Biology subjects

Law, A.

Publications and source records attributed to Law, A..

11 recordsLinked to original sources

bioRxiv2026

5β-Dihydrotestosterone reveals a mutant androgen receptor vulnerability in prostate cancer

Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) cells to rapid cycling between castrate and supraphysiologic androgen concentrations, but clinical BAT uses testosterone, which can also activate wild-type androgen receptor (AR) in androgen-responsive tissues, causing systemic side effects. 5{beta}-dihydrotestosterone (5{beta}-DHT) is a naturally occurring testosterone metabolite generally considered androgenically inactive because it binds wild-type AR weakly, yet its activity against clinically relevant AR mutants has not been systematically evaluated. Here, we tested whether 5{beta}-DHT and related 5{beta}-reduced testosterone metabolites activate AR signaling and growth programs in prostate cancer models that carry AR mutations. In C4-2 cells, 5{beta}-DHT and 3{beta}-etiocholanediol (3{beta}-ecdiol) increased canonical AR target genes, including KLK3 and TMPRSS2, with weaker activity than testosterone, whereas other 5{beta} metabolites showed limited activity. In androgen-responsive LNCaP and C4-2 models, 5{beta}-DHT and 3{beta}-ecdiol promoted cell growth under androgen-depleted conditions, and this effect was suppressed by enzalutamide, supporting AR dependence. RNA-seq confirmed that 5{beta}-DHT and 3{beta}-ecdiol induced androgen-response gene sets substantially overlapping with testosterone, albeit at lower transcriptional magnitude. Further, we found that 5{beta}-DHT, but not 3{beta}-ecdiol, suppresses cell proliferation of LNCaP, C4-2, and PC-3 cells stably expressing the clinically relevant AR gain-of-function mutants W742C and H875Y through activating AR-induced senescence-like features after high-dose exposure, consistent with the therapeutic logic of BAT. These findings identify 5{beta}-DHT as an overlooked mutant-AR agonist capable of BAT-like tumor suppression and propose it as a testosterone surrogate in BAT with potentially reduced systemic androgenic side effects. HighlightsO_LI5{beta}-DHT and 3{beta}-ecdiol promote AR-dependent prostate cancer cell growth C_LIO_LIBoth are weaker AR agonists than testosterone by RNA-seq and qPCR C_LIO_LISupraphysiologic 5{beta}-DHT suppresses growth via AR-mediated senescence C_LIO_LIGrowth suppression extends to AR mutants W742C and H875Y C_LIO_LI5{beta}-DHT may be a lower-androgenicity testosterone surrogate for BAT C_LI

cancer biology↗

bioRxiv2026

Characterization of the clade 4 non-toxigenic C. difficile isolate L-NTCD03 carrying the cfr(B) gene

Clostridioides difficile infection (CDI) is a toxin-mediated gastro-intestinal disease. Yet, C. difficile is a phylogenetically diverse species that includes many non-toxigenic strains. In general, these are understudied, despite having significant potential impact for our understanding of the colonization process and as therapeutic modalities. Here, we present an in-depth characterization - including the complete genome sequence - of the non-toxigenic C. difficile strain L-NTCD03. This strain belongs to PCR ribotype 416, clade 4 and multilocus sequence type 39. It is resistant to multiple antimicrobials, but not those used for treatment of CDI. We validated the relevance of the cfr(B) gene from this strain in antimicrobial resistance to clindamycin, linezolid, retapamulin and streptogramin A. We found the L-NTCD03 strain to be non-toxic in various assays. Altogether, L-NTCD03 is a promising candidate for developing into a live biotherapeutic product.

microbiology↗

bioRxiv2025

The cyclic nucleotide binding sites of Swiss-Cheese, the Drosophila orthologue of human PNPLA6, are required for its catalytic function

Mutations in Swiss-cheese (SWS) or its vertebrate ortholog PNLPA6, also called Neuropathy Target Esterase (NTE), cause progressive neuronal degeneration in Drosophila and mice and several complex syndromes in humans. These include mental retardation, spastic paraplegia, ataxia and blindness and several other symptoms. SWS and PNPLA6 are widely expressed in neurons and in several glial cell types in Drosophila and mice and both cell types require SWS/PNPLA6 function autonomously. SWS and PNPLA6 are structurally and functionally conserved because expression of human or mouse PNPLA6 can replace SWS in flies. These orthologues share several domains, including the highly conserved phospholipase domain that mediates its function in deacetylating phosphatidylcholine (PC) to lysophosphatidylcholine and glycerophosphocholine. In addition, they share three cyclic nucleotide binding sites and although about 10% of the known disease-causing mutations occur in these sites, their function is still unknown. We therefore generated mutations in these sites in SWS to address what consequences this has for the function of the protein. Mutating only one site (SWSG558E) results in a partially functional protein that rescues the sws knockdown and that decreases PC when overexpressed. However, mutating all three sites (SWS{Delta}CNB) renders SWS non-functional and results in an increase of PC when overexpressed, suggesting that cyclic nucleotide binding can regulate the phospholipase function.

