Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei.

Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown functions. Here, using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localization of 89% of the T. brucei proteome, a resource we call TrypTag. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultraconserved cellular architecture of eukaryotes, including the first comprehensive 'cartographic' analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

Nucleolar targeting in an early-branching eukaryote suggests a general mechanism for ribosome protein sorting.

The compartmentalised eukaryotic cell demands accurate targeting of proteins to the organelles in which they function, whether membrane-bound (like the nucleus) or non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in the early-branching eukaryote Trypanosoma brucei to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and physical protein properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins, unlike mitochondrial ribosome proteins, have more basic residues - pointing to adaptation of physicochemical properties to assist segregation.

Assessment of the iliocapsularis muscle on magnetic resonance imaging.

INTRODUCTION: Morphologic features of Iliocapsularis (IC) may aid clinical decision-making in the symptomatic hip. The relationship between IC muscle size and underlying hip pathology is emerging; however, research is limited in the imaging literature. The purpose of this study was to determine the reliability and reproducibility of measurements of the IC muscle and its MRI appearances. It also looked for any association between IC dimension and axial levels, side, gender and bony features of hip instability. METHODS: Retrospective study of 37 MRI scans were assessed by four observers. MRI axial T1 images were used to define the IC anatomy, measure the IC and rectus femoris at the femoral head centre (FHC) and adjacent levels and calculate the iliocapsularis-to-rectus femoris (IR) ratio. Measurements were repeated at least 2 weeks later. Radiographic assessment of the lateral centre edge angle, acetabular index and femoral neck-shaft angle were also conducted. RESULTS: The IC was always present, but was well-defined in only 4% of cases with fair agreement. The intraclass correlation coefficient for reliability and reproducibility was the highest for IC width 0.94 (0.91-0.96). No significant correlation was identified between the IR ratio and radiographic parameters. CONCLUSION: Iliocapsularis is visible and reliably measured on MRI despite observers considering the muscle to be not well-defined. Despite gender differences in muscle size, the IR ratio was unchanged. There is a significant difference in the IR ratio above and below the FHC; therefore, clinicians need to be aware of how this may impact the clinical use when utilising the IR ratio.

Intensive Serum Urate Lowering With Oral Urate-Lowering Therapy for Erosive Gout: A Randomized Double-Blind Controlled Trial.

OBJECTIVE: To determine whether a therapeutic approach of intensive serum urate lowering results in improved bone erosion scores in patients with erosive gout. METHODS: We undertook a 2-year, double-blind randomized controlled trial of 104 participants with erosive gout who were receiving serum urate-lowering therapy orally and who had serum urate levels of ≥0.30 mmoles/liter at baseline. Participants were randomly assigned to either an intensive serum urate target of <0.20 mmoles/liter or a standard target of <0.30 mmoles/liter (considered the standard according to rheumatology guidelines). Oral serum urate-lowering therapy was titrated to target using a standardized protocol (with the maximum approved doses of allopurinol, probenecid, febuxostat, and benzbromarone). The primary end point was the total computed tomography (CT) bone erosion score. Outcome Measures in Rheumatology (OMERACT) gout core outcome domains were secondary end points. RESULTS: Although the serum urate levels were significantly lower in the intensive target group compared to the standard target group over the study period (P = 0.002), fewer participants in the intensive target group achieved the randomized serum urate target level by year 2 (62% versus 83% of patients in the standard target group; P < 0.05). The intensive target group required higher doses of allopurinol (mean ± SD 746 ± 210 mg/day versus 497 ± 186 mg/day; P < 0.001) and received more combination therapy (P = 0.0004) compared to the standard target group. We observed small increases in CT bone erosion scores in both serum urate target groups over 2 years, with no between-group difference (P = 0.20). OMERACT core outcome domains (gout flares, tophi, pain, patient's global assessment of disease activity, health-related quality of life, and activity limitation) improved in both groups over 2 years, with no between-group differences. Adverse event and serious adverse event rates were similar between the groups. CONCLUSION: Compared to a serum urate target of <0.30 mmoles/liter, more intensive serum urate lowering is difficult to achieve with an oral urate-lowering therapy. Intensive serum urate lowering leads to a high medication burden and does not improve bone erosion scores in patients with erosive gout.

Development of a radiographic scoring system for new bone formation in gout.

BACKGROUND: Features of new bone formation (NBF) are common in tophaceous gout. The aim of this project was to develop a plain radiographic scoring system for NBF in gout. METHODS: Informed by a literature review, scoring systems were tested in 80 individual 1st and 5th metatarsophalangeal joints. Plain radiography scores were compared with computed tomography (CT) measurements of the same joints. The best-performing scoring system was then tested in paired sets of hand and foot radiographs obtained over 2 years from an additional 25 patients. Inter-reader reproducibility was assessed using intraclass correlation coefficients (ICC). NBF scores were correlated with plain radiographic erosion scores (using the gout-modified Sharp-van der Heijde system). RESULTS: Following a series of structured reviews of plain radiographs and scoring exercises, a semi-quantitative scoring system for sclerosis and spur was developed. In the individual joint analysis, the inter-observer ICC (95% CI) was 0.84 (0.76-0.89) for sclerosis and 0.81 (0.72-0.87) for spur. Plain radiographic sclerosis and spur scores correlated with CT measurements (r = 0.65-0.74, P < 0.001 for all analyses). For the hand and foot radiograph sets, the inter-observer ICC (95% CI) was 0.94 (0.90-0.98) for sclerosis score and 0.76 (0.65-0.84) for spur score. Sclerosis and spur scores correlated highly with plain radiographic erosion scores (r = 0.87 and 0.71 respectively), but not with change in erosion scores over 2 years (r = -0.04-0.15). CONCLUSION: A semi-quantitative plain radiographic scoring method for the assessment of NBF in gout is feasible, valid, and reproducible. This method may facilitate consistent measurement of NBF in gout.

