Publications by authors named "Alasdair C Ivens"

25 Publications

  • Page 1 of 1

Population genomics of Escherichia coli in livestock-keeping households across a rapidly developing urban landscape.

Nat Microbiol 2022 Apr 14;7(4):581-589. Epub 2022 Mar 14.

International Institute for Environment and Development, London, UK.

Quantitative evidence for the risk of zoonoses and the spread of antimicrobial resistance remains lacking. Here, as part of the UrbanZoo project, we sampled Escherichia coli from humans, livestock and peri-domestic wildlife in 99 households across Nairobi, Kenya, to investigate its distribution among host species in this rapidly developing urban landscape. We performed whole-genome sequencing of 1,338 E. coli isolates and found that the diversity and sharing patterns of E. coli were heavily structured by household and strongly shaped by host type. We also found evidence for inter-household and inter-host sharing and, importantly, between humans and animals, although this occurs much less frequently. Resistome similarity was differently distributed across host and household, consistent with being driven by shared exposure to antimicrobials. Our results indicate that a large, epidemiologically structured sampling framework combined with WGS is needed to uncover strain-sharing events among different host populations in complex environments and the major contributing pathways that could ultimately drive the emergence of zoonoses and the spread of antimicrobial resistance.
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http://dx.doi.org/10.1038/s41564-022-01079-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8975746PMC
April 2022

A global analysis of low-complexity regions in the proteome reveals enrichment in the C-terminus of nucleic acid binding proteins providing potential targets of phosphorylation.

Wellcome Open Res 2020 18;5:219. Epub 2020 Nov 18.

Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, EH9 3JT, UK.

Low-complexity regions (LCRs) on proteins have attracted increasing attention recently due to their role in the assembly of membraneless organelles or granules by liquid-liquid phase separation. Several examples of such granules have been shown to sequester RNA and proteins in an inactive state, providing an important mechanism for dynamic post-transcriptional gene regulation. In trypanosome parasites, post-transcriptional control overwhelmingly dominates gene regulation due to the organisation of their genome into polycistronic transcription units. The purpose of the current study was to generate a substantially more comprehensive genome-wide survey of LCRs on trypanosome proteins than currently available Using the Shannon's entropy method, provided in the R package 'entropy', we identified LCRs in the proteome of . Our analysis predicts LCRs and their positional enrichment in distinct protein cohorts and superimposes on this a range of post-translational modifications derived from available experimental datasets. We have identified 8162 LCRs present on 4914 proteins, representing 42% of the proteome, placing among the eukaryotes with the highest percentage of LCRs Our results highlight the enrichment of LCRs in the C-terminal region of predicted nucleic acid binding proteins, these acting as favoured sites for potential phosphorylation. Phosphorylation represents 51% of the post-translational modifications present on LCRs compared to 16% on the rest of the proteome. The post-translational modifications of LCRs, and in particular phosphorylation events, could contribute to post-transcriptional gene expression control and the dynamics of protein targeting to membraneless organelles in kinetoplastid parasites.
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http://dx.doi.org/10.12688/wellcomeopenres.16286.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682498PMC
November 2020

The Methyl-CpG-Binding Protein Mbd2 Regulates Susceptibility to Experimental Colitis via Control of CD11c Cells and Colonic Epithelium.

Front Immunol 2020 14;11:183. Epub 2020 Feb 14.

Faculty of Biology, Medicine and Health, Manchester Collaborative Centre for Inflammation Research, Lydia Becker Institute of Immunology and Inflammation, University of Manchester, Manchester, United Kingdom.

Methyl-CpG-binding domain-2 (Mbd2) acts as an epigenetic regulator of gene expression, by linking DNA methylation to repressive chromatin structure. Although Mbd2 is widely expressed in gastrointestinal immune cells and is implicated in regulating intestinal cancer, anti-helminth responses and colonic inflammation, the Mbd2-expressing cell types that control these responses are incompletely defined. Indeed, epigenetic control of gene expression in cells that regulate intestinal immunity is generally poorly understood, even though such mechanisms may explain the inability of standard genetic approaches to pinpoint the causes of conditions like inflammatory bowel disease. In this study we demonstrate a vital role for Mbd2 in regulating murine colonic inflammation. mice displayed dramatically worse pathology than wild type controls during dextran sulfate sodium (DSS) induced colitis, with increased inflammatory (IL-1β) monocytes. Profiling of mRNA from innate immune and epithelial cell (EC) populations suggested that Mbd2 suppresses inflammation and pathology via control of innate-epithelial cell crosstalk and T cell recruitment. Consequently, restriction of Mbd2 deficiency to CD11c dendritic cells and macrophages, or to ECs, resulted in increased DSS colitis severity. Our identification of this dual role for in regulating the inflammatory capacity of both CD11c cells and ECs highlights how epigenetic control mechanisms may limit intestinal inflammatory responses.
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http://dx.doi.org/10.3389/fimmu.2020.00183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033935PMC
March 2021

The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.

