Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions

BACKGROUND: The variant surface antigen family Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) is an important target for protective immunity and is implicated in the pathology of malaria through its ability to adhere to host endothelial receptors. The sequence diversity and organization of the 3D7 PfEMP1 repertoire was investigated on the basis of the complete genome sequence. METHODS: Using two tree-building methods we analysed the coding and non-coding sequences of 3D7 var and rif genes as well as var genes of other parasite strains. RESULTS: var genes can be sub-grouped into three major groups (group A, B and C) and two intermediate groups B/A and B/C representing transitions between the three major groups. The best defined var group, group A, comprises telomeric genes transcribed towards the telomere encoding PfEMP1s with complex domain structures different from the 4-domain type dominant of groups B and C. Two sequences belonging to the var1 and var2 subfamilies formed independent groups. A rif subgroup transcribed towards the centromere was found neighbouring var genes of group A such that the rif and var 5' regions merged. This organization appeared to be unique for the group A var genes CONCLUSION: The grouping of var genes implies that var gene recombination preferentially occurs within var gene groups and it is speculated that the groups reflect a functional diversification evolved to cope with the varying conditions of transmission and host immune response met by the parasite

Current Trends in Texas Charge Practice: Preservation of Error and Broad-Form Use.

Over the years Texas appellate courts have been wrestling with two overwhelming charge issues—charge preservation of error and broad-form use. Since the charge is the controlling document the jury uses to decide the factual issues of the case, it is of extreme importance. Before a party can complain on appeal about charge errors, the error must be preserved at trial. The Texas Rules of Civil Procedure (TRCP) have a certain set of procedures for preservation of charge error. The Texas Supreme Court amended charge preservation of error practice in State Department of Highways & Public Transportation v. Payne. In the years since, the Texas courts of review have been inconsistent in applying charge preservation of error. There are two general types of charge error: errors of omission and errors of commission. When there is a question, instruction or definition which should be included in the charge, but was not, there is an error of omission. When there is a question, instruction or definition in the charge, but it is incorrect, there is an error of commission. Conversely, under broad-form practice, questions are drafted generally and include most or all elements. Furthermore, much of the charge is contained in instructions to the general questions. Basically, the jury is asked to find conclusions without having to agree on specific facts. There are two looming issues in current Texas charge practice—preservation of error and broad-form use. At present, the TRCP generally mandates objections to be used to preserve incorrect questions, definitions and instructions within the jury charge. Whereas written requests will preserve erroneous omissions from the charge. The confusion stems from the unpredictable ways the courts have interpreted these rules. The most logical option to remedy the existing inconsistencies is to adopt new charge preservation of error rules

Current Trends in Texas Charge Practice: Preservation of Error and Broad-Form Use.

Over the years Texas appellate courts have been wrestling with two overwhelming charge issues—charge preservation of error and broad-form use. Since the charge is the controlling document the jury uses to decide the factual issues of the case, it is of extreme importance. Before a party can complain on appeal about charge errors, the error must be preserved at trial. The Texas Rules of Civil Procedure (TRCP) have a certain set of procedures for preservation of charge error. The Texas Supreme Court amended charge preservation of error practice in State Department of Highways & Public Transportation v. Payne. In the years since, the Texas courts of review have been inconsistent in applying charge preservation of error. There are two general types of charge error: errors of omission and errors of commission. When there is a question, instruction or definition which should be included in the charge, but was not, there is an error of omission. When there is a question, instruction or definition in the charge, but it is incorrect, there is an error of commission. Conversely, under broad-form practice, questions are drafted generally and include most or all elements. Furthermore, much of the charge is contained in instructions to the general questions. Basically, the jury is asked to find conclusions without having to agree on specific facts. There are two looming issues in current Texas charge practice—preservation of error and broad-form use. At present, the TRCP generally mandates objections to be used to preserve incorrect questions, definitions and instructions within the jury charge. Whereas written requests will preserve erroneous omissions from the charge. The confusion stems from the unpredictable ways the courts have interpreted these rules. The most logical option to remedy the existing inconsistencies is to adopt new charge preservation of error rules

