| 1. |
- Duong-Thi, Minh-Dao, et al.
(författare)
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Comparison of weak affinity chromatography and surface plasmon resonance in determining affinity of small molecules
- 2014
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 461, s. 57-59
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we compared affinity data from surface plasmon resonance (SPR) and weak affinity chromatography (WAC), two established techniques for determination of weak affinity (mM-mu M) small molecule-protein interactions. In the current comparison, thrombin was used as target protein. In WAC the affinity constant (K-D) was determined from retention times, and in SPR it was determined by Langmuir isotherm fitting of steady-state responses. Results indicate a strong correlation between the two methods (R-2 = 0.995, P < 0.0001). (C) 2014 Elsevier Inc. All rights reserved.
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| 2. |
- Bergström, Maria, et al.
(författare)
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Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography
- 2012
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Ingår i: Journal of chromatography. B. - : Elsevier. - 1570-0232 .- 1873-376X. ; 885, s. 66-72
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Bergström, Sten-Erik
(författare)
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Asthma and allergy in teenagers and young adults, risk-factors and T-cell regulation
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Asthma is one of the most common chronic diseases among teenagers and young adults. The prevalence of asthma among young adults in Sweden is approximately 7-10%. Despite this, only a limited number of studies have focused on asthma, allergy and allergic inflammation in this age group. The aims of this thesis are to study the consequences of asthma and allergy in teenagers and young adults, incidence and riskfactors for death due to asthma, and deterioration in asthma prior and following transfer from pediatric to adult health care. As allergic inflammation is involved in a majority of asthma patients in this age-group we have further investigated a T cell mediated inflammatory mechanism with possible implications in monitoring and modulating autoimmune and allergic diseases. Paper I: During the 1994-2003 period 37 deaths due to asthma were identified. The incidence of asthma in 1-34 year-olds decreased during the period from 1.54 to 0.53 per million. Common risk-factors were under-treatment, poor adherence to prescribed treatment and adverse psychosocial situation. An alarming finding was that 11/37 deaths was probably caused by food allergy and 8/37 were associated with exposure to pet dander. Paper II: In a 5-year prospective follow-up study to identify risk factors for deterioration of asthma following transfer from pediatric to adult health care 150 teenagers with asthma were enrolled. Skin prick test at entrance revealed that 89% were sensitized towards at least one of tested allergens. A minority performed with impaired lung function without deterioration during the five-year follow up, while bronchial hyper responsiveness (BHR) was present in 71% of the subjects at entrance and among 59% at follow-up. Risk for persistence of BHR after five years was elevated by poor adherence and attenuated by regular physical activity. Working capacity decreased significantly during the study period without any correlation to risk factors examined. Paper III: Interactions between the low-density lipoprotein receptor-related protein 1(LRP1) and thrombospondin-1 (TSP-1) is necessarily for T cell motility and that the motogenic LRP/TSP-1 mechanism antagonizes adhesion to ICAM-1 and fibronectin as well as TCR induced proliferative responses. This cascade mediates regulatory effects of IL-2 and IL-4. In addition expression of TSP-1, with known ability to protect against inflammation, was increased by IL-2. Paper IV: T cell activation induces arrest of T cell motility through down-regulation of LRP1 synthesis a concomitant up-regulation of TSP-1 synthesis providing a mechanism for enhancement of adhesion of T cells to APC´s stimulating proliferative responses. Despite this arrest of motility, co-ligation with CD28 maintains a basal motility level by enhancing transport of LRP1 to the cell surface. Paper V: Patients with allergy and psoriasis showed impaired T cell motility and decreased TSP-1 expression compared to healthy controls. IL-2 was shown to up-regulate the impaired motility in patient to the same level as in controls indicating a reversible state probably excluding a constitutional defect.
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| 4. |
- Bergström, Sten Sture, et al.
(författare)
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Concave and convex phases in ambiguous figures showing colour shifts : Mach's figure and the AMBEGUJAS phenomenon
- 2011
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Ingår i: Perception. - : SAGE Publications. - 0301-0066 .- 1468-4233. ; 40:1, s. 30-38
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Tidskriftsartikel (refereegranskat)abstract
- Two ambiguous figures (Mach's book-figure and the AMBEGUJAS phenomenon) have been studied. They show colour shifts synchronised with the shifts of their alternative phases. The perceived concave phase appeared to have chromatic surface colour, but the perceived convex phase can appear to be in coloured illumination and shadow (film colour). The two perceived reversible shapes of the Mach figure (the book and the tent) and the three perceived alternative shapes of the Mondrians used in the AMBEGUJAS phenomenon (a roof, a ceiling, and a tile) all can appear to have chromatic surface colours in their concave phases but appear to have coloured illumination and shadow in their convex phases
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| 5. |
- Duong-Thi, Minh-Dao, et al.
