| 1. |
- Corso, Giulia
(författare)
-
Purification, profiling and bioengineering of extracellular vesicles
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The nano-sized membrane enclosed extracellular vesicles (EVs) transfer macromolecular information in the form of proteins, nucleic acids and lipids across cells. They are important mediators of cell-to-cell communication, but their relatively small size makes EV isolation and down-stream analysis challenging. In this thesis, we ventured through some of the current challenges in the EV field touching upon purification, small RNA and protein content profiling and ultimately characterization of engineered vesicles at the molecular level. The isolation of EVs from the complex fluid they are surrounded by, represents the first hindrance in studying these vesicles. Ideally, the purification method should preserve the integrity and natural properties of the EVs and simultaneously deplete the vesicular portion from unwanted components. In Paper I, we describe a novel liquid chromatography technique for EV purification, that combines size separation with bind elution (BE-SEC) entrapping molecules smaller than 700 kDa within the matrix core. The BE-SEC isolation method yields high particles recovery in a reproducible and time-efficient way, without neither affecting the EVs natural surface protein signature nor their physicochemical properties. By adding a prior tangential flow filtration step, the BE-SEC could be scaled-up and the EV preparation further depleted from unwanted non-vesicular proteins and RNAs. Secondly, therapeutically engineered EVs are promising delivery vehicles and linking the administered vesicular dose to the molecular cargo concentration is of extreme relevance to achieve a desired response. Therefore, analytical methods focused on single vesicles quantification rather than ‘bulk’ analysis and improved bioengineered vesicles are of utmost importance for therapeutic applications. In Paper II, we extensively characterize a set of fluorescently labelled EV-associated proteins, employing several qualitative and quantitative methods. Using Fluorescence Correlation Spectroscopy, we quantify the number of fluorescent molecules per single loaded vesicle. Different loading efficiencies were observed for the tested proteins, with the tetraspanins (CD63, CD9 and CD81) showing the highest loading efficiency with an average of 40-60 fluorescent molecules per vesicle. To summarize, we provide a reference for selecting EV sorting domains that best fit the desired outcome, as well as an array of quantitative and qualitative methodologies to support EV engineering. In Paper III, we investigate the native RNA and protein content of EVs, with a focus on small RNAs and RNA binding proteins respectively. Across different mouse and human cell-derived EVs, a deficiency of miRNA sequences and relative depletion of ‘miRNA-related’ proteins were observed. The majority of the RNA sequences detected in EVs was represented by rRNA-, coding- and tRNA fragments, reflecting the observations in the respective protein portion, where ribosomal and translational proteins were predominantly identified. In conclusion, this thesis explores and advances some of the challenges encountered in the EV field by ameliorating the EV isolation workflow in terms of time and scalability, linking the vesicular transcriptome and proteome of EVs derived from various cell lines, and systematically comparing and quantifying the sorting efficiency of different proteins into EVs.
|
|
| 2. |
- Gorasso, Vanessa, et al.
(författare)
-
Burden of disease attributable to risk factors in European countries: a scoping literature review
- 2023
-
Ingår i: Archives of Public Health. - 0778-7367 .- 2049-3258. ; 81:1
-
Forskningsöversikt (refereegranskat)abstract
- Objectives: Within the framework of the burden of disease (BoD) approach, disease and injury burden estimates attributable to risk factors are a useful guide for policy formulation and priority setting in disease prevention. Considering the important differences in methods, and their impact on burden estimates, we conducted a scoping literature review to: (1) map the BoD assessments including risk factors performed across Europe; and (2) identify the methodological choices in comparative risk assessment (CRA) and risk assessment methods. Methods: We searched multiple literature databases, including grey literature websites and targeted public health agencies websites. Results: A total of 113 studies were included in the synthesis and further divided into independent BoD assessments (54 studies) and studies linked to the Global Burden of Disease (59 papers). Our results showed that the methods used to perform CRA varied substantially across independent European BoD studies. While there were some methodological choices that were more common than others, we did not observe patterns in terms of country, year or risk factor. Each methodological choice can affect the comparability of estimates between and within countries and/or risk factors, since they might significantly influence the quantification of the attributable burden. From our analysis we observed that the use of CRA was less common for some types of risk factors and outcomes. These included environmental and occupational risk factors, which are more likely to use bottom-up approaches for health outcomes where disease envelopes may not be available. Conclusions: Our review also highlighted misreporting, the lack of uncertainty analysis and the under-investigation of causal relationships in BoD studies. Development and use of guidelines for performing and reporting BoD studies will help understand differences, avoid misinterpretations thus improving comparability among estimates.
|
|
| 3. |
- Gupta, Dhanu, et al.
(författare)
-
Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles
- 2021
-
Ingår i: Nature Biomedical Engineering. - Stockholm : Karolinska Institutet, Dept of Laboratory Medicine. - 2157-846X.
-
Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.
|
|
| 4. |
- Muthukrishnan, Uma, 1984-, et al.
(författare)
-
The exosome membrane localization of histones is independent of DNA and upregulated in response to stress
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Extracellular histones contribute to many acute and chronic diseases but also populate the secretomes of healthy cells and biofluids. However, a secretory pathway for histones has not been described. Here we report that core and linker histones localize to multivesicular bodies and are secreted via exosomes. Histones are tightly associated with the exosome membrane, with N-terminal domains exposed, in a DNA-independent manner. Furthermore, rapid upregulation of exosomal histones occurs following heat stress, accompanied by enhanced vesicle secretion and a shift towards a population of smaller vesicles. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) complex as a possible mechanism underlying increased histone secretion.We show for the first time that membrane-associated histones are actively secreted from intact cells via the multivesicular body/exosomal pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.
|
|
| 5. |
- Sork, Helena, et al.
(författare)
-
Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome
- 2018
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding-and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.
|
|
| 6. |
- Sork, Helena, et al.
(författare)
-
Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency
- 2016
-
Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
|
|
| 7. |
- Sork, Helena, et al.
(författare)
-
Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion
- 2021
-
Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 10:6
-
Tidskriftsartikel (refereegranskat)abstract
- The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R-2 = 0.69-0.87), quantitatively an outstanding 96.2-99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 degrees C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.
|
|
| 8. |
- Abbafati, Cristiana, et al.
(författare)
-
- 2020
-
Tidskriftsartikel (refereegranskat)
|
|
