Removal of Heterologous Sequences from Plasmodium falciparum Mutants Using FLPe-Recombinase

Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the selectable marker cassette by transient transfection of a plasmid that expressed a 37°C thermostable and enhanced FLP-recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines

Problems with Using the Normal Distribution – and Ways to Improve Quality and Efficiency of Data Analysis

Background: The Gaussian or normal distribution is the most established model to characterize quantitative variation of original data. Accordingly, data are summarized using the arithmetic mean and the standard deviation, by x 6 SD, or with the standard error of the mean, x 6 SEM. This, together with corresponding bars in graphical displays has become the standard to characterize variation. Methodology/Principal Findings: Here we question the adequacy of this characterization, and of the model. The published literature provides numerous examples for which such descriptions appear inappropriate because, based on the ‘‘95 % range check’’, their distributions are obviously skewed. In these cases, the symmetric characterization is a poor description and may trigger wrong conclusions. To solve the problem, it is enlightening to regard causes of variation. Multiplicative causes are by far more important than additive ones, in general, and benefit from a multiplicative (or log-) normal approach. Fortunately, quite similar to the normal, the log-normal distribution can now be handled easily and characterized at the level of the original data with the help of both, a new sign, x /, times-divide, and notation. Analogous to x 6 SD, it connects the multiplicative (or geometric) mean x * and the multiplicative standard deviation s * in the form x * x /s*, that is advantageous and recommended. Conclusions/Significance: The corresponding shift from the symmetric to the asymmetric view will substantially increas

Gene-Specific Signatures of Elevated Non-Synonymous Substitution Rates Correlate Poorly across the Plasmodium Genus

BACKGROUND: Comparative genome analyses of parasites allow large scale investigation of selective pressures shaping their evolution. An acute limitation to such analysis of Plasmodium falciparum is that there is only very partial low-coverage genome sequence of the most closely related species, the chimpanzee parasite P. reichenowi. However, if orthologous genes have been under similar selective pressures throughout the Plasmodium genus then positive selection on the P. falciparum lineage might be predicted to some extent by analysis of other lineages. PRINCIPAL FINDINGS: Here, three independent pairs of closely related species in different sub-generic clades (P. falciparum and P. reichenowi; P. vivax and P. knowlesi; P. yoelii and P. berghei) were compared for a set of 43 candidate ligand genes considered likely to be under positive directional selection and a set of 102 control genes for which there was no selective hypothesis. The ratios of non-synonymous to synonymous substitutions (dN/dS) were significantly elevated in the candidate ligand genes compared to control genes in each of the three clades. However, the rank order correlation of dN/dS ratios for individual candidate genes was very low, less than the correlation for the control genes. SIGNIFICANCE: The inability to predict positive selection on a gene in one lineage by identifying elevated dN/dS ratios in the orthologue within another lineage needs to be noted, as it reflects that adaptive mutations are generally rare events that lead to fixation in individual lineages. Thus it is essential to complete the genome sequences of particular species of phylogenetic importance, such as P. reichenowi

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

Marine Biodiversity in the Caribbean: Regional Estimates and Distribution Patterns

This paper provides an analysis of the distribution patterns of marine biodiversity and summarizes the major activities of the Census of Marine Life program in the Caribbean region. The coastal Caribbean region is a large marine ecosystem (LME) characterized by coral reefs, mangroves, and seagrasses, but including other environments, such as sandy beaches and rocky shores. These tropical ecosystems incorporate a high diversity of associated flora and fauna, and the nations that border the Caribbean collectively encompass a major global marine biodiversity hot spot. We analyze the state of knowledge of marine biodiversity based on the geographic distribution of georeferenced species records and regional taxonomic lists. A total of 12,046 marine species are reported in this paper for the Caribbean region. These include representatives from 31 animal phyla, two plant phyla, one group of Chromista, and three groups of Protoctista. Sampling effort has been greatest in shallow, nearshore waters, where there is relatively good coverage of species records; offshore and deep environments have been less studied. Additionally, we found that the currently accepted classification of marine ecoregions of the Caribbean did not apply for the benthic distributions of five relatively well known taxonomic groups. Coastal species richness tends to concentrate along the Antillean arc (Cuba to the southernmost Antilles) and the northern coast of South America (Venezuela – Colombia), while no pattern can be observed in the deep sea with the available data. Several factors make it impossible to determine the extent to which these distribution patterns accurately reflect the true situation for marine biodiversity in general: (1) highly localized concentrations of collecting effort and a lack of collecting in many areas and ecosystems, (2) high variability among collecting methods, (3) limited taxonomic expertise for many groups, and (4) differing levels of activity in the study of different taxa

Health state utilities in patients with diabetic retinopathy, diabetic macular oedema and age-related macular degeneration: a systematic review

Background Health state utility values (HSUVs) are important in the assessment of the cost effectiveness of new interventions. In the case of visual conditions, models generally tend have tended to be built around a set of health states defined by visual acuity (VA). The aim of this review was to assess the impact of VA on HSUVs in patients with diabetic retinopathy, diabetic macular oedema or age-related macular degeneration. Methods A systematic literature search was undertaken in major bibliographic databases to identify articles reporting on the relationship between HSUVs and vision. Data were extracted for population characteristics, visual levels and estimated utilities. Evidence from reported statistical models, where available, was considered in the evaluation of vision in the better-seeing eye and the worse-seeing eye. Due to the heterogeneity of included studies, a narrative synthesis was undertaken. Results Of the 17 relevant studies, 9 studies had data that could be used in the analysis of the impact of vision on HSUVs. Visual loss was associated with a marked impact on health utilities. However, the relationship was not comparable between conditions or by measure of HSUVs. Key results included the finding that overall, self-rated time-trade off estimates were more likely to discriminate between different VA levels than EQ-5D values. Additionally, a stronger correlation was observed between HSUVs and better-seeing eye VA compared to worse-seeing eye VA. Conclusions Visual acuity has a significant impact on HSUVs. Nevertheless, care must be taken in the interpretation and use of estimates in cost-effectiveness models due to differences in measures and population diversity

International equity portfolio investment and enforcement of insider trading laws: a cross-country analysis

In this study, we examine the effects of stringent insider trading laws’ enforcement, institutions and stock market development on international equity portfolio allocation using data from 44 countries over the period 2001-2015. Our results suggest that stringent insider trading laws and their enforcement exert a positive and significant impact on international portfolio investment allocation. Further analysis indicates that the interaction between a country’s institutional quality, stock market development and enforcement of insider trading laws have a positive and significant effect on international equity portfolio allocation. The findings of this study have implications for the design of portfolio investment trading strategies and contribute to the literature on foreign equity investment decisions

Platform for Plasmodium vivax vaccine discovery and development

Plasmodium vivax is the most prevalent malaria parasite on the American continent. It generates a global burden of 80-100 million cases annually and represents a tremendous public health problem, particularly in the American and Asian continents. A malaria vaccine would be considered the most cost-effective measure against this vector-borne disease and it would contribute to a reduction in malaria cases and to eventual eradication. Although significant progress has been achieved in the search for Plasmodium falciparum antigens that could be used in a vaccine, limited progress has been made in the search for P. vivax components that might be eligible for vaccine development. This is primarily due to the lack of in vitro cultures to serve as an antigen source and to inadequate funding. While the most advanced P. falciparum vaccine candidate is currently being tested in Phase III trials in Africa, the most advanced P. vivax candidates have only advanced to Phase I trials. Herein, we describe the overall strategy and progress in P. vivax vaccine research, from antigen discovery to preclinical and clinical development and we discuss the regional potential of Latin America to develop a comprehensive platform for vaccine development