Biology subjects

McIntyre, J.

Publications and source records attributed to McIntyre, J..

10 recordsLinked to original sources

bioRxiv2024

Optimisation of single-nuclei isolation and RNA sequencing of parasitic nematodes

Single-cell/nuclei transcriptomics has revolutionised our understanding of cell, tissue, and organismal biology during health and disease of humans and model organisms. However, for non-model species, reassessment and refinement of conventional methods to address novel technical and biological challenges are needed. Here, we evaluate three established nuclei isolation protocols and, building on experiences, optimised single nuclei isolation for parasitic nematodes (SNIP). We test the versatility of SNIP across diverse species and life stages, validating it with snRNA-seq to reveal sex, stage, and tissue-specific gene expression. Optimised protocols will advance the understanding of parasitic nematodes, from fundamental biology to infection and disease.

molecular biology↗

bioRxiv2024

Chromosomal genome assembly resolves drug resistance loci in the parasitic nematode Teladorsagia circumcincta

The parasitic nematode Teladorsagia circumcincta is one of the most important pathogens of sheep and goat farming in temperate climates worldwide and can rapidly evolve resistance to drugs used to control it. To understand the genetics of drug resistance, we have generated a highly contiguous genome assembly for the UK T. circumcincta isolate, MTci2. Assembly using PacBio long-reads and Hi-C long-molecule scaffolding together with manual curation resulted in a 573 Mb assembly (N50 = 84 Mb, n = 1,286) with five autosomal and one sex-linked chromosomal-scale scaffolds consistent with its karyotype. The genome resource was further improved via annotation of 22,948 genes, with manual curation of over 3,200 of these, resulting in a robust and near complete resource (96.3% complete protein BUSCOs) to support basic and applied research on this important veterinary pathogen. Genome-wide analyses of drug resistance, combining evidence from three distinct experiments, identified selection around known candidate genes for benzimidazole, levamisole and ivermectin resistance, as well as novel regions associated with ivermectin and moxidectin resistance. These insights into contemporary and historic genetic selection further emphasise the importance of contiguous genome assemblies in interpreting genome-wide genetic variation associated with drug resistance and identified key loci to prioritise in developing diagnostic markers of anthelmintic resistance to support parasite control. AUTHOR SUMMARYUnderstanding the genetics of anthelmintic resistance is a critical part of the sustainable control of parasitic worms. Here, we have generated a chromosome-scale genome assembly for the gastrointestinal nematode, Teladorsagia circumcincta, one of the most important pathogens of sheep in temperate climates worldwide. This genome and its annotation offers a substantial improvement over existing genetic resources, and will enable new insight into the biology for this parasite and new opportunities to characterise therapeutic targets such as drug and vaccine candidates. We used this resource to map genetic variation associated with resistance to multiple anthelmintic drug classes used as the primary means of parasite control, confirming known candidate genes and variants (i.e., beta-tubulin isotypes 1 and 2 associated with benzimidazole resistance, and acr-8 associated with levamisole resistance, and pgp-9 copy number variation, of which overexpression is associated broadly with anthelmintic resistance) and revealing new regions of the genome associated with drug treatment responses (i.e. 34-38 Mb on chromosome 5 associated with ivermectin resistance). Our results highlight the importance of a contiguous genome assembly and the use of complementary experimental approaches to reveal the impact of drug-mediated selection, both historically and directly in response to treatment, on genome-wide genetic variation.

genomics↗

bioRxiv2024

Timing antigenic escape in multiple myeloma treated with T-cell redirecting immunotherapies

Recent data highlight genomic events driving antigen escape as a recurring cause of chimeric antigen receptor T-cell (CAR-T) and bispecific T-cell engager (TCE) resistance in multiple myeloma (MM). Yet, it remains unclear if these events, leading to clonal dominance at progression, result from acquisition under treatment selection or selection of pre-existing undetectable clones. This differentiation gains importance as these immunotherapies progress to earlier lines of treatment, prompting the need for innovative diagnostic testing to detect these events early on. By reconstructing phylogenetic trees and exploring chemotherapy mutational signatures as temporal barcodes in 11 relapsed refractory MM patients with available whole genome sequencing data before and after CART/TCE treatment, we demonstrated that somatic antigen escape mechanisms for BCMA- and GPRC5D-targeting therapies are acquired post-diagnosis, likely during CART/TCE treatment. Longitudinal tracking of these mutations using digital PCR in 4 patients consistently showed that genomic events promoting antigen escape were not detectable during the initial months of therapy but began to emerge nearly 1 year post therapy initiation. This finding reduces the necessity for a diagnostic panel to identify these events before CART/TCE. Instead, it underscores the importance of surveillance and identifying patients at higher risk of acquiring these events. KEY POINTSO_LIGenomic events driving antigen escape are recurrent mechanisms of resistance to CART and T-cell engagers in multiple myeloma. C_LIO_LIUsing chemotherapy mutational signatures, we demonstrated that these events are most likely acquired during treatment. C_LI

genomics↗

bioRxiv2024

Prevalent fast evolution of genes involved in heterochromatin functions

Heterochromatin is a gene-poor and repeat-rich genomic compartment universally found in eukaryotes. Despite its low transcriptional activity, heterochromatin plays important roles in maintaining genome stability, organizing chromosomes, and suppressing transposable elements (TEs). Given the importance of these functions, it is expected that the genes involved in heterochromatin regulation would be highly conserved. Yet, a handful of these genes were found to evolve rapidly. To investigate whether these previous findings are anecdotal or general to genes modulating heterochromatin, we compile an exhaustive list of 106 candidate genes involved in heterochromatin functions and investigate their evolution over short and long evolutionary time scales in Drosophila. Our analyses find that these genes exhibit significantly more frequent evolutionary changes, both in the forms of amino acid substitutions and gene copy number change, when compared to genes involved in Polycomb-based repressive chromatin. While positive selection drives amino acid changes within both structured domains with diverse functions and intrinsically disordered regions (IDRs), purifying selection may have maintained the proportions of IDRs of these proteins. Together with the observed negative associations between evolutionary rates of these genes and genomic TE abundance, we propose an evolutionary model where the fast evolution of genes involved in heterochromatin functions is an inevitable outcome of the unique functional roles of heterochromatin, while the rapid evolution of TEs may be an effect rather than cause. Our study provides an important global view of the evolution of genes involved in this critical cellular domain and provides insights into the factors driving the distinctive evolution of heterochromatin.

