Biology subjects

Chappell, L.

Publications and source records attributed to Chappell, L..

6 recordsLinked to original sources

bioRxiv2025

Fexinidazole and Corallopyronin A target Wolbachia-infected sheath cells present in filarial nematodes

The discovery of the endosymbiotic bacteria Wolbachia as an obligate symbiont of filarial nematodes has led to antibiotic-based treatments for filarial diseases. While lab and clinical studies have yielded promising results, recent animal studies reveal that Wolbachia levels may rebound following treatment with suboptimal doses of the antibiotic rifampicin. Previous work showed that a likely source of the bacterial rebound in females were dense clusters of Wolbachia in ovarian tissue. The number, size, and density of these Wolbachia clusters were not diminished despite antibiotic treatment. Here we define the cellular characteristics of the Wolbachia clusters in Brugia pahangi (wBp) and identify drugs that also target them. We have evidence that the Wolbachia clusters originate from newly formed sheath cells adjacent to the ovarian Distal Tip Cells. The dramatically enlarged volume of an infected sheath cell is strikingly similar to endosymbiont-induced bacteriocytes found in many insect species. Ultrastructural analysis reveals that the clustered Wolbachia present within the sheath cells exhibit a distinct morphology and form direct connections with the oocyte membrane and possibly the cytoplasm. This includes membrane-based channels providing a connection between Wolbachia-infected sheath cells and oocytes. We also determined that the Wolbachia within the sheath cells are either quiescent or replicating at a very low rate. Screens of known antibiotics and other drugs revealed that two drugs, Fexinidazole and Corallopyronin A, significantly reduced the number of clustered Wolbachia located within the sheath cells.

pathology↗

bioRxiv2022

Clonal diversification and histogenesis of malignant germ cell tumours

Germ cell tumours (GCTs) are a collection of benign and malignant neoplasms derived from primordial germ cells. They are uniquely able to recapitulate embryonic and extraembryonic tissues, which carries prognostic and therapeutic significance. The developmental pathways underpinning GCT initiation and histogenesis are incompletely understood. Here, we studied the relationship of histogenesis and clonal diversification in GCTs by analysing the genome and transcriptome of 547 microdissected histological units. We found that the extensive diversification of tissues and genetic subclones were not correlated. However, we identified unifying features including the retention of fetal developmental transcripts across tissues, expression changes on chromosome 12p, and a conserved somatic evolutionary sequence of whole genome duplication followed by clonal diversification. Whilst this pattern was preserved across all GCTs, the developmental timing of the duplication varied between prepubertal and postpubertal cases. In addition, tumours of younger children exhibited distinct substitution signatures, including a novel one, which may lend themselves as potential biomarkers for risk stratification. Our findings portray the extensive diversification of GCT tissues and genetic subclones as randomly distributed, whilst identifying overarching tissue and tumour transcriptional and genomic features.

cancer biology↗

bioRxiv2020

Analysis of Plasmodium vivax schizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol "DAFT-seq" to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5 and 3 untranslated regions (UTRs), some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.

genomics↗

bioRxiv2019

Refining the transcriptome of the human malaria parasite Plasmodium falciparum using amplification-free RNA-seq

Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-hour blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90-95%. We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 75% of the genome transcribed into mRNAs. These datasets allowed us to refine the 5 and 3 untranslated regions (UTRs), which can be variable, long (>1,000 nt), and often overlap those of adjacent transcripts. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. By capturing the 5 ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle resulting in an updated view of the P. falciparum transcriptome.

genomics↗

bioRxiv2019

Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells

During thymic negative selection, medullary thymic epithelial cells (mTEC) collectively express most protein coding genes, a process termed promiscuous gene expression (PGE). Although PGE is crucial for inducing central T-cell tolerance, this process has not been established definitively as being either stochastic or coordinated. To resolve this question, we sequenced the transcriptomes of 6,894 single mTEC, including 1,795 rare cells expressing either of two tissue-restricted antigens, TSPAN8 or GP2. Transcriptional heterogeneity allowed partitioning of mTEC into 15 robustly-defined subpopulations representing distinct maturational stages and subtypes. Although 50 gene co-expression groups were robustly identified, few could be explained by chromosomal location, biological pathway, or tissue specificity. Further, GP2+ mTEC were randomly dispersed spatially within medullary islands. Thus although PGE exhibits ordered co-expression, biologically it is indeterminate. This likely enhances the presentation of diverse antigens to passing thymocytes during their medullary residency, while simultaneously maintaining mTEC identity throughout PGE.

immunology↗

bioRxiv2018

Plasmodium falciparum mature schizont transcriptome variation among clinical isolates and laboratory-adapted clones

Malaria parasite genes exhibit variation in both sequence and expression level. There is much information on sequence polymorphism, but less resolution on natural variation in transcriptomes of parasites at specific developmental stages. This is largely because it is challenging to obtain highly replicated sampling of transcriptomes to overcome potentially confounding technical and biological variation. We address the issue in the major human parasite Plasmodium falciparum by obtaining RNA-seq profiles of multiple independent replicate preparations of mature schizont-stage parasites from a panel of clinical isolates recently established in culture and from long-term laboratory-adapted clones. With a goal of robustly identifying variably expressed genes, we show that increasing the numbers of biological sample replicates greatly improves the discovery rate. Generally, six independent replicates of each parasite culture is recommendable as being significantly to lower numbers, although for highly expressed genes variable expression can be detected when fewer replicates are available. A broad comparison identifies genes differing in relative expression between cultured clinical isolates and laboratory-adapted clones. Genes more highly expressed in the laboratory-adapted clones include an AP2 transcription factor gene Pf3D7_0420300 and putative methyl transferase genes. The variable expression of several known merozoite invasion ligands is confirmed, and previously uncharacterised genes are shown to be differentially expressed among clinical isolates. New RT-qPCR assays validate the variation in transcript levels of these genes, and allow quantitation of expression to be extended to a wider panel of clinical isolate samples. These variably expressed genes are new candidates for investigation as potential determinants of alternative parasite developmental pathways or targets of immunity.\n\nAuthor summaryUnderstanding parasite diversity and adaptation may require characterisation of gene expression variation, and is vital if chemotherapeutic or vaccine development is to consider new candidate targets, but it is technically challenging to generate precise data on clinical isolates. Here, we analyse the transcriptomes of mature Plasmodium falciparum schizonts using RNA-sequencing, using large numbers of biological replicate samples to minimise the impact of inter-replicate variation on observed patterns of differential expression. This identifies genes that are differentially expressed in long term laboratory-adapted parasites and recently cultured clinical isolates, as well as among different clinical isolates. In additional samples of schizonts grown in the first cycle ex vivo prior to any erythrocyte invasion, expression levels of a selected panel of these genes vary among isolates, but mean levels are similar to those in the continuously cultured clinical isolates, indicating that the latter are useful for experimental studies requiring biological replication.

cell biology↗