Оптимизация преподавания темы "Диагностика и лечение атеросклероза" на факультете повышения квалификации врачей

ФАКУЛЬТЕТЫ ПОВЫШЕНИЯ КВАЛИФИКАЦИИПОВЫШЕНИЕ КВАЛИФИКАЦИИОБРАЗОВАНИЕ МЕДИЦИНСКОЕ, ПОВЫШЕНИЕ КВАЛИФИКАЦИИПРЕПОДАВАНИЕМЕТОДИКА ПРЕПОДАВАНИЯОБРАЗОВАТЕЛЬНЫЕ ТЕХНОЛОГИ

Fast X-Ray Fluorescence Microtomography of Hydrated Biological Samples

Metals and metalloids play a key role in plant and other biological systems as some of them are essential to living organisms and all can be toxic at high concentrations. It is therefore important to understand how they are accumulated, complexed and transported within plants. In situ imaging of metal distribution at physiological relevant concentrations in highly hydrated biological systems is technically challenging. In the case of roots, this is mainly due to the possibility of artifacts arising during sample preparation such as cross sectioning. Synchrotron x-ray fluorescence microtomography has been used to obtain virtual cross sections of elemental distributions. However, traditionally this technique requires long data acquisition times. This has prohibited its application to highly hydrated biological samples which suffer both radiation damage and dehydration during extended analysis. However, recent advances in fast detectors coupled with powerful data acquisition approaches and suitable sample preparation methods can circumvent this problem. We demonstrate the heightened potential of this technique by imaging the distribution of nickel and zinc in hydrated plant roots. Although 3D tomography was still impeded by radiation damage, we successfully collected 2D tomograms of hydrated plant roots exposed to environmentally relevant metal concentrations for short periods of time. To our knowledge, this is the first published example of the possibilities offered by a new generation of fast fluorescence detectors to investigate metal and metalloid distribution in radiation-sensitive, biological samples

Disialo–trisialo bridging of transferrin is due to increased branching and fucosylation of the carbohydrate moiety

Background Carbohydrate deficient transferrin (CDT) is used for detection of alcohol abuse and follow-up. High performance liquid chromatography (HPLC) of transferrin glycoforms is highly specific for identification of alcohol abuse, but unresolved disialo- and trisialotransferrin glycoforms sometimes makes interpretation difficult. The cause of this phenomenon is unknown, cannot be explained by genetic variants of transferrin, but seems to be associated with liver disease. Methods Nineteen serum samples showing di–tri bridging when analyzed by HPLC were collected. Transferrin was purified by affinity chromatography, and N-linked oligosaccharides were released enzymatically. The N-glycans were further analyzed by high performance anion-exchange chromatography with pulsed amperometric detection and MALDI-TOF mass spectrometry. Results The HPLC-analysis showed three different types of glycoform patterns. The N-glycans from fifteen samples showed patterns with increased number of triantennary structures containing one or two fucose residues. One sample contained an increased amount of triantennary glycans without fucose. Three samples showed a glycosylation pattern similar to normal transferrin. Conclusions The di–tri bridging phenomenon was associated with alterations in transferrin glycosylation in the majority of cases. Transferrin contained a higher extent of triantennary and often fucosylated N-linked oligosaccharides. These results may be important in future diagnostic approaches to liver diseases.Funding Agencies|Medical Research Council of Southeast Sweden||</p

Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography

Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coil as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked alpha 1-3 to GIcNAc interacted with higher affinity compared to fucose linked alpha 1-6 or alpha 1-4 and the obtained dissociation constants (K-d) were in the range of 10 mu M for all AAL forms. Tetra- and pentasaccharides with fucose in alpha 1-2, alpha 1-3 or alpha 1-4 had K-d values ranging from 0.1 to 7 mM while a large alpha 1-6 fucosylated oligosaccharide had a K-d of about 20 mu M. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.Funding Agencies|Linnaeus University||Linkoping University||Medical council of Southeast Sweden|

Disialo–trisialo bridging of transferrin is due to increased branching and fucosylation of the carbohydrate moiety

Background Carbohydrate deficient transferrin (CDT) is used for detection of alcohol abuse and follow-up. High performance liquid chromatography (HPLC) of transferrin glycoforms is highly specific for identification of alcohol abuse, but unresolved disialo- and trisialotransferrin glycoforms sometimes makes interpretation difficult. The cause of this phenomenon is unknown, cannot be explained by genetic variants of transferrin, but seems to be associated with liver disease. Methods Nineteen serum samples showing di–tri bridging when analyzed by HPLC were collected. Transferrin was purified by affinity chromatography, and N-linked oligosaccharides were released enzymatically. The N-glycans were further analyzed by high performance anion-exchange chromatography with pulsed amperometric detection and MALDI-TOF mass spectrometry. Results The HPLC-analysis showed three different types of glycoform patterns. The N-glycans from fifteen samples showed patterns with increased number of triantennary structures containing one or two fucose residues. One sample contained an increased amount of triantennary glycans without fucose. Three samples showed a glycosylation pattern similar to normal transferrin. Conclusions The di–tri bridging phenomenon was associated with alterations in transferrin glycosylation in the majority of cases. Transferrin contained a higher extent of triantennary and often fucosylated N-linked oligosaccharides. These results may be important in future diagnostic approaches to liver diseases.Funding Agencies|Medical Research Council of Southeast Sweden||</p

Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography

Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coil as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked alpha 1-3 to GIcNAc interacted with higher affinity compared to fucose linked alpha 1-6 or alpha 1-4 and the obtained dissociation constants (K-d) were in the range of 10 mu M for all AAL forms. Tetra- and pentasaccharides with fucose in alpha 1-2, alpha 1-3 or alpha 1-4 had K-d values ranging from 0.1 to 7 mM while a large alpha 1-6 fucosylated oligosaccharide had a K-d of about 20 mu M. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.Funding Agencies|Linnaeus University||Linkoping University||Medical council of Southeast Sweden|

Determination of Fucose Concentration in a Lectin-Based Displacement Microfluidic Assay

We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.Funding Agencies|Health Research Council of Southeast of Sweden [FORSS-389021]; LIST grant from Linkoping University</p

Copyright: Elsevier

Disialo–trisialo bridging of transferrin is due to increased branching and fucosylation of the carbohydrate moiet