| 1. |
- Broman, Axel, et al.
(författare)
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Acoustic trapping based high throughput isolation and characterization of pathogen activated platelet derived extracellular vesicles from plasma
- 2023
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Konferensbidrag (refereegranskat)abstract
- We present the use of a high capacity and high throughput acoustic trapping platform for phenotypic characterization and functional studies of extracellular vesicles (EVs) from pathogen activated platelets. Platelet rich plasma was stimulated with bacterial M1 protein isolated from S. Pyogenes, which is known to activate platelets. The subsequently released platelet EVs were isolated from 400 μL plasma by acoustic trapping at a flowrate of 500 μL/min. We have previously reported on the acoustic trapping platform, which can process milliliter sized samples in minutes1. The EVs were then compared to EVs released by platelets stimulated with endogenous platelet activator (Thrombin) and negative control (HEPES buffer).A schematic of the sample processing can be seen in Fig. 1. Human plasma from healthy donors was incubated with HEPES buffer, Thrombin or M1 protein to stimulate platelets and induce EV release. The platelets were then removed by centrifugation, leaving EVs in plasma. The EVs were isolated and enriched by acoustic trapping and the protein content was analyzed using mass spectrometry. The EVs were also analyzed by immunoblotting, as well as immunogold labelling against CD42b and M1 protein and imaged by transmission electron microscopy (TEM). Additionally, isolated EVs were incubated with whole blood from healthy donors to investigate functional immunomodulatory effects compared to known platelet agonists (Thrombin, M1).The mass spectrometry data showed a clear distinction between isolated vesicles and plasma, Fig. 2A, with one protein cluster enriched for vesicles and one enriched for plasma samples. There was also a clear distinction between EVs from activated platelets (Thrombin, M1) and resting platelets (HEPES), Fig. 2B. Interestingly, the bacterial M1 protein was enriched in the vesicle fraction, Fig. 3A, suggesting that M1 protein binds to platelet EVs.To confirm that M1 binds to EVs, trapped samples and centrifuged samples (both pellet and supernatant) were analyzed with immunoblot against M1 protein, Fig. 3B. Clear bands are present around 54 kDa, in accordance with M1 mass, in samples containing enriched EVs. This further confirms that M1 protein binds to EVs. The TEM images showed isolated EVs for all samples, Fig. 3C. Vesicles from platelets stimulated with thrombin were found positive for CD42b. Pathogen activated platelet EVs were found positive for both CD42b and M1 protein, showing that the bacterial protein binds to platelet EVs. Although no CD42b positive EVs were found in HEPES stimulated samples in the TEM analysis, we observed a wealth of them in cytometry data.The whole blood assay showed that isolated platelet EVs stimulated platelet-neutrophil complex formation, compared to resting state, Fig. 4A. Additionally, platelet EVs stimulated IL-8 cytokine release from monocytes, Fig. 4B, suggesting functionally intact vesicles.We have demonstrated rapid isolation and enrichment of platelet EVs from plasma samples by acoustic trapping. The isolated vesicles were functionally intact, and it was possible to perform several downstream analyses, including whole blood stimulation. We found that bacterial M1 protein from S. Pyogenes binds to platelet EVs and is transported with them, a mechanism which could contribute to the rapid infectious progress in sepsis.
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| 2. |
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| 3. |
- Broman, Axel, et al.
(författare)
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Multinodal Acoustic Trapping Enables High Capacity and High Throughput Enrichment of Extracellular Vesicles and Microparticles in miRNA and MS Proteomics Studies
- 2021
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 93:8, s. 3929-3937
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Tidskriftsartikel (refereegranskat)abstract
- We report a new design of an acoustophoretic trapping device with significantly increased capacity and throughput, compared to current commercial acoustic trapping systems. Acoustic trapping enables nanoparticle and extracellular vesicle (EV) enrichment without ultracentrifugation. Current commercial acoustic trapping technology uses an acoustic single-node resonance and typically operates at flow rates <50 μL/min, which limits the processing of the larger samples. Here, we use a larger capillary that supports an acoustic multinode resonance, which increased the seed particle capacity 40 times and throughput 25-40 times compared to single-node systems. The resulting increase in capacity and throughput was demonstrated by isolation of nanogram amounts of microRNA from acoustically trapped urinary EVs within 10 min. Additionally, the improved trapping performance enabled isolation of extracellular vesicles for downstream mass spectrometry analysis. This was demonstrated by the differential protein abundance profiling of urine samples (1-3 mL), derived from the non-trapped versus trapped urine samples.