evolutionary biology↗

bioRxiv2023

Toxicokinetic Characterization of the Inter-Species Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semi-Quantification

N-(1,3-Dimethylbutyl)-N-Phenyl-P-Phenylenediamine-Quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire anti-degradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations surpassing those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semi-quantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkia lewisi) and non-fry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might more effectively detoxify 6PPD-Q. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semi-quantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being invaluable to environmental monitoring and risk assessment.

pharmacology and toxicology↗

bioRxiv2022

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant SMO and GLI into Glioma Primary Cilia

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF) enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO and GLI2 expression are more aggressive but the relationship to cilia is unclear. Previous studies showed increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here we show SMO accumulation is due to increased ciliary, but not extraciliary ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia, but not in NIH3T3 fibroblast cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449 and cyclopamine. Finally, temozolomide chemotherapy which increases ARL13B expression in glioma, stimulates SMO and GLI2 into glioma cilia, but not fibroblast cilia. Collectively, our data suggest factors that elevate ARL13B may drive drug-resistant SMO and GLI into cilia. This suggests the ARL13B-associated mechanism that leads to ciliary SMO/GLI recruitment may promote treatment resistance in glioma.

cancer biology↗

bioRxiv2021

Genomic landscape of drug response reveals novel mediators of anthelmintic resistance

Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to improving the efficacy and sustainability of parasite control. Here, we use a genetic cross in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies novel alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor, cky-1, in ivermectin resistance. This gene is within a locus under selection in ivermectin resistant populations worldwide; functional validation using knockout experiments supports a role for cky-1 overexpression in ivermectin resistance. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode, and identifies variants for the development of molecular diagnostics to combat drug resistance in the field. One-Sentence SummaryGenetic mapping of known and novel anthelmintic resistance-associated alleles in a multi-drug resistant parasitic nematode

genomics↗

bioRxiv2021

Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and recovery in a parasitic nematode

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and recovery in parasitic helminths. Author SummaryParasitic helminths (worms) infect people and animals throughout the world and are largely controlled with mass administration of anthelmintic drugs. There are a very limited number of anthelmintics available and parasitic helminths can rapidly develop resistance to these drugs. Ivermectin is a widely used anthelmintic in both humans and animals, but resistance is now widespread in the veterinary field. We crossed ivermectin resistant and ivermectin susceptible parasitic helminths and treated them with ivermectin or left them as untreated controls. This provided resistant and susceptible populations with a similar genetic background with which to study differences in gene expression associated with ivermectin resistance. We identified upregulation of a gene with no previous association with drug resistance (HCON_00155390:cky-1) in male and female worms in all resistant populations. This gene is thought to be expressed in the helminth pharynx (mouthpart) and, in mammals, plays a role in controlling nerve function and protecting nerves from damage. This is consistent with the known effects of ivermectin in inhibiting helminth feeding through pharyngeal paralysis and implicates a novel mechanism that allows resistant worms to survive treatment.

genomics↗

bioRxiv2021

EEG-Based Focus Estimation Using Neurable's Enten Headphones and Analytics Platform

We introduce Neurables research on focus using our recently developed Enten EEG headphones. First we quantify Entens performance on standard EEG protocols, including eyes-closed alpha rhythms, auditory evoked response and the P300 event-related potential paradigm. We show that Entens performance is on-par with established industry-standard hardware. We then introduce a series of experimental tasks designed to mimic how focus might be maintained or disrupted in a real-world office setting. We show that (A) these tasks induce behavioral changes that reflect underlying changes in focus levels and (B) our proprietary algorithm detects these changes across a large number of sessions without needing to adjust the model per participant or recording session. Through manipulation of our experimental protocol, we show that our algorithm is not dependent on gross EMG artifacts and it is driven by changes in EEG. Finally, we evaluated the models performance on the same subject across several days, and show that performance remained consistent over time. Our model correctly captured 80% {+/-} 4.1% of distractions present in our experiments with statistical significance. This indicates that our model generalizes across subjects, time points, and conditions. Our findings are based on EEG data collected from 132 participants across 337 sessions and 45 different experiments.

neuroscience↗

bioRxiv2020

Does gravity shape internal representations of space for human 3D perception?

Human 3D perception of visual objects is flawed by distortions, which are influenced by non-visual factors, such as gravitational vestibular signals. Whether gravity acts specifically on the visual system or at a higher, modality-independent, level of information processing remains unknown. To test these modality-specific vs modality-independent hypotheses, we performed experiments comparing visual versus haptic 3D shape perception in normo-gravity and microgravity. The results obtained for upright and supine posture in 1g show that visual and haptic perceptual anisotropies are systematically in opposing ego-centered, but not gravity-centered, directions suggesting they share a common origin. On the other hand, microgravity significantly modulates both visual and haptic perceptual distortion in the same direction. Overall, our results show a clear link between the visual and haptic perceptual distortions and demonstrate a role of gravity-related signals on a modality-independent internal representation of 3D space used to interpret incoming sensory inputs.

neuroscience↗