Biology subjects

Allan, J.

Publications and source records attributed to Allan, J..

13 recordsLinked to original sources

bioRxiv2026

Irregular nucleosome positioning governs a crystalline to liquid-like phase transition and tunes chromatin accessibility

Chromatin must fold tightly enough to protect the genome while being sufficiently accessible for DNA dependent processes such as transcription. The physical rules that balance these competing roles remain unclear, as DNA sequence encodes both biochemical information such as transcription factor binding sites, and biophysical cues that shape chromatin structure. Here, using synthetic chromatin fibres assembled from physiologically relevant DNA sequences, we show that nucleosome positioning dictates the material state of chromatin. Heterochromatin-like sequences produce compact fibres stabilised by nucleosome stacking, whereas euchromatin-like sequences generate irregular nucleosome positioning that yields disrupted, heterogeneous, and mechanically deformable fibres. Quantitative polymer modelling reveals that these irregular arrays are highly dynamic, continually sampling a broad ensemble of conformations as nucleosome stacking breaks down. We identify two previously unrecognised thresholds encoded by nucleosome positioning: minimal positional irregularity (2-3 bp) triggers a transition from an ordered paracrystalline state to a liquid-like phase, whereas an order of magnitude greater irregularity (~18 bp) is required to generate accessibility and mechanical fragility permissive for transcription factor binding. Euchromatin-like arrays reside at this accessibility threshold. These findings indicate that nucleosome positioning tunes chromatin toward or away from critical structural states that couple genome protection, chromatin dynamics, and transcriptional potential, providing a physical mechanism that helps connect DNA sequence to gene expression.

molecular biology↗

bioRxiv2026

A Toolbox for Biomanufacturing of Functionalised PHA Nanoparticles with C. necator

Microbes have the potential to manufacture plastics from sustainable feedstocks while enabling novel material properties and functions that are not easily accessible through conventional chemical synthesis. Realising this potential requires a comprehensive genetic and process engineering framework that spans chassis and bioprocess optimisation, polymer property control, and downstream functionalisation. Here we develop such a platform in Cupriavidus necator, with a focus on high-value polyhydroxyalkanoate (PHA) nanoparticles. To this end we first optimise the transformation protocol for the organism. Next, we create a library of PhaC synthase variants from C. necator, Aeromonas caviae and Brevundimonas sp. in a {Delta}phaC background, demonstrating that they allow customisation of the material properties of produced PHA particles. Our results combine data from Flow cytometry, Transmission Electron Microscopy (TEM), Fourier Transform InfraRed Spectroscopy (FTIR), and Differential Scanning Calorimetry (DSC) to show that it is possible to generate materials ranging from highly crystalline PHAs to softer P(3HB-co-3HHx) copolymers and that an A. caviae PhaC variant can double the yield of large PHA granules. To improve bioprocess sustainability, we coupled C. necator with B. subtilis in sucrose-fed co-cultures, using tetracycline tolerance differences and inoculation ratios to enhance PHA production from inexpensive, sugar-rich feedstocks. Finally, we add function to the produced PHA nanoparticles by using the molecular protein-fusion technology SpyTag-SpyCatcher, showing it is possible to efficiently capture SpyCatcher-GFP on PHA granules as a proof of concept for PHAs use as a customisable bio-based nanoparticle. Together, our work offers an innovation to produce bio-PHA nanoparticles in a customisable way, with potential applications in sustainable biomanufacturing, biosensing, drug delivery and future bioremediation technologies.

synthetic biology↗

bioRxiv2025

Embryonic stem cells do not have a globally disrupted higher-order chromatin fibre structure

Embryonic stem cells (ESCs) are thought to maintain pluripotency through global hyper-transcription, linked to an "open" chromatin structure. To investigate this idea, we analyzed higher-order chromatin fibres from mouse ESCs, their differentiated progenitors, and terminally differentiated mouse NIH3T3 fibroblasts. Bulk chromatin composition including protein:DNA ratio and nucleosome repeat length varied little between the cell types, but surprisingly biophysical analyses such as linker histone FRAP, hydrodynamic sedimentation and nuclease sensitivity also showed no significant differences in the conformation of purified higher-order chromatin fibres. To better evaluate the structure of higher-order chromatin fibres observed in cells, we developed a novel technique called SPOCC (Sedimentation Properties of Cross-Linked Chromatin). This approach revealed that ESCs and differentiated cells share similar bulk higher-order chromatin fibre structures, whilst ESCs have a slightly more disrupted structure than NIH3T3 cell chromatin. These results indicate that ESC transcriptional activity and plasticity are not driven by a fundamentally "open" higher-order chromatin conformation.

