Purification and biodistribution of extracellular vesicles

Extracellular vesicles (EVs) are nano-sized vesicles that contain bioactive lipids, RNAs and proteins, which can be transferred to recipient cells. EVs are important for physiological as well as pathological processes, such as coagulation and immune homeostasis, aiding cancer metastasis and spread of infectious diseases. Owing to their relatively small size the purification of EVs is a challenge, hence we have established and optimised workflows consisting of ultrafiltration with subsequent size exclusion liquid chromatography (UFLC)( Paper I) and bind-elute combined with size exclusion (BE-SEC) columns (Paper III) for EV purification. UF-LC allowed for purification of biophysically intact EVs with better yield and purity compared to ultracentrifugation (UC), which is the gold standard purification method in the field. The biodistribution of UF-LC EVs was different compared to vesicles isolated using UC, despite having highly similar protein composition according to proteomics analysis. We found that UF-LC vesicles accumulated less in lung, possibly owing to their higher integrity. Indeed, fluorescence correlation spectroscopy and transmission electron microscopy indicated that the high gravitational forces in UC lead to aggregation and disruption of the vesicles. The BE-SEC method is a similar method to UF-LC, however protein impurities less than 700 kDa in size are bound in the interior of the beads, thus improving simple size-based exclusion. The BE-SEC method is scalable, produces samples with better purity than UC, displaying yields exceeding 70% and demonstrates a good reproducibility between samples. Moreover, vesicles purified by BE-SEC display the same EV surface markers as UC purified EVs, and CD63-eGFP positive vesicles are taken up in recipient cells to the same extent. In summary, the BE-SEC method is a reproducible and fast alternative to UF-LC for large media volumes. Reliable purification methods are important for the implementation of therapeutically active EVs, however knowledge regarding their eventual organotropism and biodistribution is equally important. Thus, in article II we evaluated the biodistribution of EVs specifically labeled with a near-infrared dye. The main sites of accumulation of exogenously injected EVs were liver, spleen and lungs. Biodistribution profile of EVs depended strongly on injection route, and to certain extent, on EV cell type source, as dendritic cell derived EVs exhibited a more pronounced uptake in spleen compared to the other cell sources tested. We further showed that small alterations of EV surface proteins could significantly affect biodistribution as well, since EVs equipped with a brain targeting peptide on their surface increased the uptake of targeted EVs in brain. This study highlights that the biodistribution of EVs follows other nano-sized particles with uptake mainly in liver. Administration route, cell source and a targeting peptide influence the distribution, however the overall distribution is unaltered with the highest signal originating from liver. To summarise, this thesis has resulted in improvements of the EV field by systematically enhancing EV isolation workflows to achieve greater sample purity and at the same time preserving EV biophysical characteristics. Furthermore, it has laid groundwork for studying in vivo effects of exogenous vesicles. Both these aspects are particularly important for understanding EV biology more clearly and with increased detail

Supramolecular Assembly of Aminoethylene‐Lipopeptide PMO Conjugates into RNA Splice‐Switching Nanomicelles

Phosphorodiamidate morpholino oligomers (PMOs) are oligonucleotide analogs that can be used for therapeutic modulation of pre‐mRNA splicing. Similar to other classes of nucleic acid‐based therapeutics, PMOs require delivery systems for efficient transport to the intracellular target sites. Here, artificial peptides based on the oligo(ethylenamino) acid succinyl‐tetraethylenpentamine (Stp), hydrophobic modifications, and an azide group are presented, which are used for strain‐promoted azide‐alkyne cycloaddition conjugation with splice‐switching PMOs. By systematically varying the lead structure and formulation, it is determined that the type of contained fatty acid and supramolecular assembly have a critical impact on the delivery efficacy. A compound containing linolenic acid with three cis double bonds exhibits the highest splice‐switching activity and significantly increases functional protein expression in pLuc/705 reporter cells in vitro and after local administration in vivo. Structural and mechanistic studies reveal that the lipopeptide PMO conjugates form nanoparticles, which accelerate cellular uptake and that the content of unsaturated fatty acids enhances endosomal escape. In an in vitro Duchenne muscular dystrophy exon skipping model using H2K‐mdx52 dystrophic skeletal myotubes, the highly potent PMO conjugates mediate significant splice‐switching at very low nanomolar concentrations. The presented aminoethylene‐lipopeptides are thus a promising platform for the generation of PMO‐therapeutics with a favorable activity/toxicity profile

MAGIQ at the W. M. Keck Observatory: initial deployment of a new acquisition, guiding, and image quality monitoring system

The W. M. Keck Observatory has completed the development and initial deployment of MAGIQ, the Multi-function Acquisition, Guiding and Image Quality monitoring system. MAGIQ is an integrated system for acquisition, guiding and image quality measurement for the Keck telescopes. This system replaces the acquisition and guiding hardware and software for existing instruments at the Observatory and is now the standard for visible wavelength band acquisition cameras for future instrumentation. In this paper we report on the final design and implementation of this new system, which includes three major components: a visible wavelength band acquisition camera, image quality measurement capability, and software for acquisition, guiding and image quality monitoring. The overall performance is described, as well as the details of our approach to integrating low order wavefront sensing capability in order to provide closed loop control of telescope focus

Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome

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.Peer reviewe

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

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.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

SN Zwicky: uncovering a population of gravitational lens galaxies with magnified "standard candles"

We report the discovery of a very rare phenomenon, a multiply-imaged gravitationally lensed Type Ia supernova (SNe Ia), "SN Zwicky", a.k.a. SN 2022qmx, magnified nearly twenty-five times by a foreground galaxy. The system was identified as intrinsically bright thanks to the "standard candle" nature of SNe Ia. Observations with high-spatial resolution instruments resolved a system with four nearly simultaneous images, with an Einstein radius of only $\theta_E =0.167"$, corresponding to a lens mass of $8\cdot 10^9$ solar masses within a physical size below $0.8$ kiloparsecs. A smooth lens model fails to reproduce the image flux ratios, suggesting significant additional magnification from compact objects. Given the small image splitting and a relatively faint deflecting galaxy, the lensing system would not have been found through the angular separation technique generally used in large imaging surveys

MAGIQ at the W. M. Keck Observatory: initial deployment of a new acquisition, guiding, and image quality monitoring system

The W. M. Keck Observatory has completed the development and initial deployment of MAGIQ, the Multi-function Acquisition, Guiding and Image Quality monitoring system. MAGIQ is an integrated system for acquisition, guiding and image quality measurement for the Keck telescopes. This system replaces the acquisition and guiding hardware and software for existing instruments at the Observatory and is now the standard for visible wavelength band acquisition cameras for future instrumentation. In this paper we report on the final design and implementation of this new system, which includes three major components: a visible wavelength band acquisition camera, image quality measurement capability, and software for acquisition, guiding and image quality monitoring. The overall performance is described, as well as the details of our approach to integrating low order wavefront sensing capability in order to provide closed loop control of telescope focus

Osteoporosis in the community. Sensitivity of self-reported estimates and medication use of those diagnosed with the condition

Objectives: To assess the sensitivity and specificity of self-reported osteoporosis compared with dual energy X-ray absorptiometry (DXA) defined osteoporosis, and to describe medication use among participants with the condition. Methods: Data were obtained from a population-based longitudinal study and assessed for the prevalence of osteoporosis, falls, fractures and medication use. DXA scans were also undertaken. Results: Overall 3.8% (95% confidence interval (CI) 3.2 to 4.5) of respondents and 8.8% (95% CI 7.5 to 10.3) of those aged ≥ 50 years reported that they had been diagnosed with osteoporosis by a doctor. The sensitivity (those self-reporting osteoporosis and having low bone mineral density (BMD) on DXA) was low (22.7%), although the specificity was high (94.4%). Only 16.1% of those aged ≥ 50 years and with DXA-defined osteoporosis were taking bisphosphonates. Conclusions: The sensitivity of self-reporting to identify osteoporosis is low. Anti-osteoporotic medications are an important part of osteoporosis treatment but opportunities to use appropriate medications were missed and inappropriate medications were used.T. K. Gill, A. W. Taylor, C. L. Hill, P. J. Phillip

A united statement of the global chiropractic research community against the pseudoscientific claim that chiropractic care boosts immunity.

BACKGROUND: In the midst of the coronavirus pandemic, the International Chiropractors Association (ICA) posted reports claiming that chiropractic care can impact the immune system. These claims clash with recommendations from the World Health Organization and World Federation of Chiropractic. We discuss the scientific validity of the claims made in these ICA reports. MAIN BODY: We reviewed the two reports posted by the ICA on their website on March 20 and March 28, 2020. We explored the method used to develop the claim that chiropractic adjustments impact the immune system and discuss the scientific merit of that claim. We provide a response to the ICA reports and explain why this claim lacks scientific credibility and is dangerous to the public. More than 150 researchers from 11 countries reviewed and endorsed our response. CONCLUSION: In their reports, the ICA provided no valid clinical scientific evidence that chiropractic care can impact the immune system. We call on regulatory authorities and professional leaders to take robust political and regulatory action against those claiming that chiropractic adjustments have a clinical impact on the immune system

Contribution of copy number variants to schizophrenia from a genome-wide study of 41,321 subjects

Copy number variants (CNVs) have been strongly implicated in the genetic etiology of schizophrenia (SCZ). However, genome-wide investigation of the contribution of CNV to risk has been hampered by limited sample sizes. We sought to address this obstacle by applying a centralized analysis pipeline to a SCZ cohort of 21,094 cases and 20,227 controls. A global enrichment of CNV burden was observed in cases (OR=1.11, P=5.7×10−15), which persisted after excluding loci implicated in previous studies (OR=1.07, P=1.7 ×10−6). CNV burden was enriched for genes associated with synaptic function (OR = 1.68, P = 2.8 ×10−11) and neurobehavioral phenotypes in mouse (OR = 1.18, P= 7.3 ×10−5). Genome-wide significant evidence was obtained for eight loci, including 1q21.1, 2p16.3 (NRXN1), 3q29, 7q11.2, 15q13.3, distal 16p11.2, proximal 16p11.2 and 22q11.2. Suggestive support was found for eight additional candidate susceptibility and protective loci, which consisted predominantly of CNVs mediated by non-allelic homologous recombination