Biology subjects

Thomson, A.

Publications and source records attributed to Thomson, A..

12 recordsLinked to original sources

bioRxiv2026

Modeling gene regulatory perturbations via deep learning from high-throughput reporter assays

Assessing likely variant effects on phenotypes is of critical importance in diagnostic settings, and while much progress has been made in interpreting genic mutations based on our understanding of coding sequence, noncoding variants can be much more challenging to reliably interpret based on DNA sequence alone. High-throughput reporter assays such as STARR-seq and MPRA have shown utility in experimentally measuring regulatory effects of noncoding variants present in samples but provide no readout for variants not present in the assay inputs. However, whole-genome reporter assays provide copious data that can be used to train predictive models for prioritizing variants not directly observed in the experiment. We describe a retrainable predictive modeling framework, BlueSTARR, for this task, and present results of training several models with this framework on whole-genome STARR-seq data from two cell lines and one drug treatment. Using these models, we uncover a global signature across the human genome consistent with purifying selection against both loss-of-function and gain-of-function regulatory variants, with the latter showing a significant bias consistent with selection against gains of cis regulatory function in closed chromatin proximal to genes. By testing the model on synthetic enhancers with binding motifs for transcription factors GR and AP-1, we find that when trained on drug perturbation data, the model is able to learn distance-dependent and treatment-dependent binding patterns and their resulting reporter gene activation. These results demonstrate that lightweight, easily retrainable models such as ours have utility in probing latent signals present in novel experimental data. Finally, we find only modest differences in performance between different deep-learning architectures when trained on this single data modality, and while somewhat greater predictive accuracy can be achieved with much larger models trained at great expense on many terabytes of data, there is still copious room for improvement even for industrial strength, state-of-the-art models.

bioinformatics↗

bioRxiv2025

Development and validation of an exome-wide SNP genotyping array for genomic prediction, GWAS and assessment of introgressive hybridization between black and red spruces, and transferability to white and Norway spruces

Introgressive hybridization plays a major role in shaping the evolutionary dynamics and adaptive potential of forest trees. In this study, we developed and validated an exome-wide bispecific SNP genotyping array (Pmr25k) for the closely related species black spruce (Picea mariana) and red spruce (Picea rubens), two ecologically and economically important North American conifers that form a widespread hybrid zone in eastern Canada. Exome capture and sequencing of pooled red spruce samples yielded over 25,000 high-quality SNPs, which were used in conjunction with a previously developed black spruce gene SNP resource of over 97,000 high-quality SNPs, to construct the bispecific genotyping array. The final array comprised 21,573 successfully manufactured SNPs, representing 14,200 distinct gene loci, of which 85% were segregating when both species were considered together. More than 4000 segregating SNPs could also be successfully used and genotyped in each of white spruce (Picea glauca) and Norway spruce (Picea abies), highlighting the conserved nature of DNA attachment sites and presence of homologous SNPs for many gene loci. The Pmr25k array thus provides an efficient and reliable high-throughput genotyping tool to investigate introgression, genetic adaptation at the gene level, and to assist genomic-based prediction for breeding and conservation efforts in boreal spruces.

genomics↗

bioRxiv2024

De novo design of α-helical peptide channels with designer stoichiometry

Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane -helical barrels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) a low-conductance states that correlate with variations in the modeled coiled-coil barrel geometries, indicating stable transmembrane -helical barrels; and (ii) high-conductance states in which single pores change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic pores through the recruitment and expulsion of peptides, as observed in natural barrel-stave peptide pores. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry.

synthetic biology↗

bioRxiv2023

Female Alms1-deficient mice develop echocardiographic features of adult but not infantile Alström Syndrome cardiomyopathy

