| 1. |
- Bohlin, Maria, et al.
(författare)
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Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment
- 2012
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Ingår i: Electrophoresis. - Weinheim, Germany : John Wiley & Sons. - 0173-0835 .- 1522-2683. ; 33:12, s. 1695-1702
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Tidskriftsartikel (refereegranskat)abstract
- Sample adsorption to the inner surface of fused silica capillaries is a common problem inCE when analyzingmacromolecules and is harmful to the analysis. We previously utilizedthe pH hysteresis effect of fused silica to facilitate electrophoresis of the strongly adsorbingprotein β2gpI in plain-fused silica capillaries at neutral pH. In the present paper, theeffect of different pretreatments of the capillary on the adsorption of the β2-glycoproteinI has been investigated using electroosmosis markers, SDS mobilization, and imagingbased on indirect immunofluorescence microscopy for direct visualization. The amountof β2gpI adsorbed on the surface was probed using all these independent techniques afterelectrophoresis at neutral pH on capillaries pretreated with HCl, background electrolyte(BGE), and NaOH. BGE pretreatment was included as a positive control. We found that80% or more of the starting material was adsorbed to the inner surface of the silicacapillaries during electrophoresis after pretreatment with only BGE or with NaOH, butafter acidic pretreatment the loss was consistently less than 20%. NaOH most efficientlyremoves adsorbed protein between runs. A theoretical calculation of the pH change ofthe BGE showed that electrolysis affects the pH more than the deprotonation of silanolsduring electrophoresis. We conclude that acidic pretreatment of fused silica capillariesdiminishes adsorption of β2gpI by decreasing charge-dependent wall adsorption.
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| 2. |
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| 3. |
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| 4. |
- Bielecki, Johan, 1982, et al.
(författare)
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Electrospray sample injection for single-particle imaging with x-ray lasers
- 2019
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Ingår i: Science advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:5
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Tidskriftsartikel (refereegranskat)abstract
- The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.
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| 5. |
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| 6. |
- Carlsson, Gunilla, et al.
(författare)
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Falls and Fear of Falling among Persons Who Receive Housing Adaptations-Results from a Quasi-Experimental Study in Sweden
- 2017
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Ingår i: Healthcare. - : MDPI AG. - 2227-9032. ; 5:4
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Tidskriftsartikel (refereegranskat)abstract
- While health might deteriorate through the ageing and disablement process, the impacts of disability can be reduced by adapting the environment. This study aimed to investigate the effects of applying a standardized research-based strategy to housing adaptation as compared to ordinary practice with respect to falls and fear of falling. Another aim was to investigate the overall effects of housing adaptations on fall-related outcomes over time. In total, 196 clients were included at baseline, with follow-up at 3 and 6 months after the housing adaptation was implemented. The only significant difference between the two approaches was identified with respect to fear of falling at 3 months after the housing adaptation, but not after 6 months. The number of clients reporting actual falls increased over time in both sites, whereas the number of reported near-falls decreased most in the intervention site, but without significant differences. Thus, the patterns of differences between the sites are inconsistent, as are the patterns of change in fall-related outcomes. An overall conclusion is that if the goal is to improve fall-related outcomes, housing adaptation should be complemented with other interventions preventing falls and explicitly address the clients' activity limitations. In addition, longer follow-up times are necessary.
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| 7. |
- Carlsson, Gunilla H., et al.
(författare)
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Complexes of horseradish peroxidase with formate, acetate and carbon monoxide
- 2005
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:2, s. 635-642
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Tidskriftsartikel (refereegranskat)abstract
- Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the “hydrated” form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP−acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463−468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron−ligand distances of 2.3 and 1.8 Å, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 Å from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.
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| 8. |
- Carlsson, Gunilla H., et al.
