Biology subjects

King, L.

Publications and source records attributed to King, L..

16 recordsLinked to original sources

bioRxiv2026

The tuxedo sea urchin Mespilia globulus: A fast-developing and tractable model for genomic and developmental biology

Sea urchins are pivotal models in cellular and developmental biology, but their biphasic life cycle with an extended larval life limits the ability to study post-metamorphosis and adult characters. Here, we introduce a genomically-enabled model system, the tuxedo urchin Mespilia globulus, which has rapid access to late life stages in sea urchins. We describe how we cultured M. globulus in a landlocked marine facility, raised larvae under artificial conditions and closed their life cycle. We established the experimental tractability of M. globulus: we labelled transcripts by hybridization chain reaction (HCR), and knocked out pigmentation genes to produce albino larvae using CRISPR/Cas9. We generated chromosome-scale genome assemblies for two individuals representing both sexes and two color morphs (red and blue), and compared the organisation of the 21 chromosomes of M. globulus with that of other camarodont echinoid models. We determined that M. globulus showed a conservative gene repertoire lacking the gene family expansions seen in other camarodont sea urchins. We annotated the complement of genes associated with pigmentation, immune and nervous systems and profiled their expressions in tissues and organs. Finally, we surveyed sex-related regions in genomes using genome assemblies and resequencing data, finding no evidence of heteromorphic sex chromosomes in M. globulus. Our findings highlight the accessibility of this new sea urchin model for studying the metamorphosis and adult biology of sea urchins.

evolutionary biology↗

bioRxiv2025

Amoeboid-Mesenchymal Transition and the Proteolytic Control of Cancer Invasion Plasticity

Invasion plasticity allows malignant cells to toggle between collective, mesenchymal and amoeboid phenotypes while traversing extracellular matrix (ECM) barriers. Current dogma holds that collective and mesenchymal invasion programs trigger the mobilization of proteinases that digest structural barriers dominated by type I collagen, while amoeboid activity allows cancer cells to marshal mechanical forces to traverse tissues independently of ECM proteolysis. Here, we use cancer spheroid-3-dimensional matrix models, single-cell RNA sequencing, and human tissue explants to identify the mechanisms controlling mesenchymal versus amoeboid invasion. Unexpectedly, collective/mesenchymal- and amoeboid-type invasion programs - though distinct - are each characterized by active tunneling through ECM barriers, with expression of matrix-degradative metalloproteinases. CRISPR/Cas9-mediated targeting of a single membrane-anchored collagenase, MMP14/MT1-MMP, ablates tissue-invasive activity while co-regulating cancer cell transcriptional programs. Though changes in matrix architecture, nuclear rigidity, and metabolic stress as well as the presence of cancer-associated fibroblasts are proposed to support amoeboid activity, none of these changes restore invasive activity of MMP14-targeted cancer cells. While a requirement for MMP14 is bypassed in low-density collagen hydrogels, invasion by the proteinase-deleted cells is associated with nuclear envelope and DNA damage, highlighting a proteolytic requirement for maintaining nuclear integrity. Nevertheless, when cancer cells confront explants of live human breast tissue, MMP14 is again required to support invasive activity. Corroborating these results, spatial transcriptomic and immunohistological analyses of invasive human breast cancers identified clear expression of MMP14 in invasive cells that were further associated with degraded collagen, underlining the pathophysiologic importance of this proteinase in directing invasive activity in vivo.

cancer biology↗

bioRxiv2025

Dynamic uterine microenvironment drives endometrial adenocarcinoma carcinogenesis and progression