BackgroundAlstrom Syndrome (AS), a multisystem disorder caused by biallelic ALMS1 mutations, features major cardiac complications often causing early mortality. These are biphasic, including infantile dilated cardiomyopathy, and distinct adult-onset cardiomyopathy. Cardiomyocyte maturation defects, cardiac fibrosis and early atherosclerosis have all been invoked as contributors to heart failure in AS, but their relative importance and inter-relationships are unknown. MethodsCardiac function of global Alms1 knockout mice was assessed by echocardiography at postnatal day 15 (P15) and at 8 and 23 weeks of age. Echocardiography was also undertaken in female mice with Pdgfr-Cre-driven Alms1 deletion in cardiac fibroblasts and a small proportion of cardiomyocytes. Histological and transcriptional analysis of myocardium at P15 and 24 weeks of age was also performed. ResultsCardiac function was unaltered in knockout mice of both sexes at P15 and 8 weeks of age. At 23 weeks of age female but not male knockout mice showed increased left atrial area, decreased isovolumic relaxation time, and reduced ejection fraction, consistent with early restrictive cardiomyopathy. No histological or transcriptional changes could be identified in myocardium of 23-week old female Alms1 KO mice, however. Pdgfr-Cre-driven Alms1 KO in females did not recapitulate the phenotype of global KO at 23 weeks. ConclusionsAdult female, but not male, Alms1-deficient mice show echocardiographic evidence of cardiac dysfunction, consistent with the restrictive cardiomyopathy of AS. The explanation for sexual dimorphism remains unclear, but may involve metabolic or endocrine differences between sexes. No infantile cardiomyopathy was found in this study.

physiology↗

bioRxiv2023

The developmental trajectory of 1H-MRS brain metabolites from childhood to adulthood.

Human brain development is ongoing throughout childhood, with for example myelination of nerve fibres and refinement of synaptic connections continuing until early adulthood. 1H-Magnetic Resonance Spectroscopy (1H-MRS) can be used to quantify the concentrations of endogenous metabolites (e.g., glutamate and {gamma}-aminobutyric acid (GABA)) in the human brain in vivo and so can provide valuable, tractable insight into the biochemical processes that support postnatal neurodevelopment. This can feasibly provide new insight into and aid management of neurodevelopmental disorders by providing chemical markers of atypical development. This study aims to characterize the normative developmental trajectory of various brain metabolites, as measured by 1H-MRS from a midline posterior parietal voxel. We find significant non-linear trajectories for GABA+, Glx, tNAA and tCr concentrations. Glx and GABA+ concentrations steeply decrease across childhood. tNAA concentrations are relatively stable in childhood but gradually decrease from early adulthood, while tCr concentrations increase from childhood to early adulthood. tCho was the only metabolite to have a strictly linear association with age. Trajectories likely reflect fundamental neurodevelopmental processes (including local circuit refinement) which occur from childhood to early adulthood and can be associated with cognitive development; we find GABA+ concentrations significantly positively correlate with recognition memory scores across post-natal development.

neuroscience↗

bioRxiv2023

Post-infection treatment with the E protein inhibitor BIT225 reduces disease severity and increases survival of k18-hACE2 transgenic mice infected with a lethal dose of SARS-CoV-2

The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E-protein but did not inhibit endogenous calcium-induced ion channel activity or TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 h before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n=12), while all vehicle-dosed animals reached a mortality endpoint by day 9 across two studies (n=12). When treatment started at 24 h after infection, body weight loss, and mortality were also prevented (100% survival, n=5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection. AUTHORS SUMMARYAntiviral agents are highly important for the management of COVID-19. We need new antivirals, because available drugs have major drawbacks that limit their use and are threatened by drug resistance. This study demonstrates that the small molecule drug BIT225 is an inhibitor of an important viral ion channel (E-protein). E-protein is required for virus replication and is involved in eliciting inflammatory response to infection. Exacerbated inflammation is a hallmark of severe COVID-19 in mice and in humans. In a mouse model of severe SARS-CoV-2 infection, BIT225 treatment starting before or 24 hours after infection could protect all treated mice from developing disease, from experiencing weight loss and from death (100%, n=17), while all untreated mice developed severe disease, started to lose body weight from day 3 onwards and died within 9 days after infection. BIT225 treatment was associated with potent suppression of virus load, and reduced inflammation markers, consistent with effective clearance of the virus. These results are remarkable for the exceptionally high efficacy achieved with a new mechanism of action. BIT225 is a clinical stage drug candidate with an established human safety profile. These results support clinical evaluation of BIT225 for the treatment of human SARS-CoV-2 infection.

