Biology subjects

Walker, J.

Publications and source records attributed to Walker, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Shrinking in the dark: Parallel endosymbiont genome erosions are associated with repeated host transitions to an underground life

Microbial symbioses have had profound impacts on the evolution of animals. Conversely, changes in host biology may impact the evolutionary trajectory of symbionts themselves. Blattabacterium cuenoti is present in almost all cockroach species and enables hosts to subsist on a nutrient-poor diet. To investigate if host biology has impacted Blattabacterium at the genomic level, we sequenced and analysed 25 genomes from Australian soil-burrowing cockroaches (Blaberidae: Panesthiinae) which have undergone at least seven independent subterranean transitions from above-ground, wood-feeding ancestors. We find at least three independent instances of genome erosion have occurred in Blattabacterium strains exclusive to Australian soil-burrowing cockroaches. Such shrinkages have involved the repeated inactivation of genes involved in amino acid biosynthesis and nitrogen recycling, the core role of Blattabacterium in the host-symbiont relationship. The most drastic of these erosions have occurred in hosts thought to have transitioned underground the earliest relative to other lineages. As Blattabacterium is unable to fulfil its core function in such host groups, our findings suggest soil-burrowing cockroaches must acquire these nutrients from novel sources. Our study represents one of the first cases, to our knowledge, of parallel host adaptations leading to concomitant parallelism in their mutualistic symbionts, further underscoring the intimate relationship between these two partners.

genomics↗

bioRxiv2023

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

microbiology↗

bioRxiv2023

The Estrogen Receptor-Related Orphan Receptors (ERRs) Regulate Autophagy through TFEB

Autophagy is an essential self-degradative and recycling mechanism that maintains cellular homeostasis. Estrogen receptor-related orphan receptors (ERRs) play fundamental role in regulation of cardiac metabolism and function. Previously, we showed that ERR agonists improve cardiac function in models of heart failure and induce autophagy in cardiomyocytes. Here, we characterized a mechanism by which ERRs induce autophagy in cardiomyocytes. Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome pathway and has been shown to be important in cardiac autophagy. We discovered that TFEB is a direct ERR target gene whose expression is induced by ERR agonists. Activation of ERR results in increased TFEB expression in both neonatal rat ventricular myocytes and C2C12 myoblasts. ERR-dependent increases in TFEB expression results in increased expression of an array of TFEB target genes, which are critical for stimulation of autophagy. Pharmacologically targeting ERR is a promising potential method for treatment of many diseases where stimulation of autophagy may be therapeutic including heart failure.

cell biology↗

bioRxiv2023

Alpaca Single B Cell Interrogation and Heavy-Chain-Only Antibody Discovery on an Optofluidic Platform

In vivo discovery approaches for single domain antibodies such as VHH have been limited by the lack of methodologies available for camelid B cell interrogation. Here, we report a novel application of the Berkeley Lights Beacon(R) optofluidic platform to the discovery of heavy-chain-only antibodies by screening single B cells from alpacas immunized with two different targets. Custom methods for alpaca B cell enrichment, culture, on-Beacon IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH candidates from an alpaca immunized with either human prostate specific membrane antigen (PSMA) or a second blinded target, in a proof-of-concept study. PSMA-specific VHH hits discovered on the Beacon were recombinantly expressed as VHH-Fc, purified, and characterized using label-free techniques. All but one VHH-Fc bound PSMA with a single-digit nanomolar affinity, and four candidates were successfully humanized in silico using a rapid bulk humanization approach. In addition, next-generation repertoire sequencing was performed at longitudinal timepoints after immunization, uncovering additional variants within the clonal lineages of the validated hits discovered on the Beacon platform. The establishment of this single B cell VHH discovery workflow extends the powerful Beacon technology to enable rapid discovery of VHH directly from natural camelid immune repertoires.

immunology↗

bioRxiv2023

Dating in the Dark: Elevated Substitution Rates in Cave Cockroaches (Blattodea: Nocticolidae) Have Negative Impacts on Molecular Date Estimates

