Biology subjects

Walsh, J.

Publications and source records attributed to Walsh, J..

17 recordsLinked to original sources

bioRxiv2026

Broad-spectrum polerovirus resistance conferred by a potato TIR-NLR immune receptor

Potato leafroll virus (PLRV) is an economically important viral disease of potato (S. tuberosum). Genetic resistance to this phloem-limited virus is rare, and no cloned resistance (R) genes have been reported. Rladg confers resistance to PLRV in an Andean potato landrace, LOP-868 (Velasquez et al. 2007). We identified the functional Rladg gene as a homolog of the tomato TIR-NLR-encoding Bs4. Rladg interacts with the serine protease domain of the PLRV protein P1, which is essential for virus replication. This recognition is independent of the proteases enzymatic activity, and the Rladg immune receptor oligomerizes upon direct association with the protease. Like PLRV, many poleroviruses contain a serine protease. Despite their diverse amino acid sequences, these proteases are predicted to share similar structures. Rladg recognizes all ten tested polerovirus proteases, suggesting a conserved structural recognition mechanism. We propose that Rladgs broad recognition capacity could enable resistance to poleroviruses in many crop species. Rladg is the first R-gene reported to confer resistance to a phloem-limited pathogen and could provide enhanced resistance to many economically important poleroviruses.

plant biology↗

bioRxiv2026

Optimizing 3D-printed Scaffold Geometry Decreases Foreign Body Response and Enhances Allogeneic Islet Transplant Outcomes

Cellular therapy, such as beta cell transplantation for Type 1 diabetes, is a promising approach to durably alleviate disease states. Implanting cells within porous scaffolds is beneficial as they distribute the cells and mechanically support implantation; however, scaffolds can exacerbate foreign body responses (FBR). While the geometric features of a scaffold are known to impact FBR, there is limited consensus on what makes an ideal implant. Some have explored the role of pore size and interconnectivity; however, the impact of rung thickness between pores on FBR is broadly understudied. To investigate this parameter, we created a scaffold with reproducible geometric features and high biostability by combining 3D-printing with the polymer polydimethylsiloxane (PDMS). We tested 3D-printed scaffold prototypes with identical pore sizes but distinct PDMS rung thicknesses ranging from 150 to 300 {micro}m. Upon transplantation, biocompatibility screening in a mouse model revealed that scaffolds with thicker PDMS rungs led to increased intra-device fibrosis. Additional spatio-proteomic analysis revealed distinct differences in host responses to rung changes, with alterations in macrophage and adaptive immune cell markers, as well as fibrotic proteins, within scaffolds containing thicker rungs. Selecting the optimized rung size, we evaluated its efficacy in rat syngeneic and allogeneic islet transplant models. In the allogeneic model, 3D-printed scaffold islet implants demonstrated robust efficacy and stability, yielding improved outcomes compared to PDMS scaffolds without optimized geometric features. Results from this study reveal how specific geometric scaffold features critically influence FBR to biomaterial implants, accelerating or mitigating fibrotic responses, and ultimately determining transplant success.

bioengineering↗

bioRxiv2025

Epitope Expression Persists in Circulating Tumor Cells as Breast Cancers Acquire Resistance to Antibody Drug Conjugates