Endometrial cancer (EC) development is driven by the interaction between the tumor and the microenvironment. However, the immune microenvironment dynamics during this process are not clear. Here, we applied single-cell RNA sequencing (scRNA-seq) to uterine blood samples collected at hysterectomy from 15 individuals encompassing four groups--benign controls, endometrial intraepithelial neoplasia (EIN), the transition from EIN to carcinoma, and EC. Uterine blood, obtained without prior enrichment, provides a liquid biopsy of the local tumor milieu, enabling high-resolution profiling of both immune and stromal cells in a minimally invasive manner. Our analysis revealed simultaneous immune and stromal remodeling in early premalignant lesions. Notably, even in the EIN, we observed significant immune cell reprogramming alongside the emergence of protumorigenic stromal-epithelial interactions. Importantly, we discovered predictive single-cell transcriptomic signatures derived from neutrophils that stratify patients according to disease state, highlighting the potential of tumor-educated innate immune cells as biomarkers. These findings yield candidate cellular and molecular signatures, particularly from neutrophils, that could enhance early EC detection and guide therapeutic strategies. Our work provides a proof-of-concept for leveraging local liquid biopsies in single-cell oncology, offering new insights into EC initiation and paving the way for noninvasive diagnostics on the basis of single-cell transcriptomic profiles.

cancer biology↗

bioRxiv2025

Multiplexed proximity labelling proteomics identifies a non-canonical Bcl-2 family interactome associated with apoptotic priming

Bcl-2 family proteins govern the intrinsic apoptotic pathway by regulating mitochondrial outer membrane permeabilisation (MOMP), releasing apoptogenic factors into the cytosol. How close a cell is to MOMP, termed mitochondrial priming, is determined by the interactions between different Bcl-2 proteins at the outer mitochondrial membrane (OMM). However, Bcl-2 proteins can also drive diverse processes that do not result in cell death, such as incomplete MOMP, sublethal caspase activation, pro-inflammatory signalling and regulation of cellular metabolism. To understand the wider Bcl-2 family interactome and how it might be involved in these processes, we undertook an unbiased proteomic BioID screen, using both pro- and anti-apoptotic Bcl-2 family members as bait proteins. We found that most high-confidence potential interactions in non-apoptotic cells were outside the canonical Bcl-2 family interactome. Analysis of how this interactome changed in response to either BH3-mimetics or full-length BH3-only proteins revealed dynamic changes at multiple organelles and membrane contact sites in response to altered mitochondrial priming. These findings underscore the complex and varied interactions of the Bcl-2 family proteins, expanding their scope and function beyond the frequently studies intra-family interactions.

cell biology↗

bioRxiv2024

Genesis: A Modular Protein Language Modelling Approach to Immunogenicity Prediction

Neoantigen immunogenicity prediction is a highly challenging problem in the development of personalised medicines. Low reactivity rates in called neoantigens result in a difficult prediction scenario with limited training datasets. Here we describe Genesis, a modular protein language modelling approach to immunogenicity prediction for CD8+ reactive epitopes. Genesis comprises of a pMHC encoding module trained on three pMHC prediction tasks, an optional TCR encoding module and a set of context specific immunogenicity prediction head modules. Compared with state-of-the-art models for each task, Genesis encoding module performs comparably or better on pMHC binding affinity, eluted ligand prediction and stability tasks. Genesis outperforms all compared models on pMHC immunogenicity prediction (Area under the receiver operating characteristic curve=0.619, average precision: 0.514), with a 7% increase in average precision compared to the next best model. Genesis shows further improved performance on immunogenicity prediction with the integration of TCR context information. Genesis performance is further analysed for interpretability, which locates areas of weakness found across existing immunogenicity models and highlight possible biases in public datasets.

bioinformatics↗

bioRxiv2024

Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5

Despite recent success in licencing of two malaria vaccines, there continues to be an urgent need for improved vaccine immunogens as the world aims for malaria eradication. The invasion of erythrocytes by Plasmodium falciparum is an essential step in the life cycle of the parasite, preceding symptoms of disease and parasite transmission. Antibodies which target the PfRH5 protein are highly effective at preventing erythrocyte invasion and the most effective growth-inhibitory antibodies bind to a single epitope. Here we used Rosetta-based protein design to produce a focused synthetic immunogen on which this epitope is presented on a small scaffold. Structural biology and biophysics were used to demonstrate that the immunogen is correctly folded and binds neutralising monoclonal antibodies with at least nanomolar affinity. In immunised rats, the immunogen induced PfRH5-targeting antibodies that inhibit parasite growth at a thousand-fold lower concentration that those induced through immunisation with PfRH5. Finally, we show that priming with the focused immunogen and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens. One Sentence SummaryRational, structure-guided design has been used to produce a vaccine immunogen in which the epitope for the most growth-inhibitory monoclonal antibody targeting PfRH5 has been correctly presented on a synthetic scaffold for use in a vaccine to protect from blood-stage malaria.

