Biology subjects

Williams, T.

Publications and source records attributed to Williams, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Condensin-mediated restriction of retrotransposable elements facilitates brain development in Drosophila melanogaster.

Neural stem and progenitor cell (NSPC) maintenance are essential for ensuring that organisms are born with proper brain volumes and head sizes. Microcephaly is a disorder in which babies are born with significantly smaller head sizes and cortical volumes. Mutations in subunits of the DNA organizing complexes, condensins have been identified in microcephaly patients. However the molecular mechanisms by which condensin insufficiency causes microcephaly remain elusive. We previously identified conserved roles for condensins in repression of retrotransposable elements (RTEs). Here, we show that condensin subunit knockdown in NSPCs of the Drosophila larval central brain increases RTE expression and mobility which causes cell death, and significantly decreases adult head sizes and brain volumes. These findings suggest that unrestricted RTE expression and activity may lead to improper brain development in condensin insufficient organisms, and lay the foundation for future exploration of causative roles for RTEs in other microcephaly models.

neuroscience↗

bioRxiv2024

A genuinely hybrid, multiscale 3D cancer invasion and metastasis modelling framework

We introduce in this paper substantial enhancements to a previously proposed hybrid multiscale cancer invasion modelling framework to better reflect the biological reality and dynamics of cancer. These model updates contribute to a more accurate representation of cancer dynamics, they provide deeper insights and enhance our predictive capabilities. Key updates include the integration of porous medium-like diffusion for the evolution of Epithelial-like Cancer Cells and other essential cellular constituents of the system, more realistic modelling of Epithelial-Mesenchymal Transition and Mesenchymal-Epithelial Transition models with the inclusion of Transforming Growth Factor beta within the tumour microenvironment, and the introduction of Compound Poisson Process in the Stochastic Differential Equations that describe the migration behaviour of the Mesenchymal-like Cancer Cells. Another innovative feature of the model is its extension into a multi-organ metastatic framework. This framework connects various organs through a circulatory network, enabling the study of how cancer cells spread to secondary sites.

cancer biology↗

bioRxiv2023

UVAE: Integration of Heterogeneous Unpaired Data with Imbalanced Classes

We introduce the Unbiasing Variational Autoencoder (UVAE), a computational framework for the integration of unpaired biomedical data streams such as clinical flow cytometry. UVAE addresses batch effect correction and data alignment by training a semi-supervised model on partially labelled datasets, enabling simultaneous normalisation and integration of diverse data within a shared latent space. The framework implements a probabilistic model for batch effect normalisation and balances class contents during training to ensure accurate representation of underlying cell composition. We apply UVAE to integrate heterogeneous clinical flow cytometry data from COVID-19 patients. The integrated data enhances the statistical signal of cell types associated with disease severity, enables clustering of subpopulations without the impediment of batch effects, and improves the performance of longitudinal regression for predicting peak disease severity from temporal patient samples.

bioinformatics↗

bioRxiv2023

Heterogeneity in lung macrophage control of Mycobacterium tuberculosis is determined by T cells

Following Mycobacterium tuberculosis infection, alveolar macrophages are initially infected but ineffectively restrict bacterial replication. The distribution of M. tuberculosis among different cell types in the lung changes with the onset of T cell immunity when the dominant infected cellular niche shifts from alveolar to monocyte-derived macrophages (MDM). We hypothesize that changes in bacterial distribution among different cell types is driven by differences in T cell recognition of infected cells and their subsequent activation of antimicrobial effector mechanisms. We show that CD4 and CD8 T cells efficiently eliminate M. tuberculosis infection in alveolar macrophages, but they have less impact on suppressing infection in MDM, which may be a bacterial niche. Importantly, CD4 T cell responses enhance MDM recruitment to the lung. Thus, the outcome of infection depends on the interaction between the T cell subset and the infected cell; both contribute to the resolution and persistence of the infection.

immunology↗

bioRxiv2023

Base Excision Repair Pathway Regulates Transcription-Replication Conflicts in Pancreatic Ductal Adenocarcinoma

