Biology subjects

Olsen, E.

Publications and source records attributed to Olsen, E..

9 recordsLinked to original sources

bioRxiv2026

Conformational Basis of Functionally Selective Allosteric Modulation of the Angiotensin II type 1 Receptor by Small Molecules

Blockade of signaling through the angiotensin II type 1 receptor (AT1R), a prototypical G protein-coupled receptor (GPCR), by angiotensin receptor blockers (ARBs) is a major therapeutic approach to treating a wide variety of cardiovascular and renal diseases1. Like most GPCRs, the AT1R signals through two transducers, G proteins and {beta}-arrestins2,3. Previous reports have described {beta}-arrestin-biased peptide orthosteric agonists for the AT1R with potential therapeutic advantages over currently available unbiased ARBs4-6. Here we report the DNA- encoded library screening-guided isolation and pharmacological characterization of the first small molecule AT1R allosteric ligands. We use cryo-electron microscopy, double electron- electron resonance spectroscopy, molecular dynamics simulations, and targeted mutagenesis to determine their binding sites, binding modes and conformational mechanisms driving their unique and divergent modulatory effects on G protein and {beta}-arrestin pathways. Our findings uncover new mechanisms for precisely controlling the dynamic behavior of the AT1R with implications for drug development targeting this pathophysiologically important receptor family.

biochemistry↗

bioRxiv2026

Direct Counting of mRNA Copies Inside Individual Lipid Nanoparticles Using In Situ Lysis and Labeling

The optimization of mRNA-lipid nanoparticles (mRNA-LNPs) for therapeutic applications is limited in part by the inadequate characterization of mRNA payload heterogeneity. One current challenge is accurately measuring the number of mRNA copies within individual LNPs, where the standard method of intensity-based mRNA number determination is sensitive to fluorescent dye-dye interactions and heterogeneity of mRNA labeling. Here we present a single-particle microscopy method that combines direct counting of the mRNA copies per LNP with LNP size measurements. While confined in microwells, individual mRNA-LNPs are lysed to release their cargo and stained with a dye such that the number of mRNA molecules in each well can be directly counted using fluorescence microscopy. Since the method stains the mRNA cargo in situ, it enables characterization of LNPs formulated with therapeutic grade (e.g., unlabeled) mRNA. We applied this approach to two Onpattro(R)-based LNP formulations prepared using different formulation buffers, where the two formulations had different average mRNA copy number, particle size, and fraction of LNPs lacking mRNA. The ability to directly count the number of mRNA molecules in LNPs establishes a complimentary method to intensity-based mRNA number determination and supports the characterization and screening of clinically relevant LNP formulations.

biophysics↗

bioRxiv2026

SIZE DETERMINATION AND MULTIPLEXED FLUORESCENCE-BASED PHENOTYPING OF SINGLE CELL-DERIVED MEMBRANE VESICLES USING A NANOFLUIDIC DEVICE

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

biophysics↗

bioRxiv2025

Single-particle multi-parametric microscopy reveals structural, size, and payload heterogeneity in mRNA-loaded lipid nanoparticles

Deciphering the heterogeneity of mRNA-containing lipid nanoparticles (LNPs) is essential for understanding the relationship between their microscopic properties and therapeutic function. Here, by combining alternating laser excitation (ALEX) with Convex Lens-induced Confinement (CLiC) microscopy, we simultaneously measure size, multi-color fluorescence, mRNA payload, and Forster resonance energy transfer (FRET) of individual suspended LNPs containing labeled lipid and mRNA molecules. By varying formulation parameters, including ionizable lipids, formulation buffers, and molecular ratios, we investigated and correlated key microscopic properties for relevant vaccine formulations. While the per-particle lipid fluorescence was lower for empty versus mRNA-loaded particles for all formulations, the relative size of empty versus mRNA-loaded particles depended upon the formulation and intraparticle structure. When comparing CLiC-ALEX to cryogenic transmission electron microscopy measurements (Cryo-TEM), for the LNP formulations that display blebs, the fraction of bleb LNPs is in close agreement with the fraction of mRNA-containing LNPs. CLiC-ALEX also enabled quantification of the per-particle mRNA fluorescence and FRET signals, and thus the heterogeneity in the mRNA copy number and mRNA-LNP structural arrangements, where the results were compared with biophysical estimates based on the LNP formulations. These rigorous biophysical insights are critical to inform our understanding of structure-activity relationships and inform rational design of nanomedicines.

biophysics↗

bioRxiv2025

Multiparametric Characterization of Individual Suspended Nanoparticles using Confocal Fluorescence and Interferometric Scattering Microscopy with Microfluidic Confinement