Rates of nucleotide substitution vary substantially across the Tree of Life, with potentially confounding effects on phylogenetic and evolutionary analyses. A large acceleration in mitochondrial substitution rate occurs in the cockroach family Nocticolidae, which predominantly inhabit subterranean environments. To evaluate the impacts of this among-lineage rate heterogeneity on estimates of phylogenetic relationships and evolutionary timescales, we analysed nuclear ultraconserved elements (UCEs) and mitochondrial genomes from nocticolids and other cockroaches. Substitution rates were substantially elevated in nocticolid lineages compared with other cockroaches, especially in mitochondrial protein-coding genes. This disparity in evolutionary rates is likely to have led to different evolutionary relationships being supported by phylogenetic analyses of mitochondrial genomes and UCE loci. Furthermore, Bayesian dating analyses using relaxed-clock models inferred much deeper divergence times compared with a flexible local clock. Our phylogenetic analysis of UCEs, which is the first genome-scale study to include all ten major cockroach families, unites Corydiidae and Nocticolidae and places Anaplectidae as the sister lineage to the rest of Blattoidea. We uncover an extraordinary level of genetic divergence in Nocticolidae, including two highly distinct clades that separated [~]115 million years ago despite both containing representatives of the genus Nocticola. The results of our study highlight the potential impacts of high among-lineage rate variation on estimates of phylogenetic relationships and evolutionary timescales.

evolutionary biology↗

bioRxiv2022

Kinase-modulated bioluminescent indicators enable noninvasive imaging of drug activity in the brain

Aberrant kinase activity contributes to the pathogenesis of brain cancers, neurodegeneration, and neuropsychiatric diseases, but identifying kinase inhibitors that function in the brain is challenging. Drug levels in blood do not predict efficacy in the brain because the blood-brain barrier prevents entry of most compounds. Rather, assessing kinase inhibition in the brain requires tissue dissection and biochemical analysis, a time-consuming and resource-intensive process. Here, we report kinase-modulated bioluminescent indicators (KiMBIs) for non-invasive longitudinal imaging of drug activity in the brain based on a recently optimized luciferase-luciferin system. We develop an ERK KiMBI to report inhibitors of the Ras-Raf-MEK-ERK pathway, for which no bioluminescent indicators previously existed. ERK KiMBI discriminates between brain-penetrant and non-penetrant MEK inhibitors, reveals blood-tumor barrier leakiness in xenograft models, and reports MEK inhibitor pharmacodynamics in native brain tissues and intracranial xenografts. Finally, we use ERK KiMBI to screen ERK inhibitors for brain efficacy, identifying temuterkib as a promising brain-active ERK inhibitor, a result not predicted from chemical characteristics alone. Thus, KiMBIs enable the rapid identification and pharmacodynamic characterization of kinase inhibitors suitable for treating brain diseases.

pharmacology and toxicology↗

bioRxiv2022

A high-throughput COPD bronchosphere model for disease-relevant phenotypic compound screening

COPD is the third leading cause of death worldwide, but current therapies for COPD are only effective at treating the symptoms of the disease rather than targeting the underlying pathways that are driving the pathogenic changes. The lack of targeted therapies for COPD is in part due to a lack of knowledge about drivers of disease progression and the difficulty in building relevant and high throughput models that can recapitulate the phenotypic and transcriptomic changes associated with pathogenesis of COPD. To identify these drivers, we have developed a cigarette smoke extract (CSE)-treated bronchosphere assay in 384-well plate format that exhibits CSE-induced decreases in size and increase in luminal secretion of MUC5AC. Transcriptomic changes in CSE-treated bronchospheres resemble changes that occur in human smokers both with and without COPD compared to healthy groups, indicating that this model can capture human smoking signature. To identify new targets, we ran a small molecule compound deck screening with diversity in target mechanisms of action and identified hit compounds that attenuated CSE induced changes, either decreasing spheroid size or increasing secreted mucus. This work provides insight into the utility of this bronchosphere model in examining human respiratory diseases, the pathways implicated by CSE, and compounds with known mechanisms of action for therapeutic development.

