Biology subjects

Black, M.

Publications and source records attributed to Black, M..

14 recordsLinked to original sources

bioRxiv2025

LICHEN: Light-chain Immunoglobulin sequence generation Conditioned on the Heavy chain and Experimental Needs

In developing therapeutic antibodies, the heavy chain is often prioritised due to its higher variability and its central role in antigen binding. An appropriate pairing of the light sequence is however important for antibody function. Here we present LICHEN, a heavy chain conditioned light sequence generation tool that enables collaborative light sequence design by leveraging computational capabilities alongside experimental expertise. LICHEN generates light sequences which are valid (antibodylike), diverse in sequence and structure, and conditioned on a specific heavy chain. LICHEN can also condition on germline and CDRs and automatically filter generated sequences for required properties. This allows LICHEN to be used across multiple antibody development use cases. We carry out experimental validation of the method conditioning only on the heavy sequence and on the heavy sequence and binding information. Our in vitro results show that sequences created by LICHEN have effective expression yields and can retain antigen-binding.

bioinformatics↗

bioRxiv2025

3D Agent-based modeling without chemical signaling recreatescollective behaviors seen in Myxococcus xanthus colonies

Myxococcus xanthus is a soil-dwelling bacterium that lives in dense populations of millions of cells and displays an array of collective behaviors and macroscopic patterns in response to environmental conditions. The transitions between macroscopic patterns formed by this species are driven by changes in cell motility such as increasing Peclet number via increasing cell speed and reversal period. While cells are capable of passing contact-mediated signals that can trigger reversal events, we set out to study which aspects of M. xanthus collective behavior may be driven by simple individual cell behavior changes in the absence of cell signaling. We do this through 3D agent-based simulations with a bead-spring chain model. We find that when tuned to match single cell properties, this model captures multicellular structures observed at both intermediate and global scales in M. xanthus experiments. Increasing cell speed and reversal period together (as cells do when they starve), maintains a constant overall nematic structure in the population, while significantly increasing the net flow into and out of topological defects, which drives an increased aggregation that leads to fruiting body formation during starvation.

biophysics↗

bioRxiv2025

In Vivo Imaging and Tracking of VRE-Microbiota Interactions via Anaerobic Fluorescent Reporters in Extremely Drug-Resistant Bacteria

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated bloodstream infection and a dominant colonizer of the antibiotic-perturbed gut. Experimental interrogation of VREfm colonization dynamics has been constrained by three major barriers: widespread resistance to conventional selection markers in contemporary clinical isolates, instability of plasmid-based constructs, and failure of oxygen-dependent fluorescent reporters in the anaerobic intestinal environment. To address these limitations, we develop genetic tools optimized for VREfm that enable stable anaerobic fluorescent labeling and longitudinal tracking of colonization dynamics. We show that bile pigment-binding fluorescent proteins, including eUnaG2 and smURFP, generate robust signal under anaerobic conditions across diverse clinical backgrounds. Systematic analysis of resistance profiles revealed that conventional selection markers are frequently incompatible with modern isolates; accordingly, we identify puromycin as a broadly effective entry-point strategy and demonstrate that a puromycin-selectable plasmid enables reporter expression for otherwise genetically intractable extensively drug-resistant (XDR) strains. To support long-term studies without continuous selection, we establish an enhanced pheS** counterselection system for chromosomal integration at neutral loci and engineer a tandem 2xsmURFP construct to optimize single-copy fluorescence. Using these tools, we directly track competition between fluorescently labeled VREfm strains in a murine model via flow cytometric analysis of stool samples, enabling longitudinal strain-level quantification without antibiotic selection or selective plating. This work provides a unified framework for genetic manipulation and anaerobic strain tracking in XDR E. faecium, establishing a practical platform for dissecting ecological processes that govern persistence and competition in the gut. ImportanceVancomycin-resistant Enterococcus faecium (VREfm) is a dangerous and difficult-to-treat hospital pathogen. How VREfm establishes itself in the gastrointestinal tract and competes with other strains remains poorly understood, yet this knowledge is essential for preventing infection. Studying these processes has been impeded by widespread antibiotic resistance and by the failure of conventional fluorescent proteins in the oxygen-free gut environment. We describe tools that overcome these barriers and provide a practical foundation for mechanistic studies of VREfm gut colonization and persistence.