immunology↗

bioRxiv2023

Single Cell Expression Analysis of Ductal Carcinoma in Situ Identifies Alterations in Epithelial Integrity

To identify mechanisms underlying the growth of ductal carcinoma in situ (DCIS) and properties that lead to progression to invasive cancer, we performed single-cell RNA-sequencing (scRNA-seq) on DCIS lesions and matched synchronous normal breast tissue. Using inferred copy number variations (CNV), we identified neoplastic epithelial cells from the clinical specimens which contained a mixture of DCIS and normal ducts. Phylogenetic analysis based on the CNVs demonstrated intratumoral clonal heterogeneity was associated with significant gene expression differences. We also classified epithelial cells into mammary cell states and found that individual genetic clones contained a mixture of cell states suggesting an ongoing pattern of differentiation after neoplastic transformation. Cell state proportions were significantly different based on estrogen receptor (ER) expression with ER-DCIS more closely resembling the distribution in the normal breast, particularly with respect to cells with basal characteristics. Using deconvolution from bulk RNA-seq in archival DCIS specimens, we show that specific alterations in cell state proportions are associated with progression to invasive cancer. Loss of an intact basement membrane (BM) is the functional definition of invasive breast cancer (IBC) and scRNA-seq data demonstrated that ongoing transcription of key BM genes occurs in specific subsets of epithelial cell states. Examining BM in archival microinvasive breast cancers and an in vitro model of invasion, we found that passive loss of BM gene expression due to cell state proportion alterations is associated with loss of the structural integrity of the duct leading to an invasive phenotype. Our analyses provide detailed insight into DCIS biology. SIGNIFICANCESingle cell analysis reveals that preinvasive breast cancer is comprised of multiple genetic clones and there is substantial phenotypic diversity both within and between these clones. Ductal carcinoma in situ (DCIS) of the breast is a non-invasive condition commonly identified through mammographic screening. A primary diagnosis of DCIS carries little mortality risk on its own, but its presence is a risk factor for subsequent clonally related invasive breast cancer (IBC) (1-5).

cancer biology↗

bioRxiv2023

Transcriptomic contributions to a modern cytoarchitectonic parcellation of the human cerebral cortex

Transcriptomic contributions to the anatomical, functional, and network layout of the human cerebral cortex (HCC) has become a major interest in cognitive and systems neuroscience. Here, we tested if transcriptomic differences support a modern, algorithmic cytoarchitectonic parcellation of HCC. Using a data-driven approach, we identified a sparse subset of genes that differentially contributed to the cytoarchitectonic parcellation of HCC. A novel metric (cortical thickness/myelination ratio; CT/M ratio), as well as cell density, correlated with gene expression. Enrichment analyses showed that genes specific to the cytoarchitectonic parcellation of the HCC were related to molecular functions such as transmembrane transport and ion channel activity. Together, the novel relationship between transcriptomics and the CT/M ratio bridges the gap among i) gradients at the macroscale, ii) areas at the meso-scale, and iii) cell density at the microscale, as well as supports the recently proposed cortical spectrum theory.

neuroscience↗

bioRxiv2021

Socially stratified epigenetic profiles are associated with cognitive functioning in children and adolescents

