Biology subjects

Holroyd, N.

Publications and source records attributed to Holroyd, N..

At least 19 recordsLinked to original sources

bioRxiv2026

The histone code of love: epigenetics of maturation of gonads in the human blood fluke Schistosoma mansoni

Schistosoma mansoni is a parasitic flatworm that has two, genetically determined, sexes. We used aggregated data of 8 posttranslational histone modifications (ChIP-Seq), chromatin accessibility (ATAC-Seq), transcription (RNA-Seq) and genome feature annotations to decipher the histone code of genes involved in the differentiation of schistosome gonads (i.e. female ovaries and male testes). We show schistosome gonads express at least two classes of protein coding genes: H3K4me3-positive genes that display canonical features of eukaryotic protein-coding genes such as peaks of H3K4me3 at the transcription start sites (TSS) and increases in histone acetylation marks towards the transcription end site (TES), but also a non-canonical H3K9/27me3 plateau just upstream of the TSS. H3K4me3 enrichment at the TSS is highly predictive for transcription strength in these genes compared to a second class of protein coding genes (H3K4me3-negative genes) that do not display this pattern and is characterised by absence of the investigated histone marks at TSS and TES. This is indicative of the existence of hitherto unknown, potentially schistosome-specific histone marks in these genes. The absence of H3K4me3 at the TSS is not associated with inducible or stable gene expression in the gonads. Instead, gene ontology analysis indicates that H3K4me3-positive genes are related to functions which typically govern processes such as metabolism or signal transduction while H3K4me3-negative genes are dedicated to cell communication or immune responses. Second, individual histone modifications and their combinations are associated with functional features of the schistosome genome, known as "chromatin colours". In H3K4me3-positive genes, there is clear co-linearity of 3 colours, which strongly suggests a functional role for histone modifications in the control of transcription pre-initiation, promotor release, and transcription termination. Third, there are striking chromatin structure changes during maturation of the gonads in all genomic features including protein-coding and non-protein coding genes as well as repetitive sequences. The nature of these changes is different in both sexes. H3K36me3 and H3K9me3, as well as H3K23ac and H3K9ac show the strongest variations. Last, we show that pharmacological inhibition of histone demethylation activity by IOX1 leads to a concentration-dependent separation ("divorce") of schistosome couples confirming the importance of H3K36/H3K9 methylation for pairing maintenance and indicating histone demethylases as a potential drug target family. Collectively, our findings offer unprecedented insights into histone codes and chromatin dynamics governing the reproductive development of S. mansoni gonads.

developmental biology↗

bioRxiv2025

Canonical WNT signalling governs Echinococcus metacestode development

Alveolar echinococcosis (AE) is a lethal zoonosis caused by infiltrative growth of the metacestode larva of the tapeworm Echinococcus multilocularis in host organs. We previously showed that the Echinococcus metacestode is an evolutionarily unique, broadly posteriorized tissue, leading us to hypothesize that canonical WNT (cWNT) signalling--which patterns the body axis across metazoans--might be critical for metacestode formation. Here, we report effective RNAi-mediated knockdown of the E. multilocularis {beta}-catenin gene (bcat-1), the central effector of cWNT signalling, in a primary parasite cell culture system that produces metacestode vesicles. bcat-1(RNAi) cultures were markedly impaired in vesicle formation, exhibited stem-cell hyperproliferation, and displayed disrupted muscle-fibre organisation. Genome-wide transcriptomics revealed a general anteriorization of gene expression, and in situ hybridization showed an overproduction of cells expressing head-inducing factors such as sfrp upon bcat-1 knockdown. Conversely, metacestode-specific genes--including the tegumental factors muc-1, TNFR, and antigen B--as well as the posterior marker post2b were significantly downregulated, consistent with the observed vesicle-formation defects. In situ analyses further identified anterior markers--frizzled-10, nou-darake, notum, and follistatin--that were overexpressed in bcat-1(RNAi) cultures and localized to the future anterior pole at the earliest stages of protoscolex formation. Finally, pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior tissue in Echinococcus protoscoleces, killed metacestode vesicles, and reduced stem-cell proliferation at nanomolar concentrations. Together, these findings establish a central role for cWNT signalling in directing Echinococcus body-axis formation and the posteriorization events driving metacestode growth within the host, providing insight into asexual parasite proliferation mediated by this biologically unique larval stage and pointing to potential targets for chemotherapy against AE. Author SummaryAlveolar echinococcosis (AE) is a lethal disease caused by the cancer-like growth of the metacestode larva of the tapeworm Echinococcus multilocularis. From a developmental perspective, the Echinococcus metacestode is an unusual biological structure and even atypical among tapeworms. Previous work indicated that metacestode formation involves re-patterning of the body axis, eliminating head structures and producing broadly posteriorized tissue. How this is controlled at the molecular and cellular levels, however, was unknown. In this study, we perturbed expression of the {beta}-catenin gene (bcat-1), a central regulator of canonical WNT signalling, using RNA interference (RNAi). bcat-1(RNAi) parasite cultures failed to generate metacestode vesicles and instead showed stem-cell hyperproliferation and muscle-cell distortion. Genes required for posteriorized metacestode tissue were downregulated, whereas genes directing head formation in adult worms (follistatin, sfrp, fz10, ndk) were upregulated, indicating a general anteriorization of the culture system. Pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior structures in protoscoleces, reduced stem-cell proliferation, and killed metacestode tissue. These findings identify {beta}-catenin and the canonical WNT pathway as crucial regulators of the posteriorization that underlies metacestode formation. Given that WNT signalling is deregulated in many human cancers and that small-molecule inhibitors are available, our results suggest new avenues for anti-AE drug development.

