| 1. |
- von Schantz, Laura, et al.
(författare)
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Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules
- 2009
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Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Background: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.
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| 2. |
- Herring, Matthew, et al.
(författare)
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Exposing kinetic disparities between inflammasome readouts using time-resolved analysis
- 2024
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Ingår i: Heliyon. - : Elsevier. - 2405-8440. ; 10:11
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Tidskriftsartikel (refereegranskat)abstract
- The NLRP3 inflammasome is an intracellular multiprotein complex described to be involved in both an effective host response to infectious agents and various diseases. Investigation into the NLRP3 inflammasome has been extensive in the past two decades, and often revolves around the analysis of a few specific readouts, including ASC-speck formation, caspase-1 cleavage or activation, and cleavage and release of IL-1β and/or IL-18. Quantification of these readouts is commonly undertaken as an endpoint analysis, where the presence of each positive outcome is assessed independently of the others. In this study, we apply time-resolved analysis of a human macrophage model (differentiated THP-1-ASC-GFP cells) to commonly accessible methods. This approach yields the additional quantifiable metrics time-resolved absolute change and acceleration, allowing comparisons between readouts. Using this methodological approach, we reveal (potential) discrepancies between inflammasome-related readouts that otherwise might go undiscovered. The study highlights the importance of time-resolved data in general and may be further extended as well as incorporated into other areas of research.
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| 3. |
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| 4. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
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Evolution of a carbohydrate binding module into a protein-specific binder
- 2006
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Ingår i: Biomolecular Engineering. - : Elsevier BV. - 1389-0344 .- 1878-559X. ; 23:2-3, s. 111-117
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Tidskriftsartikel (refereegranskat)abstract
- A carbohydrate binding module, CBM4-2, derived front the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures. (c) 2005 Elsevier B.V. All rights reserved.
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| 5. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
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Molecular engineering of a thermostable carbohydrate-binding module
- 2006
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1029-2446 .- 1024-2422. ; 24:1-2, s. 31-37
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Konferensbidrag (refereegranskat)abstract
- Structure-function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel (TM), ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis of selected binders also helped us to identify residues important for their specificities.
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| 6. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
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A carbohydrate binding module as a diversity-carrying scaffold
- 2004
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 17:3, s. 213-221
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Tidskriftsartikel (refereegranskat)abstract
- The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
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| 7. |
- Ballerini, Lucia, et al.
(författare)
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Comparison of histomorphometrical data obtained with two different image analysis methods
- 2007
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Ingår i: Journal of materials science. Materials in medicine. - : Springer. - 0957-4530 .- 1573-4838. ; 18:8, s. 1471-1479
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Tidskriftsartikel (refereegranskat)abstract
- A common way to determine tissue acceptance of biomaterials is to perform histomorphometrical analysis on histologically stained sections from retrieved samples with surrounding tissue, using various methods. The “time and money consuming” methods and techniques used are often “in house standards”. We address light microscopic investigations of bone tissue reactions on un-decalcified cut and ground sections of threaded implants. In order to screen sections and generate results faster, the aim of this pilot project was to compare results generated with the in-house standard visual image analysis tool (i.e., quantifications and judgements done by the naked eye) with a custom made automatic image analysis program. The histomorphometrical bone area measurements revealed no significant differences between the methods but the results of the bony contacts varied significantly. The raw results were in relative agreement, i.e., the values from the two methods were proportional to each other: low bony contact values in the visual method corresponded to low values with the automatic method. With similar resolution images and further improvements of the automatic method this difference should become insignificant. A great advantage using the new automatic image analysis method is that it is time saving—analysis time can be significantly reduced.
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| 8. |
- Sul, Young-Taeg, 1960, et al.
(författare)
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A novel in vivo method for quantifying the interfacial biochemical bond strength of bone implants
- 2010
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Ingår i: Journal of the Royal Society Interface. - London, United Kingdom : Royal Society. - 1742-5689 .- 1742-5662. ; 7:42, s. 81-90
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Tidskriftsartikel (refereegranskat)abstract
- Quantifying the in vivo interfacial biochemical bond strength of bone implants is a biological challenge. We have developed a new and novel in vivo method to identify an interfacial biochemical bond in bone implants and to measure its bonding strength. This method, named biochemical bond measurement (BBM), involves a combination of the implant devices to measure true interfacial bond strength and surface property controls, and thus enables the contributions of mechanical interlocking and biochemical bonding to be distinguished from the measured strength values. We applied the BBM method to a rabbit model, and observed great differences in bone integration between the oxygen (control group) and magnesium (test group) plasma immersion ion-implanted titanium implants (0.046 versus 0.086 MPa, n=10, p=0.005). The biochemical bond in the test implants resulted in superior interfacial behaviour of the implants to bone: (i) close contact to approximately 2 μm thin amorphous interfacial tissue, (ii) pronounced mineralization of the interfacial tissue, (iii) rapid bone healing in contact, and (iv) strong integration to bone. The BBM method can be applied to in vivo experimental models not only to validate the presence of a biochemical bond at the bone–implant interface but also to measure the relative quantity of biochemical bond strength. The present study may provide new avenues for better understanding the role of a biochemical bond involved in the integration of bone implants.
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| 9. |
- Blomqvist, Johanna, et al.
(författare)
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Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis
- 2011
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Ingår i: Letters in Applied Microbiology. - Malden, USA : Wiley-Blackwell. - 0266-8254 .- 1472-765X. ; 53:1, s. 73-78
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Tidskriftsartikel (refereegranskat)abstract
- Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae.Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small-scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l(-1) (0.22 mol l(-1))]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0 42 +/- 0 01 g ethanol (g glucose)(-1) were observed for both yeasts in 1 : 10 hydrolysate. In small-scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate.Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate.Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.
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| 10. |
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