMS degradation via batch experiment was revealed. • Carbon and sulfur balance were analyzed during DMS degradation process. - Abstract: Recently, the biodegradation of volatile organic sulfur compounds (VOSCs) has become a burgeoning field, with a growing focus on the reduction of VOSCs. The reduction of VOSCs encompasses both organic emission control and odor control. Herein, Alcaligenes sp. SY1 was isolated from active sludge and found to utilize dimethyl sulfide (DMS) as a growth substrate in a mineral salt medium. Response surface methodology (RSM) analysis was applied to optimize the incubation conditions. The following conditions for optimal degradation were identified: temperature 27.03 °C; pH 7.80; inoculum salinity 0.84%; and initial DMS concentration 1585.39 μM. Under these conditions, approximately 99% of the DMS was degraded within 30 h of incubation. Two metabolic compounds were detected and identified by gas chromatography–mass spectrometry (GC–MS): dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). The DMS degradation kinetics for different concentrations were evaluated using the Haldane–Andrews model and the pseudo first-order model. The maximum specific growth rate and degradation rate of Alcaligenes sp. SY1 were 0.17 h{sup −1} and 0.63 gs gx{sup −1} h{sup −1}. A possible degradation pathway is proposed, and the results suggest that Alcaligenes sp. SY1 has the potential to control odor emissions under aerobic conditions.

Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

International Nuclear Information System (INIS)

Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na; Guo, Qinglong

2013-01-01

Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function. �

Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

Energy Technology Data Exchange (ETDEWEB)

Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na, E-mail: luna555@163.com; Guo, Qinglong, E-mail: anticancer_drug@yahoo.com.cn

2013-09-01

Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function. �

Chitin Degradation In Marine Bacteria

DEFF Research Database (Denmark)

Paulsen, Sara; Machado, Henrique; Gram, Lone

2015-01-01

Introduction: Chitin is the most abundant polymer in the marine environment and the second most abundant in nature. Chitin does not accumulate on the ocean floor, because of microbial breakdown. Chitin degrading bacteria could have potential in the utilization of chitin as a renewable carbon...... and nitrogen source in the fermentation industry.Methods: Here, whole genome sequenced marine bacteria were screened for chitin degradation using phenotypic and in silico analyses.Results: The in silico analyses revealed the presence of three to nine chitinases in each strain, however the number of chitinases...... chitin regulatory system.Conclusions: This study has provided insight into the ecology of chitin degradation in marine bacteria. It also served as a basis for choosing a more efficient chitin degrading production strain e.g. for the use of chitin waste for large-scale fermentations....

[6]-Shogaol Inhibits α-MSH-Induced Melanogenesis through the Acceleration of ERK and PI3K/Akt-Mediated MITF Degradation

Directory of Open Access Journals (Sweden)

Huey-Chun Huang

2014-01-01

Full Text Available [6]-Shogaol is the main biologically active component of ginger. Previous reports showed that [6]-shogaol has several pharmacological characteristics, such as antioxidative, anti-inflammatory, antimicrobial, and anticarcinogenic properties. However, the effects of [6]-shogaol on melanogenesis remain to be elucidated. The study aimed to evaluate the potential skin whitening mechanisms of [6]-shogaol. The effects of [6]-shogaol on cell viability, melanin content, tyrosinase activity, and the expression of the tyrosinase and microphthalmia-associated transcription factor (MITF were measured. The results revealed that [6]-shogaol effectively suppresses tyrosinase activity and the amount of melanin and that those effects are more pronounced than those of arbutin. It was also found that [6]-shogaol decreased the protein expression levels of tyrosinase-related protein 1 (TRP-1 and microphthalmia-associated transcriptional factor (MITF. In addition, the MITF mRNA levels were also effectively decreased in the presence of 20 μM [6]-shogaol. The degradation of MITF protein was inhibited by the MEK 1-inhibitor (U0126 or phosphatidylinositol-3-kinase inhibitor (PI3K inhibitor (LY294002. Further immunofluorescence staining assay implied the involvement of the proteasome in the downregulation of MITF by [6]-shogaol. Our confocal assay results also confirmed that [6]-shogaol inhibited α-melanocyte stimulating hormone- (α-MSH- induced melanogenesis through the acceleration of extracellular responsive kinase (ERK and phosphatidylinositol-3-kinase- (PI3K/Akt- mediated MITF degradation.