developmental 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↗

bioRxiv2023

Physics-informed deep generative learning for quantitative assessment of the retina

Disruption of retinal vasculature is linked to various diseases, including diabetic retinopathy and macular degeneration, leading to vision loss. We present here a novel algorithmic approach that generates highly realistic digital models of human retinal blood vessels based on established biophysical principles, including fully-connected arterial and venous trees with a single inlet and outlet. This approach, using physics-informed generative adversarial networks (PI-GAN), enables the segmentation and reconstruction of blood vessel networks that requires no human input and out-performs human labelling. Our findings highlight the potential of PI-GAN for accurate retinal vasculature characterization, with implications for improving early disease detection, monitoring disease progression, and improving patient care.

biophysics↗

bioRxiv2023

A drug repurposing screen for whipworms informed by comparative genomics

Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of[≤] 50 M against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug repurposing against whipworms. Therefore, we recommend that prior to in vivo studies, future researchers first assess drug absorption by the host, for example, using human intestinal organoids or cell lines, and drug uptake by whipworms using intestinal organoids infected with T. muris.

pharmacology and toxicology↗

bioRxiv2022

Three-dimensional imaging and single-cell transcriptomics of the human kidney implicate perturbation of lymphatics in alloimmunity

Studies of the structural and molecular features of the lymphatic vasculature, which clears fluid, macromolecules and leukocytes from the tissue microenvironment, have largely relied on animal models, with limited information in human organs beyond traditional immunohistochemical assessment. Here, we use three-dimensional imaging and single-cell RNA-sequencing to study lymphatics in the human kidney. We found a hierarchical arrangement of lymphatic vessels within human kidneys, initiating along specialised nephron epithelium in the renal cortex and displaying a distinct, kidney-specific transcriptional profile. In chronic transplant rejection we found kidney allograft lymphatic expansion alongside a loss of structural hierarchy, with human leukocyte antigen-expressing lymphatic vessels infiltrating the medulla, presenting a putative target for alloreactive antibodies. This occurred concurrently with lymphatic vessels invading and interconnecting tertiary lymphoid structures at early stages of lymphocyte colonisation. Analysis of intercellular signalling revealed upregulation of co-inhibitory molecule-mediated CD4+ T cell-lymphatic crosstalk in rejecting kidneys, potentially acting to limit local alloimmune responses. Overall, we delineate novel structural and molecular features of human kidney lymphatics and reveal perturbations to their phenotype and transcriptome in the context of alloimmunity. SUMMARYLymphatics regulate fluid balance and immune cell accumulation but are under-studied in human organs such as the kidney. Jafree and colleagues profiled human kidney lymphatics using three-dimensional imaging and single-cell RNA-sequencing, revealing structural and transcriptional perturbations in rejecting kidney transplants.

immunology↗

bioRxiv2022

Genome-wide analysis of Schistosoma mansoni reveals population structure and praziquantel drug selection pressure within Ugandan hot-spot communities

