139 research outputs found
The FORA Framework - A Fuzzy Grassroots Ontology for Online Reputation Management
No full textOnline reputation management deals with monitoring and influencing the online record of a person, an organization or a product. The Social Web offers increasingly simple ways to publish and disseminate personal or opinionated information, which can rapidly have a disastrous influence on the online reputation of some of the entities. The author focuses on the Social Web and possibilities of its integration with the Semantic Web as resource for a semi-automated tracking of online reputations using imprecise natural language terms. The inherent structure of natural language supports humans not only in communication but also in the perception of the world. Thereby fuzziness is a promising tool for transforming those human perceptions into computer artifacts. Through fuzzy grassroots ontologies, the Social Semantic Web becomes more naturally and thus can streamline online reputation management. For readers interested in the cross-over field of computer science, information systems, and social sciences, this book is an ideal source for becoming acquainted with the evolving field of fuzzy online reputation management in the Social Semantic Web area.
Identification of pollen types of beekeeping interest by non-targeted mass spectrometry
No full textBackground
The identification of pollen is important in the field of beekeeping for the determination of the botanical origin of bee products and investigations of bee diet. Until now, it has been performed by melissopalynology, the microscopic examination of pollen grains. However, this technique has some limitations, such as the necessity of experienced analysts and identification restricted to the family level for some pollen types. Although many techniques have been proposed as alternatives or complements to melissopalynology and omics techniques have been explored to gather information on the botanical origin of honey, no study has yet been conducted on a large set of pollen types.
Results
The study dataset consisted of 34 different pollen types of pellets collected by honeybees in Switzerland and analyzed in multiple biological replications, leading to 150 observations. The pollen samples were analyzed after tryptic digestion using a non-targeted mass spectrometry-based method. Liquid chromatography coupled with mass spectrometry (LC–MS) was employed to identify pollen, and melissopalynology was used as a reference method for the identification. We built an OPLS-DA prediction model for the 34 pollen types. The model clearly identified new samples in their membership group (Acer sp., n = 10) and a new pollen type at the species-specific level for Quercus sp. Less predictable results were achieved for Composita H and pollen collected directly from the plant.
Conclusion
The use of a non-targeted mass spectrometry-based method and chemometrics resulted in a promising tool for pollen identification as a replacement/supplement method to traditional melissopalynology
From food to physiology characterization and in vitro digestion of milk products and investigation of postprandial metabolism and inflammation after a high-fat meal intake in a human nutrition intervention study
No full textAccording to the World Health Organization worldwide obesity has more than doubled since 1980. The World health statistics 2012 report shows that one in six adults is obese, one in ten diabetic and one in three has a raised blood pressure. Overweight and obesity are major risk factors for cardiovascular diseases,According to the World Health Organization worldwide obesity has more than doubled since 1980. The World health statistics 2012 report shows that one in six adults is obese, one in ten diabetic and one in three has a raised blood pressure. Overweight and obesity are major risk factors for cardiovascular diseases, diabetes and some cancers. The causes of overweight and obesity are mainly a decrease in physical activity and an increased intake of energy-dense foods leading to a positive energy balance. Not only dietary patterns play a major role in the development of obesity. Food composition also directly impacts on postprandial metabolism and inflammation and, in long term, may contribute to systemic low-grade inflammation, a characteristic associated with the obese state. Thus it is not surprising that there is a growing interest in better understanding the effect of different foods and food compounds on human metabolism and health. The aim of the NutriChip project is to develop a microfluidic chip device to screen foods and their compounds for health promoting, e.g. immune-modulatory, properties. Milk products have been chosen as a food model because their consumption has been shown to be associated with decreased levels of inflammatory markers (1), and also because technological and microbiological transformations allow the realization of various products, such as yoghurt and cheese, having potentially different physiological effects. This thesis covers the biochemical (products characterization and in vitro digestion) and physiological (human nutrition study) aspects of the NutriChip project. Initially, differently heat-treated and