Development and Optimization of a Selective Whole-Genome Amplification To Study Plasmodium ovale Spp.

Since 2010, the human-infecting malaria parasite Plasmodium ovale spp. has been divided into two genetically distinct species, P. ovale wallikeri and P. ovale curtisi. In recent years, application of whole-genome sequencing (WGS) to P. ovale spp. allowed to get a better understanding of its evolutionary history and discover some specific genetic patterns. Nevertheless, WGS data from P. ovale spp. are still scarce due to several drawbacks, including a high level of human DNA contamination in blood samples, infections with commonly low parasite density, and the lack of robust in vitro culture. Here, we developed two selective whole-genome amplification (sWGA) protocols that were tested on six P. ovale wallikeri and five P. ovale curtisi mono-infection clinical samples. Blood leukodepletion by a cellulose-based filtration was used as the gold standard for intraspecies comparative genomics with sWGA. We also demonstrated the importance of genomic DNA preincubation with the endonuclease McrBC to optimize P. ovale spp. sWGA. We obtained high-quality WGS data with more than 80% of the genome covered by ≥5 reads for each sample and identified more than 5,000 unique single-nucleotide polymorphisms (SNPs) per species. We also identified some amino acid changes in pocdhfr and powdhfr for which similar mutations in P. falciparum and P. vivax are associated with pyrimethamine or cycloguanil resistance. In conclusion, we developed two sWGA protocols for P. ovale spp. WGS that will help to design much-needed large-scale P. ovale spp. population studies. IMPORTANCE Plasmodium ovale spp. has the ability to cause relapse, defined as recurring asexual parasitemia originating from liver-dormant forms. Whole-genome sequencing (WGS) data are of importance to identify putative molecular markers associated with relapse or other virulence mechanisms. Due to low parasitemia encountered in P. ovale spp. infections and no in vitro culture available, WGS of P. ovale spp. is challenging. Blood leukodepletion by filtration has been used, but no technique exists yet to increase the quantity of parasite DNA over human DNA when starting from genomic DNA extracted from whole blood. Here, we demonstrated that selective whole-genome amplification (sWGA) is an easy-to-use protocol to obtain high-quality WGS data for both P. ovale spp. species from unprocessed blood samples. The new method will facilitate P. ovale spp. population genomic studies.

Transcriptome Analysis of Plasmodium falciparum Isolates From Benin Reveals Specific Gene Expression Associated With Cerebral Malaria.

Cerebral malaria (CM) is the severest form of Plasmodium falciparum infection. Children under 5 years old are those most vulnerable to CM, and they consequently have the highest risk of malaria-related death. Parasite-associated factors leading to CM are not yet fully elucidated. We therefore sought to characterize the gene expression profile associated with CM, using RNA sequencing data from 15 CM and 15 uncomplicated malaria isolates from Benin. Cerebral malaria parasites displayed reduced circulation times, possibly related to higher cytoadherence capacity. Consistent with the latter, we detected increased var genes abundance in CM isolates. Differential expression analyses showed that distinct transcriptome profiles are signatures of malaria severity. Genes involved in adhesion, excluding variant surface antigens, were dysregulated, supporting the idea of increased cytoadhesion capacity of CM parasites. Finally, we found dysregulated expression of genes in the entry into host pathway that may reflect greater erythrocyte invasion capacity of CM parasites.

PfEMP1 A-Type ICAM-1-Binding Domains Are Not Associated with Cerebral Malaria in Beninese Children.

