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Harding, S.

Publications and source records attributed to Harding, S..

12 recordsLinked to original sources

MicroNucML: A machine learning approach for micronuclei segmentation and the refinement of nuclei-micronuclei relationships

Micronuclei (MN) are structures containing small fragments of DNA, arising from mitotic errors or failed DNA repair attempts. Therefore, MN serve as markers of genomic instability and are typically quantified either manually or through threshold-based methods, which can be tedious and inaccurate, leading to varying degrees of success and throughput. By employing a two-phase labeling approach that utilizes polygon and brush segmentation, along with refinement using SAM2, we developed a high-quality MN segmentation tool. Subsequent data augmentation, which captured heterogeneity in image quality and color diversity, enabled us to train a generalizable Mask-RCNN model optimized for small object detection, achieving state-of-the-art performance in MN detection. Finally, we applied our model to immunofluorescence data obtained from cell lines exposed to DNA damage conditions to gain biological insights into MN dynamics and their role in inducing genome instability. In summary, this work establishes an accessible resource for systematically studying genome instability with significantly greater fidelity and sensitivity, enabling insights into damage biology that were previously unresolved.

bioinformatics↗

Why do some predicted protein structures fold poorly? Benchmarking AlphaFold, ESMFold, and Boltz in maize

Protein structure prediction tools have significantly reduced the time and cost to generate protein structures and accelerated protein discovery and design. However, plant proteins are underrepresented in sequence and structural datasets used to train these programs. To quantify the downstream impact of this deficiency, we benchmarked five structure-prediction programs (AlphaFold 2, AlphaFold 3, ESMFold, Boltz-1, and Boltz-2) across 417 well-characterized Zea mays genes. These "classical" genes represent a set of well-studied genes with known genetic and phenotypic effects. We generated structures for each gene using these programs and compared how sequence, structural, and evolutionary conservation impacted the structures confidence and geometric features. Proteins lacking conserved sequence and/or structural domains had on average 25% to 43% lower confidence scores than proteins having both domains. Proteome-wide phylostratigraphy revealed that species-specific proteins had substantially lower confidence scores than proteins conserved amongst angiosperms and Eukaryotes. Boltz-1 and ESMFold structures had the highest occurrence of structures with severe geometry issues, including overlapping atoms and unlikely bond angles. We also compared computational and experimental alignments of Arabidopsis, maize, rice, wheat, and soybean proteins from the Protein Data Bank and identified structures showing incongruences with experimental data. This study challenges the assertion that protein folding has been completely solved, and urges more investigation into benchmarking and standardized evaluation frameworks to improve model performance and assessment in agricultural crops.

bioinformatics↗

CRISPR knockout of winter-biased SUT4 alters phenology, biomass accrual, and fertility of field-grown hybrid poplar

Climate uncertainty is intensifying the need for greater plasticity in carbohydrate reserve utilization to support winter survival and spring growth in woody perennials. In poplar, the single-copy SUT4, which encodes a tonoplast-localized sucrose transporter, and the SUT5/SUT6 genome duplicates, which encode plasma membrane-localized transporters, are expressed year-round, with SUT4 showing highest expression during cool seasons. Given its role in vacuolar sucrose efflux and winter-predominant expression, SUT4 may play a key role in modulating seasonal carbohydrate dynamics. While SUT4-knockdown and knockout effects have been studied under greenhouse conditions, their impact under field conditions remains unexplored. Here, we report a field-based study comparing CRISPR knockout mutants of winter-expressed SUT4 and SUT5/SUT6 in Populus tremula x alba. We show that sut4, but not sut56, mutants exhibited earlier autumn leaf senescence, delayed spring bud flush, reduced stem growth, and altered sugar partitioning in winter xylem and bark relative to controls. After two years in the field, all genotypes flowered before leaf flush in early spring; however, sut4 mutants produced sterile ovules despite developing normal-looking catkins. Metabolic profiling revealed disrupted sucrose and raffinose dynamics in elongating sut4 catkins, accompanied by transcriptomic signatures of elevated stress and downregulation of proanthocyanidin biosynthesis and circadian clock genes. These findings highlight the critical role of SUT4 in coordinating sugar allocation, stress responses, and seasonal development in poplar. Significance statementThis field study demonstrates that loss of SUT4, the most highly expressed sucrose transporter during cool seasons, disrupts phenology, growth, and fertility in poplar. Altered sugar and raffinose dynamics and transcriptomic signatures of stress and circadian clock gene dysregulation in the mutants underscore SUT4s role in coordinating sugar allocation and seasonal developmental transitions under natural environmental conditions.