microbiology↗

bioRxiv2024

A COVID-19 Rapid Antigen Test Employing Upconversion Nanoparticles

The COVID-19 pandemic has underscored the critical need for rapid and accurate diagnostic tools. Current methods, including PCR and rapid antigen tests (RAT), have limitations in speed, sensitivity, and the requirement for specialized equipment and trained personnel. Nanotechnology, particularly upconversion nanoparticles (UCNPs), offer a promising alternative due to their unique optical properties. UCNPs can convert low-energy near-infrared (NIR) light into higher-energy visible light, making them ideal for use as optical probes in single molecule detection and point of care applications. This study, initiated in early 2020, explores the opportunity of using highly doped UCNPs (40%Yb3+/4%Er3+) in lateral flow assay (LFA) for the early diagnosis of COVID-19. The UCNPs-based LFA testing demonstrated a minimum detection concentration of 100 pg/mL for SARS-CoV-2 antigen and 105 CCID50/mL for inactivated virus. Clinical trials, conducted in Malaysia and Western Australia independently, showed that the technique was at least 100 times more sensitive than commercial RAT kits, with a sensitivity of 100% and specificity of 91.34%. The development process involved multidisciplinary collaborations, resulting in the Virulizer device, an automated strip reader for point-of-care testing. This work sets a reference for future development of highly sensitive and quantitative rapid antigen tests, aiming for the Limits of Detection (LoD) in the range of sub-ng/mL.

bioengineering↗

bioRxiv2024

Investigating the mode of action for liver toxicity and wasting-like responses produced by high dose exposures to longer chain perfluoroacid substances (PFAS) using high throughput transcriptomics

Single doses of perfluoro-n-decanoic acid (PFDA) cause wasting, a progressive loss of 30 to 50% body weight, increasing liver/body weight ratios, and death within several weeks (Olson and Andersen, 1983). Repeat high doses of perfluorooctane sulfonate (PFOS) produce a subset of these responses in rats and monkeys. The mode of action (MOA) of these wasting-like syndromes is not clear, nor is it understood if these responses are limited to a subset of perfluoroacid substances (PFAS) or a common response to high dose exposure with a larger number of PFAS. To identify pathway perturbations in liver caused by PFAS, we analyzed published in vitro gene expression studies from human primary liver spheroids treated with various PFAS for treatment times up to 14 days (Rowan-Carroll et al., 2021). With treatment times of 10 to 14 days, longer-chain PFAS compounds, specifically PFOS, perfluorodecane sulfonate (PFDS) and higher doses of perfluorooctanoic acid (PFOA), downregulated large numbers of genes in pathways for steroid metabolism, fatty acid metabolism and biological oxidations. Shorter chain PFAS compounds upregulated genes in pathways for fatty acid metabolism. Although PFDA was more toxic and could only be examined at 1-day of treatment, it also downregulated genes for lipid metabolism, steroid metabolism, and biological oxidations. Shorter chain PFAS, both carboxylic and sulfonic acids, did not lead to downregulation of pathways for fatty acid or steroid metabolism. TCDD is also known to cause wasting responses in rodents and humans. In intact rats, high dose responses of longer chain PFAS produce downregulation of batteries of genes associated with fatty acid oxidation and lipogenesis similar to those seen with TCDD. Based on our results, when combined with other literature, we propose that the longer-chain PFAS impair lipogenic pathways through inhibitory interactions between PPAR{beta}, PPAR and PPAR{gamma}.

pharmacology and toxicology↗

bioRxiv2024

Empowering high-throughput high-content analysis of 3D tumor models: Open-source software for automated non-confocal image analysis

PurposeThe primary aim of this study was to develop an open-source Python-based software for the automated analysis of dynamic cell behaviors in three-dimensional tumor models using non-confocal microscopy. This research seeks to address the existing gap in accessible tools for high-throughput analysis of cancer and endothelial cell dynamics in vitro, facilitating the rapid assessment of drug sensitivity. MethodsOur approach involved annotating over 1000 2 mm Z-stacks of cancer and endothelial cell co-culture model and training machine learning models to automatically calculate cell coverage, cancer invasion depth, and microvessel dynamics. Specifically, cell coverage area was computed using focus stacking and Gaussian mixture models to generate thresholded Z-projections. Cancer invasion depth was determined using a deep neural network binary classification model, measuring the distance between Z-planes with invaded cells. Lastly, microvessel dynamics were assessed through a U-Net Xception-style deep learning model for segmentation, a disperse algorithm for network graph representation, then persistent homology to quantify microvessel length and connectivity. Finally, we reanalyzed an image set from a high-throughput drug screen involving a chemotherapy agent on a 3D cervical and endothelial co-culture model. ResultsThe software accurately measured cell coverage, cancer invasion, and microvessel length, yielding drug sensitivity IC50 values with a 95% confidence level compared to manual calculations. Additionally, it significantly reduced the image processing time from weeks down to hours. ConclusionsOur free and open source software offers an automated solution for quantifying 3D cell behavior in tumor models using non-confocal microscopy, providing the broader Cellular and Molecular Bioengineering community with an alternative to standard confocal microscopy paired with proprietary software.

bioengineering↗

bioRxiv2023

Restriction site associated DNA sequencing for tumour mutation burden estimation and mutation signature analysis

