Biology subjects

Lord, A.

Publications and source records attributed to Lord, A..

7 recordsLinked to original sources

bioRxiv2025

Active learning guides automated discovery of DNA delivery via electroporation for non-model microbes

Delivery of recombinant DNA is foundational for understanding and engineering a target organism. Electroporation can be applied to any cell type, yet identification of a working protocol for new organisms remains bespoke and laborious because no systematic framework exists, and appropriate instrumentation is lacking. Here, we describe an automated high-throughput platform which uses active learning to discover electroporation protocols for non-model microbes. We first devised a 24-condition electroporation screen, based on systematic evaluation and selection of key parameters, that can be applied to any microbe. Facilitated by a custom-built fully-programmable electroporator, we successfully identified electroporation protocols for eight non-model bacteria using this screen alone. We then combined this electroporation screen with our pooled POSSUM plasmid library to simultaneously evaluate 408 experimental conditions per organism and identified both a protocol and a replicating plasmid for five non-model Proteobacteria spanning three major classes (alpha-, beta-, and gamma-). We report the first electroporation protocols for Shewanella indica, Shewanella putrefaciens 200, Shewanella putrefaciens 95, Halomonas elongata, Piscinibacter sakaiensis, and Duganella zoogloeoides, as well as multiple alternative protocols for Shewanella amazonensis, Shewanella oneidensis, Azospirillum brasilense, Cupriavidus necator, Pseudomonas alcaliphila, and Escherichia coli. Finally, we developed an active learning pipeline to guide the selection of parameters based on gathered experimental data. Using our robotic platform, we iteratively tested 538 conditions over three iterations to improve electroporation for the emerging industrial chassis C. necator, achieving 8.6-fold higher transformation efficiency than state of the art. This work establishes a discovery platform for DNA delivery to diverse and recalcitrant microbes that can be extended broadly to non-model organisms in our biosphere.

microbiology↗

bioRxiv2025

New genomic resources for an invasive mite pest help guide the development of KASP markers for pesticide resistance screening

The redlegged earth mite, Halotydeus destructor, is an invasive pest of grain and pasture crops in Australia. Australian populations are increasingly developing resistance to broad-spectrum pesticides. Here, we assembled a chromosome-level genome for H. destructor, comprising nine chromosomes and totalling approximately 46 Mb. We also generated a genomic dataset from 190 populations collected across thousands of kilometres of its invasive Australian range and across four years (2018, 2021, 2022, and 2023), and used this dataset to develop KASP (Kompetitive allele-specific PCR) markers for identifying resistance mutations. We combined pooled whole-genome sequencing with resistance testing for organophosphate pesticides, chlorpyrifos and omethoate, and the pyrethroid pesticide bifenthrin. Target-site resistance to chlorpyrifos was strongly associated with the common ace G119S mutation. A second mutation, ace A201S, was also common, although its association with chlorpyrifos or omethoate resistance was less clear. Target-site resistance to bifenthrin was strongly associated with the common para L1024F mutation. We also detected several rare target-site mutations that likely contribute to resistance in specific populations, including F331Y in ace, and M918T, M918V, L925I, F1020S, and F1538I in para. Copy number variation in ace showed mixed associations with resistance, potentially reflecting the co-occurrence of variable gene copies and target-site mutations segregating within and among populations. Finally, we observed putative compositional and structural differences in genomic regions containing carboxylesterases with high ace-like homology, a group of genes that might be involved in organophosphate metabolism. This study advances the genomic and molecular resources available for H. destructor, and represents the most comprehensive investigation to date of resistance mechanisms and their spatiotemporal distribution in this invasive pest. Our data showed that pesticide resistance in H. destructor likely involves many genes. Nevertheless, target-site mutations are likely to remain key markers for rapid resistance diagnostics, for which our KASP markers will provide valuable tools.

genomics↗

bioRxiv2023

Giants among Cnidaria: large nuclear genomes and rearranged mitochondrial genomes in siphonophores