Nat Immunol 2019 05 1;20(5):571-580. Epub 2019 Apr 1.

Lydia Becker Institute of Immunology and Inflammation, Manchester Collaborative Centre for Inflammation Research, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.
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http://dx.doi.org/10.1038/s41590-019-0352-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8381729PMC
May 2019

Dynamics of Colon Monocyte and Macrophage Activation During Colitis.

Front Immunol 2018 27;9:2764. Epub 2018 Nov 27.

Lydia Becker Institute of Immunology and Inflammation, Manchester Collaborative Centre for Inflammation Research, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom.

Macrophages are pivotal in coordinating a range of important processes in the intestines, including controlling intracellular infections and limiting damaging inflammation against the microbiota. However, it is not clear how gut macrophages, relative to recruited blood monocytes and other myeloid cells, contribute to the intestinal inflammatory milieu, nor how macrophages and their monocyte precursors mediate recruitment of other immune cells to the inflamed intestine. Myeloid cell populations isolated from colonic inflammatory bowel disease (IBD) or murine dextran sulphate sodium (DSS) induced colitis were assessed using flow cytometry and compared to healthy controls. In addition, mRNA expression profiles in human and murine colon samples, and in macrophages and monocytes from healthy and inflamed murine colons, were analysed by quantitative PCR (qPCR) and mRNA microarray. We show that the monocyte:macrophage balance is disrupted in colon inflammation to favour recruitment of CD14HLA-DR cells in humans, and Ly6C monocytes in mice. In addition, we identify that murine blood monocytes receive systemic signals enabling increased release of IL-1β prior to egress from the blood into the colon. Further, once within the colon and relative to other myeloid cells, monocytes represent the dominant local source of both IL-1β and TNF. Finally, our data reveal that, independent of inflammation, murine colon macrophages act as a major source of and chemokines that trigger further recruitment of their pro-inflammatory monocyte precursors. Our work suggests that strategies targeting macrophage-mediated monocyte recruitment may represent a promising approach for limiting the chronic inflammation that characterises IBD.
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http://dx.doi.org/10.3389/fimmu.2018.02764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277765PMC
September 2019

HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Immunity 2017 10;47(4):739-751.e5

MRC Centre for Inflammation Research, University of Edinburgh, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK; Institute of Immunology and Infection Research, and Centre for Immunity, Infection and Evolution, School of Biological Sciences, Ashworth Laboratories, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK. Electronic address:

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.
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http://dx.doi.org/10.1016/j.immuni.2017.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655542PMC
October 2017

A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells.

Nat Commun 2015 Apr 24;6:6920. Epub 2015 Apr 24.

Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester M3 9NT, UK.

Dendritic cells (DCs) direct CD4(+) T-cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a markedly impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to the activation of CD4(+) T-cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.
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http://dx.doi.org/10.1038/ncomms7920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413429PMC
April 2015

Modulation of dendritic cell alternative activation and function by the vitamin A metabolite retinoic acid.

Int Immunol 2015 Nov 20;27(11):589-96. Epub 2015 Apr 20.

Manchester Collaborative Centre for Inflammation Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9NT, UK

The archetypal Th2 cytokine IL-4 has previously been shown to alternatively activate murine macrophages and, more recently, dendritic cells (DCs) both in vitro and in vivo. IL-4 has also been shown to induce Aldh1a2 (aldehyde dehydrogenase 1a2) expression in murine macrophages recruited to the peritoneal cavity. However, the influence of IL-4 on DC Aldh1a2 induction in vivo has not yet been addressed. In this work, we found that DCs show enhanced aldehyde dehydrogenase enzyme activity in vivo, which led us to investigate the impact of the vitamin A metabolite all-trans retinoic acid (RA) on DC alternative activation and function. Antagonism of RA receptors reduced production of resistin-like molecule alpha by DCs responding to IL-4, while addition of exogenous RA enhanced production of this marker of alternative activation. Functionally, RA increased DC induction of CD4(+) T-cell IL-10, while reducing CD4(+) T-cell IL-4 and IL-13, revealing a previously unidentified role for RA in regulating the ability of alternatively activated DCs to influence Th2 polarization.
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http://dx.doi.org/10.1093/intimm/dxv020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625886PMC
November 2015

Genomic analysis of the causative agents of coccidiosis in domestic chickens.