Effects of Allopurinol Dose Escalation on Bone Erosion and Urate Volume in Gout: A Dual-Energy Computed Tomography Imaging Study Within a Randomized, Controlled Trial.

OBJECTIVE: To examine whether allopurinol dose escalation to achieve serum urate (SU) target can influence bone erosion or monosodium urate (MSU) crystal deposition, as measured by dual-energy computed tomography (DECT) in patients with gout. METHODS: We conducted an imaging study of a 2-year randomized clinical trial that compared immediate allopurinol dose escalation to SU target with conventional dosing for 1 year followed by dose escalation to target, in gout patients who were receiving allopurinol and who had an SU level of ≥0.36 mmoles/liter. DECT scans of feet and radiographs of hands and feet were obtained at baseline, year 1, and year 2 visits. DECT scans were scored for bone erosion and urate volume. RESULTS: Paired imaging data were available for 87 patients (42 in the dose-escalation group and 45 in the control group). At year 2, the progression in the CT erosion score was higher in the control group than in the dose-escalation group (+7.8% versus +1.4%; P = 0.015). Changes in plain radiography erosion or narrowing scores did not differ between groups. Reductions in DECT urate volume were observed in both groups. At year 2, patients in the control group who had an SU level of <0.36 mmoles/liter and patients in the dose-escalation group had reduced DECT urate volume (-27.6 to -28.3%), whereas reduction in DECT urate volume was not observed in control group patients with an SU level of ≥0.36 mmoles/liter (+1.5%) (P = 0.023). CONCLUSION: These findings provide evidence that long-term urate-lowering therapy using a treat-to-SU-target strategy can influence structural damage and reduce urate crystal deposition in gout.

Cellular landmarks of Trypanosoma brucei and Leishmania mexicana.

The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in each generation. The pattern of signal from a fluorescently tagged protein can define the specific structure/organelle that this protein localises to, and can be extremely informative in phenotype analysis in experimental perturbations, life cycle tracking, post-genomic assays and functional analysis of organelles. Using the vast coverage of protein subcellular localisations provided by the TrypTag project, an ongoing project to determine the localisation of every protein encoded in the T. brucei genome, we have generated an inventory of reliable reference organelle markers for both parasites that combines epifluorescence images with a detailed description of the key features of each localisation. We believe this will be a useful comparative resource that will enable researchers to quickly and accurately pinpoint the localisation of their proteins of interest and will provide cellular markers for many types of cell biology studies. We see this as another important step in the post-genomic era analyses of these parasites, in which ever expanding datasets generate numerous candidates to analyse. Adoption of these reference proteins by the community is likely to enhance research studies and enable better comparison of data.

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens.

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.

Evidence for a genetic basis of urogenital carcinoma in the wild California sea lion.

Although neoplasia is a major cause of mortality in humans and domestic animals, it has rarely been described in wildlife species. One of the few examples is a highly prevalent urogenital carcinoma in California sea lions (CSLs). Although the aetiology of this carcinoma is clearly multifactorial, inbreeding depression, as estimated using levels of microsatellite multilocus heterozygosity, is identified as predictive for this neoplasia. On further analysis, this relationship appears to be largely driven by one marker, suggesting that a single locus might be associated with the occurrence of this disease in CSLs. In a case-control study, carcinoma was significantly associated with homozygosity at the Pv11 microsatellite locus. Pv11 was mapped to intron 9 of the heparanase 2 gene (HPSE2) locus, a very large gene encoding heparanase 2, which in humans is associated with multiple carcinomas. Correspondingly, immunohistochemical labelling in tissues was present in carcinoma cases within a single homozygous Pv11 genotype. To our knowledge, this is the first report of an individual locus being associated with cancer in any wildlife species. This adds emphasis to the study of HPSE2 in other species, including humans and will guide future studies on this sentinel species that shares much of its diet and environment with humans.

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

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.

EmaxDB: Availability of a first draft genome sequence for the apicomplexan Eimeria maxima.

Apicomplexan parasites are serious pathogens of animals and man that cause diseases including coccidiosis, malaria and toxoplasmosis. The importance of these parasites has prompted the establishment of genomic resources in support of developing effective control strategies. For the Eimeria species resources have developed most rapidly for the reference Eimeria tenella Houghton strain (http://www.genedb.org/Homepage/Etenella). The value of these resources can be enhanced by comparison with related parasites. The well characterised immunogenicity and genetic diversity associated with Eimeria maxima promote its use in genetics-led studies on coccidiosis and recommended its selection for sequencing. Using a combination of sequencing technologies a first draft assembly and annotation has been produced for an E. maxima Houghton strain-derived clone (EmaxDB; http://www.genomemalaysia.gov.my/emaxdb/). The assembly of a draft genome sequence for E. maxima provides a resource for comparative studies with Eimeria and related parasites as demonstrated here through the identification of genes predicted to encode microneme proteins in E. maxima.

Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite.

Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.

A toolbox facilitating stable transfection of Eimeria species.

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isolate stable populations of transfected Eimeria parasites will now stimulate rapid expansion of reverse genetic studies in this important coccidian.

Sequencing and analysis of chromosome 1 of Eimeria tenella reveals a unique segmental organization.

Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.