molecular biology↗

bioRxiv2024

Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

Lung structural cells, including epithelial cells and fibroblasts, form barriers against pathogens and trigger immune responses following infections such as influenza A virus. This response leads to the recruitment of innate and adaptive immune cells required for viral clearance. Some of these recruited cells remain within the lung following infection and contribute to enhanced viral control following subsequent infections. There is growing evidence that structural cells can also display long-term changes following infection or insults. Here we investigate long-term changes to mouse lung epithelial cells, fibroblasts, and endothelial cells following influenza virus infection and find that all three cell types maintain an imprint of the infection, particularly in genes associated with communication with T cells. Lung epithelial cells from IAV-infected mice display functional changes by more rapidly controlling influenza virus than cells from naive animals. This rapid anti-viral response and increased expression of molecules required to communicate with T cells demonstrates sustained and enhanced functions following infection. These data suggest lung structural cells could be effective targets for vaccines to boost durable protective immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/604410v5_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@2774e0org.highwire.dtl.DTLVardef@6a39e6org.highwire.dtl.DTLVardef@1ff5863org.highwire.dtl.DTLVardef@103df12_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILung epithelial cells, fibroblasts, and blood endothelial cells maintain an inflammatory imprint of influenza A virus (IAV) infection for at least 40 days post-infection. C_LIO_LIIn vivo re-infection leads to a more spatially restricted anti-viral response compared to primary IAV-infected animals. C_LIO_LIT cells are not required for enhanced viral control early after re-infection in vivo C_LIO_LIEx vivo lung epithelial cells from IAV-infected mice more rapidly control IAV than cells from naive animals in the absence of immune cells. C_LI

immunology↗

bioRxiv2024

Cybernetic control of a natural microbial co-culture

A key obstacle in the widespread application of microbial co-cultures in bioprocesses is their compositional instability, as faster-growing species outcompete and dominate the culture. While several synthetic biology approaches have demonstrated control over co-culture composition, there has been an increased interest in computer-based cybernetic control approaches that can offload burdensome genetic control circuitry to computers and enable dynamic control and real-time noise rejection. This work extends that approach, demonstrating a cybernetic control method that is not reliant on any genetic engineering, instead interfacing cells with computers by exploiting their natural characteristics to measure and actuate the composition. We apply this to a Pseudomonas putida (P. putida) and Escherichia coli (E. coli) co-culture grown in Chi.Bio bioreactors, first showing how composition estimates calculated from different bioreactor measurements can be combined with a system model using an extended Kalman filter to generate accurate estimates of a noisy system. We also demonstrate that because the species have different optimal temperature niches, adjusting the temperature of the culture can drive the composition in either direction. By using a proportional-integral control algorithm to calculate the temperature that would bring the measured composition towards the desired composition, we are able to track dynamic references and stabilised the co-culture for 7 days ([~]250 generations), with the experiment ending before the cells could adapt out of the control. This cybernetic framework is broadly applicable, with different microbes unique features and specific growth niches enabling robust control over diverse co-cultures.

systems biology↗

bioRxiv2024

A Machine Learning Framework to Identify the Correlates of Disease Severity in Acute Arbovirus Infection