Childrens cognitive functioning and educational performance are socially stratified. Social inequality, including classism and racism, may operate partly via epigenetic mechanisms that modulate neurocognitive development. Following preregistered analyses of data from 1,183 8-to 19-year-olds from the Texas Twin Project, we examined whether salivary DNA-methylation measures of inflammation (DNAm-CRP), cognitive functioning (Epigenetic-g), and pace of biological aging (DunedinPoAm) are socially stratified and associated with performance on tests of cognitive functions. We find that children growing up in more disadvantaged families and neighborhoods and children from marginalized racial/ethnic groups exhibit DNA-methylation profiles associated with higher chronic inflammation, lower cognitive functioning, and faster pace of biological aging. These salivary DNA-methylation profiles were associated with processing speed, general executive function, perceptual reasoning, verbal comprehension, reading, and math. Given that the DNA-methylation measures we examined were originally developed in adults, our results suggest that social inequalities may produce in children molecular signatures that, when observed in adults, are associated with chronic inflammation, advanced aging, and reduced cognitive function. Salivary DNA-methylation profiles might be useful as a surrogate endpoint in assessing the effectiveness of psychological and economic interventions that aim to reduce negative effects of childhood social inequality on lifespan development. Significance StatementChildrens cognitive functioning differs by dimensions of social inequality, such as class and race. Epigenetic mechanisms that regulate gene expression might be critically involved in the biological embedding of environmental privilege and adversity. We find that children growing up in more disadvantaged families and neighborhoods and from marginalized racial/ethnic groups exhibit higher chronic inflammation, lower cognitive functioning, and a faster pace of biological aging, as indicated by novel salivary DNA-methylation measures. These DNA-methylation measures of higher inflammation, lower cognitive functioning, and a faster pace of biological aging were, in turn, associated with performance on multiple cognitive tests. DNA-methylation measures might be useful as a surrogate endpoint in evaluation of programs to address the childhood social determinants of lifelong cognitive disparities.

genetics↗

bioRxiv2021

The Human Tumor Atlas Network (HTAN) Breast PreCancer Atlas: A multi-omic integrative analysis of ductal carcinoma in situ with clinical outcomes

Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We identified 812 genes associated with ipsilateral recurrence within 5 years from treatment and developed a classifier that was predictive of DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions were identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome. HIGHLIGHTS Development of a new classifier for DCIS recurrence or progression Outcome associated pathways identified across multiple data types and compartments Four stroma-specific signatures identified CNAs characterize DCIS subgroups associated with high risk invasive cancers

cancer biology↗

bioRxiv2020

A new method to accurately identify single nucleotide variants using small FFPE breast samples.

Most tissue collections of neoplasms are composed of formalin-fixed and paraffin-embedded (FFPE) excised tumor samples used for routine diagnostics. DNA sequencing is becoming increasingly important in cancer research and clinical management; however, it is difficult to accurately sequence DNA from FFPE samples. We developed and validated a new bioinformatic algorithm to robustly identify somatic single nucleotide variants (SNVs) from whole exome sequencing using small amounts of DNA extracted from archival FFPE samples of breast cancers. We optimized this strategy using 28 pairs of technical replicates. After optimization, the mean similarity between replicates increased 5-fold, reaching 88% (range 0-100%), with a mean of 21.4 SNVs (range 1-68) per sample, representing a markedly superior performance to existing algorithms. We found that the SNV-identification accuracy declined when there was less than 40ng of DNA available and that insertion-deletion variant calls are less reliable than single base substitutions. As the first application of the new algorithm, we compared samples of ductal carcinoma in situ (DCIS) of the breast to their adjacent invasive ductal carcinoma (IDC) samples. We observed an increased number of mutations (paired-samples sign test, p<0.05), and a higher genetic divergence in the invasive samples (paired-samples sign test, p<0.01). Our algorithm provides a significant improvement in detecting SNVs in FFPE samples over previous approaches. Key PointsO_LIThe sequencing of reduced quantities of DNA extracted from FFPE samples leads to substantial sequencing errors that require correction in order to obtain accurate detection of somatic mutations. C_LIO_LIWe developed and validated a new bioinformatic algorithm to robustly identify somatic single nucleotide variants using small amounts of DNA extracted from archival FFPE samples of breast cancers. C_LIO_LIVariant calling software packages need to be optimized to reduce the impact of sequencing errors. Our bioinformatics pipeline represents a significant methodological advance compared to the currently available bioinformatic tools used for the analysis of small FFPE samples. C_LI

genomics↗

bioRxiv2020

Hybrid assembly of an agricultural slurry virome reveals a diverse and stable community with the potential to alter the metabolism and virulence of veterinary pathogens