Genome Res 2014 Oct 11;24(10):1676-85. Epub 2014 Jul 11.

Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom;

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.
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http://dx.doi.org/10.1101/gr.168955.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199364PMC
October 2014

Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host.

PLoS Negl Trop Dis 2011 Aug 30;5(8):e1274. Epub 2011 Aug 30.

University of York, Heslington, United Kingdom.

Background: The infective schistosome cercaria develops within the intramolluscan daughter sporocyst from an undifferentiated germ ball, during which synthesis of proteins essential for infection occurs. When the aquatic cercaria locates the mammalian host it rapidly penetrates into the epidermis using glandular secretions. It then undergoes metamorphosis into the schistosomulum, including replacement of its tegument surface membranes, a process taking several days before it exits the skin. Patterns of gene expression underlying this transition have been characterised.

Methods And Principal Findings: All gene models from the S. mansoni genome (www.GeneDB.org) were incorporated into a high-density oligonucleotide array. Double-stranded cDNA from germ balls, cercariae, and day 3 schistosomula was hybridised to the array without amplification. Statistical analysis was performed using Bioconductor to reveal differentially transcribed loci. Genes were categorised on the basis of biological process, tissue association or molecular function to aid understanding of the complex processes occurring. Genes necessary for DNA replication were enriched only in the germ ball, while those involved in translation were up-regulated in the germ ball and/or day 3 schistosomulum. Different sets of developmental genes were up-regulated at each stage. A large number of genes encoding elastases and invadolysins, and some venom allergen-like proteins were up-regulated in the germ ball, those encoding cysteine and aspartic proteases in the cercaria and schistosomulum. Micro exon genes encoding variant secreted proteins were highly up-regulated in the schistosomulum along with tegument and gut-associated genes, coincident with remodelling of the parasite body. Genes encoding membrane proteins were prominently up-regulated in the cercaria and/or day 3 schistosomulum.

Conclusions/significance: Our study highlights an expanded number of transcripts encoding proteins potentially involved in skin invasion. It illuminates the process of metamorphosis into the schistosomulum and highlights the very early activation of gut-associated genes whilst revealing little change in the parasite's energy metabolism or stress responses.
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http://dx.doi.org/10.1371/journal.pntd.0001274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166049PMC
August 2011

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts.

Genome Res 2010 Aug 6;20(8):1112-21. Epub 2010 Jul 6.

Department of Biology, University of York, York YO10 5YW, United Kingdom.

Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (< or =36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.
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http://dx.doi.org/10.1101/gr.100099.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909574PMC
August 2010

Anti-schistosomal intervention targets identified by lifecycle transcriptomic analyses.

PLoS Negl Trop Dis 2009 Nov 3;3(11):e543. Epub 2009 Nov 3.

Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

Background: Novel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis.

Methodology/principal Findings: Utilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and/or adult transcripts with no known sequence similarity outside the Platyhelminthes), which were undetectable by the statistical comparisons.

Conclusions/significance: Collectively, statistical and network-based exploratory analyses of transcriptomic datasets have led to a thorough characterisation of schistosome development. Information obtained from these experiments highlighted key transcriptional programs associated with lifecycle progression and identified numerous anti-schistosomal candidate molecules including G-protein coupled receptors, tetraspanins, Dyp-type peroxidases, fucosyltransferases, leishmanolysins and the netrin/netrin receptor complex.
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http://dx.doi.org/10.1371/journal.pntd.0000543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764848PMC
November 2009

Comparative expression profiling of Leishmania: modulation in gene expression between species and in different host genetic backgrounds.

PLoS Negl Trop Dis 2009 Jul 7;3(7):e476. Epub 2009 Jul 7.

Centre for Immunology and Infection, Department of Biology/Hull York Medical School, University of York, York, United Kingdom.