fermented milk products were characterized by a proteomic approach. Various fractionation methods were used for selectively enriching minor milk and bacterial proteins in different dairy products. Proteins were separated and identified by two dimensional (2D) gel-electrophoresis and liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis. All data are collected in an interactive platform, called protein atlas, which is now publicly available on http://www.foodle.ch/de/proteinstart. Currently, the database contains more than 200 different milk proteins and about 250 bacterial proteins. For a better in depth characterization of fermented products, a method for the enrichment of living bacterial cells has been developed. This method allows the investigation of bacterial proteomes under different fermentation conditions. The method was used to monitor the adaption of the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus during milk fermentation. Some well-known stress response proteins (chaperone proteins GroEL and DnaK) and various enzymes involved in the glycolysis pathway have been identified as a proof of concept. Another experiment aimed to investigate the bacterial proteome during cheese production with special regards towards the identification of enzymes involved in the formation of the cheese flavor compound 3-methylbutanal. An aminotransferase that catalyzes the first step in the conversion pathway from the amino acid leucine to the flavor compound 3-methylbutanal could be identified. A prerequisite for screening foods for health-modulating activity was the development of a static, three-step in vitro digestion model. The aim was to keep the model as physiological as possible and ideally perform the digestion on a small scale (volume < 15 mL). The digestion model has been thoroughly validated using pasteurized whole milk. The degradation of fat, carbohydrates and proteins into their basic constituents was consistent with human physiological values found in the literature. The system has been used to digest various milk products. With the two proteins α-s1-casein and β-lactoglobulin, a representative of the caseins and whey proteins, respectively, the influence of the fat content, heat treatment and fermentation of the products on their digestibility has been monitored. Particularly we also looked at the generation of bioactive peptides during the digestion process. Towards this aim, the identified peptides in the digestion experiments were compared with bioactive peptide sequences from the literature using the statistic program R. Over 50 milk protein-derived peptides containing bioactive peptide sequences, with e.g. antihypertensive, immune- and cyto-modulatory, opioid functions, could be identified. The next step in simulating the human digestive system is the intestinal transport. Therefore, a cell culture model mimicking the last step of digestion and final absorption of the nutrients, both steps being mediated by intestinal enterocytes, was established. For this aim, Caco-2 cells were used in a Transwell system with an upper and lower compartment containing cell culture medium. Digested milk products were added on top of the Caco-2 cell monolayer and medium was collected from the basolateral chamber for identification of transported, potential bioactive peptides. The final validation of the in vitro models needs the comparison with a human nutrition intervention study. We designed and conducted a dose-response intervention study to determine the caloric dose (500 kcal, 1’000 kcal, 1’500 kcal) of a high-fat meal needed to induce a postprandial metabolic and inflammatory response in normal weight and obese subjects. In our study, we investigated postprandial metabolism and inflammation by measuring classical clinical parameters such as glucose, insulin and triglycerides, as well as the inflammation markers C-reactive protein (CRP), interleukine-6 (IL-6) and endotoxin before and at various time points after the test meals consumption. Our study provided valuable clinical, mechanistic, and methodological insights into the metabolic response of subjects, varying in their metabolic health status, to increasing doses of a high-fat meal. This represents the basis for future studies aiming to investigate health-promoting properties of foods in general, as also their capability in lowering postprandial inflammation, a normal response mechanism after food ingestion. v diabetes and some cancers. The causes of overweight and obesity are mainly a decrease in physical activity and an increased intake of energy-dense foods leading to a positive energy balance. Not only dietary patterns play a major role in the development of obesity. Food composition also directly impacts on postprandial metabolism and inflammation and, in long term, may contribute to systemic low-grade inflammation, a characteristic associated with the obese state. Thus it is not surprising that there is a growing interest in better understanding the effect of different foods and food compounds on human metabolism and health. The aim of the NutriChip project is to develop a microfluidic chip device to screen foods and their compounds for health promoting, e.g. immune-modulatory, properties. Milk products have been