Siphonophores (Cnidaria:Hydrozoa) are abundant predators found throughout the ocean and are important components in worldwide zooplankton. They range in length from a few centimeters to tens of meters. They are gelatinous, fragile, and difficult to collect, so many aspects of the biology of these 190 species remain poorly understood. To survey siphonophore genome diversity, we performed Illumina sequencing of 32 species sampled broadly across the phylogeny. Sequencing depth was sufficient to estimate nuclear genome size from k-mer spectra in 8 specimens, ranging from 0.7-4.8Gb. In 6 specimens we got heterozygosity estimates between 0.7-5.3%. Rarefaction analyses indicate k-mer peaks can be absent with as much as 30x read coverage, suggesting minimum genome sizes range from 1.0-3.8Gb in the remaining 27 samples without k-mer peaks. This work confirms most siphonophore nuclear genomes are large, but also identifies several with reduced size that are tractable targets for future siphonophore nuclear genome assembly projects. We also assembled mitochondrial genomes for 32 specimens from these new data, indicating a conserved gene order among Hydrozoa, Cystonectae and some Physonectae, also revealing the ancestral gene organization of siphonophores. There then was extensive rearrangement of mitochondrial genomes within other physonects and in Calycophorae, including the repeated loss of atp8. Though siphonophores comprise a small fraction of cnidarian species, this survey greatly expands our understanding of cnidarian genome diversity. This study further illustrates both the importance of deep phylogenetic sampling and the utility of Illumina genome skimming in understanding genomic diversity of a clade. SignificanceDescriptions of basic genome features, such as nuclear genome size and mitochondrial genome sequences, remain sparse across many clades in the tree of life, leading to over generalizations from very small sample sizes and often limiting selection of optimal species for genome assembly efforts. Here we use Illumina genome skimming to assess a variety of genome features across 35 siphonophores (Cnidaria). This deep dive within a single clade identifies six species that are optimal candidates of future genomic work, and reveals greater range in nuclear genome size and diversity of mitochondrial genome orders within siphonophores than had been described across all Cnidaria.

evolutionary biology↗

bioRxiv2021

An extremes of phenotype approach confirms significant genetic heterogeneity in patients with ulcerative colitis.

Ulcerative colitis (UC) is a major form of inflammatory bowel disease with increasing global incidence. There is significant phenotypic heterogeneity defined by a range of clinical variables including age of onset and disease extent. Clinical outcomes range from long-term remission on minimal therapy to surgical resection. Close to 70% of UC risk can be attributed to genetics and understanding the genetic mechanisms contributing to this risk and disease heterogeneity is vital for understanding disease pathogenesis and improving patient outcomes through targeted screening and therapies. This study aims to characterise the genetic heterogeneity of UC by identifying genomic risk variants specific to mild and/or severe forms of UC, exploring variations in the effect size of known risk variants and assessing the clinical value of a genetic risk score (GRS). We conducted genome-wide association (GWA) analyses in 287 patients with mild UC, 311 patients with severe UC and 583 age- and gender-matched controls. Odds ratios (OR) for mild vs control, severe vs control and combined mild and severe UC vs control were calculated. Using the combined UC data, two independent loci in the HLA region reached genome-wide significance. An additional genome-wide significant signal on chromosome 1 was identified in severe cases only. OR for known risk loci varied between mild and severe patients and were similar to previously published results. Effect estimates from the most recent UC GWA meta-analysis were used to calculate a GRS for each individual. A higher mean GRS was observed in both mild and severe UC cases compared to controls however, there was no difference between the mean GRS for mild and severe UC. Heterogeneity in effect sizes of UC associated variants between mild and severe disease burden suggests the presence of genetically distinct signatures. While large consortium data are needed to identify genome-wide significant variants, additional risk loci may be identified by targeted recruitment of individuals with a history of severe disease. Author SummaryUlcerative colitis (UC) is a chronic and often debilitating form of inflammatory bowel disease affecting approximately 0.3% of the population in industrialized economies. The disease displays significant clinical heterogeneity including age at presentation, disease severity, and the propensity to develop disease-related complications. Several previous studies have demonstrated the heritability of UC, identifying over 30 loci specific to the disease. The majority of these loci have small to modest effect sizes other than those within the Human Leucocyte Antigen (HLA) region on chromosome 6. Using stringent clinical criteria for defining mild and severe forms of UC in an extremes of phenotype approach, we undertook a genome wide association study in a dataset of 1222 participants to investigate genetic heterogeneity in this disease. We demonstrated substantial differences in genetic associations in severe UC as compared to mild UC. While over 2,000 SNPs achieved genome-wide significance in the severe UC analysis, none reached significance for mild UC. These results were reflected in significant differences in odds ratios. We identified Complement Factor B (CFB) as a potential susceptibility gene for severe UC in the Caucasian population with additional tissue gene expression demonstrating a positive correlation with disease severity.