BackgroundViruses are the most abundant biological entities on Earth, known to be crucial components of microbial ecosystems. However, there is little information on the viral community within agricultural waste. There are currently ~2.7 million dairy cattle in the UK producing 7-8% of their own bodyweight in manure daily, and 28 million tonnes annually. To avoid pollution of UK freshwaters, manure must be stored and spread in accordance with guidelines set by DEFRA. Manures are used as fertiliser, and widely spread over crop fields, yet little is known about their microbial composition. We analysed the virome of agricultural slurry over a five-month period using short and long-read sequencing. ResultsHybrid sequencing uncovered more high-quality viral genomes than long or short-reads alone; yielding 7,682 vOTUs, 174 of which were complete viral genomes. The slurry virome was highly diverse and dominated by lytic bacteriophage, the majority of which represent novel genera (~98%). Despite constant influx and efflux of slurry, the composition and diversity of the slurry virome was extremely stable over time, with 55% of vOTUs detected in all samples over a five-month period. Functional annotation revealed a diverse and abundant range of auxiliary metabolic genes and novel features present in the community. Including the agriculturally relevant virulence factor VapE, which was widely distributed across different phage genera that were predicted to infect several hosts. Furthermore, we identified an abundance of phage-encoded diversity-generating retroelements, which were previously thought to be rare on lytic viral genomes. Additionally, we identified a group of crAssphages, including lineages that were previously thought only to be found in the human gut. ConclusionsThe cattle slurry virome is complex, diverse and dominated by novel genera, many of which are not recovered using long or short-reads alone. Phages were found to encode a wide range of AMGs that are not constrained to particular groups or predicted hosts, including virulence determinants and putative ARGs. The application of agricultural slurry to land may therefore be a driver of bacterial virulence and antimicrobial resistance in the environment.

microbiology↗

bioRxiv2019

Unmasking the tissue microecology of ductal carcinoma in situ with deep learning

Despite increasing evidence supporting the clinical relevance of tumour infiltrating lymphocytes (TILs) in invasive breast cancer, TIL spatial distribution pattern surrounding ductal carcinoma in situ (DCIS) and its association with progression is not well understood. To characterize the tissue microecology of DCIS, we designed and tested a new deep learning pipeline, UNMaSk (UNet-IM-Net-SCCNN), for the automated detection and simultaneous segmentation of DCIS ducts. This new method achieved the highest sensitivity and recall over cutting-edge deep learning networks in three patient cohorts, as well as the highest concordance with DCIS identification based on CK5 staining. Following automated DCIS detection, spatial tessellation centred at each DCIS duct created the boundary in which local ecology can be studied. Single cell identification and classification was performed with an existing deep learning method to map the distribution of TILs. In a dataset comprising grade 2-3 pure DCIS and DCIS adjacent to invasive cancer (adjacent DCIS), we found that pure DCIS cases had more TILs compared to adjacent DCIS. However, TILs co-localise significantly less with DCIS ducts in pure DCIS compared with adjacent DCIS, suggesting a more inflamed tissue ecology local to adjacent DCIS cases. Our experiments demonstrate that technological developments in deep convolutional neural networks and digital pathology can enable us to automate the identification of DCIS as well as to quantify the spatial relationship with TILs, providing a new way to study immune response and identify new markers of progression, thereby improving clinical management.

pathology↗

bioRxiv2019

Bax mitochondrial residency is more critical than Bax oligomerization for apoptosis