plant biology↗

Equity in Action: A Four-Year journey towards Gender Parity and Racial Diversity in Biochemistry Hiring

Recruitment of faculty members in academic departments shapes the department for decades in both research and teaching arenas. Having a diverse department is beneficial for undergraduate and graduate students as representation of underrepresented minority groups in the professoriate can inspire a greater diversity of students to pursue higher levels of education or research-focused careers. Increased diversity benefits research directly as diverse teams have been shown to have better ideas and outcomes. In 2020, our department had lower gender diversity than would be expected based on the pool of PhD students and post-doctoral fellows in Canada. Therefore, we altered our hiring process, primarily by redacting applications, for recruitment into entry-level tenure-track faculty positions. With this change in process, female hires increased from 17% in the previous ten years (5 hires) to 80% in the subsequent four years (5 hires) with no substantial change in hiring of racially diverse individuals (50% to 40%). Overall, combined with retirements, the percentage of female faculty in the department went from 25% to 50% and the percentage of racialized faculty went from 38% to 44%. The new hires have met or exceeded expectations of success with respect to grant funding and are on track to meet or exceed expectations for other metrics of success. Thus, our intervention was very successful in increasing the diversity of our department within a short timeframe. We believe that our experience could provide other departments with a template for making substantive change, even in the absence of internal expertise in the area. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/617211v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@164c310org.highwire.dtl.DTLVardef@42585borg.highwire.dtl.DTLVardef@1b6f6eaorg.highwire.dtl.DTLVardef@10ca067_HPS_FORMAT_FIGEXP M_FIG C_FIG

scientific communication and education↗

Application of RFdiffusion to predict interspecies protein-protein interactions between fungal pathogens and cereal crops

Plant pathogenic fungi secrete small proteins known as effectors which help overcome the plant defense response and cause disease. The concept of effector-triggered immunity in plants evolved from the "gene for gene hypothesis" which describes plant resistance or susceptibility to plant pathogens based on interspecies protein-protein interactions (PPIs) between plant-derived resistance (R) genes and pathogen-derived avirulence (Avr) effector genes. Understanding the molecular interactions mediating these host-pathogen interactions in effector-triggered immunity is thus essential to managing fungal disease. In silico methods of predicting interspecies PPIs have been heavily studied to identify target genes for crop resistance. But conventional sequence-based homology methods (i.e., interlog, domain-based inference) for predicting interspecies PPIs are not as powerful as methods that also incorporate structural homology. The objective of this study was to develop a computational workflow to predict PPIs between pathogenic fungi and their cereal hosts by leveraging recent advances in artificial intelligence and structural biology. This workflow proposes the use of a generative model, RFdiffusion, to predict the structure of truncated segments of proteins likely to bind to query effector proteins. The binder structures were filtered based on the number of contacts at the effectors known binding residues. Acceptable structures were then input into FoldSeek to search the host proteome for host proteins containing similar sub-structures. Experimentally-validated PPIs between rice (Oryzae sativa cv. Japonica) and rice blast fungus (Magnaporthe oryzae) were used for workflow validation. The effects of binder length and the binding residues mode of action (i.e., residues at active/substrate recognition sites) on the binder quality and presumptive host protein matches were explored. Ultimately, 11 out of 14 experimentally validated PPIs were recovered computationally, indicating a high recall (>78%) for the workflow. The shorter binders recovered most of the PPIs, but may have produced the most false positives, as functional analyses revealed that these host proteins displayed a wide variety of functions. These findings emphasize that subject matter expertise is still required to decipher the prediction results. Yet, this framework for elucidating interactions between fungal pathogens and host proteins could provide valuable insight into mechanisms of susceptibility or resistance at a scale friendly to limited computational resources, and facilitate the development of control strategies that reduce crop diseases.