Chromium related degradation of solid oxide fuel cells; Chrom-bezogene Degradation von Festoxid-Brennstoffzellen

Energy Technology Data Exchange (ETDEWEB)

Neumann, Anita

2011-05-04

Solid Oxide Fuel Cells (SOFCs) offer a high potential for application as an auxiliary power unit (APU) for heavy goods vehicles as well as combined heat and power (CHP) systems. SOFCs are especially attractive due to their high efficiencies and the use of different fuel types. However, optimization in terms of long term stability and costs are still necessary. This work characterized the degradation of SOFCs with lanthanum strontium manganite (LSM) cathodes under chromium influence. Galvanostatic cell tests were carried out at 800 C with operation times from 250 - 3000 h and variation of the chromium source and current density. The current densities of j = 0 (A)/(cm{sup 2}), j = 0,3 (A)/(cm{sup 2}) and j = 0,5 (A)/(cm{sup 2}) were applied. The high temperature ferritic alloy Crofer22APU was used as a chromium source. Variation of the chromium source was realized by coating the Crofer22APU insert with the chromium retention layer Mn{sub 3}O{sub 4} and the cathode contact layer LCC10. Cell degradation was analyzed with regard to cell voltage, current density and area specific resistance (ASR). Microstructural alterations of the cathode as well as chromium content and distribution across the cell were investigated after completion of the cell tests. For cells with a chromium source present and operation with a nonzero current density, the course of cell degradation was divided into three phases: a run-in, weak linear degradation and strong linear degradation. A decrease of the chromium release rate by means of different coatings stretched the course of degradation along the timescale. Strong degradation, which is characterized by a significant increase in ASR as well as a decrease of current density at the operating point, was only observed when a chromium source in the setup was comb ined with operation of the cell with a non-zero current density. Operation of the cell with a chromium source but no current density caused a degradation of current density at the

A quantitative comet infection assay for influenza virus

Science.gov (United States)

Lindsay, Stephen M.; Timm, Andrea; Yin, John

2011-01-01

Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

Science.gov (United States)

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

Assay development status report for total cyanide

International Nuclear Information System (INIS)

Simpson, B.C.; Jones, T.E.; Pool, K.H.

1993-02-01

A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN - ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation

Prospects for cellular mutational assays in human populations

International Nuclear Information System (INIS)

Mendelsohn, M.L.

1984-01-01

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

Prospects for cellular mutational assays in human populations

Energy Technology Data Exchange (ETDEWEB)

Mendelsohn, M.L.

1984-06-29

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

A radiochemical assay for biotin in biological materials

International Nuclear Information System (INIS)

Hood, R.L.

1975-01-01

A radiochemical assay for biotin is described. The assay was sensitive to one nanogram and simple enough for routine biotin analyses. The assay yielded results which were comparable to those obtained from a microbiological assay using Lactobacillus plantarum. (author)

A condition-based maintenance of a dependent degradation-threshold-shock model in a system with multiple degradation processes

International Nuclear Information System (INIS)

Caballé, N.C.; Castro, I.T.; Pérez, C.J.; Lanza-Gutiérrez, J.M.

2015-01-01

This paper proposes a condition-based maintenance strategy for a system subject to two dependent causes of failure: degradation and sudden shocks. The internal degradation is reflected by the presence of multiple degradation processes in the system. Degradation processes start at random times following a Non-homogeneous Poisson process and their growths are modelled by using a gamma process. When the deterioration level of a degradation process exceeds a predetermined value, we assume that a degradation failure occurs. Furthermore, the system is subject to sudden shocks that arrive at the system following a Doubly Stochastic Poisson Process. A sudden shock provokes the total breakdown of the system. Thus, the state of the system is evaluated at inspection times and different maintenance tasks can be carried out. If the system is still working at an inspection time, a preventive maintenance task is performed if the deterioration level of a degradation process exceeds a certain threshold. A corrective maintenance task is performed if the system is down at an inspection time. A preventive (corrective) maintenance task implies the replacement of the system by a new one. Under this maintenance strategy, the expected cost rate function is obtained. A numerical example illustrates the analytical results. - Highlights: • A condition-based maintenance model is proposed. • Two dependent causes of failure are considered: deterioration and external shocks. • Deterioration is given by multiple degradation processes growing by a gamma process. • The initiation of degradation processes follows a Non-homogeneous Poisson process. • External shocks arrive at the system by using a Doubly Stochastic Poisson Process

WEATHERABILITY OF ENHANCED DEGRADABLE PLASTICS

Science.gov (United States)

The main objective of this study was to assess the performance and the asociated variability of several selected enhanced degradable plastic materials under a variety of different exposure conditions. Other objectives were to identify the major products formed during degradation ...

