Biology subjects

Leonard, M.

Publications and source records attributed to Leonard, M..

10 recordsLinked to original sources

bioRxiv2026

Decision analysis in support of proactive planning for chronic wasting disease in Vermont, USA

Chronic wasting disease (CWD), a fatal, transmissible disease in white-tailed deer (Odocoileus virginianus) and related species, is spreading across North America but has not yet been detected in Vermont, United States (U.S.). The Vermont Department of Fish and Wildlife, along with partner agencies, wants to develop a proactive prevention and response plan in anticipation of the eventual detection of the disease. Between September 2023 and September 2025, staff from the U.S. Geological Survey and the University of Vermont facilitated a structured decision-making (SDM) process with seven State and Federal agencies that have jurisdiction over some aspect of CWD management in Vermont. The aim of this process was to generate and evaluate alternative response plans against a range of long-term objectives important to the agencies. To aid in the evaluation of the alternatives, we developed a linked set of models for white-tailed deer population and disease dynamics, hunter participation and health, forest health, economic consequences, and agricultural opportunities related to the actions being contemplated as part of the response plan. We evaluated over 256 different permutations of management actions and used multi-criteria decision analysis, a branch of decision analysis designed to help decision makers navigate tradeoffs among competing objectives, to summarize the performance of those alternative strategies against the desired outcomes. Proactive actions, those designed to slow the arrival of CWD to Vermont, were moderately effective, but the most important proactive action was surveillance to detect the disease early after arrival, which triggered response actions after detection. With the insights generated by the SDM process and the results of the analyses, the participating agencies were able to identify a preferred strategy and outline the elements of a proactive response plan. This report describes the SDM process, the technical details of the modeling work, and the results of the analyses.

ecology↗

bioRxiv2026

Verticillium dahliae ubiquitin-specific proteases coordinate key developmental processes and pathogenicity

Ubiquitin is a posttranslational modifier that is conserved among eukaryotes. Ubiquitination alters stability and folding of cellular proteins. Deubiquitinases (DUBs) reverse ubiquitination and often function as part of protein complexes. There are 32 predicted DUB-encoding genes present in the soil-borne phytopathogenic fungus Verticillium dahliae. Nuclear ubiquitin-specific protease 3 (Usp3) is a member of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, whereas Usp1 is predicted to associate with the COP9 signalosome (CSN), which controls specificities of cellular E3 ubiquitin ligase activities. A proteomics approach using biotin capture and identification (BioID) supports that Usp3 regulates gene expression beyond the transcription level. Western experiments showed a dysregulation in ubiquitinated cellular proteins in corresponding deletion strains. Usp3 and Usp1 are both required for fungal development. They regulate microsclerotia formation based on different environmental cues and provide redundant functions in controlling conidiation. Absence of both corresponding genes resulted in significant impairment of conidiospore formation, which is required for fungal propagation within the plant vascular system. This paralysed spreading ability reduced virulence on tomato plants (Solanum lycopersicum). In summary, V. dahliae responds to environmental cues by Usp3- and Usp1-mediated adjustment of gene expression and protein stability. This is important for key developmental processes of the V. dahliae disease cycle and its virulence towards the host plant. Author summaryUbiquitination and deubiquitination of proteins enable cells to rapidly react to environmental cues and adjust protein stabilities and subsequently transcriptomic profiles. Usp3 is a nuclear deubiquitinase subunit of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex. Usp1 is predicted to be associated with the COP9 signalosome that regulates substrate specificities of the ubiquitin-proteasome system. BioID experiments suggest that other SAGA complex subunits, histone proteins, spliceosomal proteins, ribosomal proteins, a protein that tackles transcriptionally stalled RNAPII, and a protein that degrades mRNA with premature stop codons locate proximal to Usp3 within the cell. Deletion of USP3 led to the dysregulation of protein ubiquitination. A single deletion of USP1 did not significantly change ubiquitination profiles, however, a double deletion of USP1/3 significantly affected the ubiquitin-proteasome system. The altered ubiquitination profile correlated with a dysregulation of key developmental processes. Microsclerotia formation was decoupled from environmental cues in the {Delta}USP3 strain, whereas an additional deletion of USP1 reconnected it in a media-dependent manner. USP3 and USP1 contribute to a common governing process in conidiation, and the defect in spreading of the {Delta}USP1/3 strain is reflected by a significant reduction in plant pathogenicity.

