Exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of TP53 and two hits in DICER1 resulting in retention of 5p-derived miRNA hairpin loop sequences.

Pleuropulmonary blastoma is a rare childhood malignancy of lung mesenchymal cells that can remain dormant as epithelial cysts or progress to high-grade sarcoma. Predisposing germline loss-of-function DICER1 variants have been described. We sought to uncover additional contributors through whole exome sequencing of 15 tumor/normal pairs, followed by targeted resequencing, miRNA analysis and immunohistochemical analysis of additional tumors. In addition to frequent biallelic loss  of TP53 and mutations of NRAS or BRAF in some cases, each case had compound disruption of DICER1: a germline (12 cases) or somatic (3 cases) loss-of-function variant plus a somatic missense mutation in the RNase IIIb domain. 5p-Derived microRNA (miRNA) transcripts retained abnormal precursor miRNA loop sequences normally removed by DICER1. This work both defines a genetic interaction landscape with DICER1 mutation and provides evidence for alteration in miRNA transcripts as a consequence of DICER1 disruption in cancer.

Activation of ß-catenin/TCF targets following loss of the tumor suppressor SNF5.

The SWI/SNF chromatin remodeling complex is a master regulator of developmental cell-fate decisions, although the key target pathways are poorly characterized. Here, we interrogated the contribution of the SWI/SNF subunit and tumor suppressor SNF5 to the regulation of developmental pathways using conditional mouse and cell culture models. We find that loss of SNF5 phenocopies ß-catenin hyperactivation and that SNF5 is essential for regulating Wnt/ß-catenin pathway target expression. These data provide insight into chromatin-based mechanisms that underlie developmental regulation and elucidate the emerging theme that mutation of this tumor suppressor complex can activate developmental pathways by uncoupling them from upstream control.

Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used.

Vaccination with recombinant Leishmania donovani gamma-glutamylcysteine synthetase fusion protein protects against L. donovani infection.

Visceral leishmaniasis presents a serious health threat in many parts of the world. There is, therefore, an urgent need for an approved vaccine for clinical use to protect against infection. In this study, the ability of recombinant Leishmania donovani gamma-glutamyl cysteine synthetase protein (LdγGCS) alone or incorporated into a non-ionic surfactant vesicle (NIV) delivery system to protect against L. donovani infection was evaluated in a BALB/c mouse model. Immunization with LdγGCS alone or LdγGCS-NIV induced specific IgG1 and IgG2a antibodies compared to controls, with LdγGCS-NIV inducing significantly higher titers of both antibody classes (P < 0.05). Both formulations induced similar increases in splenocyte IFN-γ production following ex vivo antigen stimulation with LdγGCS compared with cells from control mice (P < 0.05). Similar levels of protection against infection were induced by LdγGCS alone and LdγGCS-NIV, based on their ability to suppress liver parasite burdens compared to control values (P < 0.01), indicating that using a carrier system did not enhance the protective responses induced by the recombinant protein. The results of this study indicate that LdγGCS may be a useful component in a vaccine against L. donovani.

Sequential expression of macrophage anti-microbial/inflammatory and wound healing markers following innate, alternative and classical activation.

The present study examines the temporal dynamics of macrophage activation marker expression in response to variations in stimulation. We demonstrate that markers can be categorized as 'early' (expressed most abundantly at 6 h post-stimulation) or 'late' (expressed at 24 h post-stimulation). Thus nos2 and p40 (IL-12/IL-23) are early markers of innate and classical activation, while dectin-1 and mrc-1 are early markers and fizz1 (found in inflammatory zone-1) and ym1 are late markers of alternative activation. Furthermore, argI is a late marker of both innate and alternative activation. The ability of interferon (IFN)-gamma to alter these activation markers was studied at both the protein level and gene level. As reported previously, IFN-gamma was able to drive macrophages towards the classical phenotype by enhancing nos2 gene expression and enzyme activity and p40 (IL-12/IL-23) gene expression in lipopolysaccharide (LPS)-stimulated macrophages. IFN-gamma antagonized alternative macrophage activation, as evident by reduced expression of dectin-1, mrc-1, fizz1 and ym1 mRNA transcripts. In addition, IFN-gamma antagonized arginase activity irrespective of whether macrophages were activated innately or alternatively. Our data explain some apparent contradictions in the literature, demonstrate temporal plasticity in macrophage activation states and define for the first time 'early' and 'late' markers associated with anti-microbial/inflammatory and wound healing responses, respectively.

The Toxoplasma gondii plastid replication and repair enzyme complex, PREX.

A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome.

Neuropsychiatric disease and Toxoplasma gondii infection.