Background: Genome sequencing of Leishmania species that give rise to a range of disease phenotypes in the host has revealed highly conserved gene content and synteny across the genus. Only a small number of genes are differentially distributed between the three species sequenced to date, L. major, L. infantum and L. braziliensis. It is not yet known how many of these genes are expressed in the disease-promoting intracellular amastigotes of these species or whether genes conserved between the species are differentially expressed in the host.

Methods/principal Findings: We have used customised oligonucleotide microarrays to confirm that all of the differentially distributed genes identified by genome comparisons are expressed in intracellular amastigotes, with only a few of these subject to regulation at the RNA level. In the first large-scale study of gene expression in L. braziliensis, we show that only approximately 9% of the genes analysed are regulated in their RNA expression during the L. braziliensis life cycle, a figure consistent with that observed in other Leishmania species. Comparing amastigote gene expression profiles between species confirms the proposal that Leishmania transcriptomes undergo little regulation but also identifies conserved genes that are regulated differently between species in the host. We have also investigated whether host immune competence influences parasite gene expression, by comparing RNA expression profiles in L. major amastigotes derived from either wild-type (BALB/c) or immunologically compromised (Rag2(-/-) gamma(c) (-/-)) mice. While parasite dissemination from the site of infection is enhanced in the Rag2(-/-) gamma(c) (-/-) genetic background, parasite RNA expression profiles are unperturbed.

Conclusion/significance: These findings support the hypothesis that Leishmania amastigotes are pre-adapted for intracellular survival and undergo little dynamic modulation of gene expression at the RNA level. Species-specific parasite factors contributing to virulence and pathogenicity in the host may be limited to the products of a small number of differentially distributed genes or the differential regulation of conserved genes, either of which are subject to translational and/or post-translational controls.
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http://dx.doi.org/10.1371/journal.pntd.0000476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701600PMC
July 2009

Altered patterns of gene expression underlying the enhanced immunogenicity of radiation-attenuated schistosomes.

PLoS Negl Trop Dis 2008 May 21;2(5):e240. Epub 2008 May 21.

Department of Biology, University of York, York, United Kingdom.

Background: Schistosome cercariae only elicit high levels of protective immunity against a challenge infection if they are optimally attenuated by exposure to ionising radiation that truncates their migration in the lungs. However, the underlying molecular mechanisms responsible for the altered phenotype of the irradiated parasite that primes for protection have yet to be identified.

Methodology/principal Findings: We have used a custom microarray comprising probes derived from lung-stage parasites to compare patterns of gene expression in schistosomula derived from normal and irradiated cercariae. These were transformed in vitro and cultured for four, seven, and ten days to correspond in development to the priming parasites, before RNA extraction. At these late times after the radiation insult, transcript suppression was the principal feature of the irradiated larvae. Individual gene analysis revealed that only seven were significantly down-regulated in the irradiated versus normal larvae at the three time-points; notably, four of the protein products are present in the tegument or associated with its membranes, perhaps indicating a perturbed function. Grouping of transcripts using Gene Ontology (GO) and subsequent Gene Set Enrichment Analysis (GSEA) proved more informative in teasing out subtle differences. Deficiencies in signalling pathways involving G-protein-coupled receptors suggest the parasite is less able to sense its environment. Reduction of cytoskeleton transcripts could indicate compromised structure which, coupled with a paucity of neuroreceptor transcripts, may mean the parasite is also unable to respond correctly to external stimuli.

Conclusions/significance: The transcriptional differences observed are concordant with the known extended transit of attenuated parasites through skin-draining lymph nodes and the lungs: prolonged priming of the immune system by the parasite, rather than over-expression of novel antigens, could thus explain the efficacy of the irradiated vaccine.
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http://dx.doi.org/10.1371/journal.pntd.0000240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2375114PMC
May 2008

Altered retinal microRNA expression profile in a mouse model of retinitis pigmentosa.

Genome Biol 2007 ;8(11):R248

Smurfit Institute of Genetics, Trinity College Dublin, College Green, Dublin 2, Ireland.

Background: The role played by microRNAs (miRs) as common regulators in physiologic processes such as development and various disease states was recently highlighted. Retinitis pigmentosa (RP) linked to RHO (which encodes rhodopsin) is the most frequent form of inherited retinal degeneration that leads to blindness, for which there are no current therapies. Little is known about the cellular mechanisms that connect mutations within RHO to eventual photoreceptor cell death by apoptosis.