chosen as a food model because their consumption has been shown to be associated with decreased levels of inflammatory markers (1), and also because technological and microbiological transformations allow the realization of various products, such as yoghurt and cheese, having potentially different physiological effects. This thesis covers the biochemical (products characterization and in vitro digestion) and physiological (human nutrition study) aspects of the NutriChip project. Initially, differently heat-treated and fermented milk products were characterized by a proteomic approach. Various fractionation methods were used for selectively enriching minor milk and bacterial proteins in different dairy products. Proteins were separated and identified by two dimensional (2D) gel-electrophoresis and liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis. All data are collected in an interactive platform, called protein atlas, which is now publicly available on http://www.foodle.ch/de/proteinstart. Currently, the database contains more than 200 different milk proteins and about 250 bacterial proteins. For a better in depth characterization of fermented products, a method for the enrichment of living bacterial cells has been developed. This method allows the investigation of bacterial proteomes under different fermentation conditions. The method was used to monitor the adaption of the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus during milk fermentation. Some well- known stress response proteins (chaperone proteins GroEL and DnaK) and various enzymes involved in the glycolysis pathway have been identified as a proof of concept. Another experiment aimed to investigate the bacterial proteome during cheese production with special regards towards the identification of enzymes involved in the formation of the cheese flavor compound 3-methylbutanal. An aminotransferase that catalyzes the first step in the conversion pathway from the amino acid leucine to the flavor compound 3-methylbutanal could be identified. v A prerequisite for screening foods for health-modulating activity was the development of a static, three-step in vitro digestion model. The aim was to keep the model as physiological as possible and ideally perform the digestion on a small scale (volume < 15 mL). The digestion model has been thoroughly validated using pasteurized whole milk. The degradation of fat, carbohydrates and proteins into their basic constituents was consistent with human physiological values found in the literature. The system has been used to digest various milk products. With the two proteins α-s1-casein and β-lactoglobulin, a representative of the caseins and whey proteins, respectively, the influence of the fat content, heat treatment and fermentation of the products on their digestibility has been monitored. Particularly we also looked at the generation of bioactive peptides during the digestion process. Towards this aim, the identified peptides in the digestion experiments were compared with bioactive peptide sequences from the literature using the statistic program R. Over 50 milk protein-derived peptides containing bioactive peptide sequences, with e.g. antihypertensive, immune- and cyto-modulatory, opioid functions, could be identified. The next step in simulating the human digestive system is the intestinal transport. Therefore, a cell culture model mimicking the last step of digestion and final absorption of the nutrients, both steps being mediated by intestinal enterocytes, was established. For this aim, Caco-2 cells were used in a Transwell system with an upper and lower compartment containing cell culture medium. Digested milk products were added on top of the Caco-2 cell monolayer and medium was collected from the basolateral chamber for identification of transported, potential bioactive peptides. The final validation of the in vitro models needs the comparison with a human nutrition intervention study. We designed and conducted a dose-response intervention study to determine the caloric dose (500 kcal, 1’000 kcal, 1’500 kcal) of a high-fat meal needed to induce a postprandial metabolic and inflammatory response in normal weight and obese subjects. In our study, we investigated postprandial metabolism and inflammation by measuring classical clinical parameters such as glucose, insulin and triglycerides, as well as the inflammation markers C-reactive protein (CRP), interleukine-6 (IL-6) and endotoxin before and at various time points after the test meals consumption. Our study provided valuable clinical, mechanistic, and methodological insights into the metabolic response of subjects, varying in their metabolic health status, to increasing doses of a high-fat meal. This represents the basis for future studies aiming to investigate health- promoting properties of foods in general, as also their capability in lowering postprandial inflammation, a normal response mechanism after food ingestion.LMIS
Behavior of the Chiral Herbicide Imazamox in Soils: pH-Dependent, Enantioselective Degradation, Formation and Degradation of Several Chiral Metabolites
No full textMany pesticides show a pronounced
biphasic degradation in soil,
typically with a faster initial phase, followed by a slower decline.
