SUMOylation of Paraflagellar Rod Protein, PFR1, and Its Stage-Specific Localization in Trypanosoma cruzi

BACKGROUND: The flagellate protozoan parasite, Trypanosoma cruzi, is a causative agent of Chagas disease that is transmitted by reduviid bugs to humans. The parasite exists in multiple morphological forms in both vector and host, and cell differentiation in T. cruzi is tightly associated with stage-specific protein synthesis and degradation. However, the specific molecular mechanisms responsible for this coordinated cell differentiation are unclear. METHODOLOGY/PRINCIPAL FINDINGS: The SUMO conjugation system plays an important role in specific protein expression. In T. cruzi, a subset of SUMOlylated protein candidates and the nuclear localization of SUMO have been shown. Here, we examined the biological roles of SUMO in T. cruzi. Site-directed mutagenesis analysis of SUMO consensus motifs within T. cruzi SUMO using a bacterial SUMOylation system revealed that T. cruzi SUMO can polymerize. Indirect fluorescence analysis using T. cruzi SUMO-specific antibody showed the extra-nuclear localization of SUMO on the flagellum of epimastigote and metacyclic and bloodstream trypomastigote stages. In the short-flagellate intracellular amastigote, an extra-nuclear distribution of SUMO is associated with basement of the flagellum and becomes distributed along the flagellum as amastigote transforms into trypomastigote. We examined the flagellar target protein of SUMO and show that a paraflagellar rod protein, PFR1, is SUMOylated. CONCLUSIONS: These findings indicate that SUMOylation is associated with flagellar homeostasis throughout the parasite life cycle, which may play an important role in differentiation of T. cruzi

Discourse Semantics for the Analysis of Change in Language

This paper purports to elaborate and address several issues which lie at the intersection of computational linguistics and psychology. The first issue addressed is that of the interaction between discourse and semantics by virtue of empirical linguistic and psychotherapeutic evidence. This paper then gives a formal account of the knowledge representation and reasoning processes involved in the construction of an XML knowledge base for use in the sematic analysis of psychotherapeutic transcripts. Computational methods for the automatic mark-up and inference of the psychotherapeutic phenomena under investigation are detailed in order to further develop intuitions behind a particular pragmatic theory of language known as the Metamodel. The work presented here ultimately aims to produce a sustainable system for the evaluation of the effectiveness of any given psychotherapeutic technique. The possibility exists for such a system to recognise successful therapeutic mechanisms and further still, to infer new ones, or suggest improvements, or offer novel explanations as to the success or failure of the therapy itself. The work discussed here stems from research in computational linguistics, psychotherapy, and philosophy. The corpus used is a culmination of client transcripts taken before, during, and after therapy. The particular therapeutic technique used here is known as the Metamodel (Bandler and Grinder, 1975). The Metamodel was originally proffered as a method of language analysis suitable for use by practitioners of any psychotherapeutic technique. It theorises that speech utterances are related to a clients deep structure through three primary mechanisms, namely generalisation, deletion, and distortion. Previous hand tagging of our data has proven support for such claims. It is our aim to automate the identification and reasoning process. The issues and processes involved in the automation of such tagging are discussed here. Architectural and philosophical issues relating syntax (or grammar), semantics (Larson and Segal, 1995), and pragmatics (Grice, 1989; Searle, 1969) are raised. Discourse Representation Theory (Kamp, 1981; Asher and Lascarides, 1995) is discussed and used here in order to infer discourse relations.Hosted by the Scholarly Text and Imaging Service (SETIS), the University of Sydney Library, and the Research Institute for Humanities and Social Sciences (RIHSS), the University of Sydney

Long Term Protection after Immunization with P. berghei Sporozoites Correlates with Sustained IFNγ Responses of Hepatic CD8+ Memory T Cells

Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite development before killing of blood-stages by chloroquine takes place. We assessed the longevity of protective cellular immune responses by RAS and CPS P. berghei immunization of C57BL/6j mice. Strong effector and memory (TEM) CD8+ T cell responses were induced predominantly in the liver of both RAS and CPS immunized mice while CD4+ T cells with memory phenotype remained at base line levels. Compared to unprotected naïve mice, we found high sporozoite-specific IFNγ ex vivo responses that associated with induced levels of in vivo CD8+ TEM cells in the liver but not spleen. Long term evaluation over a period of 9 months showed a decline of malaria-specific IFNγ responses in RAS and CPS mice that significantly correlated with loss of protection (r2 = 0.60, p<0.0001). The reducing IFNγ response by hepatic memory CD8+ T cells could be boosted by re-exposure to wild-type sporozoites. Our data show that sustainable protection against malaria associates with distinct intra-hepatic immune responses characterized by strong IFNγ producing CD8+ memory T cells

An Inducer of VGF Protects Cells against ER Stress-Induced Cell Death and Prolongs Survival in the Mutant SOD1 Animal Models of Familial ALS

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease, and recent evidence has suggested that endoplasmic reticulum (ER) stress signaling is involved in the pathogenesis of ALS. Here we identified a small molecule, SUN N8075, which has a marked protective effect on ER stress-induced cell death, in an in vitro cell-based screening, and its protective mechanism was mediated by an induction of VGF nerve growth factor inducible (VGF): VGF knockdown with siRNA completely abolished the protective effect of SUN N8075 against ER-induced cell death, and overexpression of VGF inhibited ER-stress-induced cell death. VGF level was lower in the spinal cords of sporadic ALS patients than in the control patients. Furthermore, SUN N8075 slowed disease progression and prolonged survival in mutant SOD1 transgenic mouse and rat models of ALS, preventing the decrease of VGF expression in the spinal cords of ALS mice. These data suggest that VGF plays a critical role in motor neuron survival and may be a potential new therapeutic target for ALS, and SUN N8075 may become a potential therapeutic candidate for treatment of ALS

A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research

Knockout of the dhfr-ts Gene in Trypanosoma cruzi Generates Attenuated Parasites Able to Confer Protection against a Virulent Challenge

Chagas disease is the clinical manifestation of the infection produced by the flagellate parasite Trypanosoma cruzi and currently there is no vaccine to prevent this disease. Therefore, different approaches or alternatives are urgently needed. Vaccination with live attenuated parasites has been used effectively in mice to reduce parasitemia and histological damage. However, the use of live parasites as inmunogens is controversial due to the risk of reversion to a virulent phenotype. In this work we genetically manipulated a naturally attenuated strain of T. cruzi in order to produce parasites with impaired replication and infectivity, using the mutation as a safety device against reversion to virulence. We show that genetically modified parasites display a lower proliferation rate in vitro and induced almost undetectable levels of T. cruzi specific CD8+ T cells when injected in mice. Furthermore, the immune response induced by these live mutant parasites confers protection against a subsequent virulent infection even a year after the original immunization