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De novo assembly of plasmodium interspersed repeat (pir) genes from Plasmodium vivax RNAseq data suggests geographic conservation of sub-family transcription.
No full textBACKGROUND: The plasmodium interspersed repeats (pir) multigene family is found across malaria parasite genomes, first discovered in the human-infecting species Plasmodium vivax, where they were initially named the virs. Their function remains unknown, although studies have suggested a role in virulence of the asexual blood stages. Sub-families of the P. vivax pir/virs have been identified, and are found in isolates from across the world, however their transcription at different localities and in different stages of the life cycle have not been quantified. Multiple transcriptomic studies of the parasite have been conducted, but many map the pir reads to existing reference genomes (as part of standard bioinformatic practice), which may miss members of the multigene family due to its inherent variability. This obscures our understanding of how the pir sub-families in P. vivax may be contributing to human/vector infection. RESULTS: To overcome the issue of hidden pir diversity from utilising a reference genome, we employed de novo transcriptome assembly to construct the pir 'reference' of different parasite isolates from published and novel RNAseq datasets. For this purpose, a pipeline was written in Nextflow, and first tested on data from the rodent-infecting P. c. chabaudi parasite to ascertain its efficacy on a sample with a full, genome-based set of pir gene sequences. The pipeline assembled hundreds of pirs from the studies included. By performing BLAST sequence identity comparisons with reference genome pirs (including P. vivax and related species) we found a clustered network of transcripts which corresponded well with prior sub-family annotations, albeit requiring some updated nomenclature. Mapping the RNAseq datasets to the de novo transcriptome references revealed that the transcription of these updated pir gene sub-families is generally consistent across the different geographical regions. From this transcriptional quantification, a time course of mosquito bloodmeals (after feeding on an infected patient) highlighted the first evidence of ookinete stage pir transcription in a human-infective malaria parasite. CONCLUSIONS: De novo transcriptome assembly is a valuable tool for understanding highly variable multigene families from Plasmodium spp., and with pipeline software these can be applied more easily and at scale. Despite a global distribution, P. vivax has a conserved pir sub-family structure-both in terms of genome copy number and transcription. We suggest that this indicates important roles of the distinct sub-families, or a genetic mechanism maintaining their preservation. Furthermore, a burst of pir transcription in the mosquito stages of development is the first glint of ookinete pir expression for a human-infective malaria parasite, suggesting a role for the gene family at a new stage of the lifecycle
Dissecting the cell cycle regulation, DNA damage sensitivity and lifespan effects of caffeine in fission yeast.
No full textCaffeine can modulate cell cycle progression, override DNA damage checkpoint signalling and increase chronological lifespan (CLS) in various model systems. Early studies suggested that caffeine inhibits the phosphatidylinositol 3-kinase-related kinase (PIKK) Rad3 to override DNA damage-induced cell cycle arrest in fission yeast. We have previously suggested that caffeine modulates cell cycle progression and lifespan by inhibiting the Target of Rapamycin Complex 1 (TORC1). Nevertheless, whether this inhibition is direct or not, has remained elusive. TORC1 controls metabolism and mitosis timing by integrating nutrients and environmental stress response (ESR) signalling. Nutritional or other stresses activate the Sty1-Ssp1-Ssp2 (AMP-activated protein kinase complex, AMPK) pathway, which inhibits TORC1 and accelerates mitosis through Sck2 inhibition. Additionally, activation of the ESR pathway can extend lifespan in fission yeast. Here, we demonstrate that caffeine indirectly activates Ssp1, Ssp2 and the AMPKβ regulatory subunit Amk2 to advance mitosis. Ssp2 is phosphorylated in an Ssp1-dependent manner following exposure to caffeine. Furthermore, Ssp1 and Amk2, are required for resistance to caffeine under conditions of prolonged genotoxic stress. The effects of caffeine on DNA damage sensitivity are uncoupled from mitosis in AMPK pathway mutants. We propose that caffeine interacts synergistically with other genotoxic agents to increase DNA damage sensitivity. Our findings show that caffeine accelerates mitotic division and is beneficial for CLS through AMPK. Direct pharmacological targeting of AMPK may serve towards healthspan and lifespan benefits beyond yeasts, given the highly conserved nature of this key regulatory cellular energy sensor
DYRK1A syndrome presenting with a familial exudative vitreoretinopathy (FEVR)-like retinovascular phenotype.