The Bax protein plays an important effector role in apoptosis by forming pores in the mitochondrial outer membrane. While doing so, Bax forms higher-order oligomers in the membrane, but it remains unclear whether this oligomer formation is essential for pore formation. Using cell-free and cellular experimental systems, we investigated two Bax C-terminus mutants, T182I and G179P. Neither mutant formed large oligomers when activated in liposomes. Nevertheless, the G179P mutant could produce membrane pores, suggesting that large oligomers are not required for permeabilization. Surprisingly, however, when G179P was transduced into Bax/Bak double knockout mouse embryonic fibroblasts, it was purely cytoplasmic and failed to mediate cell death. T182I behaved in the opposite manner. When mixed with liposomes, T182I was inefficient in both membrane insertion and permeabilization. However, transduced into cells, BaxT182I resided constitutively in mitochondria, owing to its slow retrotranslocation and mediated apoptosis as efficiently as wild-type Bax. We conclude that Baxs mitochondrial residence (regulated by targeting and retrotranslocation) is more important for apoptosis than its efficiency of membrane insertion and higher-order oligomerization.

cell biology↗

bioRxiv2019

BioID based proteomic screen identifies VDAC2 as a coordinator of Bid dependent apoptotic priming during mitosis.

Apoptotic priming controls the commitment of cells to apoptosis by determining how close they lie to mitochondrial permeabilisation. Variations in priming are important for how both healthy and cancer cells respond to chemotherapeutic agents, but how this is dynamically coordinated by Bcl-2 proteins remains unclear. The Bcl-2 family protein Bid is phosphorylated when cells enter mitosis, increasing apoptotic priming and sensitivity to anti-mitotic drugs. Here we report an unbiased proximity biotinylation (BioID) screen to identify regulators of apoptotic priming in mitosis, using Bid as bait. The screen primarily identified proteins outside of the canonical Bid interactome. Specifically, we found that voltage-dependent anion-selective channel protein 2 (VDAC2) was required for Bid phosphorylation dependent changes in apoptotic priming during mitosis. Thus, we identify an additional layer of regulation upstream of known Bid interactions that control dynamic changes in apoptotic priming. These results highlight the importance of understanding the wider Bcl-2 family interactome and its role in regulating the temporal control of apoptotic priming.

cell biology↗

bioRxiv2018

Phylogenomics, biogeography, and evolution in the American palm genus Brahea

Background and AimsSlow rates of molecular evolution at low taxonomic levels hamper studies of relationships among species, and subsequent biogeographic and evolutionary analyses. An example is the genus Brahea, which is among the most poorly understood lineages of American palms and is characterized by a wide variety of growth forms and intermediate morphological features.\n\nMethodsWe generated approximately 400 kb of genome-scale data from all three genomes for the 11 currently described species of Brahea to infer phylogenetic relationships, reconstruct ancestral growth form, estimate ancestral geographic ranges, and test for niche equivalency among closely related species with geographic overlap.\n\nKey ResultsRelationships receive strong support, and conform to previous subgeneric assignments, except for placement of the dwarf species B. moorei within subgenus Erythea. Our robust phylogenetic hypothesis reveals trends in growth form including an overall increase in height in the B. armata clade, and independent evolution of dwarf forms from taller ancestors in the B. pimo and B. dulcis clades. Ancestral range estimation reveals roles of dispersal (e.g. B. edulis on Guadalupe Island) and sympatric speciation in some cases (e.g. in the B. armata clade), but is equivocal in others (e.g. in the B. pimo clade). We find evidence of niche non-equivalency among species within the B. armata clade in northwestern Mexico, and some evidence of niche non-equivalency between B. berlandieri and B. dulcis, the former of which is synonymized under B. dulcis.\n\nConclusionsOur findings have implications for the complex biogeographic history in Central America and Mexico, suggesting that sympatric speciation and dispersal are the predominant processes of species diversification. Future studies should include population-level sampling across the genus, along with morphological and ecological information, to assess distinctness among species and, particularly, levels of gene flow, in an integrative fashion.

evolutionary biology↗