Biology subjects

Baillie, J. K.

Publications and source records attributed to Baillie, J. K..

At least 19 recordsLinked to original sources

bioRxiv2025

Identification of key host genes for influenza A virus in avian cells using a genome-wide CRISPR-Cas9 screen

Influenza A virus (IAV) causes major economic losses to the poultry industry and poses a zoonotic threat to human health. Potential pandemic outbreaks are underpinned by the ability of the virus to jump from one species to another. Host-virus interactions can dictate the success of such events and while systematic studies have successfully mapped host virus interactions in human cells, few studies have been performed in relevant animal host cell lines. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens in chicken lung epithelial cells infected with either the human-adapted PR8 vaccine strain or the avian UDL 3:5 reassortant virus encoding PR8 HA, NA and M segments. Rather than selecting solely for cell survival, we used anti-M2 antibody staining and fluorescence-activated cell sorting to capture host factors influencing multiple stages of the IAV life cycle. Across both screens, we identified 104 genes required for efficient replication in chicken cells, including 16 with strong effects (log2 fold change > 2). Comparative analysis with published human screens revealed 17 conserved host factors, 19 human-specific factors, and 42 chicken-specific factors, highlighting potential species-specific interactions. Top hits included genes involved in sialic acid biosynthesis and N-linked glycosylation--SLC35A1, SLC35A2, and the avian-specific influenza polymerase cofactor ANP32A. Functional validation demonstrated that MOGS, MGAT1, DENR, DMXL1, ENO1, IPO9, KLF6, PTAR1, and TSG101 contribute to multiple stages of the IAV life cycle. In particular, MOGS and MGAT1 were essential for N-glycan processing and modulated cell-surface sialic acid abundance, with strain- and species-specific effects. These findings define a genetic landscape of IAV dependency factors in chicken cells and suggest shared and species-specific host requirements that could impact cross-species transmission.

cell biology↗

bioRxiv2025

Disease-associated genetic variants can cause mutations in tissue-specific protein isoforms

Genetic variants can cause protein-coding mutations that result in disease. Variants are typically interpreted using the reference transcript for a gene. However, most human multi-exon genes encode alternative isoforms. Here, we show that coding exons in alternative isoforms harbour more population variants than exons of reference isoforms, consistent with their reduced evolutionary constraint, and that these variants are more likely to cause nonsynonymous coding mutations. Common and rare disease-associated variants mapping to alternative transcripts can lead to amino acid substitutions predicted to be structurally damaging in the corresponding protein isoform. The alternative transcripts to which disease-associated variants map demonstrate high tissue-specific expression, with many unannotated in reference human genomes, revealed only by long-read RNA-sequencing. As an example, we report an unannotated alternative transcript of the inflammasome regulator DPP9 that is lung epithelium-specific and which harbours a common genetic variant associated with severe COVID-19 and lung fibrosis. The variant causes a p.Leu8Pro missense mutation in an alternative first exon, predicted to disrupt the encoded alpha helix. These findings highlight the importance of considering alternative isoforms, their tissue-specific expression, and full-length transcripts in variant interpretation, with implications for uncovering underappreciated mechanisms of both common and rare disease.

genomics↗

bioRxiv2022

Hypoxia shapes the immune landscape in lung injury promoting inflammation persistence

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

cell biology↗

bioRxiv2022

Genetic control of KRAB-ZFP genes explains distal CpG-site methylation which associates with human disease phenotypes.

Kruppel-associated box zinc finger proteins (KRAB-ZFPs) are the largest gene family of transcriptional regulators in higher vertebrates. We have developed a method for inferring these factors DNA binding-sites in vivo. We achieved this by combining the genome-wide association of methylation at 573,027 CpG-sites (meQTL) in blood and KRAB-ZFP RNA expression changes (eQTL). This methods efficacy is clearly demonstrated by showing that the CpG-sites whose methylation is affected by KRAB-ZFP expression changes occur preferentially near KRAB-ZFP binding-sites in the HEK293T cell line. We found such an enrichment to be significant for 31% of factors tested. In addition, we found that binding-sites of many KRAB-ZFPs are significantly enriched (or sometimes depleted) in disease-associated CpG-sites. In total, 9% (11 of 125) of the traits tested, and 14% (32 of 222) of the factors (KRAB-ZFPs and TIF1-beta) tested, were enriched or depleted in one or more trait-factor pairing. Rheumatoid arthritis and human immunodeficiency virus (HIV) infection were associated with the largest number of KRAB-ZFP enrichments. There have been variable reports on the effects on HIV infection dynamics of transcription intermediary factor 1-beta (TIF1-beta, also known as KAP1 or TRIM28), a nuclear corepressor for KRAB-ZFPs. We provide evidence that KRAB-ZFP-independent effects of TIF1-beta are responsible for the decreased methylation of CpG-sites within its binding-sites that is observed in HIV infection. In conclusion, KRAB-ZFPs often affect CpG-site methylation within the proximity of their binding-sites, and CpG-sites within the binding-sites of KRAB-ZFPs are enriched for association with many traits, including rheumatoid arthritis and HIV infection.