Antibody-drug conjugates (ADCs) target surface proteins on cancer cells, leading to internalization and delivery of a drug payload, thereby enhancing selectivity and minimizing toxicity. ADCs against TROP2 (Sacituzumab govitecan) or HER2 (T-DXd) have demonstrated efficacy in metastatic breast cancer, yet paradoxically, outside of HER2-amplified breast cancers, expression levels of these breast cancer-enriched epitopes in tumor biopsies have not been strongly correlated with clinical response. We undertook serial quantitative imaging of circulating tumor cells (CTCs) in a prospective cohort of 35 patients treated with either of these ADCs. At the single-cell level, expression of TROP2 and HER2 within individual patients is highly heterogeneous in both CTCs and paired tumor biopsies. Measurement of these epitopes on CTCs immediately prior to ADC therapy does not predict depth of clinical response. However, absence of CTCs or >80% reduction in CTC numbers after three weeks of treatment (CTCLow) predicts durable response, compared with CTCHigh cases (TROP2: HR 5.15, P = 0.012; HER2: HR 6.01, P<0.001). Targeted epitopes are not commonly downregulated on CTCs at the time of acquired clinical resistance, and switching between TROP2- and HER2-targeting ADCs sharing similar payloads infrequently leads to second-line response. Thus, while CTC burden is correlated with response to these ADCs, the level of TROP2 or HER2 expression is poorly predictive. These findings point to sensitivity to the drug payload as a potential driver of clinical response to currently approved ADCs in breast cancer. SIGNIFICANCE STATEMENTThe clinical efficacy of ADCs may depend both on differential targeting of cancer cells, using antibodies to tumor-enriched epitopes, and on the cleavage and release of drug payloads to which cancer cells are sensitive. ADCs currently used to treat breast cancer target either of two epitopes, TROP2 or HER2, but they share chemically related payloads. While CTC numbers track with response, we find that epitope expression is not strongly predictive. The limited success of sequentially switching between TROP2- or HER2-targeting ADCs as second-line treatment, following progression on a first-line ADC, highlights the need to incorporate non-cross-resistant drug payloads on ADCs to overcome such acquired resistance.

cancer biology↗

bioRxiv2024

Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

bioengineering↗

bioRxiv2023

An exploration of mechanisms underlying Desemzia incerta colonization resistance to methicillin-resistant Staphylococcus aureus on the skin

Colonization of human skin and nares by methicillin-resistant Staphylococcus aureus (MRSA) leads to community spread of MRSA. This spread is exacerbated by transfer of MRSA between humans and livestock, particularly swine. Here we capitalized on the shared features between human and porcine skin, including shared MRSA colonization, to study novel bacterial mediators of MRSA colonization resistance. We focused on the poorly studied bacterial species Desemzia incerta, which we found to exert antimicrobial activity through a secreted product and exhibited colonization resistance against MRSA in an in vivo murine skin model. Using parallel genomic and biochemical investigation, we discovered that D. incerta secretes an antimicrobial protein. Sequential protein purification and proteomics analysis identified 24 candidate inhibitory proteins, including a promising peptidoglycan hydrolase candidate. Aided by transcriptional analysis of D. incerta and MRSA cocultures, we found that exposure to D. incerta leads to decreased MRSA biofilm production. These results emphasize the value in exploring microbial communities across a spectrum of hosts, which can lead to novel therapeutic agents as well as increased understanding of microbial competition. IMPORTANCEMethicillin-resistant Staphylococcus aureus causes significant healthcare burden and can be spread to the human population via livestock transmission. Members of the skin microbiome can prevent MRSA colonization via a poorly-understood phenomenon known as colonization resistance. Here, we studied colonization resistance of S. aureus by bacterial inhibitors previously identified from a porcine skin model. We identify a pig skin commensal, Desemzia incerta, that reduced MRSA colonization in a murine model. We employ a combination of genomic, proteomic, and transcriptomic analyses to explore the mechanisms of inhibition between D. incerta and S. aureus. We identify 24 candidate antimicrobial proteins secreted by D. incerta that could be responsible for its antimicrobial activity. We also find that exposure to D. incerta leads to decreased S. aureus biofilm formation. These findings show that the livestock transmission of MRSA can be exploited to uncover novel mechanisms of MRSA colonization resistance.