PfEMP1 is the major antigen involved in Plasmodium falciparum-infected erythrocyte sequestration in cerebrovascular endothelium. While some PfEMP1 domains have been associated with clinical phenotypes of malaria, formal associations between the expression of a specific domain and the adhesion properties of clinical isolates are limited. In this context, 73 cerebral malaria (CM) and 98 uncomplicated malaria (UM) Beninese children were recruited. We attempted to correlate the cytoadherence phenotype of Plasmodium falciparum isolates with the clinical presentation and the expression of specific PfEMP1 domains. Cytoadherence level on Hbec-5i and CHO-ICAM-1 cell lines and var genes expression were measured. We also investigated the prevalence of the ICAM-1-binding amino acid motif and dual receptor-binding domains, described as a potential determinant of cerebral malaria pathophysiology. We finally evaluated IgG levels against PfEMP1 recombinant domains (CIDRα1.4, DBLß3, and CIDRα1.4-DBLß3). CM isolates displayed higher cytoadherence levels on both cell lines, and we found a correlation between CIDRα1.4-DBLß1/3 domain expression and CHO-ICAM-1 cytoadherence level. Endothelial protein C receptor (EPCR)-binding domains were overexpressed in CM isolates compared to UM whereas no difference was found in ICAM-1-binding DBLß1/3 domain expression. Surprisingly, both CM and UM isolates expressed ICAM-1-binding motif and dual receptor-binding domains. There was no difference in IgG response against DBLß3 between CM and UM isolates expressing ICAM-1-binding DBLß1/3 domain. It raises questions about the role of this motif in CM pathophysiology, and further studies are needed, especially on the role of DBLß1/3 without the ICAM-1-binding motif.IMPORTANCE Cerebral malaria pathophysiology remains unknown despite extensive research. PfEMP1 proteins have been identified as the main Plasmodium antigen involved in cerebrovascular endothelium sequestration, but it is unclear which var gene domain is involved in Plasmodium cytoadhesion. EPCR binding is a major determinant of cerebral malaria whereas the ICAM-1-binding role is still questioned. Our study confirmed the EPCR-binding role in CM pathophysiology with a major overexpression of EPCR-binding domains in CM isolates. In contrast, ICAM-1-binding involvement appears less obvious with A-type ICAM-1-binding and dual receptor-binding domain expression in both CM and UM isolates. We did not find any variations in ICAM-1-binding motif sequences in CM compared to UM isolates. UM and CM patients infected with isolates expressing the ICAM-1-binding motif displayed similar IgG levels against DBLß3 recombinant protein. Our study raises interrogations about the role of these domains in CM physiopathology and questions their use in vaccine strategies against cerebral malaria.

Determinants of the interindividual variability in serum cytidine deaminase activity of patients with solid tumours.

AIMS: Cytidine deaminase (CDA) activity in cancer patients' serum has been proposed as a predictive biomarker for efficacy and toxicity of nucleoside analogues. However, discrepant results about its predictive value have been reported due to the high interindividual variability in CDA activity. This study aimed at identifying determinants of this interindividual variability. METHODS: From December 2014 to November 2015, 183 patients were prospectively included. Serum CDA activity, biological and clinical characteristics as well as five common single nucleotide polymorphisms (SNPs) in the CDA gene (c.-451C > T, c.-92A > G, c.-33_-31delC, c.79A > C, c.435 T > C) were analysed. Associations between clinical characteristics, pharmacogenetic variants and CDA activity were univariately tested. P < 0.1-candidate variables were analysed through a multivariate analysis. The association between CDA activity and toxicity was assessed for the 56 gemcitabine-treated patients. Intraindividual variability in CDA activity was explored in six pancreatic cancer patients treated with gemcitabine. RESULTS: Median CDA activity was 3.97 U mg-1 (range 1.53-15.49 U mg-1 ). A univariate analysis showed that CDA activity was statistically associated with Eastern Cooperative Oncology Group performance status, mild or severe malnutrition, inflammatory syndrome, leucocyte count, neutrophil count, albumin, C-reactive protein and -c.-33_-31delC single nucleotide polymorphism. A multivariate analysis identified that only neutrophil count (P < 0.0001) and severe malnutrition (P = 0.0278) were independently associated with CDA activity. Low CDA activity (<2 U mg-1 ) was not statistically associated with severe gemcitabine-related toxicities (P = 0.16). A decrease in CDA activity was observed during the longitudinal follow-up of six pancreatic cancer patients treated with gemcitabine (P = 0.03). CONCLUSIONS: These results suggest that neutrophil count and malnutrition should be considered for the interpretation of pretherapeutic CDA activity.

Distinction of Plasmodium ovale wallikeri and Plasmodium ovale curtisi using quantitative Polymerase Chain Reaction with High Resolution Melting revelation.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) have been described as two distinct species, only distinguishable by molecular methods such as PCR. Because of no well-defined endemic area and a variable clinical presentation as higher thrombocytopenia and nausea associated with Pow infection and asymptomatic forms of the pathology with Poc infection, rapid and specific identification of Plasmodium ovale curtisi and Plasmodium ovale wallikeri are needed. The aim of the study was to evaluate a new quantitative real-time PCR coupled with high resolution melting revelation (qPCR-HRM) for identification of both species. Results were compared with a nested-PCR, considered as a gold standard for Pow and Poc distinction. 356 samples including all human Plasmodium species at various parasitaemia were tested. The qPCR-HRM assay allowed Poc and Pow discrimination in 66 samples tested with a limit of detection evaluated at 1 parasite/µL. All these results were concordant with nested-PCR. Cross-reaction was absent with others blood parasites. The qPCR-HRM is a rapid and convenient technique to Poc and Pow distinction.

