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- Eklund, Malin, et al.
(author)
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Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex
- 2004
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In: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 109:3, s. 277-286
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Journal article (peer-reviewed)abstract
- Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cell ulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1(Cel6A)) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1(Cel6A) fusion protein had been previously applied. This showed that the SPA-CBM1(Cel6A) fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.
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2. |
- Kristenson, Karolina, et al.
(author)
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Peak oxygen uptake in combination with ventilatory efficiency improve risk stratification in major abdominal surgery
- 2024
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In: Physiological Reports. - : WILEY. - 2051-817X. ; 12:1
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Journal article (peer-reviewed)abstract
- This pilot study aimed to evaluate if peak VO2 and ventilatory efficiency in combination would improve preoperative risk stratification beyond only relying on peak VO2. This was a single-center retrospective cohort study including all patients who underwent cardiopulmonary exercise testing (CPET) as part of preoperative risk evaluation before major upper abdominal surgery during years 2008-2021. The primary outcome was any major cardiopulmonary complication during hospitalization. Forty-nine patients had a preoperative CPET before decision to pursue to surgery (cancer in esophagus [n = 18], stomach [6], pancreas [16], or liver [9]). Twenty-five were selected for operation. Patients who suffered any major cardiopulmonary complication had lower ventilatory efficiency (i.e., higher VE/VCO2 slope, 37.3 vs. 29.7, p = 0.031) compared to those without complications. In patients with a low aerobic capacity (i.e., peak VO2 < 20 mL/kg/min) and a VE/VCO2 slope >= 39, 80% developed a major cardiopulmonary complication. In this pilot study of patients with preoperative CPET before major upper abdominal surgery, patients who experienced a major cardiopulmonary complication had significantly lower ventilatory efficiency compared to those who did not. A low aerobic capacity in combination with low ventilatory efficiency was associated with a very high risk (80%) of having a major cardiopulmonary complication.
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3. |
- Lindblom, Hanna, et al.
(author)
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Disease prevalence and number of health care visits among members of a nationwide sports organization compared to matched controls
- 2021
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In: BMC Public Health. - : BMC. - 1471-2458. ; 21:1
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Journal article (peer-reviewed)abstract
- Background Physical activity has positive effects on several diseases and may reduce the risk of morbidity and the mortality rate. Whether the prevalence of disease and health care consumption differ between the members of sports organizations and the general population has not been established. Hence, this pilot study aimed to compare the prevalence of diseases known to be associated with physical inactivity and health care consumption in members of a large non-profit sports organization and an age-, sex- and geographically matched random sample from the general population. Methods Subjects in two Swedish cities who exercised at least once a week and had been members for at least two years in the non-profit sports organization Friskis&Svettis were invited. A randomized age-, sex- and geographically matched sample was drawn from the general population. Data on disease prevalence (by International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) codes) and health care consumption were retrieved using the members personal identification numbers through a regional health care database. Between-group differences in the prevalence of disease were compared using chi(2)-tests and logistic regression between members and controls. Health care consumption was defined as the number of visits, stratified by primary and hospital care, and was compared using chi(2)-tests and Mann-Whitney U-tests. Results In total, 3015 subjects were included in each group (response rate 11%). Controls had higher prevalence rates of musculoskeletal diseases (13.3% vs. 11.6%, p = 0.047), metabolic disease (10.4% vs. 5.4%, p < 0.001), hypertension (16.6% vs. 11.7%, p < 0.001), psychiatric diseases (8.9% vs. 7.1%, p = 0.012) and lung cancer (0.4% vs. 0%, p = 0.001) than the members. The total number of health care contacts was 22% higher in the controls than in the members, whereas the proportion of subjects with at least one health care visit was larger in the members (89% vs. 79%, p < 0.001). Conclusions The prevalence rates of lifestyle diseases related to musculoskeletal, metabolic and psychiatric diseases, hypertension and lung cancer, and the overall health care consumption, were lower among members of a sports organization than among controls. However, longitudinal studies are needed to establish a cause-effect relationship between membership and disease development.
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4. |
- Sandström, Kristofer
(author)
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Selection and use of affinity proteins developed by combinatorial engineering
- 2003
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Licentiate thesis (other academic/artistic)abstract
- In affinity protein biotechnology the selective bindingbetween a chosen protein and an interacting biomolecule isutilized for a variety of applications including bioseparation,detection and therapy. Traditionally, affinity proteinsrecruited for such applications have been derived from naturalproteins or immunoglobulins generated via immunization routes.More recently, advances in the construction and handling oflarge collections of proteins(denoted libraries) generated invitro have opened up for new routes for the development ofaffinity proteins with desired properties.In this study, phage display selection technology was usedfor the isolation of novel human CD28 (hCD28)-specific affinityproteins from a protein library constructed by combinatorialprotein engineering of a 58 aa protein domain (Z) derived fromstaphylococcal protein A (SPA). From selections using hCD28 asa target molecule, several hCD28-specific affinity proteins(denoted affibodies) could be identified and analysis of theisolated affibody variants revealed a high degree of sequencehomology between the different clones. The biosensor analysisshowed that all variants bound to hCD28 with micromolardissociation constants (KD) and no significant cross-reactivitytowards the structurally related T-cell receptor hCTLA-4 couldbe observed. The apparent binding affinity for hCD28 of one ofthe isolated affibodies was further improved through fusion toa human Fc fragment fusion partner, resulting in a homodimericversion of the affibody ligand showing avidity effects uponhCD28 binding. Further, a co-culture experiment involvingJurkat T-cells and CHO cell lines tranfected to express eitherhuman CD80 or LFA-3 on the cell surface showed that apreincubation of Jurkat cells with one of the affibody variantsresulted in a specific concentration-dependent inhibition ofthe CD80 induced IL-2 production. This indicates that thisaffibody binds to hCD28 and specifically interferes with theco-stimulation signal mediated via hCD28 and hCD80. ACD28-specific binding protein could have potential as an agentfor various immunotherapy applications. In a second study, anaffinity protein-based strategy was investigated forsite-specific anchoring of proteins onto cellulose for woodfiber engineering purposes. Here, affinity proteins derivedfrom different sources were used for the assembly of acellulosome-like complex for specific and reversible anchoringof affinity domain-tagged reporter proteins to acellulose-anchored fusion protein. A fusion protein between acellulose binding module (Cel6A CBM1) derived from the fungalTrichoderma reesei and a five-domain staphylococcal protein A(SPA) moiety was constructed to serve as a platform for thedocking of reporter proteins produced as fusion to two copiesof a SPA-binding affibody affinity protein (denoted ZSPA-1),selected by phage display technology from a Z domain basedprotein library. In a series of experiments, involving repeatedwashing and low pH elutions, affinity tagged Enhanced GreenFluorescent Protein (EGFP) and Fusarium solani pisi lipasecutinase reporter proteins were both found to be specificallydirected from solution to a region of a cellulose-based filterpaper where the SPA-CBM fusion protein previously had beenpositioned. This showed that the cellulose-anchored SPA-Cel6ACBM1 fusion protein had been stably anchored to the surfacewith retained binding activity and that the interaction betweenSPA and the ZSPA-1 affibody domain was selective.phage display, combinatorial, selection, CD28, cellulosome,cellulose, affibody
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