(författare)
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High-Throughput Fragment Screening by Affinity LC-MS
- 2013
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Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 18:2, s. 160-171
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Tidskriftsartikel (refereegranskat)abstract
- Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in < 4 h (corresponding to > 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K-D) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.
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| 6. |
- Duong-Thi, Minh-Dao, et al.
(författare)
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Weak affinity chromatography as a new approach for fragment screening in drug discovery
- 2011
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Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 414:1, s. 138-146
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Tidskriftsartikel (refereegranskat)abstract
- Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–uM in dissociation constant (KD)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23 compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10uM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.
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| 7. |
- Duong-Thi, Minh-Dao, et al.
(författare)
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Weak Affinity Chromatography for Evaluation of Stereoisomers in Early Drug Discovery
- 2013
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Ingår i: Journal of Biomolecular Screening. - : Sage Publications. - 1087-0571 .- 1552-454X. ; 18:6, s. 748-755
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Tidskriftsartikel (refereegranskat)abstract
- In early drug discovery (e.g. in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.
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| 8. |
- Landström, Jens, et al.
(författare)
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Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate-lysozyme interaction studies
- 2012
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 10:15, s. 3019-3032
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Tidskriftsartikel (refereegranskat)abstract
- By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using H-1 saturation transfer difference (STD) effects and transferred H-1,H-1-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides beta-D-Galp-(1 -> 4)-beta-D-GlcpNAc-OMe and beta-D-GlcpNAc-(1 -> 4)-beta-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/pi-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on H-1 NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.
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| 9. |
- Lundberg, Mattias
(författare)
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Residual Stresses and Fatigue of Shot Peened Cast Iron
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The complex geometry of cylinder head in heavy-duty diesel engine makes grey cast iron or compact graphite iron a perfect material choice due to its castability, thermal conductivity and damping capacity. To increase the efficiency of the engine, the fatigue property of the material needs to be improved. Shot peening is often used to increase the fatigue strength of components. The benefits are associated with the compressive stresses induced and with surface hardening. In this research project, these effects on grey and compact iron have been analyzed for different shot peening parameters using XRD, SEM and fatigue testing methods. The ultimate aim of the project is to increase the fatigue strength of cast irons by optimization of residual stresses.The XRD measurements and SEM examinations revealed that the shot peening parameters including shot size and peening intensity had significant influences on the resulted residual stresses and strain hardening while changing the coverage made little difference. Also differences in the peening results between the two materials were observed, which were ascribed to an effect of the different graphite morphology. Nevertheless, a residual stress profile similar to the one general considered to improve the fatigue strength in steels could be obtained in both grey and compact iron after shot peening.The axial fatigue testing with R=-1 on the grey iron showed that peening using large shot size and high peening intensity (heavy shot peening) resulted in a fatigue strength reduction of 15-20% in comparison with the mechanically polished surface. The negative effects are likely related to surface damage and relatively high tensile residual stresses in subsurface induced by the heavy peening. Grey cast iron has low ductility in tension and therefore tensile residual stresses may promote multiple cracking and crack networking during cyclic loading.Shot peening using much smaller shots and lower intensity (gentle shot peening) which resulted in a much smaller residual stress field gave no significant changes in fatigue strength. However, a short time annealing at 285°C of specimens being gently shot peened increased the fatigue strength roughly by 10%. The improvement could be an effect of precipitates formed due to the heat treatment, which lock the dislocation movement under cyclic loading.
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| 10. |
- Ohlson, Sten, et al.
(författare)
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Toward high-throughput drug screening on a chip-based parallell affinity separation platform
- 2010
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Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 33:17-18, s. 2575-2581
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Tidskriftsartikel (refereegranskat)abstract
- High-throughput screening of compound libraries, including the study of fragments, has become one of the cornerstones in modern drug discovery research. During this process hits are defined that may be developed into valuable leads and eventually into possible drug candidates. In this paper, we have demonstrated that parallel zonal weak affinity chromatography in microcolumns on a chip offers a possible screening format for weakly binding ligands toward a protein target. We used albumin as a model system because this transport protein is well established as a binder (both weak and strong) for drug substances. Bovine serum albumin was immobilized on microparticulate diolsilica particles and then packed into a 24-channel cartridge, which served as the separation platform. Analysis of the obtained chromatograms yielded information about affinity even in the millimolar range. Employing this approach, thousands of substances can be screened in just a day. We feel confident that zonal affinity chromatography will provide a useful technology in the future for performing high-throughput screening.
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