Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance

Background: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology. Results: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0–3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7. Conclusions: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates

Antigenic variation in Plasmodium falciparum : understanding the RIFIN protein family

RIFIN proteins are variable surface antigens, which have a central role in the survival and virulence of the malaria parasite Plasmodium falciparum. Antigenic variation is a mean for these parasites to avoid clearance by the host s immune system. However, this is often a secondary function to the main role of these proteins. In the case of RIFIN, P. falciparum s largest multicopy protein family, the main functions remain unknown. In order to elucidate a protein s function, it is crucial to understand its basic properties, including the structure of the protein family, their localization and the protein s topology. Through different methods, we have strived to simplify the RIFIN protein family into manageable entities. We have started with a simple classification of RIFIN proteins into meaningful sub-groups. We have predicted that these sub-groups are functionally distinct, although they probably share a related function. We then designed RSPred, an automatic method, based on hidden Markov models and a sorting program, to detect and classify RIFIN and STEVOR sequences according to their sub-group. Finally, using an in vitro method to determine protein topology, we have analyzed both A-RIFIN and B-RIFIN proteins for their number of transmembrane segments and their topologies. We show that both protein groups have a signal sequence targeting them to lipid bilayers and only one transmembrane domain. They both share a common topology where the bulk of the protein is exposed to the extracellular environment. With the current knowledge of RIFIN protein localizations, as well as the loss of expression of A-RIFIN but not B-RIFIN proteins in a splenectomized patient, it seems increasingly clear that B-RIFIN proteins are good targets for future studies, to decipher the functions of these variable proteins

Expression of a type B RIFIN in Plasmodium falciparum merozoites and gametes

BACKGROUND: The ability of Plasmodium falciparum to undergo antigenic variation, by switching expression among protein variants encoded by multigene families, such as var, rif and stevor, is key to the survival of this parasite in the human host. The RIFIN protein family can be divided into A and B types based on the presence or absence of a 25 amino acid motif in the semi-conserved domain. A particular type B RIFIN, PF13_0006, has previously been shown to be strongly transcribed in the asexual and sexual stages of P. falciparum in vitro. METHODS: Antibodies to recombinant PF13_0006 RIFIN were used in immunofluorescence and confocal imaging of 3D7 parasites throughout the asexual reproduction and sexual development to examine the expression of PF13_0006. Furthermore, reactivity to recombinant PF13_0006 was measured in plasma samples collected from individuals from both East and West African endemic areas. RESULTS: The PF13_0006 RIFIN variant appeared expressed by both released merozoites and gametes after emergence. 7.4% and 12.1% of individuals from East and West African endemic areas, respectively, carry plasma antibodies that recognize recombinant PF13_0006, where the antibody responses were more common among older children. CONCLUSIONS: The stage specificity of PF13_0006 suggests that the diversity of RIFIN variants has evolved to provide multiple specialized functions in different stages of the parasite life cycle. These data also suggest that RIFIN variants antigenically similar to PF13_0006 occur in African parasite populations

Impacts of Export Tax of Cocoa Beans on Indonesian Economy.

In recent years, there is a significant decline of cocoa beans in terms of exports value and share after 2010. Several studies claimed that this downward trend was caused by the introduction of an export tax on cocoa beans in 2010. Nevertheless, there are limited studies on the impacts of decreasing cocoa beans exports to the Indonesian economy. Therefore, this study aimed to simulate the impacts of the imposition of export tax on cocoa beans to the economy as well as unemployment. Methodology of this study utilised the Input-Output Table. In particular, this study calculated the impacts of export tax on cocoa beans to the changes of output, primary inputs, and unemployment in several scenarios. The main result of this study was that at extreme scenario, where the cocoa beans sector\u27s export was eliminated, the impacts on the whole economy and unemployment were insignificant. Moreover, this study found that the impacts on value added such as decreasing of profit were relatively higher than decreasing ra te on the output and others value added such as salary and wages and indirect taxes. On the other hand, this study argued that even though the introduction of export tax effectively reduced raw cocoa beans exports, there was an increasing on the exports\u27 value on the down stream industries

Characterization and gene expression analysis of the cir multi-gene family of plasmodium chabaudi chabaudi (AS)

