Biology subjects

Thompson, S.

Publications and source records attributed to Thompson, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

The Effects of Intraspecies and Interspecies Competition on Genetic Device Construction and Performance

Future genetic devices expressed in microbes need to be tested in complex environments containing multiple organisms to expand their usability. We sought to determine if genetic parts designed for monoculture are predictable when used in co-culture by testing constitutive Anderson promoters driving expression of chromoproteins from a plasmid. In Escherichia coli monoculture a high copy number origin of replication causes stochastic expression regardless of promoter strength, and high Anderson promoter strength leads to selection for inactivating mutations, resulting in inconsistent chromoprotein expression. Medium and low strength Anderson promoters function more predictably in E. coli monoculture but experience an increase in inactivating mutations when grown in co-culture over many generations with Pseudomonas aeruginosa. Expression from native promoters instead of Anderson promoters can lead to stable expression in a complex wastewater culture. Overall, we show intraspecies selection for inactivating mutations due to a competitive growth advantage for E. coli that do not express the genetic device compared to their peers that retain the functional device. We show additional interspecies selection against the functional device when E. coli is co-cultured with another organism. Together, these two selection pressures create a significant barrier to genetic device function in microbial communities that we overcome by utilize a native E. coli promoter. Future strategies for genetic device design in microorganisms that need to function in a complex microbial environment should focus on native promoters and/or strategies that give the microorganism carrying the device a selective or growth advantage. ImportanceFirst generation biotechnology focused on genetic devices designed for use in monoculture conditions. Next generation biotechnology devices will need to function in complex ecosystems with other organisms, so we sought to create conditions where the genetic device retained function when the organism carrying it is in co-culture with other organisms. We discovered that when the genetic device is a significant resource burden on the organism carrying the device mutations will be selected for due to intraspecies and interspecies selection pressures and the device will be rendered non-functional. Therefore, genetic device design for complex ecosystems in next generation biotechnology needs to balance functionality of the genetic device with the need to reduce resource burden on the organism carrying it.

synthetic biology↗

bioRxiv2025

Host microbiome determines host specificity of the human whipworm, Trichuris trichiura

Long-term whipworm-host co-evolution has resulted in tropism for the caecum of specific hosts, an organ with the densest microbial population in the body. Here, we demonstrate that the host specificity of human whipworms (Trichuris trichiura) is host microbiome-driven. We successfully establish a T. trichiura infection in a non-primate host using a humanised-microbiota mouse model. We further show, in vitro, that hatching of T. trichiura was triggered by mucosal scrapings of the caecum of human microbiota-associated mice, but not from wild-type mice, which only induced T. muris hatching. Comparative metagenomic analysis of the murine versus humanised microbiomes directly implicated specific bacterial species in T. trichiura egg hatching. Additionally, we demonstrate that host tissue does not directly determine host specificity, as T. trichiura readily infected mouse caecaloids. Our findings indicate that host-microbiome-whipworm co-evolution has resulted in exquisite bacterial-whipworm egg interactions critical for hatching and development of these parasites in their definitive hosts.

microbiology↗

bioRxiv2025

Continuous FACS sorting of double emulsion picoreactors with a 3D printed vertical mixer.

High-throughput screening and directed evolution using microfluidic picoreactors have produced high-activity enzymes. In this approach, a substrate is co-encapsulated with a candidate enzyme and individual picoreactors are sorted based on an activity reporter. While many approaches use water-in-oil droplets (single emulsions) for fluorescence-activated droplet sorting (FADS) on custom-fabricated microfluidic devices that require integrated optics and electronics, recent approaches have lowered the engineering barriers to adoption by using simple microfluidic droplet generators to produce water-in-oil-in-water droplets (double emulsion picoreactors, DEs) that can be sorted with commercial FACS (fluorescence-activated cell sorting). Despite the simplified engineering requirements, high variability in loading rates and low yield during loading are barriers to efficient DE FACS sorting. Here, we optimized surfactants to enhance DE stability and demonstrated that a 3D-printed corkscrew on the sample line acts as a vertical mixer to enable more continuous loading. With these optimized loading conditions, we analyzed 1.17 million DEs in four 10-minute sorting rounds with a mean frequency of 480 Hz (390 Hz including sample exchanges); in a mock sort of 10% fluorescent DEs, we achieved 89.2{+/-}1.1% accuracy and 78{+/-}0.9% recovery with our optimized loading protocol. Overall, improved ease-of-use and throughput for FACS sortable DEs should expand the accessibility of directed evolution in controlled in vitro environments.

