Advances in nanocarriers as drug delivery systems in Chagas disease

Chagas disease is one of the most important public health problems in Latin America due to its high mortality and morbidity levels. There is no effective treatment for this disease since drugs are usually toxic with low bioavailability. Serious efforts to achieve disease control and eventual eradication have been unsuccessful to date, emphasizing the need for rapid diagnosis, drug development, and a reliable vaccine. Novel systems for drug and vaccine administration based on nanocarriers represent a promising avenue for Chagas disease treatment. Nanoparticulate systems can reduce toxicity, and increase the efficacy and bioavailability of active compounds by prolonging release, and therefore improve the therapeutic index. Moreover, nanoparticles are able to interact with the host’s immune system, modulating the immune response to favour the elimination of pathogenic microorganisms. In addition, new advances in diagnostic assays, such as nanobiosensors, are beneficial in that they enable precise identification of the pathogen. In this review, we provide an overview of the strategies and nanocarrier-based delivery systems for antichagasic agents, such as liposomes, micelles, nanoemulsions, polymeric and non-polymeric nanoparticles. We address recent progress, with a particular focus on the advances of nanovaccines and nanodiagnostics, exploring new perspectives on Chagas disease treatment

Trypanosoma cruzi alkaline 2-DE: Optimization and application to comparative proteome analysis of flagellate life stages

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A Plasmodium falciparum FcB1-schizont-EST collection providing clues to schizont specific gene structure and polymorphism

<p>Abstract</p> <p>Background</p> <p>The <it>Plasmodium falciparum </it>genome (3D7 strain) published in 2002, revealed ~5,400 genes, mostly based on <it>in silico </it>predictions. Experimental data is therefore required for structural and functional assessments of <it>P. falciparum </it>genes and expression, and polymorphic data are further necessary to exploit genomic information to further qualify therapeutic target candidates. Here, we undertook a large scale analysis of a <it>P. falciparum </it>FcB1-schizont-EST library previously constructed by suppression subtractive hybridization (SSH) to study genes expressed during merozoite morphogenesis, with the aim of: 1) obtaining an exhaustive collection of schizont specific ESTs, 2) experimentally validating or correcting <it>P. falciparum </it>gene models and 3) pinpointing genes displaying protein polymorphism between the FcB1 and 3D7 strains.</p> <p>Results</p> <p>A total of 22,125 clones randomly picked from the SSH library were sequenced, yielding 21,805 usable ESTs that were then clustered on the <it>P. falciparum </it>genome. This allowed identification of 243 protein coding genes, including 121 previously annotated as hypothetical. Statistical analysis of GO terms, when available, indicated significant enrichment in genes involved in "entry into host-cells" and "actin cytoskeleton". Although most ESTs do not span full-length gene reading frames, detailed sequence comparison of FcB1-ESTs versus 3D7 genomic sequences allowed the confirmation of exon/intron boundaries in 29 genes, the detection of new boundaries in 14 genes and identification of protein polymorphism for 21 genes. In addition, a large number of non-protein coding ESTs were identified, mainly matching with the two A-type rRNA units (on chromosomes 5 and 7) and to a lower extent, two atypical rRNA loci (on chromosomes 1 and 8), TARE subtelomeric regions (several chromosomes) and the recently described telomerase RNA gene (chromosome 9).</p> <p>Conclusion</p> <p>This FcB1-schizont-EST analysis confirmed the actual expression of 243 protein coding genes, allowing the correction of structural annotations for a quarter of these sequences. In addition, this analysis demonstrated the actual transcription of several remarkable non-protein coding loci: 2 atypical rRNA, TARE region and telomerase RNA gene. Together with other collections of <it>P. falciparum </it>ESTs, usually generated from mixed parasite stages, this collection of FcB1-schizont-ESTs provides valuable data to gain further insight into the <it>P. falciparum </it>gene structure, polymorphism and expression.</p

Insight into the exoproteome of the tissue-derived trypomastigote form of <em>trypanosoma cruzi</em>

The protozoan parasite Trypanosoma cruzi causes Chagas disease, one of the major neglected infectious diseases. It has the potential to infect any nucleated mammalian cell. The secreted/excreted protein repertoire released by T. cruzi trypomastigotes is crucial in host-pathogen interactions. In this study, mammalian tissue culture-derived trypomastigotes (Y strain) were used to characterize the exoproteome of the infective bloodstream life form. Proteins released into the serum-free culture medium after 3h of incubation were harvested and digested with trypsin. NanoLC-MS/MS analysis resulted in the identification of 540 proteins, the largest set of released proteins identified to date in Trypanosome spp. Bioinformatic analysis predicted most identified proteins as secreted, predominantly by non-classical pathways, and involved in host-cell infection. Some proteins possess predicted GPI-anchor signals, these being mostly trans-sialidases, mucin associated surface proteins and surface glycoproteins. Moreover, we enriched phosphopeptides and glycopeptides from tryptic digests. The majority of identified glycoproteins are trans-sialidases and surface glycoproteins involved in host-parasite interaction. Conversely, most identified phosphoproteins have no Gene Ontology classification. The existence of various proteins related to similar functions in the exoproteome likely reflects this parasite’s enhanced mechanisms for adhesion, invasion and internalization of different host-cell types, and escape from immune defences

Il Cristo in forma Pietatis del Rosso Fiorentino fra devozione e bellezza

<p>Differential distribution pattern of root- identified proteins in the four conditions described: TT, 42T, 42FS and 42RS.</p

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus

Background Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. Methods After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. Results After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. Conclusions To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation

HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

The HIV-1 nucleocapsid is formed during protease (PR)-directed viral maturation, and is transformed into pre-integration complexes following reverse transcription in the cytoplasm of the infected cell. Here, we report a detailed transmission electron microscopy analysis of the impact of HIV-1 PR and reverse transcriptase (RT) on nucleocapsid plasticity, using in vitro reconstitutions. After binding to nucleic acids, NCp15, a proteolytic intermediate of nucleocapsid protein (NC), was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and p1. This allowed NC co-aggregation with its single-stranded nucleic-acid substrate. Examination of these co-aggregates for the ability of RT to catalyse reverse transcription showed an effective synthesis of double-stranded DNA that, remarkably, escaped from the aggregates more efficiently with NCp7 than with NCp9. These data offer a compelling explanation for results from previous virological studies that focused on i) Gag processing leading to nucleocapsid condensation, and ii) the disappearance of NCp7 from the HIV-1 pre-integration complexes. We propose that HIV-1 PR and RT, by controlling the nucleocapsid architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodelling process that spatiotemporally coordinates the pre-integration steps of HIV-1. Finally we suggest that nucleoprotein remodelling mechanisms are common features developed by mobile genetic elements to ensure successful replication

A Deep Insight into the Sialome of Rhodnius neglectus, a vector of chagas disease

Background Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immuneresponses. Methods/Principal Findings Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva.We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant. Conclusions/Significance The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts

Proteases of haematophagous arthropod vectors are involved in blood-feeding, yolk formation and immunity : a review

Ticks, triatomines, mosquitoes and sand flies comprise a large number of haematophagous arthropods considered vectors of human infectious diseases. While consuming blood to obtain the nutrients necessary to carry on life functions, these insects can transmit pathogenic microorganisms to the vertebrate host. Among the molecules related to the blood-feeding habit, proteases play an essential role. In this review, we provide a panorama of proteases from arthropod vectors involved in haematophagy, in digestion, in egg development and in immunity. As these molecules act in central biological processes, proteases from haematophagous vectors of infectious diseases may influence vector competence to transmit pathogens to their prey, and thus could be valuable targets for vectorial control