Redefining the expressed prototype SICAvar gene involved in Plasmodium knowlesi antigenic variation

<p>Abstract</p> <p>Background</p> <p>The <it>SICAvar </it>gene family, expressed at the surface of infected erythrocytes, is critical for antigenic variation in <it>Plasmodium knowlesi</it>. When this family was discovered, a prototypic <it>SICAvar </it>gene was characterized and defined by a 10-exon structure. The predicted 205-kDa protein lacked a convincing signal peptide, but included a series of variable cysteine-rich modules, a transmembrane domain encoded by the penultimate exon, and a cytoplasmic domain encoded by the final highly conserved exon. The <it>205 SICAvar </it>gene and its family with up to 108 possible family members, was identified prior to the sequencing of the <it>P. knowlesi </it>genome. However, in the published <it>P. knowlesi </it>database this gene remains disjointed in five fragments. This study addresses a number of structural and functional questions that are critical for understanding <it>SICAvar </it>gene expression.</p> <p>Methods</p> <p>Database mining, bioinformatics, and traditional genomic and post-genomic experimental methods including proteomic technologies are used here to confirm the genomic context and expressed structure of the prototype <it>205 SICAvar </it>gene.</p> <p><b>Results</b></p> <p>This study reveals that the <it>205 SICAvar </it>gene reported previously to have a 10-exon expressed gene structure has, in fact, 12 exons, with an unusually large and repeat-laden intron separating two newly defined upstream exons and the <it>bona fide </it>5'UTR from the remainder of the gene sequence. The initial exon encodes a PEXEL motif, which may function to localize the SICA protein in the infected erythrocyte membrane. This newly defined start of the 205 <it>SICAvar </it>sequence is positioned on chromosome 5, over 340 kb upstream from the rest of the telomerically positioned <it>SICAvar </it>gene sequence in the published genome assembly. This study, however, verifies the continuity of these sequences, a 9.5 kb transcript, and provides evidence that the 205 <it>SICAvar </it>gene is located centrally on chromosome 5.</p> <p>Conclusion</p> <p>The prototype <it>205 SICAvar </it>gene has been redefined to have a 12-exon structure. These data are important because they 1) address questions raised in the <it>P. knowlesi </it>genome database regarding <it>SICAvar </it>gene fragments, numbers and structures, 2) show that this prototype gene encodes a PEXEL motif, 3) emphasize the need for further refinement of the <it>P. knowlesi </it>genome data, and 4) retrospectively, provide evidence for recombination within centrally located <it>SICAvar </it>sequences.</p

Serologic and cytokine signatures in children with multisystem inflammatory syndrome and coronavirus disease 2019

Background: The serologic and cytokine responses of children hospitalized with multisystem inflammatory syndrome (MIS-C) vs coronavirus disease 2019 (COVID-19) are poorly understood. Methods: We performed a prospective, multicenter, cross-sectional study of hospitalized children who met the Centers for Disease Control and Prevention case definition for MIS-C (n = 118), acute COVID-19 (n = 88), or contemporaneous healthy controls (n = 24). We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) immunoglobulin G (IgG) titers and cytokine concentrations in patients and performed multivariable analysis to determine cytokine signatures associated with MIS-C. We also measured nucleocapsid IgG and convalescent RBD IgG in subsets of patients. Results: Children with MIS-C had significantly higher SARS-CoV-2 RBD IgG than children with acute COVID-19 (median, 2783 vs 146; Conclusions: MIS-C was associated with high titers of SARS-CoV-2 RBD IgG antibodies and acute hypercytokinemia with IL-6, IL-10, IL-17A, and IFN-γ

Comparative methods in R hackathon

The R statistical analysis package has emerged as a popular platform for implementation of powerful comparative methods to understand the evolution of organismal traits and diversification. A hackathon was organized to bring together active R developers as well as end-users working on the integration of comparative phylogenetic methods within R to actively address issues of data exchange standards, code interoperability, usability, documentation quality, and the breadth of functionality for comparative methods available within R. Outcomes included a new base package for phylogenetic trees and data, a public wiki with tutorials and overviews of existing packages, code to allow Mesquite and R to interact, improvement of existing packages, and increased interaction within the community

Transmedia, sofistas y el jogo bonito. Caso de estudio: Santander Fútbol Pasión

Artículo para la sección Tribuna de adComunica

Plasmodium knowlesi gene expression differs in ex vivo compared to in vitro blood-stage cultures

