Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer

Flyttfåglar - en kvalitativ studie av flyktingbarns upplevelser av krig, flykt och exil

The aim of this study was to explore refugee children’s psychological experiences of forced migration and exile in Sweden. Eleven semi structured interviews were conducted with children age 7-16. The interviews covered three main areas: life in the native country, the journey of migration, and life in Sweden. The children’s narratives were analyzed in accordance with IPA(Interpretative Phenomenological Analysis). The analysis resulted in eleven clusters of themes which covered the children’s narratives of: pre-war life and good memories; life under the threats of war; daily life during war; forced migration; arrival in Sweden; view of Sweden and the native country; life in Sweden; how they related to having family and friends left behind in their native country; how they related to the negative things about the native country; being refused asylum; and the future. The results indicated that the children had difficult experiences from the war, and sometimes also from the journey of migration. The exile in Sweden involved both positive and negative experiences. The children’s narratives showed different ways in which they related to and coped with experiences from their native country and experiences of exile in Sweden. The study showed a difference in experiences and coping strategies between the children who had been granted permanent residence and those who had been refused asylum

Nuancing the continuum from ideal to real-world implementation : a letter to the editor on Nilsen et al

Nilsen et al.’s (Implement Sci Commun 6:90, 2025) proposal to distinguish between implementation efficacy and effectiveness, and to situate implementation studies along a continuum from ideal to real-world conditions, offers a valuable conceptual advance. In this commentary, we acknowledge the contribution of their debate while highlighting potential limitations of applying a single-axis continuum to a field heavily characterized by contextual complexity. Drawing from decades of healthcare quality improvement, we argue that implementation interventions often blend efficacy-like and effectiveness-like elements, making neat classification difficult. We further suggest that oversimplification risks obscuring the realities of organizational change. Instead, we propose a double-axis model that considers both the implementation intervention and the context in which it unfolds. Economic evaluation likewise requires nuanced approaches that go beyond their proposed continuum indicator tool (“Implementation PRECIS”). To constructively extend Nilsen et al.’s contribution, we advocate for integration of the tool with existing approaches to evaluation, co-production with stakeholders, and empirical validation across diverse settings. While no implementation endeavor is ideal, advancing discourse around how efficacy and effectiveness are conceptualized can support more pragmatic, context-responsive, and sustainable improvements in healthcare

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Exosomes and Exosomal RNA – A Way of Cell-to-Cell Communication

Exosomes are nano-sized extracellular vesicles of endocytic origin participating in cell-to-cell communication, partly by the transfer of exosomal RNA between cells. These extracellular vesicles are released by most cells and found in many body fluids including plasma and urine. Exosomes differ compared to their donor cells in RNA, protein and lipid composition, and their molecular content has shown prognostic and diagnostic potential. Uveal melanoma is a tumour arising from melanocytes of the eye and despite successful control of the primary tumour, approximately one third of the patients will develop metastases, predominantly liver metastases, with poor prognosis. The overall aim of this thesis was to evaluate the role of exosomes in cell-to-cell communication and the biological role of exosomal RNA. Exosomal RNA has been extracted by different RNA isolation methods and we identified that the RNA size distribution pattern varied in multiple studies. Therefore, we aimed to determine if this RNA variation was a true variation or merely a consequence of the RNA extraction method used. We evaluated seven different RNA isolation methods using a mouse mast cell line (MC/9) that continuously releases exosomes. The results showed that the exosomal RNA yield and size distribution pattern differed substantially between different RNA isolation methods. The mRNA content and function of MC/9 cell-derived exosomes was shown to be altered depending on the culture conditions of the cells. Cells exposed to oxidative stress were shown to have the capacity to send a conditioning signal to other cells, resulting in resistance to oxidative stress in the recipient cells. Moreover, this conditioning signal was shown to be eradicated upon UV-C exposure, indicating a possible role for the exosomal RNA in this biological function. The presence of exosomes in patients with liver metastases from uveal melanoma was established with the isolation, detection and characterisation of exosomes from isolated hepatic perfusion. The results revealed melanoma-specific exosomes, which contained similar microRNA profiles between patients. Furthermore, patients with metastatic uveal melanoma were shown to have a higher concentration of exosomes in their peripheral venous blood compared to healthy controls. We conclude that exosomes play a role in cell-to-cell communication and their RNA appears to be of biological importance. Furthermore, exosomal RNA may potentially play a role in the diagnosis and prognosis of uveal melanoma