Toxoplasma gondii infects approximately 30% of the world's population, but causes overt clinical symptoms in only a small proportion of people. In recent years, the ability of the parasite to manipulate the behaviour of infected mice and rats and alter personality attributes of humans has been reported. Furthermore, a number of studies have now suggested T. gondii infection as a risk factor for the development of schizophrenia and depression in humans. As T. gondii forms cysts that are located in various anatomical sites including the brain during a chronic infection, it is well placed anatomically to mediate these effects directly. The T. gondii genome is known to contain 2 aromatic amino acid hydroxylases that potentially could directly affect dopamine and/or serotonin biosynthesis. However, stimulation of the immune response has also recently been associated with mood and behavioural alterations in humans, and compounds designed to alter mood, such as fluoxetine, have been demonstrated to alter aspects of immune function. Herein, the evidence for T.-gondii-induced behavioural changes relevant to schizophrenia and depression is reviewed. Potential mechanisms responsible for these changes in behaviour including the role of tryptophan metabolism and the hypothalamic-pituitary-adrenal axis are discussed.

Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii.

Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at -70 degrees C using dimethyl sulfoxide (DMSO; 5 or 10%) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg2+ at 4 degrees C remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at -70 degrees C with DMSO (5 or 10%) or 40% glycerol, but not 80% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at -70 degrees C.

Toward effective school-based substance abuse prevention "breaking the cycle" programme in Antigua and Barbuda.

The "Breaking the Cycle" programme, based on the Project Charlie programme, was developed for Antigua and Barbuda third grade students and was implemented in 2001. Aspects of the programme are compared with aspects recently proven effective in randomized studies in developed countries. The "Breaking the Cycle" programme includes life-skills training, teaches decision making skills, includes peer resistance training, uses trained teachers, interactive teaching methods, effective content and delivery, targets students prior to onset of drug use, teaches drug harm, teaches community values and is culturally sensitive, all aspects of successful programmes overseas. The cost of about $7 US per student would suggests cost-benefit effectiveness compared with overseas programmes. The "Breaking the Cycle" school-based drug and alcohol use prevention programme includes most aspects of evidence-based successful programmes overseas, appears cost effective and could serve as a model for programmes in the Caribbean region.

Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

DNA vaccination against the parasite enzyme gamma-glutamylcysteine synthetase confers protection against Leishmania donovani infection.

In this study the potential of using Leishmania donovani gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, gamma-GCS) as a rational target for vaccine development was determined. Mice, immunised with plasmid containing the full gene sequence for gamma-GCS (pVAXgammaGCS) or plasmid alone (pVAX control), were challenged with a high dose of L. donovani amastigotes to give a stringent test of the ability of the vaccine to protect against infection. Vaccination with pVAXgammaGCS resulted in the production of specific IgG1 and IgG2a antibodies and resulted in significantly lower liver parasite burdens compared to controls. Protection was also associated with a significant increase in cell-mediated immunity, demonstrated as an increase in nitrite production by ConA stimulated splenocytes, an increase in the percentage of splenic CD3+CD4+ cells, and enhanced granuloma maturation, compared to control values.

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Ocular toxoplasmosis: in the storm of the eye.

Ocular toxoplasmosis (OT) can occur in the children of mothers infected with Toxoplasma gondii during pregnancy. It is not limited to the congenitally infected, but can also occur following adult-acquired infection or as a result of disease reactivation in immune-compromised and pregnant individuals. Many aspects of immune privilege in the eye, including constitutive TGF-beta expression and reduced MHC class 1 expression, would appear at first to favour parasite survival. Conversely, many of the mechanisms that control parasite multiplication in other anatomical sites, such as nitric oxide expression, IFN-gamma and TNF-alpha, are known to disrupt immune privilege and are associated with ocular damage. Taking into account the opposing needs of limiting parasite multiplication and minimizing tissue destruction we review the pathogenesis of OT in the murine model.

Resistance of Leishmania donovani to sodium stibogluconate is related to the expression of host and parasite gamma-glutamylcysteine synthetase.

Sequencing studies showed that the gamma-glutamylcysteine synthetase (gamma-GCS) heavy chain genes from sodium stibogluconate (SSG)-resistant (SSG-R) and SSG-susceptible (SSG-S) Leishmania donovani strains were identical, indicating that SSG resistance was related to quantitative differences in gamma-GCS expression rather than gene interstrain polymorphisms. In vitro infection of murine macrophages with the SSG-R strain, but not the SSG-S strain, down regulated expression of host gamma-GCS, which would result in a reduction in intramacrophage glutathione (GSH) levels and promote an oxidative intramacrophage environment. This would inhibit, or minimize, the reduction of SSG pentavalent antimony to its more toxic trivalent form. Macrophage studies showed that the SSG-R strain expressed higher levels of gamma-GCS compared to the SSG-S strain, which would result in higher GSH levels, giving increased protection against oxidative stress and facilitating SSG efflux. However a similar differential effect on host and parasite gamma-GCS expression was not obtained when using tissues from infected mice. In this case gamma-GCS expression was organ and strain dependent for both the host and the parasite, indicating that environmental conditions have a profound effect on gamma-GCS expression. Consistent with the proposed mechanism from in vitro studies, increasing tissue GSH levels in the presence of SSG by cotreatment of L. donovani-infected mice with SSG solution and GSH incorporated into nonionic surfactant vesicles was more effective in reducing liver, spleen, and bone marrow parasite burdens than monotherapy with SSG. Together, these results indicate that SSG resistance is associated with manipulation of both host and parasite GSH levels by L. donovani.

