Biology subjects

Cannon, C.

Publications and source records attributed to Cannon, C..

8 recordsLinked to original sources

bioRxiv2026

Cas1 epistasis tunes conformational coupling to enhance CRISPR adaptation

CRISPR adaptation requires Cas1-Cas2 to capture prespacers, undergo conformational rearrangement and catalyse integration, but how these steps are coupled remains unclear. Using nine hyperactive Escherichia coli Cas1 substitutions as perturbational probes, we identified a prespacer-coupling module intersecting an interior conformational-coupling module. Single substitutions increased adaptation up to sixfold, whereas combinatorial reassortment generated a genotype with 103-fold greater activity than wild type. Genotype-network analysis and quantitative reconstruction revealed strong background-dependent epistasis: the same substitution could enhance activity in one genotype but impair it in another, and high activity emerged only from compatible combinations spanning both modules. These findings indicate that Cas1-Cas2 activity is constrained by compatibility among changes distributed across the Cas1 dimer, rather than by optimization of individual catalytic or DNA-binding interactions. We propose that coordinated tuning of prespacer engagement and conformational coupling governs Cas1-Cas2 activity during CRISPR adaptation.

biochemistry↗

bioRxiv2026

RecA status determines SOS- and RecBCD-dependent outcomes in CRISPR-Cas adaptation

CRISPR-Cas immunity depends on integrating DNA fragments from invading elements, yet how this process is tuned by host physiology remains poorly understood. Here, we investigate the relationship between CRISPR adaptation and the bacterial SOS DNA damage response in Escherichia coli using a highly sensitive colony-based adaptation assay. Blocking SOS with a lexA3 allele suppresses adaptation, while paradoxically, deleting recA enhances it. These findings indicate that RecA contributes positively to adaptation indirectly, through LexA cleavage and induction of SOS-regulated functions, while complete loss of RecA is associated with a RecBCD-dependent DNA-processing state that favours successful adaptation. The RecA inhibitors RecX and PsiB did not phenocopy {triangleup}recA, indicating that inhibitor-mediated perturbation of RecA in RecA-proficient cells and complete loss of RecA have different consequences for adaptation. Genome-wide maps of recovered spacers show that RecA and LexA reshape the relative distribution of chromosomal and plasmid-derived spacers, linking CRISPR adaptation to replication and DNA repair dynamics. Although Cas1-Cas2 catalyses spacer integration autonomously in vitro, our findings suggest that successful adaptation in vivo emerges from interactions between spacer acquisition, DNA repair, and bacterial stress physiology, embedding CRISPR adaptation within cellular networks that balance immune protection with the risk of autoimmunity.

microbiology↗

bioRxiv2025

Procrustean pseudo-landmark methods in Python to measure massive quantities of leaf shape data

PremiseWhen examining leaf shapes that are different from one another, it can be difficult to compare both the overall leaf shape and points along the leaf margin in biologically and statistically meaningful ways. MethodTo address this problem, we present a simple and user-friendly leaf shape analysis in Jupyter Notebook and Python that uses pseudo-landmarks and Generalized Procrustes Analysis to measure and compare the shape of any leaf. To demonstrate our analysis, we created a repository of real leaves gathered from eight experimental datasets. ResultsUsing our leaf repository, we explain how we can use pseudo-landmarks to compare all leaf shapes both within and between species using dimension reduction techniques like Principal Component Analysis and can predict leaf shapes using pseudo-landmarks through Linear Discriminant Analysis. Our leaf shape analysis also maps differences in shape as leaves grew around a rosette, showing the transition of shape across development (phyllotaxy). Finally, we showed how we can investigate the relationship between leaf shape variation and genetic diversity by combining shape with genetic data. DiscussionThrough the use of Generalized Procrustes Analysis and pseudo-landmarks, our leaf shape analysis presents a powerful tool for examining the shape of any leaf across multiple biological, ecological, evolutionary, and developmental scales.

plant biology↗

bioRxiv2025

Cancer associated fibroblasts drive epithelial to mesenchymal transition and classical to basal change in pancreatic ductal adenocarcinoma cells with loss of IL-8 expression

Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, in part resulting from cellular heterogeneity that supports overall tumorigenicity. Cancer associated fibroblasts (CAF) are key determinants of PDAC biology and response to systemic therapy. While CAF subtypes have been defined, the effects of patient-specific CAF heterogeneity and plasticity on tumor cell behavior remain unclear. Here, multi-omics was used to characterize the tumor microenvironment (TME) in tumors from patients undergoing curative-intent surgery for PDAC. In these same patients, matched tumor organoid and CAF lines were established to functionally validate the impact of CAFs on the tumor cells. CAFs were found to drive epithelial-mesenchymal transition (EMT) and a switch in tumor cell classificiaton from classical to basal subtype. Furthermore, we identified CAF-specific interleukin 8 (IL-8) as an important modulator of tumor cell subtype. Finally, we defined neighborhood relationships between tumor cell and T cell subsets. Statement of SignificanceThis multidimensional analysis highlights the diverse role CAFs have in influencing other cell types in the TME, including epithelial-derived tumor and infiltrating immune cells. Our methods provide a platform for evaluating emerging therapeutic approaches and for studying mechanisms that dictate tumor behavior in a manner that reflects patient-specific heterogeneity.