[Laboratory diagnosis of antiphospholipid syndrome: From criteria to practice]. / Diagnostic biologique du syndrome des antiphospholipides : des critères à la pratique.

The antiphospholipid syndrome is a bioclinical entity defined by thrombosis and/or obstetrical complications in the presence, at least 12 weeks apart, of antiphospholipid antibodies detected by coagulation test (lupus anticoagulant) or immunological assays (anticardiolipin, anti-ß2-glycoprotein I antibodies). Biological markers' improvement such as anti-phosphatidylserine/prothrombin and biological score should allow better patients' management and preventive therapeutic for thrombosis and obstetrical complications. This review describes different types of antibodies, link between biological profile and risk level of thrombosis events/obstetrical complications and gives practical advice to interpret biological results.

Thyroid hormone regulation of nuclear-encoded mitochondrial inner membrane polypeptides of the liver.

The effects of thyroid hormone on nuclear-encoded mitochondrial inner membrane proteins were investigated by in vitro translation of the endogenous mRNA present in a postmitochondrial fraction from the livers of rats treated in vivo with hormone. The levels of the mRNAs were estimated by quantitative immunoabsorption of the translation mixture. Total protein synthesis was increased 2.6-fold after 4 days of in vivo hormone treatment, but only 10-15% of the polypeptides were dramatically altered (greater than 5-fold). Among the most highly elevated were cytochrome c1 (greater than 10-fold increase) and the Rieske iron-sulfur protein of the cytochrome bc1 complex. Other inner membrane proteins (core protein 1, beta subunit of F1 ATPase, subunit IV of cytochrome oxidase, 3-hydroxybutyrate dehydrogenase) and non-mitochondrial proteins (rat serum albumin, beta 2-microglobulin) were not altered significantly by hormone treatment. Cytochrome c1 and the Rieske protein increased after 12 h of hormone treatment, a relatively early response in mammalian mitochondrial biogenesis. The possible significance of this response for the regulation of mitochondrial synthesis and assembly is discussed.

In vitro translation of cytosolic and peroxisomal epoxide hydrolase and catalase on liver polyribosomes from untreated and clofibrate-treated C57B1/6 mice.

Free and membrane-bound polyribosomes were isolated from the livers of untreated and clofibrate-treated male C57B1/6 mice. The in vitro translation products were investigated in a rabbit reticulocyte cell-free system by immunoprecipitation of cytosolic epoxide hydrolase, catalase and albumin. The soluble forms of epoxide hydrolase present in cytosol and in peroxisomes were found to be synthesized on free polyribosomes and could not be distinguished from each other, since only one primary translation product was found with the methods used. Clofibrate treatment was found to increase total protein synthesis, synthesis of soluble epoxide hydrolase and translational efficiency of the isolated polyribosomes.

Rhodamine 6G inhibits the matrix-catalyzed processing of precursors of rat-liver mitochondrial proteins.

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron-sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the alpha and beta subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger-molecular-mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o-phenanthroline. Rhodamine 6G inhibits matrix-catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells [Kolarov, J. & Nelson, B.D. (1984) Eur. J. Biochem. 144, 387-392].

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase.

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone.

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 micrograms/100 g body wt) of triiodothyronine (T3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T3 increases the numbers of functional respiratory chain units. T3 also induces mitochondrial protein synthesis by 50-100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T3 belong to two subsets, which were either induced or repressed by hormone. Thus, T3 stimulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.

The effects of tri-iodothyronine on the synthesis of mitochondrial proteins in isolated rat hepatocytes.

The effects of tri-iodothyronine on mitochondrial protein synthesis have been studied in in vitro labeled, isolated rat hepatocytes. Hepatocytes were isolated from hypothyroid rats or from hypothyroid rats 24 h after injecting a single, low dose of hormone (20-30 microgram/180-230 g body weight). Tri-iodothyronine increased translation on mitochondrial ribosomes by 2-3-fold, but, under our conditions, appears to have little or not effect on the general synthesis of cytoplasmically-translated mitochondrial proteins. Electrophoretic and fluorographic analysis indicated that tri-iodothyronine stimultes labeling of the four major mitochondrially-translated peptides. The hormone appears to act by inducing a general increase in translation/transcription of mitochondrially-synthesized peptides.