Biology subjects

Shepherd, S. J.

Publications and source records attributed to Shepherd, S. J..

6 recordsLinked to original sources

bioRxiv2025

Automated and parallelized microfluidic generation of large and precisely-defined lipid nanoparticle libraries

Building on the success of lipid nanoparticles (LNPs) in vaccines, LNPs are being developed for a broad set of therapeutic applications by changing both the structures of the lipids used to formulate each LNP and their relative proportions. Because lipid synthesis and in vivo screening have been parallelized using combinatorial chemistry and LNP barcoding respectively, the manual and sequential microfluidic formulation of LNPs has become the rate-limiting step in the discovery process. In this work, we present a high-throughput, automated microfluidic platform capable of generating large, precisely-defined LNP libraries in parallel at a rate of one distinct formulation every three seconds. Each formulation is defined by varying the reagent flow ratios into one of eight microscale mixers using litho-graphically encoded fluidic resistors and dynamically controlled external pressure supplies. The microfluidic chip is integrated with custom frobotic plate handling for the rapid collection of each distinct formulation. Using this platform, we produce a library of 96 formulations, which we profile physicochemically and evaluate in terms of both in vitro and in vivo transfection.

bioengineering↗

bioRxiv2024

Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery

Designing lipid nanoparticle (LNP) delivery systems with specific targeting, potency and minimal side effects is crucial for their clinical use. However, traditional characterization methods, such as dynamic light scattering, cannot accurately quantify physicochemical properties of LNPs and how they are influenced by the lipid composition and mixing method. Here we structurally characterize polydisperse LNP formulations by applying emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation, field-flow fractionation followed by multi-angle light scattering, and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering. We show that LNPs have intrinsic polydispersity in size, RNA loading, and shape, which depends on both the formulation technique and lipid composition. Lastly, we predict LNP transfection in vitro and in vivo by examining the relationship between mRNA translation and physicochemical characteristics. Solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for LNPs.

bioengineering↗

bioRxiv2024

Mylpf dosage is proportionate to fast-twitch myofibril size in the zebrafish embryo

Muscle becomes stronger by expanding sarcomeres from the cells edge to its center. Here we investigate how Mylpf (Myosin Light Chain Phosphorylatable Fast) abundance impacts sarcomere growth. The two zebrafish Mylpf genes (mylpfa and mylpfb) are exclusively expressed in fast-twitch muscle, with mylpfa expressed more abundantly than mylpfb. Mutations in both genes cause full mRNA and protein loss, and loss of sarcomere formation. The degree of sarcomere reduction in fast-twitch muscle is predicted by Mylpf protein dosage using a high-slope Hill equation. The sarcomere defect may be driven by thick filament localization, which is impaired in mylpfa-/-and obliterated in mylpfa-/-;mylpfb-/-. Slow-twitch muscle is spared, becoming larger and more active in the mutants. The mylpfa-/- defects can be rescued by Mylpfa-GFP, Mylpfb-GFP, and by human MYLPF-GFP with equivalent efficiency, but not by a MYLPF protein variant that causes Distal Arthrogryposis. These effects indicate that Mylpf dosage strengthens muscle by mediating myofilament localization, and thereby sarcomere and myofibril growth.

developmental biology↗

bioRxiv2023

Towards the Clinical Translation of a Silver Sulfide Nanoparticle Contrast Agent: Large Scale Production with a Highly Parallelized Microfluidic Chip

Ultrasmall silver sulfide nanoparticles (Ag2S-NP) have been identified as promising contrast agents for a number of modalities and in particular for dual-energy mammography. These Ag2S-NP have demonstrated marked advantages over clinically available agents with the ability to generate higher contrast with high biocompatibility. However, current synthesis methods are low-throughput and highly time-intensive, limiting the possibility of large animal studies or eventual clinical use of this potential imaging agent. We herein report the use of a scalable silicon microfluidic system (SSMS) for the large-scale synthesis of Ag2S-NP. Using SSMS chips with 1 channel, 10 parallelized channels, and 256 parallelized channels, we determined that the Ag2S-NP produced were of similar quality as measured by core size, concentration, UV-visible spectrometry, and in vitro contrast generation. Moreover, by combining parallelized chips with increasing reagent concentration, we were able to increase output by an overall factor of 3,400. We also found that in vivo imaging contrast generation was consistent across synthesis methods and confirmed renal clearance of the ultrasmall nanoparticles. Finally, we found best-in-class clearance of the Ag2S-NP occurred within 24 hours. These studies have identified a promising method for the large-scale production of Ag2S-NP, paving the way for eventual clinical translation.

bioengineering↗

bioRxiv2017

The G-box transcriptional regulatory code in Arabidopsis

Plants have significantly more transcription factor (TF) families than animals and fungi, and plant TF families tend to contain more genes--these expansions are linked to adaptation to environmental stressors (1, 2). Many TF family members bind to similar or identical sequence motifs, such as G-boxes (CACGTG), so it is difficult to predict regulatory relationships. We determine that the flanking sequences near G-boxes help determine in vitro specificity, but that this is insufficient to predict the transcription pattern of genes near G-boxes. Therefore, we construct a gene regulatory network that identifies the set of bZIPs and bHLHs that are most predictive of the gene expression of genes downstream of perfect G-boxes. This network accurately predicts transcriptional patterns and reconstructs known regulatory subnetworks. Finally, we present Ara-BOX-cis (araboxcis.org), a website that provides interactive visualisations of the G-box regulatory network, a useful resource for generating predictions for gene regulatory relations.

plant biology↗

bioRxiv2017

Integrating Patient And Whole Genome Sequencing Data To Provide Insights Into The Epidemiology Of Seasonal Influenza A(H3N2) Viruses

Genetic surveillance of seasonal influenza is largely focused upon sequencing of the haemagglutinin gene. Consequently, our understanding of the contribution of the remaining seven gene segments to the evolution and epidemiological dynamics of seasonal influenza is relatively limited. The increased availability of next generation sequencing technologies allows rapid and economic whole genome sequencing (WGS). Here, 150 influenza A(H3N2) positive clinical specimens with linked epidemiological data, from the 2014/15 season in Scotland, were sequenced directly using both Sanger sequencing of the HA1 region and WGS using the Illumina MiSeq platform. Sequences generated by both methods were highly consistent and WGS provided on average >90% whole genome coverage. As reported in other European countries during 2014/15, all strains belonged to genetic group 3C, with subgroup 3C.2a predominating. Inter-subgroup reassortants were identified (9%), including three 3C.3 viruses descended from a single reassortment event, which had persisted in the population. Significant phylogenetic associations with cases of severe acute respiratory illness observed herein warrant further investigation. Severe cases were also more likely to be associated with reassortant viruses (odds ratio: 4.4 (1.3-15.5)) and occur later in the season. These results suggest that increased levels of WGS, linked to clinical and epidemiological data, could improve influenza surveillance.

microbiology↗