neuroscience↗

bioRxiv2025

eDNA sampling reveals no negative effects of beaver recolonisation on the catchment-scale distribution of migratory fish

O_LIReintroduction of keystone species is considered part of the solution to the current biodiversity crisis. The Eurasian beaver (Castor fiber) is one such species, shaping its habitat by felling trees, building dams and creating wetlands. However, while the potential benefits to aquatic biodiversity and ecological functioning have been studied on a local scale, the impacts of beavers on catchment-scale processes such as fish migration remain understudied. C_LIO_LISequencing of environmental DNA (eDNA metabarcoding) from water samples is a cost-effective method to study species distributions across large geographical scales. Here, eDNA samples (n=426) were collected from 142 sites across the UKs oldest and largest established wild beaver population, located on Tayside, East Scotland, and analysed using a vertebrate specific metabarcoding assay. We combined presence/absence data from eDNA results with other environmental and anthropogenic variables to model the effects of beaver presence on the distribution of three migratory fish species. C_LIO_LIUsing generalised linear models, we found no effects of the current beaver presence on the distribution of Atlantic salmon or lamprey, but a positive co-occurrence with European eel at the catchment scale. Model outputs also reinforced previous findings on the impact of barriers to migration and other abiotic and biotic factors on fish species, demonstrating the effectiveness of eDNA sampling in rivers for understanding species distributions at a catchment scale. C_LIO_LIThis case study provides novel insights into the co-distribution of beavers and migratory fish, and highlights how catchment-wide eDNA monitoring can be applied by environmental managers to aid decision-making and impact assessment more generally. C_LI

ecology↗

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

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

bioRxiv2021

A novel strain of lumpy skin disease virus causes clinical disease in cattle in Hong Kong

Lumpy skin disease virus (LSDV) is an emerging poxviral pathogen of cattle that is currently spreading throughout Asia. The disease situation is of high importance for farmers and policy makers in Asia. In October 2020, feral cattle in Hong Kong developed multifocal cutaneous nodules consistent with lumpy skin disease (LSD). Gross and histological pathology further supported the diagnosis and samples were sent to the OIE Reference Laboratory at The Pirbright Institute for confirmatory testing. LSDV was detected using quantitative polymerase chain reaction (qPCR) and additional molecular analyses. This is the first report of LSD in Hong Kong. Whole genome sequencing (WGS) of the strain LSDV/HongKong/2020 and phylogenetic analysis were carried out in order to identify connections to previous outbreaks of LSD, and better understand the drivers of LSDV emergence. Analysis of the 90 core poxvirus genes revealed LSDV/HongKong/2020 was a novel strain most closely related to the live-attenuated Neethling vaccine strains of LSDV and more distantly related to wildtype LSDV isolates from Africa, the Middle East and Europe. Analysis of the more variable regions located towards the termini of the poxvirus genome revealed genes in LSDV/HongKong/2020 with different patterns of grouping when compared to previously published wildtype and vaccine strains of LSDV. This work reveals that the LSD outbreak in Hong Kong in 2020 was caused by a different strain of LSDV than the LSD epidemic in the Middle East and Europe in 2015-2018. The use of WGS is highly recommended when investigating LSDV disease outbreaks.

microbiology↗

bioRxiv2020

Host cell factors important for BHV-1 cell entry revealed by genome-wide CRISPR knockout screen

In order to identify host factors that impact Bovine Herpes Virus Type 1 (BHV-1) infection we previously applied a genome wide CRISPR knockout screen with a library covering all bovine protein coding genes. We compiled a list of both pro-viral and anti-viral proteins involved in BHV-1 replication; here we provide further analysis of those that are potentially involved in viral entry into the host cell. These entry related factors include the cell surface proteins PVR and PVRL2, a group of enzymes directly or indirectly associated with the biosynthesis of Heparan Sulfate Proteoglycans (HSPG), and proteins that reside in the Golgi apparatus engaging in intra-Golgi trafficking. For the first time, we provide evidence that PVRL2 serves a receptor for BHV-1, mediating more efficient entry than the previously identified PVR. By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we demonstrated the significance of HSPG in BHV-1 entry. Another intriguing cluster of genes, COG1, COG2 and COG4-7 encodes for six subunits of the conserved oligomeric Golgi (COG) complex. MDBK cells lacking COG6 were less infectable by BHV-1 but release newly produced virions more efficiently as evidenced by fewer but bigger plaques compared to control cells, suggesting impaired HSPG biosynthesis. To facilitate candidate validation, we devised a one-step multiplex CRISPR interference (CRISPRi) system named CRISPR3i that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified an additional 23 candidates, with many implicated in cellular entry.