bioengineering↗

bioRxiv2025

Permissive central tolerance plus defective peripheral checkpoints licence pathogenic memory B cells in CASPR2-antibody encephalitis

Autoimmunity affects 10% of the population. Within this umbrella, autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the developmental pathway of disease-causing B cells and autoantibodies. While such autoreactivities are believed to be generated during germinal centre reactions, the roles of earlier immune checkpoints in autoantigen-specific B cell tolerance are poorly understood. We address this concept in patients with CASPR2-autoantibody encephalitis and healthy controls. In both groups, comparable and high ([~]0.5%) frequencies of unmutated CASPR2-reactive naive B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with heterogenous binding kinetics. These effector molecules possessed epitope-dependent pathogenic effects in vitro neuronal cultures and in vivo. The unmutated common ancestors of these memory B cells showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results are the first to propose mechanisms underlying autoantigen-specific tolerance in humans. We identify permissive central tolerance, defective peripheral tolerance and heterogenous autoantibody binding properties as sequential pathogenic steps which licence CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches in CASPR2-antibody diseases. This paradigm is applicable across autoimmune conditions.

immunology↗

bioRxiv2024

Gut bacterial sphingolipid production modulates dysregulated skin lipid homeostasis

Sphingolipids are an essential lipid component of the skin barrier with alterations in skin sphingolipid composition associated with multiple skin disorders including psoriasis, atopic dermatitis, and ichthyosis. Contributions to skin sphingolipid abundance are not well characterized, thus the main method of modulating skin lipid levels is the topical application of creams rich with sphingolipids at the skin surface. Evidence that diet and gut microbiome function can alter skin biology proposes an intriguing potential for the modulation of skin lipid homeostasis through gut microbial metabolism, but potential mechanisms of action are not well understood. Sphingolipid synthesis by prominent gut microbes has been shown to affect intestinal, hepatic and immune functions with the potential for sphingolipid-producing bacteria to affect skin biology through altering skin sphingolipid levels. To address this question, we used bioorthogonal chemistry to label lipids from the sphingolipid-producing bacteria Bacteroides thetaiotaomicron and trace these lipids to the skin epidermis. Exposing mice to B. thetaiotaomicron strains mutant in the ability to produce sphingolipids resulted in significantly lower transfer of gut microbiome-derived lipids to the skin, while also altering skin biology and altering expression of skin barrier genes. Measurement of skin ceramide levels, a class of sphingolipids involved in skin barrier function, determined that skin sphingolipid levels were altered in the presence of gut sphingolipid-producing bacteria. Together this work demonstrates that gut bacterial lipids can transfer to the skin and provides a compelling avenue for modulating sphingolipid-dominant compartments of the skin using sphingolipid-producing bacteria of the gut microbiome.

microbiology↗

bioRxiv2024

Menadione reduces the expression of virulence- and colonisation-associated genes in Helicobacter pylori

Novel treatment options are needed for the gastric pathogen Helicobacter pylori due to its increasing antibiotic resistance. The vitamin K analogue menadione has been extensively studied due to interest in its antibacterial and anti-cancer properties. Here, we investigated the effects of menadione on H. pylori growth, viability, antibiotic resistance, motility and gene expression using clinical isolates. The minimum inhibitory concentration (MIC) of menadione was 313 {micro}M for 11/13 isolates and 156 {micro}M for 2/13 isolates. The minimum bactericidal concentrations were 1.25-2.5 mM, indicating that concentrations in the micromolar range were bacteriostatic rather than bactericidal. We were not able to experimentally evolve resistance to menadione in vitro. Sub-MIC menadione (16 {micro}M for 24 h) did not significantly inhibit bacterial growth but significantly (p < 0.05) changed the expression of 1291/1615 (79.9 %) genes encoded by strain 322A. Expression of the virulence factor genes cagA and vacA was downregulated in the presence of sub-MIC menadione, while genes involved in stress responses were upregulated. Sub-MIC menadione significantly (p < 0.0001) inhibited the motility of H. pylori, consistent with the predicted effects of the observed significant (p < 0.05) downregulation of cheY, upregulation of rpoN, and changes in expression of flagellar assembly pathway genes seen in the transcriptomic analysis. Through in-depth interrogation of transcriptomics data, we concluded that sub-MIC menadione elicits a general stress response in H. pylori with survival in the stationary phase likely mediated by upregulation of surE and rpoN. Sub-MIC menadione caused some modest increases in H. pylori susceptibility to antibiotics, but the effect was variable with strain and antibiotic type and did not reach statistical significance. Menadione (78 {micro}M) was minimally cytotoxic to human gastric adenocarcinoma (AGS) cells after 4 hours but caused significant loss of cell viability after 24 hours. Given its inhibitory effects on bacterial growth, motility and expression of virulence and colonisation- associated genes, menadione at low micromolar concentrations may have potential utility as a virulence-attenuating agent against H. pylori. Data summaryRNAseq data reported on in this study are available to download from ArrayExpress under accession E-MTAB-14439. Supplementary materials associated with this publication are available from https://figshare.com/projects/Menadione_reduces_the_expression_of_virulence-_and_colonisation-associated_genes_in_Helicobacter_pylori/219631.