(författare)
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The elusive ligand complexes of the DWARF14 strigolactone receptor
- 2018
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 69:9, s. 2345-2354
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Tidskriftsartikel (refereegranskat)abstract
- Strigolactones, a group of terpenoid lactones, control many aspects of plant growth and development, but the active forms of these plant hormones and their mode of action at the molecular level are still unknown. The strigolactone protein receptor is unusual because it has been shown to cleave the hormone and supposedly forms a covalent bond with the cleaved hormone fragment. This interaction is suggested to induce a conformational change in the receptor that primes it for subsequent interaction with partners in the signalling pathway. Substantial efforts have been invested into describing the interaction of synthetic strigolactone analogues with the receptor, resulting in a number of crystal structures. This investigation combines a re-evaluation of models in the Protein Data Bank with a search for new conditions that may permit the capture of a receptor-ligand complex. While weak difference density is frequently observed in the binding cavity, possibly due to a low-occupancy compound, the models often contain features not supported by the X-ray data. Thus, at this stage, we do not believe that any detailed deductions about the nature, conformation, or binding mode of the ligand can be made with any confidence.
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| 9. |
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| 10. |
- Daurer, Benedikt J., et al.
(författare)
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Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses
- 2017
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Ingår i: IUCrJ. - : INT UNION CRYSTALLOGRAPHY. - 2052-2525. ; 4, s. 251-262
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Tidskriftsartikel (refereegranskat)abstract
- This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of similar to 40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from similar to 35 to similar to 300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 * 10(12) photons per mu m(2) per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.
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| 11. |
- Ekstam, Lisa, et al.
(författare)
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A research-based strategy for managing housing adaptations: study protocol for a quasi-experimental trial
- 2014
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Ingår i: BMC Health Services Research. - : Springer Science and Business Media LLC. - 1472-6963. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: The primary aim of this paper is to describe the design of a project evaluating the effects of using a research-based strategy for managing housing adaptations (HAs). The evaluation targets clients' perspectives in terms of activity, participation, usability, fear of falling, fall incidence, use of mobility devices, and health-related quality of life, and determines the societal effects of HAs in terms of costs. Additional aims of the project are to explore and describe this strategy in relation to experiences and expectations (a) among clients and cohabitants and (b) occupational therapists in ordinary practice. Methods/design: This study is a quasi-experimental trial applying a multiphase design, combining quantitative and qualitative data. At the experimental sites, the occupational therapists (OTs) apply the intervention, i.e. a standardized research-based strategy for HA case management. At the control site, the occupational therapists are following their regular routine in relation to HA. Three municipalities in south Sweden will be included based on their population, their geographical dispersion, and their similar organizational structures for HA administration. Identical data on outcomes is being collected at all the sites at the same four time points: before the HA and then 3, 6, and 12 months after the HA. The data-collection methods are semi-structured qualitative interviews, observations, clinical assessments, and certificates related to each client's HA. Primary outcomes are the usability of the home and the client's independence in daily activities (ADL). Cross-sectional and longitudinal data analyses will be conducted as well as statistical analyses, health-economic analyses, and qualitative analyses. Qualitative and quantitative data will be sequentially analyzed, and case studies will be developed. Discussion: The intervention in this study has been developed and tested through many years of research and in collaboration with practitioners. This process includes methodological development and testing research aimed at identifying the most important outcomes and research targeting current HA case-management procedures in Swedish municipalities. When the study is completed, the results will be used for further optimization of the practice strategy for HA, in close collaboration with the data-collecting OTs.
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| 12. |
- Hantke, Max F., et al.
(författare)
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A data set from flash X-ray imaging of carboxysomes
- 2016
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Ingår i: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
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| 13. |
- Hantke, Max F., et al.
(författare)
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High-throughput imaging of heterogeneous cell organelles with an X-ray laser
- 2014
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Ingår i: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 8:12, s. 943-949
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Tidskriftsartikel (refereegranskat)abstract
- We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.
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| 14. |
- Jonasson, Stina B., et al.
