| 1. |
- Ahuja, Sanjay, et al.
(author)
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Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein I domains in Escherichia coli
- 2006
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In: Malaria Journal. - : Springer Science and Business Media LLC. - 1475-2875. ; 5
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Journal article (peer-reviewed)abstract
- Background: Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL ( Duffy binding like), CIDR ( cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally. Methods: A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli. Results: Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1 alpha (46.8%) > CIDR2 beta (42.9%) > DBL2-4 gamma (31.7%) > DBL2 beta + C2 (30.6%) > DBL1 alpha (24.9%) > DBL2-7 epsilon (23.1%) > DBL2-5 delta (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions. Conclusion: The acidic terminal segment ( ATS) and CIDR alpha/beta domain types are suitable for recombinant expression in E. coli while all DBL subtypes (alpha, beta, gamma, delta, epsilon) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.
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| 2. |
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| 3. |
- Brolin, M, et al.
(author)
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Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites
- 2009
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In: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 10:10, s. R117-
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Journal article (peer-reviewed)abstract
- Background: Single nucleotide polymorphisms are common in duplicated genes, causing functional preservation, alteration or silencing. The Plasmodium falciparum genes var2csa and Pf332 are duplicated in the haploid genome of the HB3 parasite line. Whereas the molecular function of Pf332 remains to be elucidated, VAR2CSA is known to be the main adhesin in placental parasite sequestration. Sequence variations introduced upon duplication of these genes provide discriminative possibilities to analyze allele-specific transcription with a bearing towards understanding gene dosage impact on parasite biology. Results: We demonstrate an approach combining real-time PCR allelic discrimination and discriminative RNA-FISH to distinguish between highly similar gene copies in P. falciparum parasites. The duplicated var2csa variants are simultaneously transcribed, both on a population level and intriguingly also in individual cells, with nuclear co-localization of the active genes and corresponding transcripts. This indicates transcriptional functionality of duplicated genes, challenges the dogma of mutually exclusive var gene transcription and suggests mechanisms behind antigenic variation, at least in respect to the duplicated and highly similar var2csa genes. Conclusions: Allelic discrimination assays have traditionally been applied to study zygosity in diploid genomes. The assays presented here are instead successfully applied to the identification and evaluation of transcriptional activity of duplicated genes in the haploid genome of the P. falciparum parasite. Allelic discrimination and gene or transcript localization by FISH not only provide insights into transcriptional regulation of genes such as the virulence associated var genes, but also suggest that this sensitive and precise approach could be used for further investigation of genome dynamics and gene regulation.
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| 4. |
- Elfstrand, Lidia, et al.
(author)
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From starch to starch microspheres: Factors controlling the microspheres quality
- 2006
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In: Stärke. - : Wiley. - 0038-9056. ; 58:8, s. 381-390
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Journal article (peer-reviewed)abstract
- Starch microparticle is a dosage form suitable for encapsulation of protein drugs. The microparticle characteristics can be influential factors for consecutive sustained drug release from the starch matrix and impact its ability to be coated by poly(D,L-lactide-co-glycolide). In this study, six types of starch microparticles were investigated and characterized. Desired qualities of the microspheres such as spherical form of the particles, well-defined and sharp particle contours were coincident with coarse surface morphology, sharp and distinct peaks in DSC and X-ray graphs. It was concluded that the quality of the starch microparticles was influenced by molecular properties of the starch material as well as type of buffer, and these factors were related to recrystallization kinetics, amount and quality of ordered/crystalline structure of final starch microspheres and their microscopic appearance.
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| 5. |
- Elfstrand, Lidia, et al.
(author)
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Recrystallization of waxy maize starch during manufacturing of starch microspheres for drug delivery: Optimization by experimental design
- 2007
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In: Carbohydrate Polymers. - : Elsevier BV. - 0144-8617. ; 68:3, s. 568-576
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Journal article (peer-reviewed)abstract
- Gelatinized starch/water dispersions with a water content of 70% w/w were studied in order to evaluate the influence of the incubation time at two fixed temperatures (6 and 37 degrees C) on the recrystallization behavior. The starch material was produced from waxy maize starch by acid hydrolysis followed by a mechanical treatment, i.e. a high pressure homogenization. Empirical models for the recrystallization of the model starch dispersions were investigated by using a central composite circumscribed (CCC) design in two factors involving I I trials. The rebuilt crystalline structure was estimated by measuring the melting transition parameters with differential scanning calorimetry (DSC). The designed models for the melting enthalpy, the melting transition temperatures, and the melting interval as the responses were reproducible and predictable and the results were analyzed by using a response surface modeling. Operative conditions for the formation of the most thermally stable crystalline structure with the highest possible crystallinity were determined. It was also concluded that the decisive factor for the crystallinity of the given starch material was a prolonged storage at 6 'C, while thermal characteristics of the crystallites could be controlled by storage at 37 degrees C. An interaction effect was observed between the incubation time at these two experimental temperatures regarding enthalpy values and the melting interval of the samples. (c) 2006 Elsevier Ltd. All rights reserved.