Background: The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due to antigenic variation. In this model, pir genes are called cir s and may be involved in this mechanism, allowing evasion of host immune responses. In order to fully understand the role(s) of CIR proteins during P. chabaudi infection, a detailed characterization of the cir gene family was required. Results: The cir repertoire was annotated and a detailed bioinformatic characterization of the encoded CIR proteins was performed. Two major sub-families were identified, which have been named A and B. Members of each sub-family displayed different amino acid motifs, and were thus predicted to have undergone functional divergence. In addition, the expression of the entire cir repertoire was analyzed via RNA sequencing and microarray. Up to 40% of the cir gene repertoire was expressed in the parasite population during infection, and dominant cir transcripts could be identified. In addition, some differences were observed in the pattern of expression between the cir subgroups at the peak of P. chabaudi infection. Finally, specific cir genes were expressed at different time points during asexual blood stages. Conclusions: In conclusion, the large number of cir genes and their expression throughout the intraerythrocytic cycle of development indicates that CIR proteins are likely to be important for parasite survival. In particular, the detection of dominant cir transcripts at the peak of P. chabaudi infection supports the idea that CIR proteins are expressed, and could perform important functions in the biology of this parasite. Further application of the methodologies described here may allow the elucidation of CIR sub-family A and B protein functions, including their contribution to antigenic variation and immune evasion

RSpred, a set of Hidden Markov Models to detect and classify the RIFIN and STEVOR proteins of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Many parasites use multicopy protein families to avoid their host's immune system through a strategy called antigenic variation. RIFIN and STEVOR proteins are variable surface antigens uniquely found in the malaria parasites <it>Plasmodium falciparum </it>and <it>P. reichenowi</it>. Although these two protein families are different, they have more similarity to each other than to any other proteins described to date. As a result, they have been grouped together in one Pfam domain. However, a recent study has described the sub-division of the RIFIN protein family into several functionally distinct groups. These sub-groups require phylogenetic analysis to sort out, which is not practical for large-scale projects, such as the sequencing of patient isolates and meta-genomic analysis.</p> <p>Results</p> <p>We have manually curated the <it>rif </it>and <it>stevor </it>gene repertoires of two <it>Plasmodium falciparum </it>genomes, isolates DD2 and HB3. We have identified 25% of mis-annotated and ~30 missing <it>rif </it>and <it>stevor </it>genes. Using these data sets, as well as sequences from the well curated reference genome (isolate 3D7) and field isolate data from Uniprot, we have developed a tool named RSpred. The tool, based on a set of hidden Markov models and an evaluation program, automatically identifies STEVOR and RIFIN sequences as well as the sub-groups: A-RIFIN, B-RIFIN, B1-RIFIN and B2-RIFIN. In addition to these groups, we distinguish a small subset of STEVOR proteins that we named STEVOR-like, as they either differ remarkably from typical STEVOR proteins or are too fragmented to reach a high enough score. When compared to Pfam and TIGRFAMs, RSpred proves to be a more robust and more sensitive method. We have applied RSpred to the proteomes of several <it>P. falciparum </it>strains, <it>P. reichenowi, P. vivax</it>, <it>P. knowlesi </it>and the rodent malaria species. All groups were found in the <it>P. falciparum </it>strains, and also in the <it>P. reichenowi </it>parasite, whereas none were predicted in the other species.</p> <p>Conclusions</p> <p>We have generated a tool for the sorting of RIFIN and STEVOR proteins, large antigenic variant protein groups, into homogeneous sub-families. Assigning functions to such protein families requires their subdivision into meaningful groups such as we have shown for the RIFIN protein family. RSpred removes the need for complicated and time consuming phylogenetic analysis methods. It will benefit both research groups sequencing whole genomes as well as others working with field isolates. RSpred is freely accessible via <url>http://www.ifm.liu.se/bioinfo/</url>.</p

Determinants of Micro and Small Enterprise Food Industry Market Expansion in Indonesia