Most viral diseases display a variable clinical outcome due to differences in virus strain virulence and/or individual host susceptibility to infection. Understanding the biological mechanisms differentiating a viral infection displaying severe clinical manifestations from its milder forms can provide the intellectual framework toward therapies and early prognostic markers. This is especially true in arbovirus infections, where most clinical cases are present as mild febrile illness. Here, we used a naturally occurring vector-borne viral disease of ruminants, bluetongue, as an experimental system to uncover the fundamental mechanisms of virus-host interactions resulting in distinct clinical outcomes. As with most viral diseases, clinical symptoms in bluetongue can vary dramatically. We reproduced experimentally distinct clinical forms of bluetongue infection in sheep using three bluetongue virus (BTV) strains (BTV-1IT2006, BTV-1IT2013 and BTV-8FRA2017). Infected animals displayed clinical signs varying from clinically unapparent, to mild and severe disease. We collected and integrated clinical, haematological, virological, and histopathological data resulting in the analyses of 332 individual parameters from each infected and uninfected control animal. We subsequently used machine learning to identify the key viral and host processes associated with disease pathogenesis. We identified five different fundamental processes affecting the severity of bluetongue: (i) virus load and replication in target organs, (ii) modulation of the host type-I IFN response, (iii) pro-inflammatory responses, (iv) vascular damage, and (v) immunosuppression. Overall, our study using an agnostic machine learning approach, can be used to prioritise the different pathogenetic mechanisms affecting the disease outcome of an arbovirus infection.

microbiology↗

bioRxiv2023

The SARS-CoV-2 Omicron sub-variant BA.2.86 is attenuated in hamsters

SARS-CoV-2 variants have emerged throughout the COVID-19 pandemic. There is a need to risk-assess newly emerged variants in near "real-time" to estimate their potential threat to public health. The recently emerged Omicron sub-variant BA.2.86 raised concerns as it carries a high number of mutations compared to its predecessors. Here, we assessed the virulence of BA.2.86 in hamsters. We compared the pathogenesis of BA.2.86 and BA.2.75, as the latter is one of the most virulent Omicron sub-variants in this animal model. Using digital pathology pipelines, we quantified the extent of pulmonary lesions measuring T cell and macrophage infiltrates, in addition to alveolar epithelial hyperplasia. We also assessed body weight loss, clinical symptoms, virus load in oropharyngeal swabs, and virus replication in the respiratory tract. Our data show that BA.2.86 displays an attenuated phenotype in hamsters, suggesting that it poses no greater risk to public health than its parental Omicron sub-variants. Article summary lineThe newly emerged Omicron sub-variant BA.2.86 is attenuated in hamsters.

microbiology↗

bioRxiv2023

Lycopene Production in Dedicated Novel Chasses for Lignocellulosic Waste Material Utilisation Capable of Sustained Coculture

Municipal solid waste (MSW) represents tonnes of material that, for the most part, is relegated to landfill. Synthetic biology proposes solutions to many of the challenges faced by humanity today, but many approaches are confined to use in classical chassis organisms. In MSW there are a variety of potentially toxic materials such as glues, dyes, and preservatives that could pose a challenge to its capitalisation when using these commonplace chassis. We have isolated a bank of strains that utilise paper and cardboard waste from a relevant waste environment. From these we have identified three strains that are capable of utilising cellulose as a sole carbon source. We have analysed how they utilise cellulose and hemicelluloses, both alone and in coculture. This revealed insights to how they might be used in synthetic consortia which were then produced under laboratory conditions. Production of complete genome sequences of these strains provides genetic insight to how these processes may be occurring at the metabolic level, and how they could be augmented using synthetic biology. To this end, we have produced protocols for transforming plasmids into these strains and have produced high value metabolites from this material. HighlightsO_LIFully annotated genomes were produced from novel mesophilic aerobic strains isolated from lignocellulosic solid waste C_LIO_LILycopene was produced directly from relevant solid waste substrates by genetically modified variants of these strains C_LIO_LIOptimised carbon source blends influence coculture compositions of specific strains C_LI

synthetic biology↗

bioRxiv2023

Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning

SARS-CoV-2 has continued to evolve throughout the COVID-19 pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2 and BA.2.75. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.

microbiology↗

bioRxiv2023

A miniature cellulosome with novel interaction modes

Cellulosomes are efficient enzymatic nanomachines which have arisen for the degradation of cellulosic biomass. They are found abundantly in soil-dwelling microbes and bacteria which thrive in the stomachs of ruminant mammals. Two protein domains, cohesins and dockerins, characterise cellulosomes. These domains interact with each other to form enormous complexes with as many as 130 individual proteins. Annotation of the genome of Cellulosilyticum lenotcellum DSM 5427 revealed one single cohesin and one single dockerin domain. This pales in comparison to most cellulosomal organisms. We have recombinantly produced the proteins which bear these domains and demonstrated cellulase activity, which is enhanced by interaction between the two proteins. Moreover we have identified additional novel interacting partners for this unique cellulosome complex. This broadens the repertoire and definitions of cellulosomes overall.