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| 6. |
- Joannin, Nicolas, et al.
(author)
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Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family.
- 2008
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In: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 9, s. 19-
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Journal article (peer-reviewed)abstract
- BACKGROUND: 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. RESULTS: 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 Plasmodium falciparum. 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 Plasmodium 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. CONCLUSION: 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.
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| 7. |
- Mok, Bobo W., et al.
(author)
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Default Pathway of var2csa Switching and Translational Repression in Plasmodium falciparum
- 2008
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:4, s. e1982-
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Journal article (peer-reviewed)abstract
- Antigenic variation is a subtle process of fundamental importance to the survival of a microbial pathogen. In Plasmodium falciparum malaria, PfEMP1 is the major variable antigen and adhesin expressed at the surface of the infected erythrocyte, which is encoded for by members of a family of 60 var-genes. Peri-nuclear repositioning and epigenetic mechanisms control their mono-allelic expression. The switching of PfEMP1 depends in part on variable transition rates and short-lived immune responses to shared minor epitopes. Here we show var-genes to switch to a common gene that is highly transcribed, but sparsely translated into PfEMP1 and not expressed at the erythrocyte surface. Highly clonal and adhesive P. falciparum, which expressed distinct var-genes and the corresponding PfEMP1s at onset, were propagated without enrichment or panning. The parasites successively and spontaneously switched to transcribe a shared var-gene (var2csa) matched by the loss of PfEMP1 surface expression and host cell-binding. The var2csa gene repositioned in the peri-nuclear area upon activation, away from the telomeric clusters and heterochromatin to transcribe spliced, full-length RNA. Despite abundant transcripts, the level of intracellular PfEMP1 was low suggesting post-transcriptional mechanisms to partake in protein expression. In vivo, off-switching and translational repression may constitute one pathway, among others, coordinating PfEMP1 expression.
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| 8. |
- Pettersson, Fredrik, et al.
(author)
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Whole-body imaging of sequestration of Plasmodium falciparum in the rat.
- 2005
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In: Infect Immun. - 0019-9567. ; 73:11, s. 7736-46
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Journal article (peer-reviewed)abstract
- The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with 99mtechnetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria.
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| 9. |
- Ribacke, Ulf, et al.
(author)
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Genome wide gene amplifications and deletions in Plasmodium falciparum
- 2007
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In: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 155:1, s. 33-44
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Journal article (peer-reviewed)abstract
- The extent to which duplications and deletions occur in the Plasmodium falciparum genome, outside of the subtelomeres, and their contribution to the virulence of the malaria parasite is not known. Here we show the presence of multiple genome wide copy number polymorphisms (CNPs) covering 82 genes, the most extensive spanning a cumulative size of 110 kilobases. CNPs were identified in both laboratory strains and fresh clinical isolates using a 70-mer oligonucleotide microarray in conjunction with fluorescent in situ hybridizations and real-time quantitative PCR. The CNPs were found on all chromosomes except on chromosomes 6 and 8 and involved a total of 50 genes with increased copy numbers and 32 genes with decreased copy numbers relative to the 3D7 parasite. The genes, amplified in up to six copies, encode molecules involved in cell cycle regulation. cell division, drug resistance, erythrocyte invasion, sexual differentiation and unknown functions. These together with previous findings, suggest that the malaria parasite employs gene duplications and deletions as general strategies to enhance its survival and spread. Further analysis of the impact of discovered genetic differences and the underlying mechanisms is likely to generate a better understanding of the biology and the virulence of the malaria parasite.
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| 10. |
- Vogt, Anna M., et al.
(author)
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Release of sequestered malaria parasites upon injection of a glycosaminoglycan
- 2006
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 2:9, s. 853-863
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Journal article (peer-reviewed)abstract
- Severe human malaria is attributable to an excessive sequestration of Plasmodium falciparum-infected and uninfected erythrocytes in vital organs. Strains of P. falciparum that form rosettes and employ heparan sulfate as a host receptor are associated with development of severe forms of malaria. Heparin, which is similar to heparan sulfate in that it is composed of the same building blocks, was previously used in the treatment of severe malaria, but it was discontinued due to the occurrence of serious side effects such as intracranial bleedings. Here we report to have depolymerized heparin by periodate treatment to generate novel glycans (dGAG) that lack anticoagulant-activity. The dGAGs disrupt rosettes, inhibit merozoite invasion of erythrocytes and endothelial binding of P. falciparum-infected erythrocytes in vitro, and reduce sequestration in in vivo models of severe malaria. An intravenous injection of dGAGs blocks up to 80% of infected erythrocytes from binding in the micro-vasculature of the rat and releases already sequestered parasites into circulation. P. falciparum-infected human erythrocytes that sequester in the non-human primate Macaca fascicularis were similarly found to be released in to the circulation upon a single injection of 500 mu g of dGAG. We suggest dGAGs to be promising candidates for adjunct therapy in severe malaria.
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