Oncogenic mutations (such as in KRAS) can dysregulate transcription and replication leading to transcription conflicts (TRCs). Unresolved TRCs can cause lethal DNA damage. Here, we sought to investigate the oncogene dependency of TRCs and TRC regulatory pathways in pancreatic ductal adenocarcinoma (PDAC). Human PDAC demonstrated 30-120-fold higher levels of TRC genomic signatures compared to breast, colon and lung cancer (p<0.001). TRCs were significantly enriched in human PDAC cells (Panc-1, BxPC3, MiaPaca2) compared to immortalized human pancreatic ductal epithelial (HPNE). Ectopic oncogenic KRAS(G12D) in HPNE cells enhanced TRCs, and TRC-related DNA:RNA hybrids (R-loops). Inhibition of KRAS or downstream effectors abrogated TRCs in Panc1 and MiaPaca2 cells. An siRNA screen identified several factors in the base-excision repair pathway as regulators of TRCs. In pharmacologic validation, inhibitors of APE1 endonuclease in BER pathway (Methoxyamine and CRT) enhanced TRCs. Mechanistic studies revealed that BER pathway inhibition severely altered RNA polymerase II dynamics at nascent DNA; causing RNAPII trapping and contributing to enhanced TRCs. The ensuing DNA damage activated Chk2-ATR pathway but not Chk1-ATM pathway. Co-treatment with ATR inhibitor (VX970) and BER inhibitor (methoxyamine) at clinically relevant doses, synergistically enhanced DNA damage and reduced cell proliferation in PDAC cells. The study uncovers a novel role of BER pathway defects and oxidative DNA damage in promoting TRCs. Our studies provide mechanistic insights into the regulation of TRCs in PDAC which has implications for genome instability and therapy in PDAC.

cancer biology↗

bioRxiv2023

Sequence characterization of T, Bip, and Phbw demonstrates the role of MYB-bHLH-WD40 complexes and temperature in common bean seed color pattern formation

Seed colors and color patterns are critical for the survival of wild plants and the consumer appeal of crops. In common bean, a major global staple, these patterns are also critical for determining market classes, yet the genetic and environmental control of many pigmentation patterns remains unresolved. In this study, we genetically mapped variation for three important seed pattern loci, T, Bip, and phbw, which co-segregated completely with PvTTG1, PvMYC1, and PvTT8, respectively. Proteins encoded by these genes are predicted to work together in MYB-bHLH-WD40 (MBW) complexes, propagating flavonoid biosynthesis across the seed coat. Whole-genome sequencing of 37 diverse accessions identified putative mutations in each gene, including seven unique parallel mutations in T (PvTTG1) and a non-synonymous SNP in a conserved residue in bipana (PvMYC1). A 612 bp intron deletion in phbw (PvTT8) eliminated motifs conserved since the origins of the Papilionoidea and corresponded to a 20-fold reduction in transcript abundance. Mutations in MBW candidate genes for Z (PvTT2) and Sellatus (WDR) were also identified. In multi-location field trials with seven varieties with partial seed coat patterning, pigmented seed coat area was highly unstable and correlated with temperature, with up to 11-fold differences in pigmented area between the warmest and the coolest environments. In controlled growth chamber conditions, an increase of 4 {degrees}C was sufficient to cause pigmentation on an additional 21% of the seed coat area. Our results shed light on the fundamental activation of flavonoid biosynthesis in common bean and will be instrumental for maximizing consumer appeal in this nutritious staple crop. Summary- Seed colors and patterns are critical for the survival of wild plants, and are important in differentiating crop market classes, but the genetic control of these in the staple crop common bean (Phaseolus vulgaris) is largely unknown. - The genetic, transcriptional, and environmental basis of common bean seed color patterning was explored through QTL mapping, whole-genome sequencing, RT-qPCR, and automated pigmentation quantification of seed grown in multi-location field trials and growth chamber environments. - MYB-bHLH-WD40 complex-forming genes PvTTG1, PvMYC1, and PvTT8 co-segregated completely with the color patterning genes T, Bip, and phbw. Mutations were identified in each gene, including seven unique parallel mutations in T (PvTTG1), a non-synonymous SNP in a conserved residue in bipana (PvMYC1), and an intron deletion in phbw (PvTT8) eliminating highly conserved motifs and corresponding to 20-fold lower PvTT8 transcript abundance. Mutations in MBW candidate genes Z (PvTT2) and Sellatus (WDR) were also identified. In multi-location field trials, pigmented seed coat area was highly unstable and corresponded to temperature. In growth chamber conditions, an increase of 4 {degrees}C caused pigmentation on an additional 21% of the seed coat area. - Our results highlight the critical interaction between MYB-bHLH-WD40 complex components and temperature in establishing seed pattern diversity.