One of the indicators that might upgrade micro and small enterprise is the ability to expand their market target outside their location or district. The objective of this article was to analyze the determinants of market expansion for small and medium enterprise especially in the food industry in Indonesia. Market expansion is defined as selling products or services outside the district where the enterprise is located. Secondary data were utilized in the research by using the Micro and Small Enterprise Survey conducted by Statistics Indonesia in 2014 with the data of 21,380 firms. Two analysis were conducted, firstly using the logit analysis in order to differentiate between enterprises selling their products inside and outside the district. The second analysis used Tobit analysis of which the dependent variable is the share of product sold outside the district. Independent variables used in both equations are similar. The results indicated that higher education level, number of labor, value of production, number of enterprise with external finance, number of enterprise located in Java and male-owned firms resulted in higher probability of selling their product outside the district. Moreover, the same variables will also increase the share of product sold outside the district. From the two equations, it can be concluded that the government policy must be addressed in two aspects in order to upgrade the small and medium enterprises, the first is increasing the scale of the enterprises and secondly, fostering financial inclusion for these enterprises.Keywords: micro and small enterprise, logit analysis, tobit analysis, food industry, market expansio

Revisiting the Plasmodium falciparum RIFIN family: from comparative genomics to 3D-model prediction

<p>Abstract</p> <p>Background</p> <p>Subtelomeric <it>RIFIN </it>genes constitute the most abundant multigene family in <it>Plasmodium falciparum</it>. <it>RIFIN </it>products are targets for the human immune response and contribute to the antigenic variability of the parasite. They are transmembrane proteins grouped into two sub-families (RIF_A and RIF_B). Although recent data show that RIF_A and RIF_B have different sub-cellular localisations and possibly different functions, the same structural organisation has been proposed for members of the two sub-families. Despite recent advances, our knowledge of the regulation of <it>RIFIN </it>gene expression is still poor and the biological role of the protein products remain obscure.</p> <p>Results</p> <p>Comparative studies on <it>RIFINs </it>in three clones of <it>P. falciparum </it>(3D7, HB3 and Dd2) by Multidimensional scaling (MDS) showed that gene sequences evolve differently in the 5'upstream, coding, and 3'downstream regions, and suggested a possible role of highly conserved 3' downstream sequences. Despite the expected polymorphism, we found that the overall structure of <it>RIFIN </it>repertoires is conserved among clones suggesting a balance between genetic drift and homogenisation mechanisms which guarantees emergence of novel variants but preserves the functionality of genes. Protein sequences from a <it>bona fide </it>set of 3D7 RIFINs were submitted to predictors of secondary structure elements. In contrast with the previously proposed structural organisation, no signal peptide and only one transmembrane helix were predicted for the majority of RIF_As. Finally, we developed a strategy to obtain a reliable 3D-model for RIF_As. We generated 265 possible structures from 53 non-redundant sequences, from which clustering and quality assessments selected two models as the most representative for putative RIFIN protein structures.</p> <p>Conclusion</p> <p>First, comparative analyses of <it>RIFIN </it>repertoires in different clones of <it>P. falciparum </it>provide insights on evolutionary mechanisms shaping the multigene family. Secondly, we found that members of the two sub-families RIF_As and RIF_Bs have different structural organization in accordance with recent experimental results. Finally, representative models for RIF_As have an "Armadillo-like" fold which is known to promote protein-protein interactions in diverse contexts.</p

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype

Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family

<p>Abstract</p> <p>Background</p> <p>Parasitic protozoans possess many multicopy gene families which have central roles in parasite survival and virulence. The number and variability of members of these gene families often make it difficult to predict possible functions of the encoded proteins. The families of extra-cellular proteins that are exposed to a host immune response have been driven via immune selection to become antigenically variant, and thereby avoid immune recognition while maintaining protein function to establish a chronic infection.</p> <p>Results</p> <p>We have combined phylogenetic and function shift analyses to study the evolution of the RIFIN proteins, which are antigenically variant and are encoded by the largest multicopy gene family in <it>Plasmodium falciparum</it>. We show that this family can be subdivided into two major groups that we named A- and B-RIFIN proteins. This suggested sub-grouping is supported by a recently published study that showed that, despite the presence of the <it>Plasmodium </it>export (PEXEL) motif in all RIFIN variants, proteins from each group have different cellular localizations during the intraerythrocytic life cycle of the parasite. In the present study we show that function shift analysis, a novel technique to predict functional divergence between sub-groups of a protein family, indicates that RIFINs have undergone neo- or sub-functionalization.</p> <p>Conclusion</p> <p>These results question the general trend of clustering large antigenically variant protein groups into homogenous families. Assigning functions to protein families requires their subdivision into meaningful groups such as we have shown for the RIFIN protein family. Using phylogenetic and function shift analysis methods, we identify new directions for the investigation of this broad and complex group of proteins.</p