Genome-wide measures of genetic disruption such as tumour mutation burden (TMB) and mutation signatures are emerging as useful biomarkers to stratify patients for treatment. Clinicians commonly use cancer gene panels for tumour mutation burden estimation, and whole genome sequencing is the gold standard for mutation signature analysis. However, the accuracy and cost associated with these assays limits their utility at scale. Using in silico library simulations we demonstrate that restriction enzyme associated DNA sequencing (RADseq) may be a cost-effective solution to improve accuracy of TMB estimation and derivation of mutation profiles when compared to a derived FDA approved cancer gene panel TMB score. Using simulated immune checkpoint blockade (ICB) trials, we show that inaccurate tumour mutation burden estimation leads to a reduction in power for deriving an optimal TMB cutoff to stratify patients for immune checkpoint blockade treatment. Additionally, prioritisation of APOBEC hypermutated tumours in these trials optimises TMB cutoff determination for breast cancer. Finally, the utility of RADseq in an experimental setting is also demonstrated, based on characterisation of an APOBEC mutation signature in an APOBEC3A transfected mouse cell line. In conclusion, our work demonstrates that RADseq has the potential to be used as a cost-effective, accurate solution for TMB estimation and mutation signature analysis by both clinicians and basic researchers.

genomics↗

bioRxiv2023

TRIB1 modulates transcriptional programming in breast cancer cells to regulate cell proliferation

The pseudokinase Tribbles Homolog 1 (TRIB1) is a known driver of tumorigenesis in acute myeloid leukemia and is encoded upstream of the oncogene MYC at the 8q24 locus. We observed that TRIB1/MYC co-amplification is associated with decreased relapse-free and overall survival in breast cancer patients, but the role of TRIB1 in this disease has not been well characterized. TRIB1 knockdown in multiple breast cancer cell lines inhibited cell proliferation and suppressed MYC expression, implicating TRIB1 in breast cancer cell proliferation. Transcriptomic and cell cycle analysis revealed cell cycle regulation as the likely mechanism through which TRIB1 influences breast cancer cell proliferation. TRIB1 knockdown also resulted in significant changes in both estrogen receptor (ER) and {beta}-catenin associated transcription. Interrogating the TRIB1 interactome in breast cancer cells by qPLEX-RIME reinforced the known association between TRIB1 and ubiquitination, while revealing a range of previously undescribed TRIB1 associated factors. Further analysis of the association between TRIB1, {beta}-catenin and FERMT2 suggests TRIB1 may regulate {beta}-catenin activity by controlling the levels of both {beta}-catenin, and its co-factor FERMT2. Together, these results suggest that coregulation of {beta}-catenin and ER-driven transcription by TRIB1, facilitates regulation of MYC expression and breast cancer cell proliferation. SignificanceThe pseudokinase TRIB1 is frequently co-amplified in breast cancers with the potent oncogene MYC, although the functional consequences of this event are not well understood. This study demonstrates TRIB1 is a regulator of cell cycle progression and MYC expression in breast cancer cells. It also profiles TRIB1-associated proteins in breast cancer cells, demonstrating conservation of TRIB1s canonical interaction with COP1 and reveals associations with members of the wider ubiquitination machinery, a range of transcriptional regulators and chromatin remodelers. The data presented provide insight into the function of TRIB1 in breast cancer and the role of TRIB1 in transcriptional regulation.

cancer biology↗

bioRxiv2023

The fate of intracoelomic acoustic transmitters in Atlantic Salmon (Salmo salar) post-smolts and wider considerations for casual factors driving tag retention and mortality in fishes

Acoustic telemetry is a widely used method in assaying behavioural dynamics in fishes. Telemetry tags are often surgically implanted in the coelom of the animal and are assumed to have minimal rates of post-release mortality and tag shedding. However, fish are capable expelling tags and mortalities do occur following release, with the mechanism(s) underlying these effects not well understood. The purpose of this research was to address causal factors underlying tagging mortality and tag expulsion in fishes. We conducted an empirical assessment of tag retention and post-surgical mortality rates in post-smolt Atlantic salmon (Salmo salar) fitted with a dummy acoustic tag over a 92 day monitoring period. This was complimented with a meta-analysis of factors affecting tag retention and post-surgical mortality rates in the wider literature. Post-smolt salmon exhibited low rates of mortality following tag implantation ([≤] 5.1%) but had high rates of tag expulsion (54.8%) and impaired growth and a foreign body response was evident. The meta-analysis showed that mortality was generally low across all studies (12.4%) and was largely unaffected by model cofactors. Tag retention rates were high among the studies investigated here (86.7%) and had a weak negative relationship with tag:body mass ratios. Our results suggest that while mortality is often low among tagging studies, including this one, caution must be exercised in assessing stationary tags as they may represent an expelled tag rather than a mortality event. Our results also indicate that tag dimensions are not nearly important as the tag:body mass ratio.