Results: Global miR expression profiling using miR microarray technology and quantitative real-time RT-PCR (qPCR) was performed in mouse retinas. RNA samples from retina of a mouse model of RP carrying a mutant Pro347Ser RHO transgene and from wild-type retina, brain and a whole-body representation (prepared by pooling total RNA from eight different mouse organs) exhibited notably different miR profiles. Expression of retina-specific and recently described retinal miRs was semi-quantitatively demonstrated in wild-type mouse retina. Alterations greater than twofold were found in the expression of nine miRs in Pro347Ser as compared with wild-type retina (P < 0.05). Expression of miR-1 and miR-133 decreased by more than 2.5-fold (P < 0.001), whereas expression of miR-96 and miR-183 increased by more than 3-fold (P < 0.001) in Pro347Ser retinas, as validated by qPCR. Potential retinal targets for these miRs were predicted in silico.

Conclusion: This is the first miR microarray study to focus on evaluating altered miR expression in retinal disease. Additionally, novel retinal preference for miR-376a and miR-691 was identified. The results obtained contribute toward elucidating the function of miRs in normal and diseased retina. Modulation of expression of retinal miRs may represent a future therapeutic strategy for retinopathies such as RP.
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http://dx.doi.org/10.1186/gb-2007-8-11-r248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258196PMC
August 2008

Microarray analysis identifies genes preferentially expressed in the lung schistosomulum of Schistosoma mansoni.

Int J Parasitol 2006 Jan 15;36(1):1-8. Epub 2005 Nov 15.

Department of Biology, University of York, PO Box 373, York YO10 5YW, UK.

The lung schistosomulum of Schistosoma mansoni is a validated target of protective immunity elicited in vaccinated mice. To identify genes expressed at this stage we constructed a microarray, representing 3088 contigs and singlets, with cDNA derived from in vitro cultured larvae and used it to screen RNA from seven life-cycle stages. Clustering of genes by expression profile across the life cycle revealed a number of membrane, membrane-associated and secreted proteins up-regulated at the lung stage, that may represent potential immune targets. Two promising secreted molecules have homology to antigens with vaccine and/or immunomodulatory potential in other helminths.
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http://dx.doi.org/10.1016/j.ijpara.2005.10.008DOI Listing
January 2006

The genome of the kinetoplastid parasite, Leishmania major.

Science 2005 Jul;309(5733):436-42

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK.

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.
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http://dx.doi.org/10.1126/science.1112680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1470643PMC
July 2005

Comparative genomics of trypanosomatid parasitic protozoa.

Science 2005 Jul;309(5733):404-9

Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA.

A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that-along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions-have shaped the genomes of each parasite. Contrary to recent reports, our analyses reveal no evidence that these species are descended from an ancestor that contained a photosynthetic endosymbiont.
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http://dx.doi.org/10.1126/science.1112181DOI Listing
July 2005

Integration of tools and resources for display and analysis of genomic data for protozoan parasites.

Int J Parasitol 2005 Apr;35(5):481-93

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Centralisation of tools for analysis of genomic data is paramount in ensuring that research is always carried out on the latest currently available data. As such, World Wide Web sites providing a range of online analyses and displays of data can play a crucial role in guaranteeing consistency of in silico work. In this respect, the protozoan parasite research community is served by several resources, either focussing on data and tools for one species or taking a broader view and providing tools for analysis of data from many species, thereby facilitating comparative studies. In this paper, we give a broad overview of the online resources available. We then focus on the GeneDB project, detailing the features and tools currently available through it. Finally, we discuss data curation and its importance in keeping genomic data 'relevant' to the research community.
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http://dx.doi.org/10.1016/j.ijpara.2005.01.011DOI Listing
April 2005

Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome.

Mol Biochem Parasitol 2004 Jul;136(1):87-100

Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK.