cancer biology↗

bioRxiv2024

Caspase-11 drives macrophage hyperinflammation in models of Polg-related mitochondrial disease

Mitochondrial diseases (MtD) represent a significant public health challenge due to their heterogenous clinical presentation, often severe and progressive symptoms, and lack of effective therapies. Environmental exposures, such bacterial and viral infection, can further compromise mitochondrial function and exacerbate the progression of MtD. Infections in MtD patients more frequently progress to sepsis, pneumonia, and other detrimental inflammatory endpoints. However, the underlying immune alterations that enhance immunopathology in MtD remain unclear, constituting a key gap in knowledge that complicates treatment and increases mortality in this vulnerable population. Here we employ in vitro and in vivo approaches to clarify the molecular and cellular basis for innate immune hyperactivity in models of polymerase gamma (Polg)-related MtD. We reveal that type I interferon (IFN-I)-mediated upregulation of caspase-11 and guanylate-binding proteins (GBPs) increase macrophage sensing of the opportunistic microbe Pseudomonas aeruginosa (PA) in Polg mutant mice. Furthermore, we show that excessive cytokine secretion and activation of pyroptotic cell death pathways contribute to lung inflammation and morbidity after infection with PA. Our work sheds new light on innate immune dysregulation in MtD and reveals potential targets for limiting infection- and inflammation-related complications in Polg-related MtD.

immunology↗

bioRxiv2023

Assembly and analysis of the genome of Notholithocarpus densiflorus

Tanoak (Notholithocarpus densiflorus) is an evergreen tree in the Fagaceae family found in California and southern Oregon. Historically, tanoak acorns were an important food source for Native American tribes and the bark was used extensively in the leather tanning process. Long considered a disjunct relictual element of the Asian stone oaks (Lithocarpus spp.), phylogenetic analysis has determined that the tanoak is an example of convergent evolution. Tanoaks are deeply divergent from oaks (Quercus) of the Pacific Northwest and comprise a new genus with a single species. These trees are highly susceptible to sudden oak death (SOD), a plant pathogen (Phytophthora ramorum) that has caused widespread mortality of tanoaks. Here, we set out to assemble the genome and perform comparative studies among a number of individuals that demonstrated varying levels of susceptibility to SOD. First, we sequenced and de novo assembled a draft reference genome of N. densiflorus using co-barcoded library processing methods and an MGI DNBSEQ-G400 sequencer. To increase the contiguity of the final assembly, we also sequenced Oxford Nanopore (ONT) long reads to 30X coverage. To our knowledge, the draft genome reported here is one of the more contiguous and complete genomes of a tree species published until now, with a contig N50 of [~]1.2 Mb and a scaffold N50 of [~]2.1 Mb. In addition, we sequenced 11 genetically distinct individuals and mapped these onto the draft reference genome enabling the discovery of almost 25 million single nucleotide polymorphisms and [~]4.4 million small insertions and deletions. Finally, using co-barcoded data we were able to generate complete haplotype coverage of all 11 genomes.

genomics↗

bioRxiv2021

Host protease activity on bacterial pathogens promotes complement- and antibiotic-directed killing

Our understanding of how the host immune system thwarts bacterial evasive mechanisms remains incomplete. Here, we show that host protease neutrophil elastase acts on Acinetobacter baumannii and Pseudomonas aeruginosa to destroy factors that prevent serum-associated, complement-directed killing. The protease activity also enhances bacterial susceptibility to antibiotics in sera. These findings implicate a new paradigm where host protease activity on bacteria acts synergistically with the host complement system and antibiotics to defeat bacterial pathogens.

microbiology↗

bioRxiv2020

HIV Diversity Considerations in the Application of the Intact Proviral DNA Assay (IPDA)

Opening Paragraph (serves as abstract for submission) and BodyThe Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method for intact HIV reservoir quantification1. This duplexed droplet digital PCR (ddPCR) assay simultaneously targets the HIV Packaging Signal ({Psi}) and the Rev Responsive Element (RRE) within Envelope (env) to distinguish genomically intact proviruses against a large background of defective ones2. The IPDA requires less time, resources, and biological material than the gold standard for replication-competent HIV reservoir measurement, the Quantitative Viral Outgrowth Assay (QVOA)3, and is being adopted in research and clinical studies4-7. In our cohort of HIV-1 subtype B-infected individuals from North America however, the IPDA yielded a 28% failure rate due to HIV polymorphism. We further demonstrate that within-host HIV diversity can lead the IPDA to underestimate intact HIV reservoir size, which could negatively impact clinical trial results interpretation. While the IPDA represents an important methodological advance, HIV diversity should be addressed before its widespread adoption.

genetics↗