microbiology↗

bioRxiv2023

A suite of engineered mice for interrogating psychedelic drug actions

Psychedelic drugs like lysergic acid diethylamide (LSD) and psilocybin have emerged as potentially transformative therapeutics for many neuropsychiatric diseases, including depression, anxiety, post-traumatic stress disorder, migraine, and cluster headaches. LSD and psilocybin exert their psychedelic effects via activation of the 5-hydroxytryptamine 2A receptor (HTR2A). Here we provide a suite of engineered mice useful for clarifying the role of HTR2A and HTR2A-expressing neurons in psychedelic drug actions. We first generated Htr2a-EGFP-CT-IRES-CreERT2 mice (CT:C-terminus) to independently identify both HTR2A-EGFP-CT receptors and HTR2A-containing cells thereby providing a detailed anatomical map of HTR2A and identifying cell types that express HTR2A. We also generated a humanized Htr2a mouse line and an additional constitutive Htr2A-Cre mouse line. Psychedelics induced a variety of known behavioral changes in our mice validating their utility for behavioral studies. Finally, electrophysiology studies revealed that extracellular 5-HT elicited a HTR2A-mediated robust increase in firing of genetically-identified pyramidal neurons--consistent with a plasma membrane localization and mode of action. These mouse lines represent invaluable tools for elucidating the molecular, cellular, pharmacological, physiological, behavioral, and other actions of psychedelic drugs in vivo.

neuroscience↗

bioRxiv2022

On the causes, consequences, and avoidance of PCR duplicates: towards a theory of library complexity

AO_SCPLOWBSTRACTC_SCPLOWLibrary preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artifact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing datasets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a minor role for this artifact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any dataset containing PCR duplicates. We discuss how amplification-related artifacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.

molecular biology↗

bioRxiv2022

Continuous transcriptome analysis reveals novel patterns of early gene expression in Drosophila embryos.

Early organismal development consists of transformative events that lay the foundation for body formation and long-term phenotype. Despite this understanding, the rapid progression of events and the limited material available are major barriers to studying the earliest stages. The size and accessibility of Drosophila embryos overcome some of these limitations, and several studies characterizing early transcriptional events have been reported. Unfortunately, manual embryo staging, and elaborate protocols make the techniques employed in these studies prone to human and technical errors and incompatible with routine laboratory use. Herein, we present a straight-forward and operationally simple methodology for studying the early transcription ([≤]3 hours) in Drosophila. This method relies on single-embryo RNA-sequencing and transcriptome ordering along a developmental trajectory (pseudo-time), thereby avoiding the need for the staging of the embryos. The obtained high-resolution and time-sensitive mRNA expression profiles uncovered the exact onset of transcription and degradation of transcripts and revealed an earlier transcription start for several zygotic genes. In addition, degradation patterns suggest that maternal mRNA decay is independent of mRNA levels. By classifying each embryo as male or female, we were also able to study sex-biased transcription between the beginning of zygotic transcription to gastrulation and identified 120 differentially expressed mRNAs. Using sex-specific transcription signatures, embryos can be sexed directly, eliminating the need for Y-chromosome genotyping. Herein, we report an accessible, single-embryo sequencing approach for high-resolution, time-sensitive transcriptome analysis. Our data provide an unparalleled resolution of gene expression during early development and enhance the current understanding of early transcriptional processes.

genomics↗

bioRxiv2022

Harnessing diversity and antagonism within the pig skin microbiota to identify novel mediators of colonization resistance to methicillin-resistant Staphylococcus aureus

The microbiota mediates multiple aspects of skin barrier function, including colonization resistance to pathogens such as Staphylococcus aureus. The endogenous skin microbiota limits S. aureus colonization via competition and direct inhibition. Novel mechanisms of colonization resistance are promising therapeutic targets for drug-resistant infections, such as those caused by methicillin-resistant S. aureus (MRSA). Here, we developed and characterized a swine model of topical microbiome perturbation and MRSA colonization. As in other model systems, topical antimicrobial treatment had little discernable effect on community diversity though the overall microbial load was sensitive to any type of intervention, including swabbing. In parallel, we established a porcine skin culture collection and screened 7,700 isolates for MRSA inhibition. Inhibitory isolates were represented across all major phyla of the pig skin microbiota and did not have a strong preference for inhibiting closely related species, suggesting that relatedness is not a condition of antagonism. Using genomic and phenotypic criteria, we curated 3 isolates to investigate whether prophylactic colonization would inhibit MRSA colonization in vivo. The 3-member consortium together, but not individually, provided protection against MRSA colonization, suggesting cooperation and/or synergy among the strains. These findings reveal the porcine skin as an underexplored reservoir of skin commensal species with the potential to prevent MRSA colonization and infection.