Kunskapsbaserad omvårdnad och implementering

Forskningsresultat fyller en viktig funktion för att utveckla och bibehålla säker, effektiv och personcentrerad vård och omsorg. Trots kontinuerlig forskning är ett känt faktum att det kan ta lång tid från att ny kunskap framställs tills att nödvändiga forskningsresultat nyttiggörs i hälso- och sjukvård och omsorg. Studier visar att det kan ta upp till 17 år innan forskningsresultat får genomslag och blir till rutin i vårdens vardag. Implementeringsforskning är ett tvärprofessionellt forskningsområde där flera forskande sjuksköterskor har gjort betydelsefulla bidrag. Varför tar det så lång tid att implementera en omvårdnadsåtgärd eller omvårdnadsmetod och hur går det till att implementera ny eller annan relevant kunskap kommer att belysas i detta kapitel. Andra frågor som tas upp här är om det går att underlätta implementering och i så fall hur det kan möjliggöras.</p

Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of different methods

Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA. Different methods were used to extract exosomal and cellular RNA. All methods evaluated extracted high quality and purity RNA as determined by RNA integrity number (RIN) and OD values for cellular RNA using capillary electrophoresis and spectrophotometer. Interestingly, the exosomal RNA yield differed substantially between the different RNA isolation methods. There was also a difference in the exosomal RNA patterns in the electropherograms, indicating that the tested methods extract exosomal RNA with different size distribution. A pure column based approach resulted in the highest RNA yield and the broadest RNA size distribution, whereas phenol and combined phenol and column based approaches lost primarily large RNAs. Moreover, the use of phenol and combined techniques resulted in reduced yield of exosomal RNA, with a more narrow size distribution pattern resulting in an enrichment of small RNA including microRNA. In conclusion, the current study presents a unique comparison of seven different methods for extraction of exosomal RNA. As the different isolation methods give extensive variation in exosomal RNA yield and patterns, it is crucial to select an isolation approach depending on the research question at hand

Reduction of Surgical Complications in Dogs and Cats by the Use of a Surgical Safety Checklist

ObjectiveTo examine whether the use of a surgical safety checklist (SSC) could reduce the incidence of complications after small animal surgery.Study DesignProspective clinical study. Animals: Client-owned dogs and cats (n=520).MethodsConsecutive cases were enrolled in the study, the first 300 cases without implementation of the surgical checklist (SSC-), followed by 220 cases with implementation of the checklist (SSC+). The checklist was adapted from the WHO surgical checklist and consisted of three different check points: (1) before induction of anaesthesia (sign in), (2) before surgical incision (time out), and (3) before recovery (sign out). In-hospital outcomes were prospectively recorded, and complications within 6 weeks were retrospectively recorded by reviewing medical records and by telephone interviews with owners. The severity of each recorded complication was graded as minor, moderate, or severe. Comparisons were made between SSC- and SSC+ outcomes.ResultsThere were significantly more complications in SSC- animals than SSC+ animals (SSC- 52/300 vs. SSC+ 15/220, P=.0003). There was a significantly higher frequency of SSI (P=.045) and wound healing complications (P=.0006) for SSC- animals than SSC+ animals.ConclusionThe frequency and severity of postoperative complications was significantly decreased after introduction of a surgical checklist. All veterinary hospitals should consider using a surgical checklist. Compliance with implementation of the checklist is important for success