cell biology↗

bioRxiv2022

A high-throughput 3D cantilever array to model airway smooth muscle hypercontractility in asthma

Asthma is often characterized by tissue-level mechanical phenotypes that include remodeling of the airway and an increase in airway tightening driven by the underlying smooth muscle. Existing therapies only provide symptom relief and do not improve the baseline narrowing of the airway or halt progression of the disease. To investigate such targeted therapeutics, there is a need for models that can recapitulate the 3D environment present in this tissue, provide phenotypic readouts of contractility, and be easily integrated into existing assay plate designs and laboratory automation used in drug discovery campaigns. To address this, we have developed DEFLCT, a high-throughput plate insert that can be paired with standard labware to easily generate high volumes of microscale tissues in vitro for screening applications. Using this platform, we exposed primary human airway smooth muscle cell-derived microtissues to a panel of six of inflammatory cytokines present in the asthmatic niche, identifying TGF-{beta}1 and IL-13 as strong contractile modulators. RNAseq analysis further demonstrated enrichment of contractile and remodeling-relevant pathways in TGF-{beta}1 and IL-13 treated tissues as well as pathways generally associated with asthma. Taken together, these data establish a disease relevant, 3D tissue model for the asthmatic airway which combines niche specific inflammatory cues and complex mechanical readouts that can be utilized in drug discovery efforts.

bioengineering↗

bioRxiv2022

Introgression of the Triticum timopheevii genome into wheat detected by chromosome-specific KASP markers

Triticum timopheevii (2n=28, AtAtGG) is a tetraploid wild relative species with great potential to increase the genetic diversity of hexaploid wheat Triticum aestivum (2n=42, AABBDD) for various important agronomic traits. A breeding scheme that propagated advanced backcrossed populations of wheat-T. timopheevii introgression lines through further backcrossing and self-fertilisation resulted in the generation of 99 introgression lines (ILs) that carried 309 homozygous segments from the At and G subgenomes of T. timopheevii. These introgressions contained 89 and 74 unique segments from the At and G subgenomes, respectively. These overlapping segments covered 98.9% of the T. timopheevii genome that has now been introgressed into bread wheat cv. Paragon including the entirety of all T. timopheevii chromosomes via varying sized segments except for chromosomes 3At, 4G and 6G. Homozygous ILs contained between one and eight of these introgressions with an average of three per introgression line. These homozygous introgressions were detected through the development of a set of 480 chromosome-specific Kompetitive allele specific PCR (KASP) markers that are well-distributed across the wheat genome. Of these, 149 were developed in this study based on single nucleotide polymorphisms (SNPs) discovered through whole genome sequencing of T. timopheevii. A majority of these KASP markers were also found to be T. timopheevii subgenome specific with 182 detecting At subgenome and 275 detecting G subgenome segments. These markers showed that 98% of the At segments had recombined with the A genome of wheat and 74% of the G genome segments had recombined with the B genome of wheat with the rest recombining with the D genome of wheat. These results were validated through multi-colour in situ hybridisation analysis. Together these homozygous wheat-T. timopheevii ILs and chromosome-specific KASP markers provide an invaluable resource to wheat breeders for trait discovery to combat biotic and abiotic stress factors affecting wheat production due to climate change.

plant biology↗

bioRxiv2022

Realtime morphological characterization and sorting of unlabeled viable cells using deep learning

Phenotyping of single cells has dramatically lagged advances in molecular characterization and remains a manual, subjective, and destructive process. We introduce COSMOS, a platform for phenotyping and enrichment of cells based on deep learning interpretation of high-content morphology data in realtime. By training models on an atlas of >1.5 billion images, we demonstrate enrichment of unlabeled cells up to 33,000 fold. We apply COSMOS to multicellular tissue biopsy samples demonstrating that it can identify malignant cells with similar accuracy to molecular approaches while enriching viable cells for functional evaluation. We show high-dimensional embedding vectors of morphology generated by COSMOS without any need for complex sample pre-processing, gating, or bioinformatics capabilities, which enables discovery of cellular phenotypes, and integration of morphology into multi-dimensional analyses. One sentence summaryA novel platform capable of high-throughput imaging and gently sorting cells using deep morphological assessment.