microbiology↗

bioRxiv2024

ER Aggregation Causes Synaptic Protein Imbalance in Retinitis Pigmentosa Mutant Photoreceptor Neurons

Rod photoreceptor neurons in the retina detect scotopic light through the visual pigment rhodopsin (Rho) in their outer segments (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health. Given the importance of protein trafficking to the OS, less is known about the trafficking of rod synaptic proteins. Furthermore, the subcellular impact of Rho mislocalization on rod synapses (i.e., "spherules") has not been investigated. In this study we used super-resolution and electron microscopies, along with proteomics, to perform a subcellular analysis of Rho synaptic mislocalization in P23H-Rho-RFP mutant mice. We discovered that mutant P23H-Rho-RFP protein mislocalized in distinct ER aggregations within the spherule cytoplasm, which we confirmed with AAV overexpression. Additionally, we found synaptic protein abundance differences in P23H-Rho-RFP mice. By comparison, Rho mislocalized along the spherule plasma membrane in WT and rd10 mutant rods, in which there was no synaptic protein disruption. Throughout the study, we also identified a network of ER membranes within WT rod presynaptic spherules. Together, our findings indicate that photoreceptor synaptic proteins are sensitive to ER dysregulation. Summary StatementThis study examines the impact of rhodopsin mislocalization on rod photoreceptor synaptic structures and synaptic protein levels using P23H rhodopsin and other retinitis pigmentosa mouse models.

neuroscience↗

bioRxiv2024

Cell-free immuno-profiling on a genetically programmed biochip

Emerging cell-free synthetic biology approaches provide biosafe, cheap, and versatile genetic tools to advance therapeutic research and development. Combined with micro-fabrication technology, we developed a platform to quantitatively reconstitute interactions of cell-free synthesized antigens with antibodies and human receptors in miniaturized compartments on a silicon chip. Photolithographic surface patterning of protein traps and on chip expression from high density gene brushes generated a continuous surface density gradient of fluorescently labeled antigens. Antibodies binding to the antigen gradient generate a full binding curve in each single compartment for affinity determination. We used the SARS-CoV-2 antigens as a model to profile the specificity and affinity of monoclonal antibodies to > 30 viral epitopes synthesized simultaneously on one chip in a genotype-phenotype linked compartments. We further profiled polyclonal antibodies in minute volumes of human sera, revealing patient-specific epitope profiles that are difficult to detect by conventional approaches. Cell-free co-synthesis of the human ACE2 receptor with the viral Receptor-Binding-Domain yielded relative binding affinities to different SARS-CoV-2 variants. This rapid, quantitative, and on-chip genetically programmed approach allows to study complex protein-protein interactions independent of protein purification steps for human immuno-profiling with a fast response time for combating emerging pathogens.

bioengineering↗

bioRxiv2024

The Visual Experience Evaluation Tool: A Myopia Research Instrument for Quantifying Visual Experience

Current myopia research has demonstrated the role of extended visual experience in healthy ocular development. Optical cues and the spectrum, intensity, and temporal characteristics of light landing on the retina are all known factors affecting the development of the eye. However, there is still limited understanding as to which of these extrinsic factors are most important or how they interplay with intrinsic physical and neural differences between individuals. Part of the problem is inadequate tooling. Our team at Reality Labs Research created the Visual Environment Evaluation Tool (VEET), a non-commercial research instrument, to accelerate myopia research. In this paper, we describe the VEETs physical design, sensor suite and capabilities, and the associated software which makes it well-suited for research of quantified visual experience.