genetics↗

bioRxiv2020

Near-infrared Spectroscopy evaluations for rapid differentiation of carbapenem resistant Enterobacteriaceae from susceptible strains

Antimicrobial resistance (AMR) caused by Carbapenem-Resistant Enterobacteriaceae (CRE) is a global threat. Accurate identification of these bacterial species with associated AMR is critical for their management. While highly accurate methods to detect CRE are available, they are costly, timely and require expert skills making their application infeasible in low-resource settings. Here, we investigated the potential of Near-infrared Spectroscopy (NIRS) for a range of applications; i) the detection and differentiation of isolates of two pathogenic Enterobacteriaceae species, Klebsiella pneumoniae and Escherichia coli and, ii) the differentiation of carbapenem resistant and susceptible K. pneumoniae. NIRS has successfully differentiated between K. pneumoniae and E. coli isolates with a predictive accuracy of 89.04% (95% CI; 88.7-89.4%). K. pneumoniae isolates harbouring carbapenem resistance determinants were differentiated from susceptible K. pneumoniae strains with an accuracy of 85% (95% CI; 84.2-86.1%). To our knowledge, this is the largest demonstration of a proof of concept for the utility and feasibility of NIRS for rapidly differentiating between K. pneumoniae from E.coli as well as from carbapenem resistant K. pneumoniae from susceptible strains.

microbiology↗

bioRxiv2019

Caenorhabditis elegans PIEZO Channel Coordinates Multiple Reproductive Tissues to Govern Ovulation

The PIEZO proteins are involved in a wide range of developmental and physiological processes. Human PIEZO1 and PIEZO2 are newly identified excitatory mechano-sensitive proteins; they are non-selective ion channels that exhibit a preference for calcium in response to mechanical stimuli. To further understand the function of these proteins, we investigated the roles of pezo-1, the sole PIEZO ortholog in C. elegans. pezo-1 is expressed throughout development in C. elegans, with strong expression in reproductive tissues. A number of deletion alleles as well as a putative gain-of-function mutant caused severe defects in reproduction. A reduced brood size was observed in the strains depleted of PEZO-1. In vivo observations show that oocytes undergo a variety of transit defects as they enter and exit the spermatheca during ovulation. Post ovulation oocytes were frequently damaged during spermathecal contraction. Calcium signaling in the spermatheca is normal during ovulation in pezo-1 mutants, however, pezo-1 interacts genetically with known regulators of calcium signaling. Lastly, loss of PEZO-1 caused defective sperm navigation after being pushed out of the spermatheca during ovulation. Mating with males rescued these reproductive deficiencies in our pezo-1 mutants. These findings suggest that PEZO-1 may act in different reproductive tissues to promote proper ovulation and fertilization in C. elegans.

developmental biology↗

bioRxiv2018

DOT1L suppresses nuclear RNAi originating from enhancer elements in Caenorhabditis elegans

Methylation of histone H3 on lysine 79 (H3K79) by DOT1L is associated with actively transcribed genes. Earlier, we described that DOT-1.1, the Caenorhabditis elegans DOT1L homologue, cooperates with the chromatin-binding protein ZFP-1 (AF10 homologue) to negatively modulate transcription of highly and widely expressed target genes. Also, reduction in ZFP-1 levels has long been associated with lower efficiency of RNA interference (RNAi) triggered by exogenous double-stranded RNA (dsRNA), but the reason for this is not clear. Here, we demonstrate that DOT1L suppresses bidirectional transcription, including that producing enhancer RNAs, thereby preventing dsRNA formation and ectopic RNAi. This ectopic elevation of endogenous dsRNA may engage the Dicer complex and, therefore, limit efficiency of exogenous RNAi. Our insight provides a novel perspective on the underlying mechanisms of DOT1L function in development, neural activity, and cancer.

genomics↗