MOSFET Degradation Under RF Stress

NARCIS (Netherlands)

Sasse, G.T.; Kuper, F.G.; Schmitz, Jurriaan

2008-01-01

We report on the degradation of MOS transistors under RF stress. Hot-carrier degradation, negative-bias temperature instability, and gate dielectric breakdown are investigated. The findings are compared to established voltage- and field-driven models. The experimental results indicate that the

Biocompatibility and bond degradation of poly-acrylic acid coated copper iodide-adhesives.

Science.gov (United States)

ALGhanem, Adi; Fernandes, Gabriela; Visser, Michelle; Dziak, Rosemary; Renné, Walter G; Sabatini, Camila

2017-09-01

To investigate the effect of poly-acrylic acid (PAA) copper iodide (CuI) adhesives on bond degradation, tensile strength, and biocompatibility. PAA-CuI particles were incorporated into Optibond XTR, Optibond Solo and XP Bond in 0.1 and 0.5mg/ml. Clearfil SE Protect, an MDPB-containing adhesive, was used as control. The adhesives were applied to human dentin, polymerized and restored with composite in 2mm-increments. Resin-dentin beams (0.9±0.1mm 2 ) were evaluated for micro-tensile bond strength after 24h, 6 months and 1year. Hourglass specimens (10×2×1mm) were evaluated for ultimate tensile strength (UTS). Cell metabolic function of human gingival fibroblast cells exposed to adhesive discs (8×1mm) was assessed with MTT assay. Copper release from adhesive discs (5×1mm) was evaluated with UV-vis spectrophotometer after immersion in 0.9% NaCl for 1, 3, 5, 7, 10, 14, 21 and 30 days. SEM, EDX and XRF were conducted for microstructure characterization. XTR and Solo did not show degradation when modified with PAA-CuI regardless of the concentration. The UTS for adhesives containing PAA-CuI remained unaltered relative to the controls. The percent viable cells were reduced for Solo 0.5mg/ml and XP 0.1 or 0.5mg/ml PAA-CuI. XP demonstrated the highest ion release. For all groups, the highest release was observed at days 1 and 14. PAA-CuI particles prevented the bond degradation of XTR and Solo after 1year without an effect on the UTS for any adhesive. Cell viability was affected for some adhesives. A similar pattern of copper release was demonstrated for all adhesives. Copyright © 2017. Published by Elsevier Ltd.

Gamma Radiolytic Degradation of Heptachlor in Methanol and Monitoring of Degradation by HPLC

International Nuclear Information System (INIS)

Riaz, M.; Butt, S.B.

2014-01-01

Removal of known insecticide Heptachlor (HPTC) in methanol solution by gamma-rays under varied experimental conditions has been optimized. Air saturated solution of HPTC was irradiated at x-rays dose from 1 to 10 kGys. The extent of radiolytic degradation was monitored by reversed phase high performance liquid chromatography (HPLC) coupled with UV detector. At dose of 10 kGys gamma 98 % of HPTC was degraded. The degradation of HPTC occurs by CH/sub 3/O and CH/sub 2/OH radicals generated by methanol radiolysis. It is concluded that gamma-rays can remove Persistent Organic Pollutants (POPs) form environmental matrices. It can decrease the harmful properties of these POPs by their transformation into less resistant fragments to biological / natural elimination in the aquatic atmosphere. (author)

Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

Energy Technology Data Exchange (ETDEWEB)

Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Gye Won [Department of Pharmaceutical Engineering, Konyang University, Nonsan 320-711 (Korea, Republic of); Yun, Mi-Young [Department of Beauty Health Care, Daejeon University, Daejeon 305-764 (Korea, Republic of); Cuong, Nguyen Manh [Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi (Viet Nam); Shin, Jae-Gook [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Song, Gyu-Yong, E-mail: gysong@cnu.ac.kr [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Oh, Sangtaek, E-mail: ohsa@inje.ac.kr [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)

2010-01-01

Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

Linearization of the Bradford Protein Assay

OpenAIRE

Ernst, Orna; Zor, Tsaffrir

2010-01-01

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

New automated pellet/powder assay system