genomics↗

bioRxiv2022

Systematic comparison of ranking aggregation methods for gene lists in experimental results

A common experimental output in biomedical science is a list of genes implicated in a given biological process or disease. The results of a group of studies answering the same, or similar, questions can be combined by meta-analysis to find a consensus or a more reliable answer. Ranking aggregation methods can be used to combine gene lists from various sources in meta-analyses. Evaluating a ranking aggregation method on a specific type of dataset before using it is required to support the reliability of the result since the property of a dataset can influence the performance of an algorithm. Evaluation of aggregation methods is usually based on a simulated database especially for the algorithms designed for gene lists because of the lack of a known truth for real data. However, simulated datasets tend to be too small compared to experimental data and neglect key features, including heterogeneity of quality, relevance and the inclusion of unranked lists. In this study, a group of existing methods and their variations which are suitable for meta-analysis of gene lists are compared using simulated and real data. Simulated data was used to explore the performance of the aggregation methods as a function of emulating the common scenarios of real genomics data, with various heterogeneity of quality, noise level, and a mix of unranked and ranked data using 20000 possible entities. In addition to the evaluation with simulated data, a comparison using real genomic data on the SARS-CoV-2 virus, cancer (NSCLC), and bacteria (macrophage apoptosis) was performed. We summarise our evaluation results in terms of a simple flowchart to select a ranking aggregation method for genomics data.

bioinformatics↗

bioRxiv2021

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

microbiology↗

bioRxiv2021

Pandemic, epidemic, endemic: B cell repertoire analysis reveals unique anti-viral responses to SARS-CoV-2, Ebola and Respiratory Syncytial Virus

Immunoglobulin gene heterogeneity reflects the diversity and focus of the humoral immune response towards different infections, enabling inference of B cell development processes. Detailed compositional and lineage analysis of long read IGH repertoire sequencing, combining examples of pandemic, epidemic and endemic viral infections with control and vaccination samples, demonstrates general responses including increased use of IGHV4-39 in both EBOV and COVID-19 infection cohorts. We also show unique characteristics absent in RSV infection or yellow fever vaccine samples: EBOV survivors show unprecedented high levels of class switching events while COVID-19 repertoires from acute disease appear underdeveloped. Despite the high levels of clonal expansion in COVID-19 IgG1 repertoires there is a striking lack of evidence of germinal centre mutation and selection. Given the differences in COVID-19 morbidity and mortality with age, it is also pertinent that we find significant differences in repertoire characteristics between young and old patients. Our data supports the hypothesis that a primary viral challenge can result in a strong but immature humoral response where failures in selection of the repertoire risks off-target effects.

immunology↗

bioRxiv2021

Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-{kappa}B signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

microbiology↗

bioRxiv2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

immunology↗

bioRxiv2021

Sequence analysis of SARS-CoV-2 in nasopharyngeal samples from patients with COVID-19 illustrates population variation and diverse phenotypes, placing the in vitro growth properties of B.1.1.7 and B.1.351 lineage viruses in context.

New variants of SARS-CoV-2 are continuing to emerge and dominate the regional and global sequence landscapes. Several variants have been labelled as Variants of Concern (VOCs) because of perceptions or evidence that these may have a transmission advantage, increased risk of morbidly and/or mortality or immune evasion in the context of prior infection or vaccination. Placing the VOCs in context and also the underlying variability of SARS-CoV-2 is essential in understanding virus evolution and selection pressures. Sequences of SARS-CoV-2 in nasopharyngeal swabs from hospitalised patients in the UK were determined and virus isolated. The data indicated the virus existed as a population with a consensus level and non-synonymous changes at a minor variant. For example, viruses containing the nsp12 P323L variation from the Wuhan reference sequence, contained minor variants at the position including P and F and other amino acids. These populations were generally preserved when isolates were amplified in cell culture. In order to place VOCs B.1.1.7 (the UK Kent variant) and B.1.351 (the South African variant) in context their growth was compared to a spread of other clinical isolates. The data indicated that the growth in cell culture of the B.1.1.7 VOC was no different from other variants, suggesting that its apparent transmission advantage was not down to replicating more quickly. Growth of B.1.351 was towards the higher end of the variants. Overall, the study suggested that studying the biology of SARS-CoV-2 is complicated by population dynamics and that these need to be considered with new variants. ImportanceSARS-CoV-2 is the causative agent of COVID-19. The virus has spread across the planet causing a global pandemic. In common with other coronaviruses, SARS-CoV-2 genetic material (genomes) can become quite diverse as a consequence of replicating inside cells. This has given rise to multiple variants from the original virus that infected humans. These variants may have different properties and in the context of a widespread vaccination program may render vaccines less ineffective. Our research confirms the degree of genetic diversity of SARS-CoV-2 in patients. By isolating viruses from these patients, we show that there is a 100-fold range in growth of even normal variants. Interestingly, by comparing this to the pattern seen with two Variants of Concern (UK and South African variants), we show that at least in cells the ability of the B.1.1.7 variant to grow is not substantially different to many of the previous variants.