molecular biology↗

bioRxiv2023

OPTIMAL: An OPTimsed Imaging Mass cytometry AnaLysis framework for Segmentation and Data Exploration

Analysis of Imaging Mass Cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single cell segmentation and sub-optimal approaches for data visualisation and exploration. This can lead to inaccurate identification of cell phenotypes, states or spatial relationships compared to reference data from single cell suspension technologies. To this end we have developed the "OPTIMAL" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualisation/clustering and spatial neighbourhood analysis. Using a panel of 27 metal-tagged antibodies recognising well characterised phenotypic and functional markers to stain the same FFPE human tonsil sample Tissue Microarray (TMA) over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, five different dimensionality reduction algorithms and two clustering methods. Finally we assessed the optimal approach for performing neighbourhood analysis. We found that single cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bi-variate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximised the statistical separation between negative and positive signal distributions and a simple Z-score normalisation step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing Phenograph in terms of cell type identification. We also found that neighbourhood analysis was influenced by the method used for finding neighbouring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output, allows for single cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

bioinformatics↗

bioRxiv2021

Synaptic targets of functionally specialized R7 and R8 photoreceptors in the central eye and dorsal rim area of Drosophila

Color and polarization provide complementary information about the world and are detected by specialized photoreceptors. However, the downstream neural circuits that process these distinct modalities are incompletely understood in any animal. Using electron microscopy, we have systematically reconstructed the synaptic targets of the photoreceptors specialized to detect color and skylight polarization in Drosophila, and we have used light microscopy to confirm many of our findings. We identified known and novel downstream targets that are selective for different wavelengths or polarized light, and followed their projections to other areas in the optic lobes and the central brain. Our results revealed many synapses along the photoreceptor axons between brain regions, new pathways in the optic lobes, and spatially segregated projections to central brain regions. Strikingly, photoreceptors in the polarization-sensitive dorsal rim area target fewer cell types, and lack strong connections to the lobula, a neuropil involved in color processing. Our reconstruction identifies shared wiring and modality-specific specializations for color and polarization vision, and provides a comprehensive view of the first steps of the pathways processing color and polarized light inputs.

neuroscience↗

bioRxiv2021

Vitrifying multiple embryos in different arrangements does not alter the cooling rate

Vitrification is the most common method of cryopreservation of gametes in fertility clinics due to its improved survival rates compared to slow freezing techniques. For the Open Cryotop(R) vitrification device, the number of oocytes, or embryos, mounted onto a single device can vary. In this work, a mathematical model is developed for the cooling of oocytes and embryos (samples). The model is solved computationally, to investigate whether varying the number of samples mounted onto the Open Cryotop(R) affects the cooling rates, and consequently the survival rates, of vitrified samples. Several realistic spatial arrangements of samples are examined, determining their temperature over time. In this way we quantify the effect of spatial arrangement on the cooling rate. Our results indicate that neither the spatial arrangement nor the number of mounted samples has a large effect on cooling rates, so long as the volume of the cryoprotectant remains minimal. The time taken for cooling is found to be on the order of half a second, or less, regardless of the spatial arrangement or number of mounted samples. Hence, rapid cooling can be achieved for any number or arrangement of samples, as long as device manufacturer guidelines are adhered to.

developmental biology↗

bioRxiv2020

Phosphorylation-dependent sub-functionalization of the calcium-dependent protein kinase CPK28

Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+-sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1), and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intra- and inter-molecular auto-phosphorylation on Ser318 and can additionally be trans-phosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem-elongation, indicating pathway specific requirements for phosphorylation-based Ca2+-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

plant biology↗

bioRxiv2019

Eosinophil deficiency promotes aberrant repair and adverse remodelling following acute myocardial infarction