microbiology↗

bioRxiv2022

Spatial variation in population genomic responses to over a century of anthropogenic change within a tidal marsh songbird

Combating the current biodiversity crisis requires the accurate documentation of population responses to human-induced ecological change. To this end, museum collections preserve a record of population responses to anthropogenic change that can provide critical baseline data on patterns of genetic diversity, connectivity, and population structure. We leveraged spatially-replicated time series of specimens to document population genomic responses to the destruction of nearly 90% of coastal habitats occupied by the Savannah sparrow (Passerculus sandwichensis) in California. Spatial-temporal analyses of genetic diversity from 219 sparrows collected between 1889-2017 showed that the amount of habitat lost was not predictive of genetic diversity loss. Despite experiencing the greatest levels of habitat loss, we found that genetic diversity in the San Francisco Bay Area remained relatively high. Over the past century, immigration into the Bay Area from interior populations has also increased. This may have minimized genetic diversity declines, but likely led to the erosion of divergence at loci associated with tidal marsh adaptation. Tracing the genomic trajectories of multiple populations over time provided unique insights into how shifting patterns of gene flow through time in response to human-induced habitat loss may contribute to negative fitness consequences.

evolutionary biology↗

bioRxiv2022

Piezo buffers mechanical stress via modulation of intracellular Ca2+ handling in the Drosophila heart

Throughout its lifetime the heart is buffeted continuously by dynamic mechanical forces resulting from contraction of the heart muscle itself and fluctuations in haemodynamic load and pressure. These forces are in flux on a beat-by-beat basis, resulting from changes in posture, physical activity or emotional state, and over longer timescales due to altered physiology (e.g. pregnancy) or as a consequence of ageing or disease (e.g. hypertension). It has been known for over a century of the hearts ability to sense differences in haemodynamic load and adjust contractile force accordingly1-4. These adaptive behaviours are important for cardiovascular homeostasis, but the mechanism(s) underpinning them are incompletely understood. Here we present evidence that the mechanically-activated ion channel, Piezo, is an important component of the Drosophila hearts ability to adapt to mechanical force. We find Piezo is a sarcoplasmic reticulum (SR)-resident channel and is part of a mechanism that regulates Ca2+ handling in cardiomyocytes in response to mechanical stress. Our data support a simple model in which Drosophila Piezo transduces mechanical force such as stretch into a Ca2+ signal, originating from the SR, that modulates cardiomyocyte contraction. We show that Piezo mutant hearts fail to buffer mechanical stress, have altered Ca2+ handling, become prone to arrhythmias and undergo pathological remodelling.

physiology↗

bioRxiv2021

Single-molecule analysis of the entire perfringolysin O poreformation pathway

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings ([~]35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

biophysics↗

bioRxiv2020

Development of a method for revertable CRISPR/Cas9-based mutagenesis in cell culture

CRISPR/Cas9 mutagenesis is a revolutionary tool for genetics in organismal and cell culture systems. One notable caveat with this system is the potential for phenotype-inducing off-target/background mutations. There has been considerable success in modifying the methodology to minimize these potential confounds. Here we have developed a tool to functionally demonstrate that a targeted mutation of interest is responsible for the phenotype observed. This approach creates revertable mutations in cell culture systems using CRISPR/Cas9-induced homology-directed repair (HDR) to insert a LoxP-flanked transcriptional stop sequence into an early intron of a target gene. This method has the potential to be used in multiplexed and inducible scenarios to restore gene function within a given experiment.