plant biology↗

bioRxiv2023

Metagenomics untangles metabolic adaptations of Antarctic endolithic 1 bacteria at the fringe of habitability

BackgroundEndolithic niches offer an ultimate refuge, supplying buffered conditions for microorganisms that dwell inside rock airspaces. Yet, survival and growth strategies of Antarctic endolithic microbes residing in Earths driest and coldest desert remains virtually unknown. ResultsFrom 109 endolithic microbiomes, 4,539 metagenome-assembled genomes were generated, 49.3% of which were novel candidate bacterial species. We present evidence that trace gas oxidation and atmospheric chemosynthesis may be the prevalent strategies supporting metabolic activity and persistence of these ecosystems at the fringe of life and the limits of habitability. ConclusionsThese results represent the foundation to untangle adaptability at the edge of sustainability on Earth and on other dry Earth-like planetary bodies such as Mars.

microbiology↗

bioRxiv2023

Diving efficiency at depth and pre-breeding foraging effort increase with hemoglobin levels in gentoo penguins

Individual differences in oxygen storage and carrying capacity have been associated with fitness-related traits and, for air-breathing aquatic animals, to diving ability and foraging success. In winter, many seabirds must replenish the energy reserves they have depleted during the breeding period. Thus, winter foraging efficiency can influence their upcoming breeding behaviour. Using gentoo penguins (Pygoscelis papua), we investigate (1) if inter-individual variation in diving efficiency (proportion of time spent at the bottom) is associated with indices of oxygen storage and carrying capacity (haemoglobin, haematocrit, body mass), and (2) if measures of pre-breeding foraging effort (mean trip duration, total time at sea, and vertical distance travelled) are associated with these oxygen indices and breeding status. Haemoglobin was positively correlated with diving efficiency, particularly for deeper dives, and only penguins with high haemoglobin levels frequently dove at depth [≥] 140 m. Such differences could affect resource access. However, potentially because reaching deep offshore waters requires travelling more than foraging nearshore, vertical distance travelled pre-breeding increased with haemoglobin levels. The relationship with haematocrit was non-linear, suggesting that commonly-used analyses may be inappropriate for this index. We found that early-laying penguins spent less time at sea prior to nesting than non-breeding penguins, suggesting that more efficient foragers lay earlier. Given that diving efficiency at depth is linked to aerobic capacity, anthropogenic activities taking place in either nearshore or offshore waters (e.g., shallow water fisheries, offshore oil rigs) may have differing impacts on individuals. Further understanding these links could help the conservation of diving species.

ecology↗

bioRxiv2023

Morphogen-driven human iPSCs differentiation in 3D in vitro models of gastrulation is precluded by physical confinement.

In early human development, gastrulation is tightly associated with lineage specification. The interplay between mechanical forces and biochemical signals during these processes is poorly understood. Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model of gastrulation that uses spheroids formed from human induced pluripotent stem cells (hiPSCs). First, we compare self-renewing versus differentiating media conditions in free-floating cultures, and observe the emergence of organised tri-germ layers. In these unconfined cultures, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead. Our study combines high-content imaging, synthetic hydrogels and hiPSCs-derived models of early development to define the drivers causing changes in shape and emergence of germ layers.

cell biology↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗

bioRxiv2022

Improving Therapeutic Protein Secretion in the Probiotic Yeast Saccharomyces boulardii using a Multifactorial Engineering Approach