(författare)
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Experiences of fear of falling in persons with Parkinson's disease - A qualitative study
- 2018
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Ingår i: BMC Geriatrics. - : Springer Science and Business Media LLC. - 1471-2318. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Fear of falling is common among persons with Parkinson's disease and is negatively associated with quality of life. However a lack of in-depth understanding of fear of falling as a phenomenon persists. This qualitative study aimed to explore the experiences of fear of falling in persons with Parkinson's disease. Methods: Individual interviews were performed with twelve persons with Parkinson's disease (median age 70 years, median Parkinson duration 9 years, 50% women). The interviews were semi-structured and followed a study-specific interview guide. The transcribed interviews were analyzed using qualitative content analysis. Results: Fear of falling was experienced as a disturbing factor in everyday life. It generated a feeling of vulnerability and made daily activities and everyday environments seem potentially hazardous. Persons also missed performing previous activities. The fear of falling was a varying experience, fueled by an awareness of falls and near falls, Parkinson-related symptoms and disabilities, and by others in their environment. The persons adopted different strategies to handle their fear of falling. Activities were adapted, avoided, performed with help, or carried out despite their fear of falling. Conclusions: The experiences of fear of falling were complex, multifaceted and varied over time and in relation to different activities and environments. This indicates that interventions targeting fear of falling need to be individually tailored for persons with Parkinson's disease and should focus on several aspects, such as Parkinson-related symptoms and disabilities, activities and environmental factors. This study provides new information that increases the understanding of fear of falling, which has implications for researchers as well as clinicians working with persons with Parkinson's disease and fear of falling.
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| 15. |
- Jonasson, Stina, et al.
(författare)
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Content Analysis of Four Fear of Falling Rating Scales by Linking to the International Classification of Functioning, Disability and Health.
- 2013
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Ingår i: PM&R. - : Wiley. - 1934-1563 .- 1934-1482. ; 5:7, s. 573-582
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To gain a deeper understanding of the content of 4 fear of falling (FOF) rating scales by linking them to the International Classification of Functioning, Disability and Health (ICF). DESIGN: Linking study according to the ICF linking rules. SETTING: Not applicable. PATIENTS: Not applicable. METHODS: The rating scales were the Falls Efficacy Scale-International (FES-I), the Swedish version of the Falls Efficacy Scale (FES[S]), the Activities-specific Balance Confidence Scale (ABC), and the modified Survey of Activities and Fear of Falling in the Elderly (SAFFE). The process followed the established and updated linking rules. Three linkers independently identified all meaningful concepts in the rating scales and linked them to the most precise ICF categories. The linkers then discussed their results to reach consensus. If consensus was not attained, the linkers pursued the discussions with a fourth person to reach consensus. MAIN OUTCOME MEASUREMENTS: Not applicable. RESULTS: Most meaningful concepts from the overall questions were linked to the ICF component of body functions. Of the 62 items, all but one meaningful concept were linked to the component of activities and participation. All 4 rating scales covered the chapters of mobility and domestic life and had most linkages to the mobility chapter. CONCLUSIONS: The linking process revealed similarities and differences between the 4 FOF rating scales, as well as methodologic challenges in linking instruments to the ICF. By providing a content description that allows for a direct comparison of the rating scales, the results may be helpful when choosing an appropriate rating scale assessing FOF in clinical practice and research. A further head-to-head comparison through psychometric analyses is required to recommend appropriate FOF rating scales. Studies are also needed to investigate how the overall question and response categories of a rating scale affect respondents' answers.
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| 16. |
- Ludvigsson, Johnny, et al.
(författare)
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GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus
- 2012
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Ingår i: New England Journal of Medicine. - : Massachusetts Medical Society. - 0028-4793 .- 1533-4406. ; 366:5, s. 433-442
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The 65-kD isoform of glutamic acid decarboxylase (GAD65) is a major autoantigen in type 1 diabetes. We hypothesized that alum-formulated GAD65 (GAD-alum) can preserve beta-cell function in patients with recent-onset type 1 diabetes.METHODS: We studied 334 patients, 10 to 20 years of age, with type 1 diabetes, fasting C-peptide levels of more than 0.3 ng per milliliter (0.1 nmol per liter), and detectable serum GAD65 autoantibodies. Within 3 months after diagnosis, patients were randomly assigned to receive one of three study treatments: four doses of GAD-alum, two doses of GAD-alum followed by two doses of placebo, or four doses of placebo. The primary outcome was the change in the stimulated serum C-peptide level (after a mixed-meal tolerance test) between the baseline visit and the 15-month visit. Secondary outcomes included the glycated hemoglobin level, mean daily insulin dose, rate of hypoglycemia, and fasting and maximum stimulated C-peptide levels.RESULTS: The stimulated C-peptide level declined to a similar degree in all study groups, and the primary outcome at 15 months did not differ significantly between the combined active-drug groups and the placebo group (P=0.10). The use of GAD-alum as compared with placebo did not affect the insulin dose, glycated hemoglobin level, or hypoglycemia rate. Adverse events were infrequent and mild in the three groups, with no significant differences.CONCLUSIONS: Treatment with GAD-alum did not significantly reduce the loss of stimulated C peptide or improve clinical outcomes over a 15-month period.