cell biology↗

bioRxiv2021

Genome-wide association study and functional validation implicates JADE1 in tauopathy

Primary age-related tauopathy (PART) is a neurodegenerative tauopathy with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-{beta} (A{beta}) deposition in plaques. The pathogenesis of PART is unknown, but evidence suggests it is associated with genes that promote tau pathology as well as others that protect from A{beta} toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n=647) using Braak neurofibrillary tangle stage as a quantitative trait adjusting for sex, age, genotyping platform, and principal components. We found significant associations with some candidate loci associated with AD and progressive supranuclear palsy, a primary tauopathy (SLC24A4, MS4A6A, HS3ST1, MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brain from tauopathies containing isoforms with four microtubule-binding domain repeats (4R) and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation revealed a direct and specific binding of JADE1 protein to tau containing four (4R) and no N-terminal inserts (0N4R) in post-mortem human PART brain tissue. Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a mediator of neurofibrillary degeneration.

neuroscience↗

bioRxiv2021

Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to transiently modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging and knockdown can be transient due to rapid crRNA degradation. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes enables transient gene expression modulation in primary CD4+ and CD8+ T-cells. This system represents a robust and efficient method to transiently modulate transcripts without genetic manipulation.

bioengineering↗

bioRxiv2021

Defining 'Destructive fishing': a lack of consensus inhibits effective global policy

The term "destructive fishing" appears in multiple international policy instruments intended to improve outcomes for marine biodiversity, coastal communities and sustainable fisheries. However, the meaning of "destructive fishing" is often vague, limiting effectiveness in policy. Therefore, in this study we systematically reviewed the use of "destructive fishing" in three record types: academic literature, media articles, and policy documents between 1976-2020. A more detailed analysis was performed on sub-sets of these records, considering the extent to which the term is characterised, geographic distribution of use, and specific impacts and practices associated with the term. We found that use of "destructive fishing" relative to the generic term fisheries has increased since the 1990s. Records focused predominantly on fishing practices in South-eastern Asia, followed by Southern Asia and Europe. The term was characterised in detail in only 15% of records. Habitat damage and blast/poison fishing were the most associated ecological impacts and gear/practices, respectively. Bottom trawling and unspecified net fishing were regularly linked to destructive fishing. Importantly, the three record types use the term differently. Academic literature tends to specifically articulate the negative impacts, while media articles focus generally on associated gears/practices. Significant regional variation also exists in how the term is used and what phenomena it is applied to. This study provides evidence and recommendations to inform stakeholders in any future pursuit of a unified definition of "destructive fishing" to support more meaningful implementation of global sustainability goals.

ecology↗

bioRxiv2021

Chronic wireless neural population recordings with common marmosets

Marmosets are an increasingly important model system for neuroscience in part due to genetic tractability and enhanced cortical accessibility, due to a lissencephalic neocortex. However, many of the techniques generally employed to record neural activity in primates inhibit the expression of natural behaviors in marmosets precluding neurophysiological insights. To address this challenge, we developed methods for recording neural population activity in unrestrained marmosets across multiple ethological behaviors, multiple brain states, and over multiple years. Notably, our flexible methodological design allows for replacing electrode arrays and removal of implants providing alternative experimental endpoints. We validate the method by recording sensorimotor cortical population activity in freely moving marmosets across their natural behavioral repertoire and during sleep. HIGHLIGHTS- Simultaneous and chronic wireless neural population recordings in multiple freely moving marmosets - Neural recording approach enables studies of natural repertoire of behaviors and sleep - Methyl-methacrylate free surgical approach designed to promote biocompatibility and longitudinal success of the implant - Modular headstage configuration requires minimal daily animal handling for daily neural recordings - Alternative experimental endpoints: implant removal, healing, and electrode array replacement