cell biology↗

bioRxiv2020

Dynamics of Tpm1.8 domains on actin filaments with single moleculeresolution

Tropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

biophysics↗

bioRxiv2019

Divergence in hormone signalling links local adaptation and hybrid failure

Natural selection is a significant driver of speciation. Yet it remains largely unknown whether local adaptation can drive speciation through the evolution of hybrid sterility between populations. Here, we show that adaptive divergence in shoot gravitropism, the ability of a plants shoot to bend upwards in response to the downward pull of gravity, contributes to the evolution of hybrid sterility in an Australian wildflower, Senecio lautus. We find that shoot gravitropism has evolved multiple times in association with plant height between adjacent populations inhabiting contrasting environments, suggesting that these traits have evolved by natural selection. We directly tested this prediction using a hybrid population subjected to eight rounds of recombination and three rounds of selection in the field. It revealed that shoot gravitropism responds to natural selection in the expected direction of the locally adapted population. This provided an ideal platform to test whether genetic differences in gravitropism contribute to hybrid sterility in S. lautus. Using this advanced hybrid population, we discovered that crossing individuals with extreme differences in gravitropism reduce their ability to produce seed by 21%, providing strong evidence that this adaptive trait is genetically correlated with hybrid sterility. Our results suggest that natural selection can drive the evolution of locally adaptive traits that also create hybrid sterility, thus indicating an evolutionary connection between local adaptation and the origin of new species. Significance statementNew species originate as populations become reproductively isolated from one another. Despite recent progress in uncovering the genetic basis of reproductive isolation, it remains unclear whether intrinsic reproductive barriers, such as hybrid sterility, evolve as a by-product of local adaptation to contrasting environments or evolve through non-ecological processes, such as meiotic drive. Here, we show that differences in a plants response to the pull of gravity have repeatedly evolved amongst coastal populations of an Australian wildflower, thus implicating a role of natural selection in their evolution. We found a strong genetic correlation between variation in this adaptive trait and hybrid sterility, suggesting that intrinsic reproductive barriers contribute to the origin of new species as populations adapt to heterogeneous environments.

evolutionary biology↗

bioRxiv2019

Fluorescent biosensor for real-time interaction dynamics of host proteins with HIV-1 capsid tubes

The human immunodeficiency virus 1 (HIV-1) capsid serves as a binding platform for proteins and small molecules from the host cell that regulate various steps in the virus life cycle. However, there are currently no quantitative methods that use assembled capsid lattices for measuring host-pathogen interaction dynamics. Here we developed a single molecule fluorescence biosensor using self-assembled capsid tubes as biorecognition elements and imaged capsid binders using total internal reflection fluorescence microscopy in a microfluidic setup. The method is highly sensitive in its ability to observe and quantify binding, obtain dissociation constants, extract kinetics with an extended application of using more complex analytes that can accelerate characterisation of novel capsid binders.

biophysics↗

bioRxiv2018

Divergent selection and drift shape the genomes of two avian sister species spanning a saline-freshwater ecotone

The role of species divergence due to ecologically-based divergent selection - or ecological speciation - in generating and maintaining biodiversity is a central question in evolutionary biology. Comparison of the genomes of phylogenetically related taxa spanning a selective habitat gradient enables discovery of divergent signatures of selection and thereby provides valuable insight into the role of divergent ecological selection in speciation. Tidal marsh ecosystems provide tractable opportunities for studying organisms adaptations to selective pressures that underlie ecological divergence. Sharp environmental gradients across the saline-freshwater ecotone within tidal marshes present extreme adaptive challenges to terrestrial vertebrates. Here we sequence 20 whole genomes of two avian sister species endemic to tidal marshes - the Saltmarsh Sparrow (Ammodramus caudacutus) and Nelsons Sparrow (A. nelsoni) - to evaluate the influence of selective and demographic processes in shaping genome-wide patterns of divergence. Genome-wide divergence between these two recently diverged sister species was notably high (genome-wide FST = 0.32). Against a background of high genome-wide divergence, regions of elevated divergence were widespread throughout the genome, as opposed to focused within islands of differentiation. These patterns may be the result of genetic drift acting during past tidal march colonization events in addition to divergent selection to different environments. We identified several candidate genes that exhibited elevated divergence between Saltmarsh and Nelsons sparrows, including genes linked to osmotic regulation, circadian rhythm, and plumage melanism - all putative candidates linked to adaptation to tidal marsh environments. These findings provide new insights into the roles of divergent selection and genetic drift in generating and maintaining biodiversity.

evolutionary biology↗