neuroscience↗

bioRxiv2024

Protein Language Models in Directed Evolution

The dominant paradigms for integrating machine-learning into protein engineering are de novo protein design and guided directed evolution. Guiding directed evolution requires a model of protein fitness, but most models are only evaluated in silico on datasets comprising few mutations. Due to the limited number of mutations in these datasets, it is unclear how well these models can guide directed evolution efforts. We demonstrate in vitro how zero-shot and few-shot protein language models of fitness can be used to guide two rounds of directed evolution with simulated annealing. Our few-shot simulated annealing approach recommended enzyme variants with 1.62 x improved PET degradation over 72 h period, outperforming the top engineered variant from the literature, which was 1.40 x fitter than wild-type. In the second round, 240 in vitro examples were used for training, 32 homologous sequences were used for evolutionary context and 176 variants were evaluated for improved PET degradation, achieving a hit-rate of 39 % of variants fitter than wild-type.

bioinformatics↗

bioRxiv2024

The consequences of mismatched buffers in spectral cell sorting

Although spectral flow cytometry has become a ubiquitous tool for cell analysis, the use of spectral cytometry on cell sorters requires additional considerations arising from the unique requirements of sorting workflows. Here, we show that care should be taken when ascertaining the purity of a sort on a spectral cell sorter, as the mismatch of buffers used for initial sample suspension and the buffers used for sort collection can affect the unmixing of the data, potentially giving rise to erroneous purity check results.

cell biology↗

bioRxiv2024

FACS-Sortable Picoreactors for Ultra High-throughput Screening of Catalysts in Biphasic Environments

Biphasic environments can enable successful chemical reactions where any single solvent results in poor substrate solubility or poor catalyst reactivity. For screening biphasic reactions at high-throughput, a platform based on microfluidic double emulsions could use widely available FACS (Fluorescence Activated Cell Sorting) machines to screen millions of picoliter reactors in a few hours. However, encapsulating biphasic reactions within double emulsions to form FACS-sortable droplet picoreactors requires optimized solvent phases and surfactants to produce triple emulsion droplets that are stable over multi-hour assays and compatible with desired reaction conditions. This work demonstrates such FACS-sortable triple emulsion picoreactors with a fluorocarbon shell and biphasic octanol-in-water core. First, surfactants were screened to stabilize octanol-in-water emulsions for the picoreactor core. With these optimized conditions, stable triple emulsion picoreactors were produced (>70% of droplets survived to 24 hours), and the ability to produce protein in the biphasic core was demonstrated via cell-free protein synthesis. Finally, triple emulsion picoreactors were sorted based on fluorescence using commercial FACS sorters at >100 Hz with 75-80% of droplets recovered. These triple emulsion picoreactors have potential for future screening bead-encoded catalyst libraries, including enzymes such as lipases for biofuel production.

bioengineering↗

bioRxiv2024

Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins

Integral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPs in vitro or labeling IIMPs without disrupting their function in vivo. Here, we report TXTL-QCMD to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes or oscillating interactions by the E. coli Min system. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system unamenable to labeling, we discovered that ZorA and ZorB integrate together within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.

biophysics↗

bioRxiv2023

Characterising the contribution of auditory and somatosensory inputs to TMS-evoked potentials following stimulation of prefrontal, premotor and parietal cortex

Transcranial magnetic stimulation (TMS) results in a series of evoked potentials (TEPs) in electroencephalography (EEG) recordings. However, it remains unclear whether these responses reflect neural activity resulting from transcranial stimulation of the cortex, or from the sensory experiences of TMS. Across three experiments (total n = 135), we recorded EEG activity following TMS to the dorsolateral prefrontal cortex, premotor cortex, and parietal cortex as well as a sensory control condition (stimulation of the shoulder or electrical stimulation of the scalp with a click sound). We found that TEPs showed a stereotypical frontocentral N100/P200 complex following TMS of all cortical sites and control conditions, regardless of TMS intensity or the type of sensory control. In contrast, earlier TEPs (<60 ms) showed site-specific characteristics which were largest at the site of stimulation. Self-reported sensory experiences differed across sites, with prefrontal stimulation resulting in stronger auditory (click sound perception) and somatosensory input (scalp muscle twitch, discomfort) than premotor or parietal stimulation, a pattern that was reflected in the amplitude of later (N100/P200), but not earlier (<60 ms) TEP peak amplitudes. Later TEPs were also larger in individuals who experienced stronger click sound perception and, to a lesser extent, TMS-evoked scalp muscle twitches. Increasing click sound perception by removing auditory masking increased N100/P200 amplitudes without altering earlier peaks, an effect which was more prominent at sites with more successful masking. Together, these findings suggest that the frontocentral N100/P200 complex represents a generalised sensory response resulting from TMS-related auditory and somatosensory input. In contrast, early TEP peaks likely reflect activity resulting from transcranial stimulation of the cortex. The results have important implications for designing and interpreting TEP studies, especially when comparing TEPs between stimulation sites and participant groups showing differences in sensory experiences following TMS.