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| 17. |
- Malmgren Fänge, Agneta, et al.
(författare)
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The TECH@HOME study, a technological intervention to reduce caregiver burden for informal caregivers of people with dementia: study protocol for a randomized controlled trial
- 2017
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Ingår i: Trials. - : Springer Science and Business Media LLC. - 1745-6215. ; 18:63
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Tidskriftsartikel (refereegranskat)abstract
- Background: It is estimated that global dementia rates will more than triple by 2050 and result in a staggering economic burden on families and societies. Dementia carries significant physical, psychological and social challenges for individuals and caregivers. Informal caregiving is common and increasing as more people with dementia are being cared for at home instead of in nursing homes. Caregiver burden is associated with lower perceived health, lower social coherence, and increased risk of morbidity and mortality. The aim of this trial is to evaluate the effects of information and communication technology (ICT) on caregiver burden among informal caregivers of people with dementia by reducing the need for supervision. Methods/design: This randomized controlled trial aims to recruit 320 dyads composed of people with dementia living in community settings and their primary informal caregivers. In the intervention group, people with dementia will have a home monitoring kit installed in their home while dyads in the control group will receive usual care. The ICT kit includes home-leaving sensors, smoke and water leak sensors, bed sensors, and automatic lights that monitor the individual's behavior. Alerts (text message and/or phone call) will be sent to the caregiver if anything unusual occurs. All study dyads will receive three home visits by project administrators who have received project-specific training in order to harmonize data collection. Home visits will take place at enrollment and 3 and 12 months following installation of the ICT kit. At every home visit, a standardized questionnaire will be administered to all dyads to assess their health, quality of life and resource utilization. The primary outcome of this trial is the amount of informal care support provided by primary informal caregivers to people with dementia. Discussion: This is the first randomized controlled trial exploring the implementation of ICT for people with dementia in a large sample in Sweden and one of the first at the international level. Results hold the potential to inform regional and national policy-makers in Sweden and beyond about the cost-effectiveness of ICT and its impact on caregiver burden.
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| 18. |
- Nilsson, Maria H., et al.
(författare)
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Uncovering indications of the international classification of functioning, disability, and health from the 39-item Parkinson's Disease Questionnaire
- 2010
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Ingår i: Parkinson's Disease. - 2090-8083 .- 2042-0080. ; , s. 984673-
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Tidskriftsartikel (refereegranskat)abstract
- The 39-item Parkinson's disease questionnaire (PDQ-39) is the most widely used patient-reported rating scale in Parkinson's disease (PD). However, recent studies have questioned its validity and it is unclear what scores represent. This study explored the possibility of regrouping PDQ-39 items into scales representing the International Classification of Functioning, Disability, and Health (ICF) components of Body Functions and Structures (BF), Activities and Participation (AP), and Environmental (E) factors. An iterative process using Rasch analysis produced five new items sets, two each for the BF and AP components and one representing E. Four of these were found to represent clinically meaningful variables: Emotional Impairment (BF), Gross Motor Disability (AP), Fine Motor Disability (AP), and Socioattitudinal Environment (E) with acceptable reliability (0.73–0.96) and fit to the Rasch model (total item-trait chi-square, 8.28–33.2; P>.05). These new ICF-based scales offer a means to reanalyze PDQ-39 data from an ICF perspective and to study its health components using a widely available health status questionnaire for people with PD.