Detailed characterization of the size, mass, payload, and structure of suspended mRNA-lipid nanoparticles (LNPs) is necessary to improve our understanding of how these heterogenous properties influence therapeutic efficacy and potency. Methods currently in use face limitations in reporting ensemble-average particle properties or requiring dedicated home-built microscopes that are beyond the reach of nanoparticle developers. In this work, we overcome these limitations by combining a commercially available confocal microscope and a convex lens-induced confinement (CLiC) instrument to achieve simultaneous characterization and correlation of the size, mass, refractive index, and nucleic acid payload of individual LNPs. We established the accuracy and precision of our method using nanosized beads and used it to investigate the size, payload, and water content of LNPs in different solvent pH. By employing readily available microscopy tools, we open the door to widespread adoption of our quantitative, in-solution nanoparticle characterization method.

biophysics↗

bioRxiv2025

Long-term, age-associated activity quantification in the DE50-MD dog model of Duchenne muscular dystrophy (DMD)

Animal models with a clinically relevant phenotype remain important for robust evaluation of novel therapeutics for the fatal, X-linked genetic disorder, Duchenne Muscular Dystrophy (DMD). Demonstration of functional improvement is crucial for both patients and regulatory authorities. DMD is associated with a decline in musculoskeletal function with progressive paresis, muscle atrophy and fibrosis: phenotypic features that are also seen in the DE50-MD canine model of DMD. Here we investigate non-invasive methods to quantify changes in activity and behaviour in DE50-MD dogs, using collar-based, tri-axial accelerometers. We measured activity in affected DE50-MD male dogs (3-8 per age point) and littermate wild-type (WT) male controls (3-13 per age point) at monthly intervals from 3 to 18 months of age using Axivity-AX3 accelerometers attached ventrally on each dogs collar. Data were recorded for 48 hours while dogs remained in their kennels with outside runs following their normal routine. Acceleration vector magnitudes were used to derive various activity indicators over a 24-hour period. Mixed model analyses were used to examine differences between affected and WT groups at different ages. DE50-MD dogs activity indicators were significantly higher for % time spent at rest (p<0.001) and significantly lower for all other activity indicators (all p<0.05), when compared to age-matched WT dogs. Sample size calculations reveal that these non-invasive and objective biomarkers offer significant promise for preclinical testing of therapeutics in this model of DMD. Our approach reveals opportunities for cross-model standardisation of activity monitoring methods, applicable to both research and companion animal settings. Summary statementThe DE50-MD dog model of Duchenne muscular dystrophy shows significant age-associated reduction in activity quantified through non-invasive, wearable accelerometers. Activity metrics tested show promise for objective assessment of activity patterns for preclinical trials.

neuroscience↗

bioRxiv2024

Time Resolved Inspection of Ionizable-Lipid Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Endosomal Membrane Mimic

This study investigates pH-triggered fusion dynamics of lipid nanoparticles (LNPs) with an endosomal membrane mimic, addressing mechanistic aspects of a crucial yet elusive process for effective mRNA delivery. Utilizing time-resolved total internal reflection fluorescence (TIRF) imaging, we observed a delayed onset of LNP fusion upon pH drop, lasting seconds to minutes depending on pH and LNP size. Once initiated, LNP fusion and cargo escape occurred rapidly within tens to hundreds of milliseconds. While LNP disintegration is observed to lead to release of a significant portion of mRNA into the acidic environment, some mRNA molecules remained mobile on the endosomal membrane mimic due to deprotonation-resistant complex salt formation. Comparison of the fusion efficiency of two LNP formulations correlated with protein translation in human primary cell transfection data, emphasizing the importance of biophysical investigations in understanding ionizable-lipid-containing LNP-assisted mRNA delivery mechanisms and providing insights for optimizing mRNA-LNP design for enhanced endosomal escape.

biophysics↗

bioRxiv2023

No evidence for enhanced disease with polyclonal SARS-CoV-2 antibody in the ferret model.

Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-enhanced disease (AED) as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for AED, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.

microbiology↗

bioRxiv2019

Distribution and diversity of fish species exposed to artisanal fishery along the Sudanese Red Sea coast.

The semi-enclosed Red Sea harbours one of the longest coral-reef ecosystems on the planet. The {approx} 850 km section of the western shore, comprising the coastline of the Red Sea State of the Republic of Sudan, has however been sparsely studied. Sudans coral reef fishery provides livelihoods to fishers and business opportunities by means of local and regional trade, however, the knowledge level of the state of the natural resources and the impacts of fisheries are poorly known. Here we report the results from the first three comprehensive fisheries research surveys spanning the entire Sudanese coast in 2012-13, representing a new baseline for the western coast fisheries resources. The surveys covered the entire coast from inshore down to about 150 m bottom depth using a combination of baited traps, gillnets and handlines to sample the various reef habitats and fish assemblages. The results demonstrate a uniform latitudinal species distribution with peak catch per unit effort rates in and around the Dungonab Bay area in the north and the outer Suakin archipelago in the south. Functional diversity (Raos Q index) was found to be highest in and around the Dungonab Bay area, thus coming through as a regional hot-spot of biodiversity. The results form a baseline for future research and monitoring, thus representing key input for an ecosystem approach to management of Sudans coastal artisanal fisheries.

ecology↗