No full textINTRODUCTION: The DYRK1A gene plays a crucial role in central nervous system development, with haploinsufficiency leading to DYRK1A-related intellectual disability syndrome. Ocular manifestations are common in DYRK1A syndrome and include refractive error, strabismus and optic nerve hypoplasia. Retinal involvement, however is less frequently reported and remains uncharacterised. METHODS: We conducted comprehensive ocular and systemic evaluations in two unrelated individuals with familial exudative vitreoretinopathy (FEVR)-like presentations and de novo DYRK1A variants. Genetic testing included whole genome sequencing with variant interpretation based on clinical guidelines. RESULTS: Patient 1 had a previously reported recurrent pathogenic DYRK1A variant [c.1282C>T; p.(Arg428Ter)], whilst Patient 2 had a novel missense likely pathogenic variant [c.857T>C; p.(Leu286Pro)]. Both patients demonstrated systemic features consistent with DYRK1A syndrome. DISCUSSION: These cases confirm vitreoretinal involvement as an associated finding in DYRK1A syndrome and highlight FEVR-like retinovascular abnormalities as a potential diagnostic clue for the condition in individuals with neurodevelopmental disorders.</p
Probing the molecular determinants of Ty1 retrotransposon restriction specificity in yeast.
No full textThe evolutionary history of retrotransposons and their hosts shapes the dynamics of transposition and restriction. The Pseudoviridae of yeast includes multiple Ty1 LTR-retrotransposon subfamilies. Saccharomyces cerevisiae prevents uncontrolled retrotransposition of Ty1 subfamilies using distinct mechanisms: canonical Ty1 is inhibited by a self-encoded restriction factor, p22/p18, whereas Ty1' is inhibited by an endogenized restriction factor, Drt2. The minimal inhibitory fragment of both restriction factors (p18m and Drt2m) is a conserved C-terminal capsid domain. Here, we use biophysical and genetic approaches to demonstrate that p18m and Drt2m are highly specific to their subfamilies. Although the crystal structures of p18m and Drt2m are similar, three divergent residues found in a conserved hydrophobic interface direct restriction specificity. By mutating these three residues, we re-target each restriction factor to the opposite transposon. Our work highlights how a common lattice-poisoning mechanism of restriction evolved from independent evolutionary trajectories in closely related retrotransposon subfamilies. These data raise the possibility that similar capsid-capsid interactions may exist in other transposons/viruses and that highly specific inhibitors could be engineered to target capsid interfaces
The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks
No full textUnderstanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart
Drosophila AHR limits tumor growth and stem cell proliferation in the intestine
No full textBackground
The aryl hydrocarbon receptor (AHR) plays important roles in intestinal homeostasis, limiting tumour growth and promoting differentiation in the intestinal epithelium. Spineless, the Drosophila homolog of AHR, has only been studied in the context of development but not in the adult intestine.
Methods
The role of Spineless in the Drosophila midgut was studied by overexpression or inactivation of Spineless in infection and tumour models and RNA sequencing of sorted midgut progenitor cells.
Results
We show that spineless is upregulated in the adult intestinal epithelium after infection with Pseudomonas entomophila (P.e.). Spineless inactivation increased stem cell proliferation following infection-induced injury. Spineless overexpression limited intestinal stem cell proliferation and reduced survival after infection. In two tumour models, using either Notch RNAi or constitutively active Yorkie, Spineless suppressed tumour growth and doubled the lifespan of tumour-bearing flies. At the transcriptional level it reversed the gene expression changes induced in Yorkie tumours, counteracting cell proliferation and altered metabolism.
Conclusions
These findings demonstrate a new role for Spineless in the adult Drosophila midgut and highlight the evolutionarily conserved functions of AHR/Spineless in the control of proliferation and differentiation of the intestinal epithelium
HIF2α: the interface between oxygen-sensing systems in physiology and pathology.
No full textMore than 100 years after the original descriptions of altitude adaptation, it is now clear that many of these responses are mediated by a specific isoform of the transcription factor hypoxia-inducible factor (HIF-2α). Here, we review this work, including connectivity with the oxygen chemosensitive response itself, and with paraganglioma, a tumour often affecting chemosensitive tissues
A new approach to large multiomics data integration.