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| 4. |
- Broman, Axel, et al.
(författare)
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Multinodal high throughput acoustic trapping of exosomes from urine samples
- 2019
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Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 2-3
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Konferensbidrag (refereegranskat)abstract
- We report a new design of an acoustophoretic trapping device with greatly increased capacity and throughput, compared to current commercial systems. Acoustic trapping enables nanoparticle and exosome enrichment without the need for ultracentrifugation. The commercial acoustic trapping technology, AcouTrap (AcouSort AB), typically operates at flow rates < 50 μl/min which limits processing of larger samples. Using a larger capillary that supports an acoustic multi-node resonance, capacity and throughput was increased a factor 40 compared to AcouTrap. It was possible to capture and enrich exosomes from urine samples at 30 times higher flow rate than previously reported.
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| 5. |
- Broman, Axel, et al.
(författare)
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Rapid multinodal acoustic trapping of extracellular vesicles for downstream mass spectrometry analysis
- 2020
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Ingår i: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9781733419017 ; , s. 148-149
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Konferensbidrag (refereegranskat)abstract
- We report the use of multinodal acoustic trapping for high throughput and high capacity capturing of EV's (extracellular vesicles) for quantitative mass spectrometry analysis. The multinode trapping unit was shown to isolate sufficient amount of EV's from dilute biological samples (urine and cell culture supernatant) at flow rates of 500 ul/min within minutes, enabling EV proteome profiling. This was shown by differential protein expression analysis of urine and the urine EV fraction. Differential protein profiling of trapped EVs from stimulated versus non-stimulated platelets also demonstrated an easy access to differential expression in the EV-proteome.
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| 6. |
- Havers, Megan, et al.
(författare)
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Advancement and obstacles in microfluidics-based isolation of extracellular vesicles
- 2023
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 415:7, s. 1265-1285
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Forskningsöversikt (refereegranskat)abstract
- There is a great need for techniques which enable reproducible separation of extracellular vesicles (EVs) from biofluids with high recovery, purity and throughput. The development of new techniques for isolation of EVs from minute sample volumes is instrumental in enabling EV-based biomarker profiling in large biobank cohorts and paves the way to improved diagnostic profiles in precision medicine. Recent advances in microfluidics-based devices offer a toolbox for separating EVs from small sample volumes. Microfluidic devices that have been used in EV isolation utilise different fundamental principles and rely largely on benefits of scaling laws as the biofluid processing is miniaturised to chip level. Here, we review the progress in the practicality and performance of both passive devices (such as mechanical filtering and hydrodynamic focusing) and active devices (using magnetic, electric or acoustic fields). As it stands, many microfluidic devices isolate intact EV populations at higher purities than centrifugation, precipitation or size-exclusion chromatography. However, this comes at a cost. We address challenges (in particular low throughput, clogging risks and ability to process biofluids) and highlight the need for more improvements in microfluidic devices. Finally, we conclude that there is a need to refine and standardise these lab-on-a-chip techniques to meet the growing interest in the diagnostic and therapeutic value of purified EVs.
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| 7. |
- Högberg, Johan, 1994, et al.
(författare)
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Is absolute or relative knee flexor strength related to patient- reported outcomes in patients treated with ACL reconstruction with a hamstring tendon autograft? An analysis of eccentric Nordic hamstring strength and seated concentric isokinetic strength
- 2023
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Ingår i: Knee. - : Elsevier BV. - 0968-0160. ; 41, s. 161-170
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a need for better understanding of how knee flexor strength influence patient-reported outcomes (PROs) after anterior cruciate ligament (ACL) reconstruction. Our aim was to investigate the relationship between the eccentric NordBord test and the seated concentric Biodex test with PROs, during the first year of rehabilitation after ACL reconstruction with hamstring tendon (HT) autograft.Methods: Patients with an index ACL reconstruction with an HT autograft participating in a rehabilitation registry were screened for inclusion. Outcomes of interest were the correla-tion between absolute (N/kg or Nm/kg) and relative (limb symmetry index) knee flexor strength measured in the NordBord and Biodex with the results of PROs. The significance level was set at p < 0.05 and Pearson's correlation coefficient was used.Results: 137 patients were included (47% women) with a mean age of 24.8 +/- 8.4 years. There were non-significant and weak correlations between relative strength for all PROs. Significant and weak correlations between absolute strength in the Biodex with the Knee Self-Efficacy Scale18 (K-SES18) present at 4 and 8 months, and for the ACL-Return to Sport after Injury scale (ACL-RSI) at 12 months was observed, accounting for 8.4-15.7% of the variance. Significant and weak correlations between absolute strength in the Nordbord with the Knee injury and Osteoarthritis Outcome Scale subscale Sports and Recreation at 4 months, the K-SES18 present and the ACL-RSI at 8 months were observed, accounting for 9.4-14.4% of the variance.