BackgroundEosinophil count predicts outcome following myocardial infarction (MI) and eosinophils regulate tissue repair and regeneration in extra-cardiac settings.\n\nObjectivesTo investigate the role of eosinophils in regulating inflammation, repair and remodelling following MI.\n\nMethodsBlood eosinophil count was assessed in 732 patients undergoing primary percutaneous coronary intervention for ST-segment elevation MI (STEMI). Experimental MI was induced in wild-type (WT) and eosinophil-depleted mice ({Delta}dblGATA or anti-Siglec F antibody treated). Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of infarct digests.\n\nResultsBlood eosinophil count declined in the hours following STEMI in patients and following MI induction in mice. Eosinophils were subsequently identified in the myocardium of patients and mice. Genetic eosinophil depletion in mice increased LV dilatation (end-systolic area: 29.0{+/-}2.2cm2 v 21.6{+/-}1.6cm2; p=0.02) and reduced ejection fraction (22.0{+/-}3.6% v 34.3{+/-}4.0% in p=0.04,) in {Delta}dblGATA v WT following MI (n=8-9/group), an outcome reproduced by pharmacological depletion. {Delta}dblGATA mice had increased scar size with disrupted collagen deposition and altered expression of the collagen cross-linking genes plod2 and lox. CD206+pro-repair macrophages were less prevalent in the infarct zone of {Delta}dblGATA mice (27.0{+/-}2.3% v WT: 39.2.4{+/-}3.5%; p=0.01, n=9-11/group) but were restored by replenishment with bone marrow-derived eosinophils. Anti-inflammatory cytokine concentrations were reduced in {Delta}dblGATA mice and IL-4 complex administration 24h after MI rescued adverse remodelling.\n\nConclusionsEosinophils are recruited to the heart following MI and are required for effective repair and to prevent adverse remodelling. IL-4 therapy has potential to limit detrimental outcomes when eosinophil availability is low.\n\nHIGHLIGHTSA drop in eosinophil blood count is associated with recruitment of eosinophils to the heart during repair following clinical and experimental myocardial infarction.\n\nGenetic & pharmacological eosinophil depletion leads to increased adverse remodelling in experimental MI.\n\nEosinophils are required for aquisition of an anti-inflammatory macrophage phenotype and a shift to resolution of inflammation during infarct repair.\n\nInterleukin-4 therapy is able to rescue the adverse remodelling phenotype in conditions of eosinophil deficiency.\n\nCondensed abstractIn ST-segment elevation myocardial infarction (STEMI) of both patients and mice, there was a decline in blood eosinophil count, with activated eosinophils recruited to the infarct zone. Eosinophil deficiency resulted in attenuated anti-inflammatory pro-repair macrophage polarization, enhanced myocardial inflammation, increased scar size and deterioration of myocardial structure and function. Adverse cardiac remodelling in the setting of eosinophil deficiency was prevented by IL-4 therapy.

immunology↗

bioRxiv2019

Does alpha phase modulate visual target detection? Three experiments with tACS phase-based stimulus presentation.

In recent years the influence of alpha (7-13 Hz) phase on visual processing has received a lot of attention. Magneto-/encephalography (M/EEG) studies showed that alpha phase indexes visual excitability and task performance. If occipital alpha phase is functionally relevant, the phase of occipital alpha-frequency transcranial alternating current stimulation (tACS) could modulate visual processing. Visual stimuli presented at different pre-determined, experimentally controlled, phases of the entraining tACS signal should then result in an oscillatory pattern of visual performance. We studied this in a series of experiments. In experiment one, we applied 10 Hz tACS to right occipital cortex (O2) and used independent psychophysical staircases to obtain contrast thresholds for detection of visual gratings in left or right hemifield, in six equidistant tACS phase conditions. In experiments two and three, tACS was at EEG-based individual peak alpha frequency. In experiment two, we measured detection rates for gratings with (pseudo-)fixed contrast levels. In experiment three, participants detected brief luminance changes in a custom-built LED device, at eight equidistant alpha phases. In none of the experiments did the primary outcome measure over phase conditions consistently reflect a one-cycle sinusoid as predicted. However, post-hoc analyses of reaction times (RT) suggested that tACS alpha phase did modulate RT in both experiments 1 and 2 (not measured in experiment 3). This observation is in line with the idea that alpha phase causally gates visual inputs through cortical excitability modulation.

neuroscience↗