plant biology↗

Fusarium Protein Toolkit: AI-powered tools to combat fungal threats to agriculture

BackgroundThe fungal genus Fusarium poses significant threats to food security and safety worldwide because it consists of numerous species that cause destructive diseases in crops, as well as mycotoxin contamination. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets to control crop diseases and mycotoxin contamination incited by Fusarium. DescriptionIn response to these challenges, we developed the Fusarium Protein Toolkit (FPT, https://fusarium.maizegdb.org/), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. FPT offers a comprehensive approach to understanding and mitigating the detrimental effects of Fusarium on agriculture. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum, which causes Fusarium head blight and trichothecene mycotoxin contamination of cereals, and F. verticillioides, which causes ear rot and fumonisin mycotoxin contamination of maize. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome constructed from 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework. ConclusionFPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium, the most agriculturally important group of mycotoxin-producing plant pathogens. FPT will deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets that can be used to develop control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats affected by climate change. By providing a novel approach to interrogate Fusarium-induced crop diseases, FPT is a crucial step toward safeguarding food security and safety worldwide.

bioinformatics↗

Phage-induced efflux down-regulation boosts antibiotic efficacy

The interactions between a virus and its host vary in space and time and are affected by the presence of molecules that alter the physiology of either the host or the virus. Determining the molecular mechanisms underpinning these interactions is paramount for predicting the fate of bacterial and phage populations and for designing rational phage-antibiotic therapies. We study the interactions between stationary phase Burkholderia thailandensis and the phage {Phi}Bp-AMP1. Although heterogeneous genetic resistance to phage rapidly emerges in B. thailandensis, the presence of phage enhances the efficacy of three major antibiotic classes, the quinolones, the beta-lactams and the tetracyclines, but antagonizes tetrahydrofolate synthesis inhibitors. We discovered that enhanced antibiotic efficacy is underpinned by reduced antibiotic efflux in the presence of phage. This new phage-antibiotic therapy allows for eradication of stationary phase bacteria, whilst requiring reduced antibiotic concentrations, which is crucial for treating infections in sites where it is difficult to achieve high antibiotic concentrations.

microbiology↗

High-quality complete genome resource of plant pathogenic bacterium Pectobacterium atrosepticum strain Green1 isolated from potato (Solanum tuberosum L.) in Greenland

Pectobacterium atrosepticum is a narrow host range pectinolytic plant pathogenic bacterium causing blackleg of potato (Solanum tuberosum L.) worldwide. Till present, several P. atrosepticum genomes have been sequenced and characterized in detail; all of these genomes have come, however, from P. atrosepticum strains isolates from plants grown in temperate zones, not from hosts cultivated under different climatic conditions. Herewith, we present the first complete, high-quality genome of the P. atrosepticum strain Green1 isolated from potato plants grown under subarctic climate in Greenland. The genome of P. atrosepticum strain Green1 consists of one chromosome of 4,959,719 bp., with a GC content of 51% and no plasmids. The genome contains 4531 annotated features, including 4179 protein-coding genes (CDSs), 22 rRNA genes, 70 tRNA genes, 8 ncRNA genes, 2 CRISPRs and 126 pseudogenes. We believe that the information of this first, high-quality, complete, closed genome of P. atrosepticum strain isolated from host plant grown in subarctic agricultural region will provide resources for comparative genomic studies and for analyses targeting climatic adaptation and ecological fitness mechanisms present in P. atrosepticum.

microbiology↗

Drug screening to identify compounds to act as co-therapies for the treatment of pathogenic Burkholderia

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics, and is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to most currently available antibiotics. The most efficacious treatment for melioidosis requires at least two weeks of intravenous treatment with ceftazidime or meropenem. This places a large treatment burden on the predominantly middle income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z factor of 0.68. We screened a library of 61,250 compounds, and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six \"best in class\" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, preliminary data also demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy against these bacteria.

microbiology↗

Nocturnal expression of Crassulacean acid metabolism (CAM) genes predates the origin of CAM in the genus Yucca.