microbiology↗

bioRxiv2020

Characterising pre-clinical sub-phenotypic models of Acute Respiratory Distress Syndrome: an experimental ovine study.

The Acute Respiratory Distress Syndrome (ARDS) describes a heterogenous population of patients with acute severe respiratory failure. However, contemporary advances have begun to identify distinct sub-phenotypes that exist within its broader envelope. These sub-phenotypes have varied outcomes and respond differently to several previously studied interventions. A more precise understanding of their pathobiology and an ability to prospectively identify them, may allow for the development of precision therapies in ARDS. Historically, animal models have played a key role in translational research, although few studies have so far assessed either the ability of animal models to replicate these sub-phenotypes or investigated the presence of sub-phenotypes within animal models. Here, in three ovine models of ARDS, using combinations of oleic acid and intravenous, or intratracheal lipopolysaccharide, we demonstrate the presence of sub-phenotypes which qualitatively resemble those found in clinical cohorts. Principal Components Analysis and partitional clustering reveal two clusters, differentiated by markers of shock, inflammation, and lung injury. This study provides the first preliminary evidence of ARDS phenotypes in pre-clinical models and develops a methodology for investigating this phenomenon in future studies.

physiology↗

bioRxiv2020

Subtractive CRISPR screen identifies factors involved in non-canonical LC3 lipidation.

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. LC3 is attached to membranes by covalent lipidation that depends on recruitment of the ATG5-12-16L1 complex. Non-canonical LC3 lipidation requires the C-terminal WD40 domain of ATG16L1 that is dispensable for canonical autophagy. We devised a subtractive CRISPR knock-out screening strategy to investigate the requirements for non-canonical LC3-lipidation. This correctly identified the enzyme complexes directly responsible for LC3-lipidation. We additionally identified the RALGAP complex as important for M2-induced, but not ionophore drug induced LC3 lipidation. In contrast, we identified ATG4D as responsible for LC3 recycling in M2-induced and basal LC3-lipidation. Identification of a vacuolar ATPase subunit in the screen suggested a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug induced LC3-lipidation leads to association of the vacuolar ATPase and ATG16L1 and can be antagonised by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.

cell biology↗

bioRxiv2020

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFN{gamma} based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4+ and/or CD8+ epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8+ T cells than spike-specific CD8+ T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8+ to CD4+ T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.

immunology↗

bioRxiv2019

Upstream translation initiation expands the coding capacity of segmented negative-strand RNA viruses

Segmented negative-strand RNA viruses (sNSVs) include the influenza viruses, the bunyaviruses, and other major pathogens of humans, other animals and plants. The genomes of these viruses are extremely short. In response to this severe genetic constraint, sNSVs use a variety of strategies to maximise their coding potential. Because the eukaryotic hosts parasitized by sNSVs can regulate gene expression through low levels of translation initiation upstream of their canonical open reading frames (ORFs), we asked whether sNSVs could use upstream translation initiation to expand their own genetic repertoires. Consistent with this hypothesis, we showed that influenza A viruses (IAVs) and bunyaviruses were capable of upstream translation initiation. Using a combination of reporter assays and viral infections, we found that upstream translation in IAVs can initiate in two unusual ways: through non-AUG initiation in virally encoded untranslated regions, and through the appropriation of an AUG-containing leader sequence from host mRNAs through viral cap-snatching, a process we termed start-snatching. Finally, while upstream translation of cellular genes is mainly regulatory, for sNSVs it also has the potential to create novel viral gene products. If in frame with a viral ORF, this creates N-extensions of canonical viral proteins. If not, it allows the expression of cryptic overlapping ORFs, which we found were highly conserved in IAV and widely distributed in peribunyaviruses. Thus, by exploiting their hosts capacity for upstream translation initiation, sNSVs can expand still further the coding potential of their extremely compact RNA genomes.