neuroscience↗

bioRxiv2023

The Genetic Landscape of a Metabolic Interaction

Enzyme abundance, catalytic activity, and ultimately sequence are all shaped by the need of growing cells to maintain metabolic flux while minimizing accumulation of deleterious intermediates. While much prior work has explored the constraints on protein sequence and evolution induced by physical protein-protein interactions, the sequence-level constraints emerging from non-binding functional interactions in metabolism remain unclear. To quantify how variation in the activity of one enzyme constrains the biochemical parameters and sequence of another, we focused on dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), a pair of enzymes catalyzing consecutive reactions in folate metabolism. We used deep mutational scanning to quantify the growth rate effect of 2,696 DHFR single mutations in 3 TYMS backgrounds under conditions selected to emphasize biochemical epistasis. Our data are well-described by a relatively simple enzyme velocity to growth rate model that quantifies how metabolic context tunes enzyme mutational tolerance. Together our results reveal the structural distribution of epistasis in a metabolic enzyme and establish a foundation for the design of multi-enzyme systems.

biochemistry↗

bioRxiv2023

Single cell transcriptomics of the human parasite Schistosoma mansoni first intra-molluscan stage reveals tentative tegumental and stem cell regulators.

BackgroundSchistosomiasis is a major Neglected Tropical Disease, caused by the infection with blood flukes in the genus Schistosoma. To complete the life cycle, the parasite undergoes asexual and sexual reproduction within an intermediate snail host and a definitive mammalian host, respectively. The intra-molluscan phase provides a critical amplification step that ensures a successful transmission. However, the cellular and molecular mechanisms underlying the development of the intra-molluscan stages remain poorly understood. MethodsS. mansoni mother sporocysts were dissociated into single cell suspensions, and live cells enriched and sequenced using the single cell 10X Genomics Chromium platform. We defined somatic and stem/germinal cell clusters, identified cell type-enriched Gene Ontology (GO) terms, and predicted transcription factor binding sites for key marker genes. ResultsSix cell clusters comprising stem/germinal, two tegument, muscle, neuron, and parenchyma were identified and validated by Fluorescence in situ Hybridisation (FISH). GO term analysis predicted key biological processes for each of the clusters. Using the Self-Assembling Manifold (SAM) algorithm, three sub-clusters were identified within the stem/germinal cell population. Furthermore, transcription factor binding sites and putative transcription factors were predicted for stem/germinal and tegument clusters. ConclusionsWe report a spatially validated single cell transcriptomic analysis of the first intra-molluscan stage of S. mansoni. Key cell regulators were identified, paving the way for future analyses to unveil their role during the parasite development and interaction with its intermediate host.

developmental biology↗

bioRxiv2022

Back to the Future: Unleashing your cytometers spectral potential

With the recent growth in spectral flow cytometry many laboratories are investing in new spectral flow cytometers in order to maximise the information gathered about every cell. This study hypothesised that traditional cytometers already within many laboratories may be used as spectral cytometers and have shown using a range of different cytometers that data acquired may be unmixed after acquisition.

bioengineering↗

bioRxiv2022

Identification of a regulatory pathway governing TRAF1 via an arthritis-associated non-coding variant

TRAF1/C5 was among the first loci shown to confer risk for inflammatory arthritis in the absence of an associated coding variant, but its genetic mechanism remains undefined. Using ImmunoChip data from 3,939 juvenile idiopathic arthritis (JIA) patients and 14,412 controls, we identified 132 plausible common non-coding variants, reduced serially by SNP-seq, electrophoretic mobility shift, and luciferase studies to the single variant rs7034653 in the third intron of TRAF1. Genetically manipulated experimental cells and primary monocytes from genotyped donors establish that the risk G allele reduces binding of Fos-Related Antigen 2 (FRA2), resulting in reduced TRAF1 expression and enhanced TNF production. Conditioning on this variant eliminates attributable risk for rheumatoid arthritis, implicating a mechanism shared across the arthritis spectrum. These findings reveal that rs7034653, FRA2, and TRAF1 mediate a pathway through which a non-coding causal variant drives risk of inflammatory arthritis in children and adults.

genetics↗