biochemistry↗

bioRxiv2021

A human iPSC derived bone marrow milieu identifies a novel target against niche-conferred leukaemia proliferation and treatment resistance

Leukaemia cells re-program their microenvironment to provide proliferation support and protection from standard chemotherapy, molecularly targeted therapies, and immunotherapy. Although much is becoming known about molecules that drive niche-dependent treatment resistance; means of targeting these in the clinics has remained a key obstacle. To address this challenge, we have developed human induced pluripotent stem cell engineered niches ex vivo to reveal insights into druggable cancer-niche dependencies. We show that mesenchymal (iMSC) and vascular niche-like (iANG) cells support ex vivo proliferation of patient-derived leukaemia cells, impact dormancy and mediate therapy resistance. iMSC protected both non-cycling and cycling blasts against dexamethasone treatment while iANG protected only dormant blasts. Leukaemia proliferation and protection from dexamethasone induced-apoptosis was dependent on direct cell-cell contact and mediated by CDH2. To explore the therapeutic potential of disrupting this cell-cell interaction, we tested the CDH2 antagonist ADH-1 (previously in phase I / II for solid tumours) in a very aggressive patient-derived xenograft leukaemia mouse model. ADH-1 showed high in vivo efficacy. ADH-1/ dexamethasone combination therapy was superior to dexamethasone alone with no ADH1 conferred additional toxicity. These findings provide a proof-of-concept starting point to develop novel, potentially safer therapeutics that target niche-mediated cancer cell dependencies in haematological malignancies. SummaryCDH2 mediated niche-dependent cancer proliferation and treatment resistance is clinically targetable via ADH-1, a low toxic agent that could be potentially repurposed for future clinical trials in acute leukaemia.

cancer biology↗

bioRxiv2021

Language barriers in global bird conservation

Multiple languages being spoken within a species distribution can impede communication among conservation stakeholders, the compilation of scientific information, and the development of effective conservation actions. Here, we investigate the number of official languages spoken within the distributions of 10,863 bird species to identify which ones might be particularly affected by consequences of language barriers. We show that 1587 species have 10 languages or more spoken within their distributions. Threatened, migratory and wide-ranging species have especially many languages spoken within their distribution. Particularly high numbers of species with many languages within their distribution are found in Eastern Europe, Russia and central and western Asia. Global conservation efforts would benefit from implementing guidelines to overcome language barriers, especially in regions with high species and language diversity.

ecology↗

bioRxiv2019

Genome-wide association analyses identify variants in IRF4 associated with Acute Myeloid Leukemia and Myelodysplastic Syndrome susceptibility

The role of common genetic variation in susceptibility to acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS), a group of rare clonal hematologic disorders characterized by dysplastic hematopoiesis and high mortality, remains unclear. We performed AML and MDS genome-wide association studies (GWAS) in the DISCOVeRY-BMT cohorts (2309 cases and 2814 controls). Association analysis based on subsets (ASSET) was used to conduct a summary statistics SNP-based analysis of MDS and AML subtypes. For each AML and MDS case and control we used PrediXcan to estimate the component of gene expression determined by their genetic profile and correlate this imputed gene expression level with risk of developing disease in a transcriptome-wide association study (TWAS). ASSET identified an increased risk for de novo AML and MDS (OR=1.38, 95% CI, 1.26-1.51, Pmeta=2.8x10-12) in patients carrying the T allele at rs12203592 in Interferon Regulatory Factor 4 (IRF4), a transcription factor which regulates myeloid and lymphoid hematopoietic differentiation. Our TWAS analyses showed increased IRF4 gene expression is associated with increased risk of de novo AML and MDS (OR=3.90, 95% CI, 2.36-6.44, Pmeta =1.0x10-7). The identification of IRF4 by both GWAS and TWAS contributes valuable insight on the role of genetic variation in AML and MDS susceptibility.

genomics↗