neuroscience↗

bioRxiv2021

Extensive N4 cytosine methylation is essential for Marchantia sperm function

4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance, and even presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwort Marchantia polymorpha involves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC), a well-known eukaryotic DNA modification, expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC is catalyzed by two novel methyltransferases (MpDN4MT1a and MpDN4MT1b) specifically expressed during late spermiogenesis. Deletion of MpDN4MT1s eliminates 4mC, alters the sperm transcriptome, and produces sperm with swimming defects. Our results reveal extensive 4mC in a eukaryote and define a new family of eukaryotic methyltransferases, thereby expanding the repertoire of functional eukaryotic DNA modifications.

developmental biology↗

bioRxiv2021

Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis

The plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and facilitates meiosis. Why reprogramming is limited to the germline and how specific genes are chosen is unknown. Here, we demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by germline-specific siRNAs transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) via activity of the tapetum-specific chromatin remodeler CLASSY3. Remarkably, tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Finally, we demonstrate that these nurse cell-derived siRNAs (niRNAs) silence germline transposons, thereby safeguarding genome integrity. Our results reveal that tapetal niRNAs are sufficient to reconstitute germline methylation patterns and drive extensive, functional methylation reprogramming analogous to piRNA-mediated reprogramming in animal germlines. O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/428150v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1e985c0org.highwire.dtl.DTLVardef@4c065corg.highwire.dtl.DTLVardef@1358a5forg.highwire.dtl.DTLVardef@f9c03_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

TMIGD1, a putative tumor suppressor, induces G2-M cell cycle checkpoint arrest in colon cancer cells

Colorectal cancer (CRC) is a leading non-familial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis remains incompletely understood. In this study, we demonstrate that cell adhesion molecule transmembrane and immunoglobulin domain containing1 (TMIGD1) is highly expressed in mouse and human normal intestinal epithelial cells. We have developed TMIGD1 knockout mice and show that the loss of TMIGD1 in mice results in the development of adenomas in small intestine and colon. Additionally, the loss of TMIGD1 in mouse impaired intestinal epithelium brush border formation, junctional polarity and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation, cell migration, arrests cell cycle at G2/M phase and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, we demonstrate that TMIGD1 is progressively downregulated in sporadic human CRC and correlates with poor overall survival. Our findings identify TMIGD1 as a novel tumor suppressor gene and provide insights into the pathogenesis of colorectal cancer and possibilities as a potential therapeutic target.

cancer biology↗

bioRxiv2020

Intragenic conflict in phylogenomic datasets

Most phylogenetic analyses assume that a single evolutionary history underlies one gene. However, both biological processes and errors in dataset assembly can violate this assumption causing intragenic conflict. The extent to which this conflict is present in empirical datasets is not well documented. However, if common, it would have far-reaching implications for phylogenetic analyses. Here, we examined several large phylogenomic datasets from diverse taxa using a fast and simple method to identify well supported intragenic conflict. We found conflict to be highly variable between datasets, from 1% to more than 92% of genes investigated. To better characterize patterns of conflict, we analyzed four genes with no obvious data assembly errors in more detail. Analyses on simulated data highlighted that alignment error may be one major source of conflict. Whether as part of data analysis pipelines or in order to explore potential biologically compelling intragenic processes, analyses of within gene signal should become common. The method presented here provides a relatively fast means for identifying conflicts that is agnostic to the generating process. Datasets identified with high intragenic conflict may either have significant errors in dataset assembly or represent conflict generated by biological processes. Conflicts that are the result of error should be identified and discarded or corrected. For those conflicts that are the result of biological processes, these analyses contribute to the growing consensus that, similar to genomes, genes themselves may exhibit multiple conflicting evolutionary histories across the tree of life.

evolutionary biology↗