Populations within schistosomiasis control areas, especially those in Africa, are recommended to receive regular mass drug administration (MDA) with praziquantel (PZQ) as the main strategy for controlling the disease. The impact of PZQ treatment on schistosome genetics remains poorly understood, and is limited by a lack of high-resolution genetic data on the population structure of parasites within these control areas. We generated whole-genome sequence data from 174 individual miracidia collected from both children and adults from fishing communities on islands in Lake Victoria in Uganda that had received either annual or quarterly MDA with PZQ over four years, including samples collected immediately before and four weeks after treatment. Genome variation within and between samples was characterised and we investigated genomic signatures of natural selection acting on these populations that could be due to PZQ treatment. The parasite population on these islands was more diverse than found in nearby villages on the lake shore. We saw little or no genetic differentiation between villages, or between the groups of villages with different treatment intensity, but slightly higher genetic diversity within the pre-treatment compared to post-treatment parasite populations. We identified classes of genes significantly enriched within regions of the genome with evidence of recent positive selection among post-treatment and intensively treated parasite populations. The differential selection observed in post-treatment and pre-treatment parasite populations could be linked to any reduced susceptibility of parasites to praziquantel treatment. Author summarySchistosomiasis is caused by parasitic helminths of the genus Schistosoma. Schistosoma mansoni is the primary cause of intestinal schistosomiasis, a devastating and widespread parasitic infection that causes morbidity, death and socio-economic impact on endemic communities across the world and especially sub-Saharan Africa. Using whole-genome sequencing, we were able to elucidate the parasite population within Lake Victoria island fishing communities in Uganda which are among the major hotspots for schistosomiasis. We further assessed genetic markers that might be linked to recent observations concerning reduced susceptibility to praziquantel, the major drug used in the treatment of this disease. Whole-genome data on the population genetics of S. mansoni in an African setting will provide a strong basis for future functional genomics or transcriptomic studies that will be key to identifying drug targets, improving existing drugs or developing new therapeutic interventions.

genomics↗

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

Assembled chromosomes of the blood fluke Schistosoma mansoni provide insight into the evolution of its ZW sex-determination system

BackgroundSchistosoma mansoni is a flatworm that causes a neglected tropical disease affecting millions worldwide. Most flatworms are hermaphrodites but schistosomes have genotypically determined male (ZZ) and female (ZW) sexes. Sex is essential for pathology and transmission, however, the molecular determinants of sex remain unknown and is limited by poorly resolved sex chromosomes in previous genome assemblies. ResultsWe assembled the 391.4 Mb S. mansoni genome into individual, single-scaffold chromosomes, including Z and W. Manual curation resulted in a vastly improved gene annotation, resolved gene and repeat arrays, trans-splicing, and almost all UTRs. The sex chromosomes each comprise pseudoautosomal regions and single sex-specific regions. The Z-specific region contains 932 genes, but on W all but 29 of these genes have been lost and the presence of five pseudogenes indicates that degeneration of W is ongoing. Synteny analysis reveals an ancient chromosomal fusion corresponding to the oldest part of Z, where only a single gene--encoding the large subunit of pre-mRNA splicing factor U2AF--has retained an intact copy on W. The sex-specific copies of U2AF have divergent N-termini and show sex-biased gene expression. ConclusionOur assembly with fully resolved chromosomes provides evidence of an evolutionary path taken to create the Z and W sex chromosomes of schistosomes. Sex-linked divergence of the single U2AF gene, which has been present in the sex-specific regions longer than any other extant gene with distinct male and female specific copies and expression, may have been a pivotal step in the evolution of gonorchorism and genotypic sex determination of schistosomes.

genomics↗

bioRxiv2021

The parasitic nematode Strongyloides ratti exists as populations of long-lived asexual lineages.

Nematodes are important parasites of people and animals and in natural ecosystems they are a major ecological force. Strongyloides ratti is a common parasitic nematode of wild rats and we have investigated its population genetics using single worm, whole genome sequencing. We find that S. ratti populations in the UK consist of mixtures of asexual lineages that are widely dispersed across a host population. These parasite lineages are likely very old and may have originated in Asia from where rats originated. Genes that underly the parasitic phase of the parasites life cycle are hyperdiverse, compared with the rest of the genome, and this may allow the parasites to maximise their fitness in a diverse host population. These patterns of parasitic nematode population genetics have not been found before and may also apply to Strongyloides spp. that infect people, which will affect how we should approach their control.