HighlightAlthough large differences in metabolism exist between C3 and CAM species, we find that many CAM genes have shared expression patterns regardless of photosynthetic pathway, suggesting ancestral propensity for CAM.\n\nAbstractCrassulacean acid metabolism (CAM) is a carbon-concentrating mechanism that has evolved numerous times across flowering plants and is thought to be an adaptation to water limited environments. CAM has been investigated from physiological and biochemical perspectives, but little is known about how plants evolve from C3 to CAM at the genetic or metabolic level. Here we take a comparative approach in analyzing time-course data of C3, CAM, and C3+CAM intermediate Yucca (Asparagaceae) species. RNA samples were collected over a 24-hour period from both well-watered and drought-stressed plants and were clustered based on time-dependent expression patterns. Metabolomic data reveals differences in carbohydrate metabolism and antioxidant response between the CAM and C3 species, suggesting changes to metabolic pathways are important for CAM evolution and function. However, all three species share expression profiles of canonical CAM pathway genes, regardless of photosynthetic pathway. Despite differences in transcript and metabolite profiles between the C3 and CAM species, shared time-structured expression of CAM genes in both CAM and C3 Yucca species suggests ancestral expression patterns required for CAM may have predated its origin in Yucca.

plant biology↗

A role for phylloquinone biosynthesis in the plasma membrane as revealed in a non-photosynthetic parasitic plant

Phylloquinone is a lipophilic naphthoquinone found predominantly in chloroplasts and best known for its function in photosystem I electron transport and disulfide bridge formation of photosystem II subunits. Phylloquinone has also been detected in plasma membrane preparations of heterotrophic tissues with potential transmembrane redox function, but the molecular basis for this noncanonical pathway is unknown. Here we provide evidence of plasma membrane phylloquinone biosynthesis in a nonphotosynthetic holoparasite Phelipanche aegyptiaca. A nonphotosynthetic and nonplastidial role for phylloquinone is supported by transcription of phylloquinone biosynthetic genes during seed germination and haustorium development, by plasma membrane-localization of alternative terminal enzymes, and by detection of phylloquinone in germinated seeds. Comparative gene network analysis with photosynthetically competent parasites revealed a bias of Phelipanche phylloquinone genes toward coexpression with oxidoreductases involved in plasma membrane electron transport. Genes encoding the plasma membrane phylloquinone pathway are also present in several photoautotrophic taxa of Asterids, suggesting an ancient origin of multifunctionality. Our findings suggest that nonphotosynthetic holoparasites exploit alternative targeting of phylloquinone for transmembrane redox signaling associated with parasitism.

plant biology↗

DNA damage in 3D constricted migration or after lamin-A depletion in 2D: shared mechanisms of repair factor mis-localization under nuclear stress

Cells that migrate through small, rigid pores and that have normal levels of the nuclear structure protein lamin-A exhibit an increase in DNA damage, which is also observed with lamin-A depletion in diseases such as cancer and with many lamin-A mutations. Here we show nuclear envelope rupture is a shared feature that increases in standard culture after lamin-A knockdown, which causes nuclear loss of multiple DNA repair factors and increased DNA damage. Some repair factors are merely mis-localized to cytoplasm whereas others are partially depleted unless rescued by lamin-A expression. Compared to standard cultures on rigid glass coverslips, the growth of lamin-A low cells on soft matrix relaxes cytoskeletal stress on the nucleus, suppresses the mis-localization of DNA repair factors, and minimizes DNA damage nearly to wildtype levels. Conversely, constricted migration of the lamin-A low cells causes abnormally high levels of DNA damage, consistent with sustained loss of repair factors. The findings add insight into why monogenic progeroid syndromes that often associate with increased DNA damage and predominantly impact cells in stiff tissues result from mutations only in lamin-A or DNA repair factors.

cell biology↗