Development and application of a positive–negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.     

Prioritizing emerging zoonoses in the Netherlands

Background

To support the development of early warning and surveillance systems of emerging zoonoses, we present a general method to prioritize pathogens using a quantitative, stochastic multi-criteria model, parameterized for the Netherlands.

Methodology/Principal Findings

A risk score was based on seven criteria, reflecting assessments of the epidemiology and impact of these pathogens on society. Criteria were weighed, based on the preferences of a panel of judges with a background in infectious disease control.

Conclusions/Significance

Pathogens with the highest risk for the Netherlands included pathogens in the livestock reservoir with a high actual human disease burden (e.g. Campylobacter spp., Toxoplasma gondii, Coxiella burnetii) or a low current but higher historic burden (e.g. Mycobacterium bovis), rare zoonotic pathogens in domestic animals with severe disease manifestations in humans (e.g. BSE prion, Capnocytophaga canimorsus) as well as arthropod-borne and wildlife associated pathogens which may pose a severe risk in future (e.g. Japanese encephalitis virus and West-Nile virus). These agents are key targets for development of early warning and surveillance.     

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.     

The invasion of urban forest by dengue vectors in Rio de Janeiro.

The invasion of a secondary forest within the city of Rio de Janeiro by Aedes aegypti and Ae. albopictus was evaluated from July 1997 to June 1998 through collections of immature stages in ovitraps set at 1 m, 10 m, 100 m, 500 m, and 1,000 m into the forest from houses on the periphery. Both mosquito species were much more abundant close to houses (1-10 m). Aedes aegypti was not collected beyond 100 m, while Ae. albopictus was the most abundant species overall and in ovitraps at all distances from houses. Abundances of Ae. albopictus were significantly correlated with time-lagged rainfall and with abundances of Ae. aegypti. Co-occurrences of Ae. albopictus in traps with Ae. aegypti and Limatus durhami, but not with Culex dolosus, were more likely close to houses. The results suggest that the urban forest is a refuge for both Aedes species, but especially for Ae. albopictus, whose abundance both near houses and in the forest raises concern that this invader may transmit arboviruses to humans that are presently restricted to the sylvan environment.     

Improving efficacy of Box gravid traps for collecting Culex quinquefasciatus.

In outdoor screen cages, one and two-choice assays were conducted with gravid Cx. quinquefasciatus to assess and improve the efficacy of the Box gravid trap. Subsequently, in a field trial, a modified Box gravid trap was compared with three other gravid traps: the CDC gravid trap, the CFG gravid trap, and a novel design of a sticky gravid trap. A major disadvantage of the Box gravid trap, a relatively low trapping efficacy, was overcome with a simple modification to the trap configuration. By replacing the solid lid of the collecting tray with mosquito netting, the air velocity through the trap inlet was doubled and the quality of the resting sites for caught females was improved. As a result, the modified Box gravid trap caught and retained twice as many Cx. quinquefasciatus as its predecessor in two-choice bioassays in the screened cages and proved as effective as the commercially available CDC gravid trap in the field, but without damaging the collected specimens. Captures with the Box and CDC gravid traps were significantly greater than those of either the Counter Flow Geometry gravid trap or a sticky gravid trap.     

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as 'Hit and Run' and 'vector recycling' which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.     

Sequestration and Tissue Accumulation of Human Malaria Parasites: Can We Learn Anything from Rodent Models of Malaria?

The sequestration of Plasmodium falciparum-infected red blood cells (irbcs) in the microvasculature of organs is associated with severe disease; correspondingly, the molecular basis of irbc adherence is an active area of study. In contrast to P. falciparum, much less is known about sequestration in other Plasmodium parasites, including those species that are used as models to study severe malaria. Here, we review the cytoadherence properties of irbcs of the rodent parasite Plasmodium berghei ANKA, where schizonts demonstrate a clear sequestration phenotype. Real-time in vivo imaging of transgenic P. berghei parasites in rodents has revealed a CD36-dependent sequestration in lungs and adipose tissue. In the absence of direct orthologs of the P. falciparum proteins that mediate binding to human CD36, the P. berghei proteins and/or mechanisms of rodent CD36 binding are as yet unknown. In addition to CD36-dependent schizont sequestration, irbcs accumulate during severe disease in different tissues, including the brain. The role of sequestration is discussed in the context of disease as are the general (dis)similarities of P. berghei and P. falciparum sequestration.     

Selective Visual Attention during Mirror Exposure in Anorexia and Bulimia Nervosa

Objective

Cognitive theories suggest that body dissatisfaction results from the activation of maladaptive appearance schemata, which guide mental processes such as selective attention to shape and weight-related information. In line with this, the present study hypothesized that patients with anorexia nervosa (AN) and bulimia nervosa (BN) are characterized by increased visual attention for the most dissatisfying/ugly body part compared to their most satisfying/beautiful body part, while a more balanced viewing pattern was expected for controls without eating disorders (CG).

Method

Eye movements were recorded in a group of patients with AN (n = 16), BN (n = 16) and a CG (n = 16) in an ecologically valid setting, i.e., during a 3-min mirror exposure.

Results

Evidence was found that patients with AN and BN display longer and more frequent gazes towards the most dissatisfying relative to the most satisfying and towards their most ugly compared to their most beautiful body parts, whereas the CG showed a more balanced gaze pattern.

Discussion

The results converge with theoretical models that emphasize the role of information processing in the maintenance of body dissatisfaction. Given the etiological importance of body dissatisfaction in the development of eating disorders, future studies should focus on the modification of the reported patterns.