For chiral compounds, a biphasic decline of the total concentration
may result from enantioselective degradation. In this study with the
chiral herbicide imazamox, biphasic degradation was observed in most
of the 18 soils investigated. In neutral soils, degradation was, in
fact, enantioselective with faster degradation of (+)-imazamox. In
slightly acidic soils, differences between enantiomers were not pronounced,
and in strongly acidic soils, degradation was again enantioselective,
but with reversed preference. Additional experiments with pure enantiomers
indicated no interconversion. Enantioselective degradation thus contributed
to the biphasic decline of the total concentration in certain soils.
However, this was not the only factor since degradation of the individual
enantiomers was biphasic in itself. In addition to the observed correlation
between enantioselectivity and pH, degradation was generally faster
in neutral than in acidic soils with half-lives ranging from only
2 to >120 days. Half-lives were also determined for two known metabolites
and a further chiral metabolite, the structure of which was characterized
by high resolution tandem mass spectrometry. As for the parent compound,
half-lives of the metabolites varied considerably in the different
soils
Turning placenta into brain: placental mesenchymal stem cells differentiate into neurons and oligodendrocytes
No full textObjective
We aimed to induce neural stem (NSC) and progenitor cells (NPC) from human placental tissues.
Study Design
Placental stem cells from first-trimester placental chorionic villi and term chorion were isolated. Neural differentiation was initiated with plating on collagen, retinoic acid, and/or human brain-derived neurotrophic factor and epidermal and fibroblast growth factor. Differentiation into neurons, oligodendrocytes, and astrocytes was monitored by immunohistochemistry. Two-dimensional polyacrylamide gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry were used to identify proteins involved in the differentiation.
Results
Differentiated cells were mostly immediately postmitotic with some more but not fully mature postmitotic neurons. Neurons had dopaminergic or serotonergic character. Some cells differentiated into predominantly immature oligodendrocytes. Upon differentiation, neuron-specific proteins were up-regulated, whereas placental proteins were reduced.
Conclusion
Stem cells derived from human placenta can be differentiated into neural progenitors
Purification and functional reconstitution of the human Wilson copper ATPase, ATP7B
No full textAbstractWilson disease is a disorder of copper metabolism, due to inherited mutations in the Wilson copper ATPase gene ATP7B. To purify and study the function of the ATPase, the enzyme was truncated by five of the six metal binding domains and endowed with an N-terminal histidine-tag for affinity purification. This construct, Δ1–5WNDP, was able to functionally complement a yeast strain defective in its native copper ATPase CCC2. Δ1–5WNDP was purified by Ni-affinity chromatography and reconstituted into proteoliposomes. This allowed, for the first time, the functional study of the Wilson ATPase in a purified, reconstituted system
Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking
No full textThe CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived
CopY-like copper inducible repressors are putative 'winged helix' proteins
No full textCopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of 'winged helix' proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same alpha-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the 'winged helix' type
Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae
No full textQuantitative Characterization of Digestion Processes
No full textThe growing interest in understanding the molecular mechanisms of digestive processes led to an increasing number of different in vivo and in vitro studies focusing on the understanding of food digestion and the physiological impacts on the human body. For this purpose, dedicated analytical methods were developed to characterize and quantify the different intermediate and final products of nutrient hydrolysis. However, one major issue is the comparability of analytical results between different experimental setups and individual labs. The COST action Infogest network (http://www.cost-infogest.eu/) specifically defined the experimental conditions and the corresponding enzymatic tests for a harmonized in vitro digestion protocol based on physiological data from literature. The new protocol increased the reproducibility of results between labs and between different studies. Moreover, the analytical methods developed in parallel, allow for the verification of the progress in nutrient hydrolysis and the variations between different experiments. This book chapter gives a short introduction on the in vitro digestion protocol and describes the analytical methods suited for the assessment of protein digestion during IVD. Moreover, methods for the quantification of lipids and carbohydrates are described. The final part of this chapter offers a selection of analytical results showing the relationship between food digestion and nutrition
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