As genomic sequencing of Leishmania nears completion, functional analyses that provide a global genetic perspective on biological processes are important. Despite polycistronic transcription, RNA transcript abundance can be measured using microarrays. To provide a resource to evaluate cDNA arrays, we undertook 5' expressed sequence tag analysis of 2183 full-length randomly selected cDNAs from Leishmania major promastigote (days 3, 7, 10 of culture in vitro), and lesion-derived amastigote libraries. PCR-amplified inserts from 1830 of these cDNA representing 1001 unique genes were spotted onto microarrays, and compared internally with PCR-amplified open reading frames (ORFs) from 904 genes representing 842 unique genes annotated in the L. major genome. Microarrays were screened with RNA from procyclic, metacyclic and amastigote populations of L. major. Redundant clones on the array gave highly reproducible results, providing confidence in identification of stage-specific gene expression. Four hundred and thirty unique (i.e. non-redundant) stage-specific genes were identified. A higher percentage of stage-specific gene expression was observed in amastigotes ( approximately 35%) compared to metacyclics ( approximately 12%) for both cDNAs and ORFs, but cDNAs provided a richer source of regulated genes than currently annotated ORFs from the Leishmania genome. In mapping cDNAs onto the Leishmania genome, we noted that approximately 42% aligned to regions not recognised as genes using current predictive annotation tools. These genes are highly represented in our stage-specific genes, and therefore represent important drug targets and vaccine candidates. Careful annotation of cDNAs onto the Leishmania genome will be important before producing the next generation of oligonucleotide arrays based on annotated genes of the genomic sequencing project.
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http://dx.doi.org/10.1016/j.molbiopara.2004.03.004DOI Listing
July 2004

GeneDB: a resource for prokaryotic and eukaryotic organisms.

Nucleic Acids Res 2004 Jan;32(Database issue):D339-43

The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

GeneDB (http://www.genedb.org/) is a genome database for prokaryotic and eukaryotic organisms. The resource provides a portal through which data generated by the Pathogen Sequencing Unit at the Wellcome Trust Sanger Institute and other collaborating sequencing centres can be made publicly available. It combines data from finished and ongoing genome and expressed sequence tag (EST) projects with curated annotation, that can be searched, sorted and downloaded, using a single web based resource. The current release stores 11 datasets of which six are curated and maintained by biologists, who review and incorporate information from the scientific literature, public databases and the respective research communities.
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http://dx.doi.org/10.1093/nar/gkh007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC308742PMC
January 2004

Leishmania major chromosome 3 contains two long convergent polycistronic gene clusters separated by a tRNA gene.

Nucleic Acids Res 2003 Jul;31(14):4201-10

Seattle Biomedical Research Institute, 4 Nickerson Street, Seattle, WA 98109-1651, USA.

Leishmania parasites (order Kinetoplastida, family Trypanosomatidae) cause a spectrum of human diseases ranging from asymptomatic to lethal. The approximately 33.6 Mb genome is distributed among 36 chromosome pairs that range in size from approximately 0.3 to 2.8 Mb. The complete nucleotide sequence of Leishmania major Friedlin chromosome 1 revealed 79 protein-coding genes organized into two divergent polycistronic gene clusters with the mRNAs transcribed towards the telomeres. We report here the complete nucleotide sequence of chromosome 3 (384 518 bp) and an analysis revealing 95 putative protein-coding ORFs. The ORFs are primarily organized into two large convergent polycistronic gene clusters (i.e. transcribed from the telomeres). In addition, a single gene at the left end is transcribed divergently towards the telomere, and a tRNA gene separates the two convergent gene clusters. Numerous genes have been identified, including those for metabolic enzymes, kinases, transporters, ribosomal proteins, spliceosome components, helicases, an RNA-binding protein and a DNA primase subunit.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC167632PMC
http://dx.doi.org/10.1093/nar/gkg469DOI Listing
July 2003

Boudicca, a retrovirus-like long terminal repeat retrotransposon from the genome of the human blood fluke Schistosoma mansoni.

J Virol 2003 Jun;77(11):6153-66

Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.

The genome of Schistosoma mansoni contains a proviral form of a retrovirus-like long terminal repeat (LTR) retrotransposon, designated BOUDICCA: Sequence and structural characterization of the new mobile genetic element, which was found in bacterial artificial chromosomes prepared from S. mansoni genomic DNA, revealed the presence of three putative open reading frames (ORFs) bounded by direct LTRs of 328 bp in length. ORF1 encoded a retrovirus-like major homology region and a Cys/His box motif, also present in Gag polyproteins of related retrotransposons and retroviruses. ORF2 encoded enzymatic domains and motifs characteristic of a retrovirus-like polyprotein, including aspartic protease, reverse transcriptase, RNase H, and integrase, in that order, a domain order similar to that of the gypsy/Ty3 retrotransposons. An additional ORF at the 3' end of the retrotransposon may encode an envelope protein. Phylogenetic comparison based on the reverse transcriptase domain of ORF2 confirmed that Boudicca was a gypsy-like retrotransposon and showed that it was most closely related to CsRn1 from the Oriental liver fluke Clonorchis sinensis and to kabuki from Bombyx mori. Bioinformatics approaches together with Southern hybridization analysis of genomic DNA of S. mansoni and the screening of a bacterial artificial chromosome library representing approximately 8-fold coverage of the S. mansoni genome revealed that numerous copies of Boudicca were interspersed throughout the schistosome genome. By reverse transcription-PCR, mRNA transcripts were detected in the sporocyst, cercaria, and adult developmental stages of S. mansoni, indicating that Boudicca is actively transcribed in this trematode.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154989PMC
http://dx.doi.org/10.1128/jvi.77.11.6153-6166.2003DOI Listing
June 2003