microbiology↗

bioRxiv2020

Genome-wide CRISPR knockout screen reveals membrane tethering complexes EARP and GARP important for Bovine Herpes Virus Type 1 replication

We produced a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome and used it to identify host genes important for Bovine Herpes Virus Type 1 (BHV-1) replication. By infecting library transduced MDBK cells with a GFP tagged BHV-1 virus and FACS sorting them based on their GFP intensity, we identified a list of pro-viral and anti-viral candidate host genes that might affect various aspects of the virus biology, such as cell entry, RNA transcription and viral protein trafficking. Among them were VPS51, VPS52 and VPS53 that encode for subunits of two membrane tethering complexes EARP and GARP. Simultaneous loss of both complexes in MDBKs resulted in a significant reduction in the production of infectious cell free BHV-1 virions, suggesting the vital roles they play during capsid re-envelopment with endocytosed membrane tubules prior to endosomal recycling mediated cellular egress. We also observed potential capsid retention and aggregation in the nuclei of these cells, indicating that they might also indirectly affect capsid egress from the nucleus. The btCRISPRko.v1 library generated here greatly expanded our capability in BHV-1 related host gene discovery; we hope it will facilitate efforts intended to study interactions between the host and other pathogens in cattle and also basic host cell biology.

microbiology↗

bioRxiv2019

Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

O_LIEnvironmental DNA (eDNA) metabarcoding is largely used to survey aquatic communities, but can also provide data on terrestrial taxa utilising aquatic habitats. However, the entry, dispersal, and detection of terrestrial species DNA within waterbodies is understudied. C_LIO_LIWe evaluated eDNA metabarcoding of pond water for monitoring semi-aquatic, ground-dwelling, and arboreal mammals, and examined spatiotemporal variation in mammal eDNA signals using experiments in captive and wild conditions. C_LIO_LIWe selected nine focal species of conservation and management concern: European water vole, European otter, Eurasian beaver, European hedgehog, European badger, red deer, Eurasian lynx, red squirrel, and European pine marten. We hypothesised that eDNA signals (i.e. proportional read counts) would be stronger for semi-aquatic than terrestrial species, and at sites where mammals exhibited behaviours (e.g. swimming, urination). We tested this by sampling waterbodies in enclosures of captive focal species at specific sites where behaviours had been observed ( directed sampling) and at equidistant intervals along the shoreline ( stratified sampling). We then surveyed natural ponds (N = 6) where focal species were present using stratified water sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-specific primers. C_LIO_LIAll focal species were detected in captivity. eDNA signal strength did not differ between directed and stratified samples across or within species, between species lifestyles (i.e. semi-aquatic, ground-dwelling, arboreal), or according to behaviours. Therefore, eDNA was evenly distributed within artificial waterbodies. Conversely, eDNA was unevenly distributed in natural ponds. eDNA metabarcoding, camera trapping, and field signs detected beaver, red deer, and roe deer. Badger and red fox were recorded with cameras and field signs, but not eDNA metabarcoding. However, eDNA metabarcoding detected small mammals missed by cameras and field signs, e.g. water vole. Terrestrial mammal eDNA signals were weaker and detected in fewer samples than semi-aquatic mammal eDNA signals. C_LIO_LIeDNA metabarcoding has potential for inclusion in mammal monitoring schemes by enabling large-scale, multi-species distribution assessment for priority and difficult to survey species, and could provide early indication of range expansions or contractions. However, eDNA surveys need high spatiotemporal resolution and metabarcoding biases require further investigation before this tool is routinely implemented. C_LI

molecular biology↗

bioRxiv2016

Structural and Functional analysis of the GABARAP Interaction Motif (GIM).

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence (W/F-V-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed eleven-fold more specific for GABARAP than LC3B. Selective mutation of the X1 and X2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of the canonical p62/SQSTM1-LIR into our newly defined GIM, by introducing two valine residues, enhances p62/SQSTM1 interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the continually expanding array of autophagy receptor and adaptor proteins and their in vivo functions.

biochemistry↗