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| 19. |
- Nilsson, Maria H, et al.
(författare)
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Uncovering indicators of the international classification of functioning, disability, and health from the 39-item Parkinson's disease questionnaire.
- 2010
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Ingår i: Parkinson's Disease. - : Hindawi Limited. - 2042-0080 .- 2090-8083. ; 2010
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Tidskriftsartikel (refereegranskat)abstract
- The 39-item Parkinson's disease questionnaire (PDQ-39) is the most widely used patient-reported rating scale in Parkinson's disease (PD). However, recent studies have questioned its validity and it is unclear what scores represent. This study explored the possibility of regrouping PDQ-39 items into scales representing the International Classification of Functioning, Disability, and Health (ICF) components of Body Functions and Structures (BF), Activities and Participation (AP), and Environmental (E) factors. An iterative process using Rasch analysis produced five new items sets, two each for the BF and AP components and one representing E. Four of these were found to represent clinically meaningful variables: Emotional Impairment (BF), Gross Motor Disability (AP), Fine Motor Disability (AP), and Socioattitudinal Environment (E) with acceptable reliability (0.73-0.96) and fit to the Rasch model (total item-trait chi-square, 8.28-33.2; P > .05). These new ICF-based scales offer a means to reanalyze PDQ-39 data from an ICF perspective and to study its health components using a widely available health status questionnaire for people with PD.
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| 20. |
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| 21. |
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| 22. |
- van der Schot, Gijs, et al.
(författare)
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Imaging single cells in a beam of live cyanobacteria with an X-ray laser
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.
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| 23. |
- van der Schot, Gijs, et al.
(författare)
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Open data set of live cyanobacterial cells imaged using an X-ray laser
- 2016
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Ingår i: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Structural studies on living cells by conventional methods are limited to low resolution because radiation damage kills cells long before the necessary dose for high resolution can be delivered. X-ray free-electron lasers circumvent this problem by outrunning key damage processes with an ultra-short and extremely bright coherent X-ray pulse. Diffraction-before-destruction experiments provide high-resolution data from cells that are alive when the femtosecond X-ray pulse traverses the sample. This paper presents two data sets from micron-sized cyanobacteria obtained at the Linac Coherent Light Source, containing a total of 199,000 diffraction patterns. Utilizing this type of diffraction data will require the development of new analysis methods and algorithms for studying structure and structural variability in large populations of cells and to create abstract models. Such studies will allow us to understand living cells and populations of cells in new ways. New X-ray lasers, like the European XFEL, will produce billions of pulses per day, and could open new areas in structural sciences.
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| 24. |
- Wilmot, Carrie M., et al.
(författare)
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Defining redox state of X-ray crystal structures by single-crystal ultraviolet visible microspectrophotometry
- 2002
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 353, s. 301-318
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Tidskriftsartikel (refereegranskat)abstract
- Exciting results have been emerging from the field of single-crystal X-ray crystallography, giving unprecedented detail of freeze-trapped reaction intermediates from important classes of macromolecules that contain chromophores. These structures have been coupled with single-crystal UV-visible microspectrophotometry. This has defined the distinct catalytic intermediates present in the crystal structures, allowing the correlation of electronic transitions with the observed structural transitions. Of particular note is that many of these structures have been generated “on the fly” during kinetic turnover in the crystal. Most enzymatic reactions proceed through distinct catalytic intermediates that, under favorable conditions, may accumulate transiently in the crystal during turnover. In some cases, the physical constraints of the contacts within crystals may also lead to a significant slowing of the reaction at certain points along the pathway where conformational changes are required. This can lead to a transient build-up of spectrally distinct intermediates in the crystal that can be trapped by flash freezing in liquid nitrogen, allowing a complete single-crystal data set to be collected to the highest possible resolution at a later time. Similar build-up of intermediates may be achieved by altering the pH, temperature, or the solvent environment around the protein in the crystal, or by producing engineered variants that build up an intermediate of interest. The chapter focuses on the technical considerations required to carry out UV-visible microspectroscopy of single crystals.
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