Leishmania RAB7: characterisation of terminal endocytic stages in an intracellular parasite.

Mol Biochem Parasitol 2002 Aug;123(2):105-13

Wellcome Trust Laboratory for Molecular Parasitology and Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK.

Leishmania species are intracellular parasites that inhabit a parasitophorous vacuole (PV) within host macrophages and engage with the host endo-membrane network to avoid clearance from the cell. Intracellular Leishmania amastigotes exhibit a high degree of proteolytic/lysosomal activity that may assist degradation of MHC class II molecules and subsequent interruption of antigen presentation. As an aid to further analysis of the endosomal/lysosomal events that could facilitate this process, we have characterised a Leishmania homologue of the late endosomal marker, Rab7, thought to be involved in the terminal steps of endocytosis and lysosomal delivery. The Leishmania major Rab7 (LmRAB7) protein is expressed throughout the life-cycle, shows 73 and 64% identity to Trypanosoma cruzi and Trypanosoma brucei Rab7s (TcRAB7 and TbRAB7), respectively, and includes a kinetoplastid-specific insertion. The recombinant protein binds GTP and polyclonal antibodies raised against this antigen recognise structures in the region of the cell between the nucleus and kinetoplast. By immunoelectron microscopy of axenic amastigotes, Leishmania mexicana Rab7 (LmexRAB7) is found juxtaposed to and overlapping membrane structures labelled for the megasomal marker, cysteine proteinase B, confirming a late-endosomal/lysosomal localisation.
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http://dx.doi.org/10.1016/s0166-6851(02)00133-0DOI Listing
August 2002

Molecular cloning and characterization of two new isoforms of the protein kinase A catalytic subunit from the human parasite Leishmania.

Gene 2002 Apr;288(1-2):65-75

Department of Parasitology, Hebrew University - Hadassah Medical School, Jerusalem 91220, Israel.

Leishmania are protozoan parasites that cause extensive morbidity and mortality in humans. Genes for two new isoforms of the protein kinase A catalytic subunit (PKAC) in Leishmania, Lmpkac2a and Lmpkac2b, were cloned and characterized. The predicted open reading frames for these isoforms are 93.4% identical over 338 amino acids (aa). The conserved PK catalytic cores (subdomains I-XI) are identical, while the carboxy-terminal extensions differ by only two aa. However, LmPKAC2 shares only 62% identity over the 255 aa catalytic core region with the previously described LmPKAC1 (c-lpk2). Unlike LmPKAC1, the location of the FXXF motif at the carboxy-terminus is conserved in both LmPKAC2 isoforms; however, the aa sequence, LXXF, in isoform-2a is unusual. The leishmanial isoforms can be distinguished by their NH(2)-terminal extensions, which show minimal similarity at the primary sequence level. Structural analysis of the three enzymes based on the crystal structure of mammalian PKAs predicts that both LmPKAC2 isoforms, unlike LmPKAC1, have identical alpha-helix structures in the NH(2)-terminal extension. Lmpkac2 genes are located on chromosome 35 just downstream from the leishmanial prp8 gene. This genomic organization is conserved in two species of Leishmania and Crithidia fasciculata and allowed for the partial analysis of Cfpkac2a. Phylogenetic analysis groups the two LmPKAC2 isoforms together and separately from LmPKAC1, which is more similar to the Euglena gracilis PKAC, EPK2. These findings provide the basis for additional studies on the role of the PKA family in parasite differentiation and virulence.
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http://dx.doi.org/10.1016/s0378-1119(02)00403-1DOI Listing
April 2002
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