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sbs innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e., to the expression cassette of the secreted protein) and trans- (i.e., to the Sb genome) that enhance Sbs ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by 6-fold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 - 463 mg/L. Then, guided by prior knowledge of S. cerevisiaes secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of >10-fold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.

synthetic biology↗

bioRxiv2022

Molecularly distinct memory CD4+ T cells are induced by SARS-CoV-2 infection and mRNA vaccination

Adaptive immune responses are induced by vaccination and infection, yet little is known about how CD4+ T cell memory differs when primed in these two contexts. Notably, viral infection is generally associated with higher levels of systemic inflammation than is vaccination. To assess whether the inflammatory milieu at the time of CD4+ T cell priming has long-term effects on memory, we compared Spike-specific memory CD4+ T cells in 22 individuals around the time of the participants third SARS-CoV-2 mRNA vaccination, with stratification by whether the participants first exposure to Spike was via virus or mRNA vaccine. Multimodal single-cell profiling of Spike-specific CD4+ T cells revealed 755 differentially expressed genes that distinguished infection- and vaccine-primed memory CD4+ T cells. Spike-specific CD4+ T cells from infection-primed individuals had strong enrichment for cytotoxicity and interferon signaling genes, whereas Spike-specific CD4+ T cells from vaccine-primed individuals were enriched for proliferative pathways by gene set enrichment analysis. Moreover, Spike-specific memory CD4+ T cells established by infection had distinct epigenetic landscapes driven by enrichment of IRF-family transcription factors, relative to T cells established by mRNA vaccination. This transcriptional imprint was minimally altered following subsequent mRNA vaccination or breakthrough infection, reflecting the strong bias induced by the inflammatory environment during initial memory differentiation. Together, these data suggest that the inflammatory context during CD4+ T cell priming is durably imprinted in the memory state at transcriptional and epigenetic levels, which has implications for personalization of vaccination based on prior infection history. One Sentence SummarySARS-CoV-2 infection versus SARS-CoV-2 mRNA vaccination prime durable transcriptionally and epigenetically distinct Spike-specific CD4+ T cell memory landscapes.

immunology↗

bioRxiv2022

Towards optimised extracellular vesicle proteomics from cerebrospinal fluid

The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean {+/-} standard deviation. Using proteomics, SEC fractions 3-5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4-5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6ml, 3ml, 1ml, and 0.5ml) to evaluate the effect on the proteomic profile. Even with a 0.5ml starting volume, 743{+/-}77 or 345{+/-}88 proteins were identified depending on whether matches between runs was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5ml of canine CSF.

cell biology↗

bioRxiv2022

Host genetic background is a barrier to vaccine-induced protection against infection

The heterogeneity of immune responses observed in humans is difficult to model in standard inbred laboratory mice. To capture the diversity inherent in mice and better understand how host variation affects BCG-induced immunity against Mycobacterium tuberculosis, 24 unique Collaborative Cross (CC) recombinant inbred mouse strains and the C57BL/6 reference strain were vaccinated with or without BCG, and then challenged with low-dose aerosolized virulent M. tuberculosis. In contrast to standard lab strains, BCG protected only half of the CC strains tested. Furthermore, BCG efficacy is dissociable from inherent susceptibility to TB. As these strains differed primarily in the genes and alleles they inherited from the CC founder strains, we conclude that the host genetic background has a major influence on whether BCG confers protection against M. tuberculosis infection and indicates that host genetics should be considered as an important barrier to vaccine-mediated protection. Importantly, we wished to identify the components of the immune response stimulated by BCG, which were subsequently recalled after Mtb infection and associated with protection. The T cell immune response following BCG vaccination and Mtb challenge was extensively characterized. Although considerable diversity was observed, BCG vaccination had little impact on the composition of T cells recruited and maintained in the lung after infection. Instead, the variability was largely shaped by the genetic background. We developed models to detect vaccine-induced differences, which identified immune signatures associated with BCG-elicited protection against TB. Importantly, even when categorized as susceptible vs. resistant, and protected vs. unprotected, many of the protected CC strains had unique flavors of immunity, indicating multiple paths to protection. Thus, CC mice can be used to define correlates of protection and to identify vaccine strategies that protect a larger fraction of genetically diverse individuals instead of optimizing protection for a single genotype.