genetics↗

bioRxiv2021

Daily rhythms in the transcriptomes of the human parasite Schistosoma mansoni

BackgroundThe consequences of the earths daily rotation have led to 24-hour biological rhythms in most organisms. Even parasites have daily rhythms, which, when in synchrony with host rhythms, optimize their fitness. Understanding these rhythms may enable development of novel control strategies that take advantage of rhythmic vulnerabilities. Recent work on blood-dwelling protozoan parasites has revealed daily rhythms in gene expression, physiology, drug sensitivity and the presence of an intrinsic circadian clock. However, similar studies on metazoan parasites are lacking. The aims of this study were to investigate if a metazoan parasite has daily molecular oscillations, whether they give us insight into how these longer-lived organisms can survive host daily cycles over a life-span of many years and to determine whether canonical metazoan circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni, that lives in the vasculature for decades and causes the serious neglected tropical disease schistosomiasis. ResultsUsing round-the-clock transcriptomics of male and female adult worms we discovered that [~]2% of its genes followed a daily pattern of expression. Rhythmic processes included a night-time stress response and a day-time metabolic rush hour. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include drug targets and a vaccine candidate. These 24hr rhythms may be driven by host rhythms and/or generated by a circadian clock. However, core clock genes are missing and orthologues of secondary clock genes show no 24hr rhythmicity in transcript abundance. ConclusionsThe daily rhythms identified here reveal temporally compartmentalised internal processes and host interactions over the daily cycle, including processes relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other Metazoa. Most importantly, knowing which gene transcripts oscillate at this temporal scale is relevant to functional genomic studies that will lead to the development and delivery of therapeutics against schistosomiasis.

systems biology↗

bioRxiv2021

Multiscale three-dimensional imaging of intact human organs down to the cellular scale using hierarchical phase-contrast tomography

Human organs are complex, three-dimensional and multiscale systems. Spatially mapping the human body down through its hierarchy, from entire organs to their individual functional units and specialised cells, is a major obstacle to fully understanding health and disease. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique utilising the European Synchrotron Radiation Facilitys Extremely Brilliant Source: the worlds first high-energy 4th generation X-ray source. HiP-CT enabled three-dimensional and non-destructive imaging at near-micron resolution in soft tissues at one hundred thousand times the voxel size whilst maintaining the organs structure. We applied HiP-CT to image five intact human parenchymal organs: brain, lung, heart, kidney and spleen. These were hierarchically assessed with HiP-CT, providing a structural overview of the whole organ alongside detail of the organs individual functional units and cells. The potential applications of HiP-CT were demonstrated through quantification and morphometry of glomeruli in an intact human kidney, and identification of regional changes to the architecture of the air-tissue interface and alveolar morphology in the lung of a deceased COVID-19 patient. Overall, we show that HiP-CT is a powerful tool which can provide a comprehensive picture of structural information for whole intact human organs, encompassing precise details on functional units and their constituent cells to better understand human health and disease.

physiology↗

bioRxiv2020

Strongyle-resistant sheep express their potential across environments and leave limited scope for parasite plasticity

IntroductionDrug-resistant parasites threaten livestock production. Breeding more resistant hosts could be a sustainable control strategy. Environmental variation may however alter the expression of genetic potential and directional selection toward host resistance could initiate an arms race between the host and its parasites. Methods and ResultsWe created sheep lines with high or low resistance to Haemonchus contortus. We first exposed both lines to chronic stress or to the infection by another parasite Trichostrongylus colubriformis, to test for genotype-by-environment and genotype-by-parasite species interactions respectively. Overall, between-line divergence remained significant across environmental perturbations. But we found that the impact of chronic stress on H. contortus infection varied among families and that divergence was reduced during infection by T. colubriformis. Second, we quantified genomic and transcriptomic differences in H. contortus worms collected from both lines to identify components of an arms race. We found no evidence of genetic differentiation between worms from each line. But survival to more resistant hosts was associated with enhanced expression of cuticle collagen coding genes. DiscussionBreeding for resistance hence remains a sustainable strategy that requires to anticipate the effects of environmental perturbations and to monitor worm populations.

evolutionary biology↗

bioRxiv2020

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni

At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in S. mansoni, to produce somatic mutations in the omega-1 ({omega}1) gene. Here, we employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing SULT-OR. Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.