molecular biology↗

bioRxiv2019

Comprehensive characterisation of molecular host-pathogen interactions in influenza A virus-infected human macrophages

Macrophages in the lung detect and respond to influenza A virus (IAV), determining the nature of the immune response. Using terminal depth 5-RNA sequencing (CAGE) we quantify transcriptional activity of both host and pathogen over a 24-hour timecourse of IAV infection in primary human monocyte-derived macrophages (MDM). We use a systems approach to describe the transcriptional landscape of the host response to IAV contrasted with bacterial lipopolysaccharide treated MDMs, observing a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation. Systematic comparison of host RNA sequences incorporated into viral mRNA (\"snatched\") against a complete survey of background RNA in the host cell enables an unbiased quantification of over-represented features of snatched host RNAs. We detect preferential snatching of RNAs associated with snRNA transcription and demonstrate that cap-snatching avoids transcripts encoding host ribosomal proteins, which are required by IAV for replication.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC=\"FIGDIR/small/670919v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (34K):\norg.highwire.dtl.DTLVardef@b05478org.highwire.dtl.DTLVardef@79a519org.highwire.dtl.DTLVardef@462739org.highwire.dtl.DTLVardef@1b91eb6_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract(A) Overview of bioinformatics pipeline. (B) Host gene expression reveals that human macrophages exposed to IAV exhibit sustained production of key inflammatory mediators and failure to induce expression of feedback inhibitors of inflammation. (C) Unbiased comparison with total background RNA expression demonstrates that IAV cap-snatching has a strong preference for, and aversion to, different groups of host transcripts.

immunology↗

bioRxiv2019

Proteome-by-phenome Mendelian Randomisation detects 38 proteins with causal roles in human diseases and traits

Target identification remains a crucial challenge in drug development. To enable unbiased detection of proteins and pathways that have a causal role in disease pathogenesis or progression, we propose proteome-by-phenome Mendelian Randomisation (P2MR). We first detected genetic variants associated with plasma concentration of 249 proteins. We then used 64 replicated variants in two-sample Mendelian Randomisation to quantify evidence of a causal role for each protein across 846 phenotypes: this yielded 509 robust protein-outcome links. P2MR provides substantial promise for drug target prioritisation. We provide confirmatory evidence for a causal role for the proteins encoded at multiple cardiovascular disease risk loci (FGF5, IL6R, LPL, LTA), and discovered that intestinal fatty acid binding protein (FABP2) contributes to disease pathogenesis. Additionally, we find and replicate evidence for a causal role of tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1; SIRPA) in schizophrenia. Our results provide specific prediction of the effects of changes of plasma protein concentration on complex phenotypes in humans.

genomics↗

bioRxiv2019

Multiomic definition of generalizable endotypes in human acute pancreatitis.

Acute pancreatitis (AP) is sudden onset pancreas inflammation that causes multiple organ dysfunction syndrome (MODS) and death in certain individuals who develop AP yet minimal systemic inflammation in others. Here, we show that this observed diversity in systemic response and outcome is accompagnied by diversity in molecular subtypes that can be identified using computational analysis of clinical and multiomic data. We integrated co-incident whole blood transcriptomic, plasma proteomic, and serum metabolomic data at serial time points from a cohort of patients presenting with AP and systematically evaluated four different metrics for patient similarity, using unbiased mathematical, biological and clinical measures of internal and external validity. Our results identify four distinct and stable AP endotypes that are characterized by pathway and biomarker combination stereotypes into hypermetabolic, hepatopancreaticobiliary, catabolic and innate immune endotypes. The catabolic endotype in AP strikingly recapitulates a disease endotype previously reported in acute respiratory distress syndrome, a recognized complication of AP. Our findings demonstrate that clinically-relevant and generalizable endotypes exist in AP.

systems biology↗

bioRxiv2018

Predictive validity of effective shunt fraction in critically ill patients

Accurate measurement of pulmonary oxygenation is important for classification of disease severity and quantification of outcomes in clinical studies. We compared predictive validity of established tension-based methods with two new measures of shunt fraction: (1) a non-invasive effective shunt (ES); and (2) inferred values from an integrated mathematical model of gas exchange (DB). Median absolute error (MAE) values for the four measures considered were: alveolar-arterial difference, 7.30kPa; PaO2/FIO2 ratio, 2.41kPa; DB, 2.13kPa; ES: 1.88kPa. ES performed significantly better than other measures (p<10-10 in all comparisons). While the simplicity of P/F is suitable for routine use, the superior predictive validity of ES should make this measure the preferred choice where physiological accuracy is important, such as for use as surrogate outcome in clinical research.

physiology↗