microbiology↗

bioRxiv2024

Connectome-driven neural inventory of a complete visual system

Vision provides animals with detailed information about their surroundings, conveying diverse features such as color, form, and movement across the visual scene. Computing these parallel spatial features requires a large and diverse network of neurons, such that in animals as distant as flies and humans, visual regions comprise half the brains volume. These visual brain regions often reveal remarkable structure-function relationships, with neurons organized along spatial maps with shapes that directly relate to their roles in visual processing. To unravel the stunning diversity of a complex visual system, a careful mapping of the neural architecture matched to tools for targeted exploration of that circuitry is essential. Here, we report a new connectome of the right optic lobe from a male Drosophila central nervous system FIB-SEM volume and a comprehensive inventory of the flys visual neurons. We developed a computational framework to quantify the anatomy of visual neurons, establishing a basis for interpreting how their shapes relate to spatial vision. By integrating this analysis with connectivity information, neurotransmitter identity, and expert curation, we classified the [~]53,000 neurons into 727 types, about half of which are systematically described and named for the first time. Finally, we share an extensive collection of split-GAL4 lines matched to our neuron type catalog. Together, this comprehensive set of tools and data unlock new possibilities for systematic investigations of vision in Drosophila, a foundation for a deeper understanding of sensory processing.

neuroscience↗

bioRxiv2023

HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways

HUWE1 is a large, enigmatic HECT domain ubiquitin ligase implicated in the regulation of diverse pathways including DNA repair, apoptosis, and differentiation. How HUWE1 engages its structurally diverse substrates and how HUWE1 activity is regulated are unknown. Using unbiased quantitative proteomics, we identify C16orf72/HAPSTR1 as a dedicated HUWE1 substrate despite HUWE1 targeting substrates in a largely cell type-specific manner. The established physical and genetic interactions between HUWE1 and C16orf72/HAPSTR1 suggest that HAPSTR1 positively regulates HUWE1 function. Here, we show that HAPSTR1 is both a HUWE1 substrate and is required for HUWE1 nuclear localization to facilitate HUWE1 nuclear substrate targeting. HUWE1 or HAPSTR1 loss of function triggers a broad transcriptional stress response. We show that nuclear HUWE1 is both critical for modulating stress signaling pathways, which include p53 and NF-{kappa}B-mediated signaling and required for cell proliferation. Combined, our results define a role for HAPSTR1 in gating critical nuclear HUWE1 functions.

molecular biology↗

bioRxiv2021

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

Since multiple ribosomes can engage a single mRNA, nonuniform ribosome progression can result in collisions. Ribosome collisions during translation elongation elicit a multifaceted ribosome-associated quality control (RQC) response. Despite advanced mechanistic understanding of translation initiation, a parallel RQC pathway that acts on collided preinitiation complexes has not been described. Here, we show that blocking progression of scanning or elongating ribosomes past the start codon triggers uS3 and uS5 ribosomal ubiquitylation. We demonstrate that conditions that activate the integrated stress response can also induce preinitiation complex collisions. The ubiquitin ligase, RNF10, and the deubiquitylating enzyme, USP10, are the key regulators of uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in an autophagy-independent manner. This study identifies a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on pervasive ribosome collisions during translation initiation to modulate translation activity and capacity.

cell biology↗

bioRxiv2020

Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

Ribosomes are highly abundant cellular machines that perform the essential task of translating the genetic code into proteins. Cellular translation activity is finely tuned and proteostasis insults, such as those incurred upon viral infection, activate stress signaling pathways that result in translation reprogramming. Viral infection selectively shuts down host mRNA while redistributing ribosomes for selective translation of viral mRNAs. The intricacies of this selective ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome associated quality control (RQC) factor ZNF598, a sensor for collided ribosomes, as a critical factor for vaccinia virus mRNA translation. Collided ribosomes are sensed by ZNF598, which ubiquitylates 40S subunit proteins uS10 and eS10 and thereby initiates RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or contain a ubiquitylation deficient version of uS10. Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that host translation of vaccinia virus mRNAs is compromised in cells that lack RQC activity as compared to control cells whereas there was little evidence of differences in host or viral transcription. Additionally, vaccinia virus infection resulted in a loss of cellular RQC activity, suggesting that ribosomes engaged in viral protein production recruit ZNF598 away from its function in host translation. Thus, co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

molecular biology↗

bioRxiv2020

V. longisporum elicits media-dependent secretome responses with a further capacity to distinguish between plant-related environments

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteome of the allodiploid Verticillium longisporum was analyzed upon cultivation in different media. Secreted fungal proteins were identified by label free LC-MS/MS screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 223 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 144 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known effectors, metallopeptidases and carbohydrate-active enzymes. The most abundant and uniquely enriched proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the CAZys Gla1, Amy1 and Cbd1. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the NLP2 and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization. Author SummaryVerticillium spp. infect hundreds of different plants world-wide leading to enormous economic losses. Verticillium wilt is a disease of the vasculature. The fungus colonizes the xylem of its host plant where it exploits the vascular system to colonize the whole plant. Therefore, the fungus spends part of its lifetime in this nutrient-low and imbalanced environment where it is inaccessible for disease control treatments. This lifestyle as well requires the fungus to react to plant defense responses by secreting specific effector molecules to establish a successful infection. We addressed the differences in media-dependent secretion responses of Verticillium longisporum. We identified a broad response pattern induced by several media, and a similar response (but with some distinct differences) for the plant-related environments: the pectin-rich medium SXM and xylem sap from the host rapeseed. Importantly, we show that the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3 are xylem sap-specific proteins that are required for full V. dahliae pathogenicity on tomato. These factors play a role during the colonization phase and represent potential targets for new control strategies for Verticillium wilt.