Background: Plasmodium knowlesi is one of five Plasmodium species known to cause malaria in humans and can result in severe illness and death. While a zoonosis in humans, this simian malaria parasite species infects macaque monkeys and serves as an experimental model for in vivo, ex vivo and in vitro studies. It has underpinned malaria discoveries relating to host-pathogen interactions, the immune response and immune evasion strategies. This study investigated differences in P. knowlesi gene expression in samples from ex vivo and in vitro cultures. Methods: Gene expression profiles were generated using microarrays to compare the stage-specific transcripts detected for a clone of P. knowlesi propagated in the blood of a rhesus macaque host and then grown in an ex-vivo culture, and the same clone adapted to long-term in vitro culture. Parasite samples covering one blood-stage cycle were analysed at four-hour intervals. cDNA was generated and hybridized to an oligoarray representing the P. knowlesi genome. Two replicate experiments were developed from in vitro cultures. Expression values were filtered, normalized, and analysed using R and Perl language and applied to a sine wave model to determine changes in equilibrium and amplitude. Differentially expressed genes from ex vivo and in vitro time points were detected using limma R/Bioconductor and gene set enrichment analysis (GSEA). Results: Major differences were noted between the ex vivo and in vitro time courses in overall gene expression and the length of the cycle (25.5 hours ex vivo; 33.5 hours in vitro). GSEA of genes up-regulated ex vivo showed an enrichment of various genes including SICAvar, ribosomal- associated and histone acetylation pathway genes. In contrast, certain genes involved in metabolism and cell growth, such as porphobilinogen deaminase and tyrosine phosphatase, and one SICAvar gene, were significantly up-regulated in vitro. Conclusions: This study demonstrates how gene expression in P. knowlesi blood-stage parasites can differ dramatically depending on whether the parasites are grown in vivo, with only one cycle of development ex vivo, or as an adapted isolate in long-term in vitro culture. These data bring emphasis to the importance of studying the parasite, its biology and disease manifestations in the context of the host.NMRC (Natl Medical Research Council, S’pore)Published versio

GO-term enrichment analysis of <i>P</i>. <i>vivax</i> proteins in proteomes 1 and 2.

<p>GO-term enrichment analysis of <i>P</i>. <i>vivax</i> proteins in proteomes 1 and 2.</p

A large scale <i>Plasmodium vivax- Saimiri boliviensis</i> trophozoite-schizont transition proteome

<div><p><i>Plasmodium vivax</i> is a complex protozoan parasite with over 6,500 genes and stage-specific differential expression. Much of the unique biology of this pathogen remains unknown, including how it modifies and restructures the host reticulocyte. Using a recently published <i>P</i>. <i>vivax</i> reference genome, we report the proteome from two biological replicates of infected <i>Saimiri boliviensis</i> host reticulocytes undergoing transition from the late trophozoite to early schizont stages. Using five database search engines, we identified a total of 2000 <i>P</i>. <i>vivax</i> and 3487 <i>S</i>. <i>boliviensis</i> proteins, making this the most comprehensive <i>P</i>. <i>vivax</i> proteome to date. PlasmoDB GO-term enrichment analysis of proteins identified at least twice by a search engine highlighted core metabolic processes and molecular functions such as glycolysis, translation and protein folding, cell components such as ribosomes, proteasomes and the Golgi apparatus, and a number of vesicle and trafficking related clusters. Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 enriched functional annotation clusters of <i>S</i>. <i>boliviensis</i> proteins highlighted vesicle and trafficking-related clusters, elements of the cytoskeleton, oxidative processes and response to oxidative stress, macromolecular complexes such as the proteasome and ribosome, metabolism, translation, and cell death. Host and parasite proteins potentially involved in cell adhesion were also identified. Over 25% of the <i>P</i>. <i>vivax</i> proteins have no functional annotation; this group includes 45 VIR members of the large PIR family. A number of host and pathogen proteins contained highly oxidized or nitrated residues, extending prior trophozoite-enriched stage observations from <i>S</i>. <i>boliviensis</i> infections, and supporting the possibility of oxidative stress in relation to the disease. This proteome significantly expands the size and complexity of the known <i>P</i>. <i>vivax</i> and Saimiri host iRBC proteomes, and provides in-depth data that will be valuable for ongoing research on this parasite’s biology and pathogenesis.</p></div

Giemsa-stained <i>P</i>. <i>vivax</i> iRBC isolated from biological replicate 1.

<p>Thin smears were prepared from Percoll-gradient enriched iRBC, and show early nucleated schizonts (iRBC 1, 4 and 5) and large late-stage trophozoites (iRBC 2 and 3).</p

Functional categories of <i>P</i>. <i>vivax</i> and <i>S</i>. <i>boliviensis</i> trophozoite-schizont transition iRBC proteins.

<p>Major categories for both organisms include metabolism, translation, transcription, proteolysis and trafficking. Over 25% of <i>P</i>. <i>vivax</i> proteins (hypothetical and PIR proteins) have no annotated function. Numerous cytoskeletal proteins, particularly actin-related, are identified for <i>S</i>. <i>boliviensis</i>. Details of individual proteins, each identified more than once by a database search engine in the two combined biological replicate proteomes, are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182561#pone.0182561.s005" target="_blank">S5 Table</a>. These pie charts are similar to those of trophozoite-stage iRBC proteins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182561#pone.0182561.ref044" target="_blank">44</a>], however the <i>P</i>. <i>vivax</i> hypothetical protein fraction has decreased. New <i>P</i>. <i>vivax</i> categories include PIR/VIR proteins and Plasmodium exported proteins.</p

DAVID-derived <i>S</i>. <i>boliviensis</i> functional annotation clusters in proteomes 1 and 2.

<p>DAVID-derived <i>S</i>. <i>boliviensis</i> functional annotation clusters in proteomes 1 and 2.</p