Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains

Background

Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes.

Methods/Principal Findings

Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported.

Conclusions/Significance

The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.     

Plasmodium vivax Diversity and Population Structure across Four Continents

Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11 microsatellite markers was either generated or compiled from 841 isolates from four continents collected in 1999–2008. Diversity was highest in South-East Asia (mean allelic richness 10.0–12.8), intermediate in the South Pacific (8.1–9.9) Madagascar and Sudan (7.9–8.4), and lowest in South America and Central Asia (5.5–7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60–80% in Latin American populations, suggesting that typing of 2–6 markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (F _ST>0.2). Outside Central Asia F _ST values were highest (0.11–0.16) between South American and all other populations, and lowest (0.04–0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations.     

Determination of urinary vanillactic acid and plasma dihydroxyphenylalanine as markers of non-secreting neuroblastoma by high-performance liquid chromatography with electrochemical detection

An accurate and precise isocratic high-performance liquid chromatographic technique for the analysis of urinary vanillactic acid (VLA) and plasma dihydroxyphenylalanine (DOPA), especially at low concentrations (pmol/l) for VLA and nmol/l for DOPA), is described. The compounds were purified in a single step, (on an anion exchanger for VLA and on aluminium oxide for DOPA), separated by ion-pair reversed-phase liquid chromatography, and detected electrochemically. A single analysis was complete within 18 min. Mean recoveries of 103 and 81% were obtained for VLA and DOPA, respectively, and the limits of detection were 42 and 76 pmol/l, respectively. The mean values of the intra-assay coefficient of variation were 14 and 7.1% for VLA and DOPA, respectively, and the mean values of the inter-assay coefficient of variation were 15.7 and 11.6%, respectively. Modifications of the retention times (between 2 and 42 min) induced by changes in the eluent were determined. Reference values for normal children and children with neuroblastoma or various tumours are given.     

Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases

Prevention of food spoilage and food poisoning pathogens is usually achieved by use of chemical preservatives which have negative impacts including: human health hazards of the chemical applications, chemical residues in food & feed chains and acquisition of microbial resistance to the used chemicals. Because of such concerns, the necessity to find a potentially effective, healthy safer and natural alternative preservatives is increased. Within these texts, Plant extracts have been used to control food poisoning diseases and preserve foodstuff. Antimicrobial activity of five plant extracts were investigated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi using agar disc diffusion technique. Ethanolic extracts of Punica granatum, Syzygium aromaticum, Zingiber officinales and Thymus vulgaris were potentially effective with variable efficiency against the tested bacterial strains at concentration of 10 mg/ml while extract of Cuminum cyminum was only effective against S. aureus respectively. P. granatum and S. aromaticum ethanolic extracts were the most effective plant extracts and showed bacteriostatic and bactericidal activities against the highly susceptible strains of food borne pathogenic bacteria (S. aureus and P. aeruginosa) with MIC's ranged from 2.5 to 5.0 mg/ml and MBC of 5.0 and 10 mg/ml except P. aeruginosa which was less sensitive and its MBC reached to 12.5 mg/ml of S. aromaticum respectively. These plant extracts which proved to be potentially effective can be used as natural alternative preventives to control food poisoning diseases and preserve food stuff avoiding healthy hazards of chemically antimicrobial agent applications.     

Embryo apoptosis identification: Oocyte grade or cleavage stage?

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced.Abbreviations: ART, assisted reproductive technologies; BO, Brackett and Oliphant; BSA, bovine serum albumin; CaI, calcium ionophore; CC, cumulus cells; COC, cumulus–oocyte complex; CO₂, carbon dioxide; CR1aa, Charles Rosenkran’s 1 amino acid; DNA, deoxyribonucleic acid; DO, denuded oocyte; EA, early apoptosis; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FSH, follicle stimulating hormone; GSH, glutathione; hpi, hours post insemination; IVC, in vitro culture; IVF, in vitro fertilization; IVM, in vitro maturation; IVP, in vitro produced; LA, late apoptosis; LH, luteinizing hormone; PBS, phosphate buffered saline; PI, propidium iodide; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling.     

Effect of additional carbon source and moisture level on xylanase production by Cochliobolus sativus in solid fermentation

The fungus Cochliobolus sativus has been shown to be an efficient producer ofxylanase from an industrial point ofview. The addition of extra carbon sources and the initial moisture content of the solid-state fermentation were found to have a marked influence on the xylanase production by C. sativus Cs6 strain. Xylan and starch resulted in an increased xylanase production (1469.4 and 1396.56 U/g, respectively) after 8 days of incubation. Optimal initial moisture content for xylanase production was 80%. The cultivation systems can easily be modified to enhance the productivity of the enzyme formation by C. sativus Cs6, which will facilitate the scale up processes for mass production.     

Freezing of fresh Barhi dates for quality preservation during frozen storage

Fresh harvested dates are perishable and there is a need for extending their shelf life while preserving their fresh like quality characteristics. This study evaluates three different freezing methods, namely cryogenic freezing (CF) using liquid nitrogen; individual quick freezing (IQF) and conventional slow freezing (CSF) in preserving the quality and stability of dates during frozen storage. Fresh dates were frozen utilizing the three methods. The produced frozen dates were frozen stored for nine months. The color values, textural parameters, and nutrition qualities were measured for fresh dates before freezing and for the frozen dates every three months during the frozen storage. The frozen dates’ color values were affected by the freezing method and the frozen storage period. There are substantial differences in the quality of the frozen fruits in favor of cryogenic freezing followed by individual quick freezing compared to the conventional slow freezing. The results revealed large disparity among the times of freezing of the three methods. The freezing time accounted to 10 min for CF, and around 80 min for IQF, and 1800 min for CSF method.