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| 8. |
- Palm, Frida, et al.
(författare)
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Phenotypic characterization of acoustically enriched extracellular vesicles from pathogen-activated platelets
- 2023
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Ingår i: Journal of Innate Immunity. - : S. Karger. - 1662-811X .- 1662-8128. ; 15:1, s. 599-613
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are derived from the membrane of platelets and released in the circulation upon activation or injury. Analogous to the parent cell, platelet derived EVs play an important role in hemostasis and immune responses by transfer of bioactive cargo from the parent cells. Platelet activation and release of EVs increases in several pathological inflammatory diseases, such as sepsis. We have previously reported that the M1 protein released from the bacterial pathogen Streptococcus pyogenes directly mediates platelet activation. In this study, EVs were isolated from these pathogen-activated platelets using acoustic trapping and their inflammation phenotype was characterized using quantitative mass spectrometry-based proteomics and cell-based models of inflammation. We determined that M1 protein mediated release of platelet derived EVs that contained the M1 protein. The isolated EVs derived from pathogen-activated platelets contained a similar protein cargo to those from physiologically activated platelets (thrombin), and included platelet membrane proteins, granule proteins and cytoskeletal proteins, coagulation factors and immune mediators. Immunomodulatory cargo, complement proteins and IgG3, were significantly enriched in EVs isolated from M1 protein-stimulated platelets. Acoustically enriched EVs were functionally intact and exhibited proinflammatory effects on addition to blood, including platelet-neutrophil complex formation, neutrophil activation, and cytokine release. Collectively, our findings reveal novel aspects of pathogen-mediated platelet activation during invasive streptococcal infection.
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| 9. |
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| 10. |
- Sjöqvist, Axel S.L. 1990, et al.
(författare)
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Shock metamorphism and hydrothermal alteration of mafic impact ejecta from the Lockne impact structure, Sweden
- 2017
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Ingår i: GFF. - : Informa UK Limited. - 1103-5897 .- 2000-0863. ; 139:2, s. 119-128
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Tidskriftsartikel (refereegranskat)abstract
- © 2016 Geologiska FöreningenThe local geology at Kloxåsen is characterised by ejecta deposits from the 458 Ma Lockne marine impact. The Kloxåsen ejecta are located on a Caledonian parautochthonous unit, approximately 7 km from the centre of the 7.5-km-wide Lockne crater structure. The ejecta were deposited on the seafloor and were covered with seawater immediately after the impact event. Of special interest is a mafic impact breccia within the ejecta, which before the impact was Åsby dolerite that belongs to the Jämtland suite of the 1.25 Ga Central Scandinavian Dolerite Group. The mafic impact breccia occurs mainly as a coherent thin domain within a larger block of granitic breccia, which we interpret as a result of the in situ brecciation of a dolerite sill within granitic bedrock. Shock pressure in the doleritic breccia was low, in the order of 0.4 GPa, constrained by the presence of mechanically twinned clinopyroxene. Low shock pressure and brecciation corresponds well to the spall zone of an impact crater, where ejecta originate from. Whereas spalled ejecta can also show signs of having been exposed to high shock pressures, including shocked quartz, evidence for this was not found in the Kloxåsen ejecta. The breccia has been hydrothermally altered, but the ejecta are too far removed from the crater to have been affected by hydrothermal circulation in relation to Lockne’s impact event. Fluid inclusion analyses suggest that most of the alteration happened later, during the Caledonian orogeny. Geochemical analyses reflect observed mineral alterations well, such as serpentinisation of olivine.
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