physiology↗

bioRxiv2022

RBFOX2 deregulation promotes pancreatic cancer metastasis through alternative splicing

RNA splicing is an important biological process associated with cancer initiation and progression, yet in pancreatic cancer the role and regulation of splicing is not well understood. From a forward genetic screen in a mouse model of pancreatic cancer, we identified an enrichment of RNA binding proteins (RBPs) associated with the spliceosome. Here, we link deregulation of RBFOX2, an RBP of the FOX family, to pancreatic cancer progression and liver metastasis. We show that RBFOX2 regulation in pancreatic cancer occurs at both the RNA and protein level, and that nuclear localization of RBFOX2 is significantly reduced in poorly differentiated PDAC. Deregulation of RBFOX2 in PDAC is associated with an enrichment of exon exclusion events in transcripts encoding proteins involved in cytoskeletal remodeling and invadopodia programs that potentiate metastatic potential in vivo. Using splice-switching antisense oligonucleotides (AONs) and inducible cDNA isoforms, we demonstrate that RBFOX2 mediated exon exclusion in ABI1 controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells, and that ABI1 splice-switching enhances cellular phenotypes associated with cancer cell stemness. Together, our data identify a novel role for RBFOX2 deregulation in promoting PDAC progression through alternative splicing regulation.

cancer biology↗

bioRxiv2021

Structural and mechanistic basis for protein glutamylation by the kinase fold

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

biochemistry↗

bioRxiv2019

Modeling adaptive therapy in non-muscle invasive bladder cancer

Bladder cancer is the 9th most commonly diagnosed cancer. Nearly half of patients with early stage bladder cancer treated with the immune-stimulating agent BCG have disease recurrence, while 13% progress to invasive bladder cancer. Here we explored the potential of tumor mutational heterogeneity and the role of pro- and anti-inflammatory cytokines to identify different subtypes of bladder cancer that may predict therapeutic response to BCG. Further, we used mathematical modeling of dosing strategies to infer tumor response to varying doses and time schedules f BCG administration. As a proof-of-concept, present adaptive therapy scheduling of BCG as a viable strategy to control tumor size and minimize recurrence.

cancer biology↗

bioRxiv2018

Accurate Reference-Free Somatic Variant-Calling by Integrating Genomic, Sequencing and Population Data

The detection of somatic single nucleotide variants (SNVs) is critical in both research and clinical applications. Studies of human cancer typically use matched normal (reference) samples from a distant tissue to increase SNV prediction accuracy. This process both doubles sequencing costs and poses challenges when reference samples are not readily available, such as for many cell-lines. To address these challenges, we created S22S: an approach for the prediction of somatic mutations without need for matched reference tissue. S22S takes underlying sequence data, augments them with genomic background context and population frequency information, and classifies SNVs as somatic or non-somatic. We validated S22S using primary tumor/normal pairs from four tumor types, spanning two different sequencing technologies. S22S robustly identifies somatic SNVs, with the area under the precision recall curve reaching 0.97 in kidney clear cell carcinoma, comparable to the best tumor/normal analysis pipelines. S22S is freely available at http://labs.oicr.on.ca/Boutros-lab/software/s22s.

bioinformatics↗

bioRxiv2017

Recurrent loss of heterozygosity correlates with clinical outcome in pancreatic neuroendocrine cancer

Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Analysis of gene mutation, copy number and RNA expression of 57 sporadic pNETs showed that pNET genomes are dominated by aneuploidy. Remarkably, ~25% of pNETs had genomes characterized by recurrent loss of heterozygosity (LoH) of the same 10 chromosomes, accompanied by bi-allelic MEN1 inactivation, and these cases had generally poor clinical outcome. Another ~25% of all pNETs had chromosome 11 LoH and bi-allelic MEN1 inactivation, lacking the recurrent LoH pattern - these had universally good clinical outcome. Some level of aneuploidy was common, and overall ~80% of pNETs had LoH of [≥]1 chromosome. This aneuploidy led to changes in RNA expression at the level of whole chromosomes and allowed pathogenic germline variants (e.g. ATM) to be expressed unopposed, inactivating downstream tumor suppressor pathways. Some pNETs appear to utilize VHL gene methylation or mutation to activate pseudo-hypoxia. Contrary to expectation neither tumor morphology within well-differentiated pNETs nor single gene mutation had significant associations with clinical outcome, nor did expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. MEN1 was the only statistically significant recurrently mutated driver gene in pNETs. Only one pNET had clearly oncogenic and actionable SNVs (in PTEN and FLCN) confirmed by corroborating RNA expression changes. The two distinct patterns of aneuploidy described here, associated with markedly poor and good clinical outcome respectively, define a novel oncogenic mechanism and the first route to genomic precision oncology for this tumor type.

cancer biology↗