immunology↗

bioRxiv2022

Choice of spatial discretisation influences the progression of viral infection within multicellular tissues

There has been an increasing recognition of the utility of models of the spatial dynamics of viral spread within tissues. Multicellular models, where cells are represented as discrete regions of space coupled to a virus density surface, are a popular approach to capture these dynamics. Conventionally, such models are simulated by discretising the viral surface and depending on the rate of viral diffusion and other considerations, a finer or coarser discretisation may be used. The impact that this choice may have on the behaviour of the system has not been studied. Here we demonstrate that, if rates of viral diffusion are small, then the choice of spatial discretisation of the viral surface can have quantitative and even qualitative influence on model outputs. We investigate in detail the mechanisms driving these phenomena and discuss the constraints on the design and implementation of multicellular viral dynamics models for different parameter configurations. Author summaryHere we analyse the impact that the resolution of spatial discretisation has on the behaviour of spatial models of viral dynamics. We show that the extent of this influence depends on the size of the diffusion coefficient, and that, if the diffusion coefficient is sufficiently small - for example, small enough to describe tight plaque-forming dynamics - the choice of discretisation of the viral surface relative to the cell grid can appreciably change key predictions of the model. We show that the resolution of spatial discretisation modulates the timescale of infection, and identify the mechanisms by which this occurs. This work provides theory to inform the development of multicellular models of viral dynamics, which is in short supply in the literature.

immunology↗

bioRxiv2021

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N=10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase (www.facebase.org, doi.org/10.25550/3-HXMC) and GitHub (https://github.com/jaydevine/MusMorph).

genetics↗

bioRxiv2021

Relating genome completeness to functional predictions

Genome and transcriptome assemblies vary in their quality in terms of the connectedness of the assemblies and the amount of biological information captured. Interpreting de novo assemblies from new, poorly characterized, organisms in the context of complex traits can be challenging because, in the absence of a reference, it is difficult to know how much information is enough to claim the presence or absence of a trait. This study uses randomly downsampled proteome files to compare a genome completeness metric, BUSCO, to functional predictions of the complex trait of phagocytosis in known phagocytotic organisms broadly across the eukaryotic tree of life. We find that as additional proteins are added, BUSCO scores increase incrementally, while the phagocytosis prediction follows a sigmoidal curve. Generalizing our findings, we suggest a threshold of the number of BUSCOs detected above which one would expect an accurate prediction, positive or negative, of the complex trait of phagocytosis. While these findings are specific to a single trait, the methods can be extended to consider additional functional traits and predictive frameworks.

genomics↗

bioRxiv2021

AP-2α and AP-2β cooperatively function in the craniofacial surface ectoderm to regulate chromatin and gene expression dynamics during facial development.

The facial surface ectoderm is essential for normal development of the underlying cranial neural crest cell populations, providing signals that direct appropriate growth, patterning, and morphogenesis. Despite the importance of the ectoderm as a signaling center, the molecular cues and genetic programs implemented within this tissue are understudied. Here we show that removal of two members of the AP-2 transcription factor family, AP-2 and AP-2{beta}, within the early embryonic ectoderm leads to major alterations in the mouse craniofacial complex. Significantly, there are clefts in both the upper face and mandible, accompanied by fusion of the upper and lower jaws in the hinge region. Comparison of ATAC-seq and RNA-seq analyses between controls and mutants revealed significant changes in chromatin accessibility and gene expression centered on multiple AP-2 binding motifs associated with enhancer elements within these ectodermal lineages. In particular, loss of these AP-2 proteins affects both skin differentiation as well as multiple signaling pathways, most notably the WNT pathway. The role of reduced Wnt signaling throughput in the mutant phenotype was further confirmed using reporter assays and rescue experiments involving Wnt1 ligand overexpression. Collectively, these findings highlight a conserved ancestral function for AP-2 transcription factors in ectodermal development and signaling, and provide a framework from which to understand the gene regulatory network operating within this tissue that directs vertebrate craniofacial development.

developmental biology↗