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of <it>Plasmodium falciparum </it>adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development.</p> <p>Methods</p> <p>A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy</p> <p>Results</p> <p>Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1.</p> <p>Conclusion</p> <p>A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.</p

Failing boys and moral panics: perspectives on the underachievement debate

The paper re-examines the underachievement debate from the perspective of the ‘discourse of derision’ that surrounds much writing in this area. It considers the contradictions and inconsistencies which underpin much of the discourse – from a reinterpretation of examination scores, to the conflation of the concepts of ‘under’ and ‘low’ achievement and finally to the lack of consensus on a means of defining and measuring the term underachievement. In doing so, this paper suggests a more innovative approach for understanding, re-evaluating and perhaps rejecting the notion of underachievement

Treatment rationale for dogs poisoned with aldicarb (carbamate pesticide)

The treatment rationale for dogs poisoned by aldicarb is reviewed from a pharmacological perspective. The illegal use of aldicarb to maliciously poison dogs is a major problem in some parts of the world. In South Africa, it is probably the most common canine poisoning treated by companion animal veterinarians. Aldicarb poisoning is an emergency and veterinarians need to be able to diagnose it and start with effective treatment immediately to ensure a reasonable prognosis. Successful treatment depends on the timely use of an anti-muscarinic drug (e.g. atropine). Additional supportive treatment options, including fluid therapy, diphenhydramine, benzodiazepines and the prevention of further absorption (activated charcoal) should also be considered. Possible complications after treatment are also briefly discussed.http://www.journals.co.za/ej/ejour_savet.htmlmn201

Failure of diplodiatoxin to induce diplodiosis in juvenile goats

Diplodiosis is an important neuromycotoxicosis of ruminants in South Africa when grazing on harvested maize fields in winter. It is believed to be caused by mycotoxin(s) synthesised by Stenocarpella (Diplodia) maydis. Although several metabolites have been isolated from S. maydis culture material, none of these have been administered to ruminants to reproduce the disease. The objectives of this study were to isolate diplodiatoxin and to administer it to juvenile goats. Diplodiatoxin, considered as a major metabolite, was purified from S. maydis-infected maize cultures (Coligny 2007 isolate). Following intravenous administration of 2 mg and 4 mg diplodiatoxin/kg body weight for five consecutive days to two juvenile goats, no clinical signs reminiscent of diplodiosis were observed. Based on previous experimental results and if diplodiatoxin was the causative compound, the dosage regimen employed was seemingly appropriate to induce diplodiosis. In addition, intraruminal administration of 2 mg/kg diplodiatoxin to one goat for three consecutive days also did not induce clinical signs. It appears as if diplodiatoxin alone is not the causative compound. Other metabolites and/or mixtures of diplodiatoxin and other mycotoxins, when available in sufficient quantities, should also be evaluated.The Maize Trust of South Africahttp://www.ojvr.orgam2021Paraclinical SciencesProduction Animal Studie

Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>The PFD1235w <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and <it>Escherichia coli </it>based system was used to express single and double domains encoded by the <it>pfd1235w var </it>gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7_PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed.</p> <p>Methods</p> <p>The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and <it>E. coli</it>-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7_PFD1235w-IE.</p> <p>Results</p> <p>All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the <it>E. coli </it>system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the <it>E. coli </it>produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7_PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay.</p> <p>Conclusions</p> <p>The baculovirus based insect cell system was distinctly superior to the <it>E. coli </it>expression system in producing a larger number of different recombinant PFD1235w protein domains and these were significantly easier to purify at a useful yield. However, proteins produced in both systems were able to induce antibodies in rats, which can recognize the native PFD1235w on the surface of IE.</p

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