Toxoplasma gondii dense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif.

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory-secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine 'KKXX' endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

Intestinal pathology during acute toxoplasmosis is IL-4 dependent and unrelated to parasite burden.

The role of interleukin-4 (IL-4) during the course of Toxoplasma gondii infection was studied using IL-4-/- mice and their wild-type (WT) counterparts on a C57BL/6 background. Following oral infection with T. gondii tissue cysts an exacerbative role for IL-4 was demonstrated and IL-4-/- mice were found to be more resistant to infection than WT mice as measured by significantly reduced mortality. Furthermore pathology in the small intestine was less severe in IL-4-/- mice although conversely liver pathology was greater than in wild-type mice. Significantly, plasma IL-12 and IFN-gamma levels, which peaked at days 6 and 8, respectively, were higher in IL-4-/- mice. The exacerbatory role of IL-4 in the intestine was found by competitive RT-PCR not to be associated with increased parasite burdens but was related to comparative expression of IL-10.

A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation.

The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or replaced in a number of lineages.

Delivery of antimicrobials into parasites.

To eliminate apicomplexan parasites, inhibitory compounds must cross host cell, parasitophorous vacuole, and parasite membranes and cyst walls, making delivery challenging. Here, we show that short oligomers of arginine enter Toxoplasma gondii tachyzoites and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP reductase (ENR), conjugated to arginine oligomers enters extracellular tachyzoites, host cells, tachyzoites inside parasitophorous vacuoles within host cells, extracellular bradyzoites, and bradyzoites within cysts. We identify, clone, and sequence T. gondii enr and produce and characterize enzymatically active, recombinant ENR. This enzyme has the requisite amino acids to bind triclosan. Triclosan released after conjugation to octaarginine via a readily hydrolyzable ester linkage inhibits ENR activity, tachyzoites in vitro, and tachyzoites in mice. Delivery of an inhibitor to a microorganism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to sequestered parasites, a model system to characterize transport across multiple membrane barriers and structures, a widely applicable paradigm for treatment of active and encysted apicomplexan and other infections, and a generic proof of principle for a mechanism of medicine delivery.

Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa.

Current treatments for diseases caused by apicomplexan and trypanosomatid parasites are inadequate due to toxicity, the development of drug resistance and an inability to eliminate all life cycle stages of these parasites from the host. New therapeutics agents are urgently required. It has recently been demonstrated that type II fatty acid biosynthesis occurs in the plastid of Plasmodium falciparum and Toxoplasma gondii and inhibitors of this pathway such as triclosan and thiolactomycin restrict their growth. Furthermore, Trypanosoma brucei has recently been demonstrated to use type II fatty acid biosynthesis for myristate synthesis and to be susceptible to thiolactomycin. As this pathway is absent from mammals, it may provide an excellent target for novel antimicrobial agents to combat these diverse parasites. Leishmania and Trypanosoma parasites produce ergosterol-related sterols by a biosynthetic pathway similar to that operating in pathogenic fungi and their growth is susceptible to sterol biosynthesis inhibitors. Thus, inhibition of squalene 2,3-epoxidase by terbinafine, 14alpha-methylsterol 14-demethylase by azole and triazole compounds and delta(24)-sterol methyl transferase by azasterols all cause a depletion of normal sterols and an accumulation of abnormal amounts of sterol precursors with cytostatic or cytoxic consequences. However, Leishmania parasites can survive with greatly altered sterol profiles induced by continuous treatment with low concentrations of some inhibitors and they also have some ability to utilise and metabolise host sterol. These properties may permit the parasites to evade treatment with sterol biosynthesis inhibitors in some clinical situations and need to be taken into account in the design of future drugs.

Construction and validation of a polycompetitor construct (SWITCH) for use in competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii.

The obligate intracellular protozoan parasite, Toxoplasma gondii exists as 2 life-cycle forms in intermediate hosts. The rapidly dividing tachyzoites responsible for acute disease, present in the first 14 days of infection, give rise to slowly dividing bradyzoites that reside in tissue cysts. Reactivation of disease is associated with conversion of bradyzoites to tachyzoites. A sensitive method for detection and assessment of the number of each life-cycle stage would be useful for following these events. Herein we describe the construction and validation of a plasmid (pSWITCH) containing a polycompetitor construct (SWITCH) for use in competitive reverse transcriptase-PCR (cRT-PCR). pSWITCH contains competitors for SAG2A and LDH2 genes, which are exclusively expressed by tachyzoite and bradyzoite stages respectively, and for beta-tubulin, a gene expressed by both stages. Using cRT-PCR, samples can first be accurately normalized for expression of the housekeeping gene, beta-tubulin and then the relative levels of SAG2A and LDH2 expression compared to follow stage conversion. The abundance of transcripts for other genes of interest can then be followed during this process as demonstrated here for the SAG2-related family of genes. This technique offers a powerful tool for studying the processes involved in tachyzoite and bradyzoite interconversion.