genetics↗

bioRxiv2019

Light activates the translational regulatory GCN2 kinase via reactive oxygen species emanating from the chloroplast

Cytosolic mRNA translation is subject to global and mRNA-specific controls. Phosphorylation of translation initiation factor eIF2 anchors a reversible switch that represses translation globally. The stress-responsive GCN2 kinase is the only known kinase for eIF2 in Arabidopsis. Here we show that conditions that generate reactive oxygen species (ROS) in the chloroplast, such as dark-light transitions, high light, and the herbicide methyl viologen all rapidly activated the GCN2 kinase, whereas mitochondrial and ER stress did not. In addition, GCN2 activation was light dependent and mitigated by photosynthesis inhibitors and ROS quenchers. Accordingly, seedling growth of multiple gcn2 mutant alleles was retarded under conditions of excess light, implicating the GCN2-eIF2 pathway in responses to light and associated ROS. Once activated, the GCN2 kinase preferentially suppressed the ribosome loading of mRNAs for functions such as mitochondrial ATP synthesis, the chloroplast thylakoids, vesicle trafficking, and translation. The transcriptome of gcn2 mutants was sensitized to abiotic stress, including oxidative stress, as well as innate immune responses. Accordingly, gcn2 displayed defects in immune priming by the fungal elicitor, chitin. In conclusion, we provide evidence that reactive oxygen species produced by the photosynthetic apparatus help to activate the highly conserved GCN2 kinase, leading to eIF2 phosphorylation and thus affecting the status of the cytosolic protein synthesis apparatus.

plant biology↗

bioRxiv2018

Multi-Method Molecular Characterisation of Human Dust-Mite-associated Allergic Asthma

Asthma is a chronic inflammatory disorder of the airways. Disease presentation varies greatly in terms of cause, development, severity, and response to medication, and thus the condition has been subdivided into a number of asthma phenotypes. There is still an unmet need for the identification of phenotype-specific markers and accompanying molecular tools that facilitate the classification of asthma phenotype. To this end, we utilised a range of molecular tools to characterise a well-defined group of adults with poorly controlled asthma associated with house dust mite (HDM) allergy, relative to non-asthmatic control subjects. Circulating messenger RNA (mRNA) and microRNA (miRNA) were sequenced and quantified, and a differential expression analysis of the two RNA populations performed to determine how gene expression and regulation varied in the disease state. Further, a number of circulating proteins (IL-4, 5, 10, 17A, Eotaxin, GM-CSF, IFNy, MCP-1, TARC, TNFa, Total IgE, and Endotoxin) were quantified to determine whether the protein profiles differed significantly dependent on disease state. Finally, assessment of the circulating \"blood microbiome\" was performed using 16S rRNA amplification and sequencing. Asthmatic subjects displayed a range of significant alterations to circulating gene expression and regulation, relative to healthy control subjects, that may influence systemic immune activity. Notably, several circulating mRNAs were detected in the plasma in a condition-specific manner, and many more were found to be expressed at altered levels. Proteomic analysis revealed increased levels of inflammatory proteins within the serum, and decreased levels of the bacterial endotoxin protein in the asthma state. Comparison of blood microbiome composition revealed a significant increase in the Firmicutes phylum with asthma that was associated with a concomitant reduction in the Proteobacteria phylum. This study provides a valuable insight into the systemic changes evident in the HDM-associated asthma, identifies a range of molecules that are present in the circulation in a condition-specific manner (with clear biomarker potential), and highlights a range of hypotheses for further study.

molecular biology↗

bioRxiv2018

USP21 and OTUD3 Antagonize Regulatory Ribosomal Ubiquitylation and Ribosome-Associated Quality Control

Defects within mRNAs or nascent chains that halt ribosomal progression can trigger ribosome-associated quality control (RQC) pathways that facilitate mRNA and nascent polypeptide destruction as well as ribosome recycling. Failure to remove defective mRNAs or nascent chains can lead to the accumulation of cytotoxic protein aggregates and proteotoxic stress. We previously established that the E3 ligase ZNF598 catalyzes regulatory ribosomal ubiquitylation of specific 40S ribosomal proteins required for downstream RQC events. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and facilitate ribosomal deubiquitylation following RQC activation. Overexpression of either USP21 or OTUD3 enhances readthrough of stall-inducing sequences as compared to knock-in cells lacking individual RRub sites suggesting that combinatorial ubiquitylation of RPS10 (eS10) and RPS20 (uS10) is required for optimal resolution of RQC events and that deubiquitylating enzymes can limit RQC activation.

molecular biology↗