molecular biology↗

bioRxiv2020

Complete representation of a tapeworm genome reveals chromosomes capped by centromeres, necessitating a dual role in segregation and protection

BackgroundChromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published but little is yet known about the biology of their chromosomes. Here we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference-quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution. ResultsLong-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader transsplicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes. ConclusionsDespite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

genomics↗

bioRxiv2020

Multi-fluorescence high-resolution episcopic microscopy (MF-HREM) for three dimensional imaging of adult murine organs

Three-dimensional microscopy of large biological samples (>0.5 cm3) is transforming biological research. Many existing techniques require trade-offs between image resolution, sample size and method complexity. A simple robust instrument with the potential to perform large volume 3D imaging currently exists in the form of the Optical HREM, however the development of the instrument to date is limited to single fluorescent wavelength imaging with non-specific eosin staining. This work presents developments to realize the potential of the HREM to become Multi-fluorescent High Resolution Episcopic Microscopy (MF-HREM). MF-HREM is a serial-sectioning and block-facing wide-field fluorescence imaging technique, which does not require tissue clearing or optical sectioning. Multiple developments are detailed in sample preparation and image post-processing to enable multiple specific stains in large samples, and show how these enable segmentation and quantification of the data. The application of MF-HREM is demonstrated in a variety of biological contexts: 3D imaging of whole tumor vascular networks and tumor cell invasion in xenograft tumors up to 7.5 mm3 at resolutions of 2.75 m, quantification of glomeruli volume in the adult mouse kidney, and quantification of vascular networks and white matter track orientation in adult mouse brain.

biophysics↗

bioRxiv2020

Extensive genomic and transcriptomic variation defines the chromosome-scale assembly of Haemonchus contortus, a model gastrointestinal worm

BackgroundHaemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants, and has become the key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Two draft genome assemblies for H. contortus were reported in 2013, however, both were highly fragmented, incomplete, and differed from one another in important respects. While the introduction of long-read sequencing has significantly increased the rate of production and contiguity of de novo genome assemblies broadly, achieving high quality genome assemblies for small, genetically diverse, outcrossing eukaryotic organisms such as H. contortus remains a significant challenge. ResultsHere, we report using PacBio long read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome. We show a remarkable pattern of almost complete conservation of chromosome content (synteny) with Caenorhabditis elegans, but almost no conservation of gene order. Long-read transcriptome sequence data has allowed us to define coordinated transcriptional regulation throughout the life cycle of the parasite, and refine our understanding of cis- and trans-splicing relative to that observed in C. elegans. Finally, we use this assembly to give a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally. ConclusionsThe H. contortus MHco3(ISE).N1 genome assembly presented here represents the most contiguous and resolved nematode assembly outside of the Caenorhabditis genus to date, together with one of the highest-quality set of predicted gene features. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes, and extends the experimental tractability of this model parasitic nematode in understanding pathogen biology, drug discovery and vaccine development, and important adaptive traits such as drug resistance.

genomics↗

bioRxiv2019

Population genomic evidence that human and animal infections in Africa come from the same populations of Dracunculus medinensis

BackgroundGuinea worm - Dracunculus medinensis - was historically one of the major parasites of humans and has been known since antiquity. Now, Guinea worm is on the brink of eradication, as efforts to interrupt transmission have reduced the annual burden of disease from millions of infections per year in the 1980s to only 30 human cases reported globally last year. Despite the enormous success of eradication efforts to date, one complication has arisen. Over the last few years, hundreds of dogs have been found infected with this previously apparently anthroponotic parasite, almost all in Chad. Moreover, the relative numbers of infections in humans and dogs suggests that dogs may be key in maintaining transmission in that country.\n\nResultsIn an effort to shed light on this peculiar epidemiology of Guinea worm in Chad, we have sequenced and compared the genomes of worms from dog, human and other animal infections. Confirming previous work with other molecular markers, we show that all of these worms are D. medinensis, and that the same population of worms are causing both infections, can confirm the suspected transmission between host species and detect signs of a population bottleneck due to the eradication efforts. The diversity of worms in Chad appears to exclude the possibility that there were no, or very few, worms present in the country during a 10-year absence of reported cases.\n\nConclusionsThis work reinforces the importance of adequate surveillance of both human and dog populations in the Guinea worm eradication campaign and suggests that control programs should stay aware of the possible emergence of unusual epidemiology as they approach elimination.

genomics↗