No full textData reduction and data mining are common practices for handling large-scale data from wide-ranging sources, but high-dimensional omics and imaging data sets present difficult challenges for feature extraction and data mining due to the large number of features that cannot be simultaneously examined. The sample numbers and variables in these methods are constantly growing as new technologies are developed, and computational analysis needs to evolve to keep up with growing demand. In recent years, there has been a rapid uptake of nonlinear dimensionality reduction via methods such as t-distributed stochastic neighbor embedding and uniform manifold approximation and projection. These approaches have revolutionized our ability to visualize and interpret high-dimensional data and have rapidly become preferred methods for analysis of data sets containing an extremely high number of variables. Further to this is the emerging interest in combining information from multiple omics sources to gain a more holistic view of systems biology. Current state-of-the-art algorithms can perform data mining, visualization, and classification on routine data sets but struggle when data sets grow above a certain size. We present a new approach to large and multiomic data integration to extract, mine, and integrate large multiomics data sets that were previously considered prohibitively large. Here, we demonstrate the use of deep learning on subsampled nonlinear dimensionality reduction using t-SNE and UMAP to extract features from large complex data sets including mass spectrometry imaging and chromosome conformation capture. We then go on to demonstrate how this method can be used to learn embeddings from the fusion of different omics data, allowing metabolomics data to be projected into a reduced transcriptomics representation
Mycobacterium tuberculosis biology, pathogenicity and interaction with the host.
No full textSince the release of the first Mycobacterium tuberculosis genome in 1998, major advances have been made in understanding the biology of this pathogen, the leading infectious cause of death in modern human history. In this Review, we outline the physiological and metabolic features thought to underpin the survival, evasion and subversion strategies employed by M. tuberculosis as it drives a cycle of transmission, infection and disease in its obligate human host. We also consider adaptations to key host innate immune effectors, including the roles of granulocytes, phagosomal damage and repair, autophagy and cell death in determining host-mycobacterium outcomes. Given the increasing awareness of the importance of asymptomatic M. tuberculosis infection and transmission, we advocate for the need to ensure greater intersection between laboratory and clinical research, taking into account the environmental context in which natural infection and disease occur. We identify knowledge gaps in the field and reflect on the opportunities and challenges for integrating host, bacterium and environment into future investigations to inform intervention strategies to control tuberculosis disease
Discordance between measures of Mycobacterium tuberculosis sensitization and type 2 diabetes mellitus in the United States (NHANES): A population-based cohort study.
No full textOBJECTIVE: We examined how latent TB infection (LTBI), evaluated by cell-mediated immune responses to Mycobacterium tuberculosis (Mtb) antigens, impacts glucose metabolism in US adults. METHODS: Mtb sensitization was evaluated by interferon-γ (IFN-γ) release assay (IGRA+: assay reactivity) and tuberculin skin testing (TST+: skin induration ≥10 mm), and categorized as: IGRA-/TST- (TB uninfected controls); IGRA-/TST+; IGRA+/TST-; or IGRA+/TST+. Diabetes was ascertained by fasting plasma glucose (FPG) ≥7.0 mmol/L, HbA1c ≥6.5% and/or antidiabetic medication. Adjusted generalized additive models examined nonlinear effects of skin induration and IFN-γ reactivity on FPG and HbA1c; and LTBI on diabetes prevalence. RESULTS: Among 1787 (IGRA-/TST-), 101 (IGRA-/TST+), 92 (IGRA+/TST-), and 99 (IGRA+/TST+) adults, skin induration linearly associated with FPG [effective degrees of freedom (EDF) =1.01; p<0.001] and non-linearly with HbA1c [EDF=1.76; p=0.003]. IFN-γ reactivity correlated with neither FPG [p=0.58] nor HbA1c [p=0.94]. Relatedly, adjusted diabetes prevalence was greater in IGRA-/TST+ [24.9%; p=0.048] and IGRA+/TST+ [27.3%; p=0.004] but not IGRA+/TST- [15.9%; p=0.69] individuals than among controls [15.3%]. CONCLUSIONS: LTBI associated with glycemic measures and diabetes when assessed by skin induration, but not IFN-γ release. This suggests an association with innate immune activation rather than acquired T-cell response, as determined by ex vivo IFN-γ release assay