Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased Susceptibility to Pentamidine and Melarsoprol

The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.     

Tree Height Reduction After Selective Logging in a Tropical Forest

By harvesting scattered large trees, selective logging increases light availability and thereby stimulates growth and crown expansion at early‐life stage among remnant trees. We assessed the effects of logging on total and merchantable bole (i.e., lowest branch at crown base) heights on 952 tropical canopy trees in French Guiana. We observed reductions in both total (mean, ?2.3 m) and bole (mean, ?2.0 m) heights more than a decade after selective logging. Depending on local logging intensity, height reductions resulted in 2–13 percent decreases in aboveground tree biomass and 3–17 percent decreases in bole volume. These results highlight the adverse effects of logging at both tree and stand levels. This decrease in height is a further threat to future provision of key environmental services, such as timber production and carbon sequestration.     

Volatile Compounds of Viola odorata Absolutes: Identification of Odorant Active Markers to Distinguish Plants Originating from France and Egypt

Absolutes isolated from Viola odorata leaves, valuable materials for the flavor and fragrance industry, were studied. Violets are mainly cultivated in France and Egypt and extracted locally. The absolutes of the two origins showed different olfactory profiles both in top and heart notes, as evidenced by sensory analysis. The aims of this study were i) to characterize the volatile compounds, ii) to determine the odorant‐active ones, and iii) to identify some markers of the plant origin. Two complementary analytical methods were used for these purposes, i.e., headspace solid‐phase microextraction (HS‐SPME) using different fiber coatings followed by GC/MS analysis and gas chromatography – olfactometry/mass spectrometry (GC‐O/MS) applied to violet leaf extracts. From a total of 70 identified compounds, 61 have never been reported so far for this species, 17 compounds were characterized by both techniques (with seven among them known from the literature), 23 compounds were solely identified by HS‐SPME GC/MS (among them only two being already mentioned as components of violet absolutes in the literature), and, finally, 30 compounds were only identified by GC‐O/MS. According to the HS‐SPME GC/MS analyses, ethyl hexanoate and (2E,6Z)‐nona‐2,6‐dienol were specific volatile compounds of the sample with French origin, while (E,E)‐hepta‐2,4‐dienal, hexanoic acid, limonene, tridecane, and eugenol were specific of the samples with Egyptian origin. Additional compounds that were not detected by HS‐SPME GC/MS analysis were revealed by GC‐O analyses, some of them being markers of origin. Pent‐1‐en‐3‐ol, 3‐methylbut‐2‐enal, 2‐methoxy‐3‐(1‐methylethyl)pyrazine, 4‐ethylbenzaldehyde, β‐phenethyl formate, and 2‐methoxy‐3‐(2‐methylpropyl)pyrazine revealed to be odorant markers of the French sample, whereas cis‐rose oxide, trans‐rose oxide, and 3,5,5‐trimethylcyclohex‐2‐enone were odorant markers of the Egyptian samples.     

The Relation of Hepcidin to Iron Disorders,Inflammation and Hemoglobin in Chronic Kidney Disease

The metabolism of hepcidin is profoundly modified in chronic kidney disease (CKD). We investigated its relation to iron disorders, inflammation and hemoglobin (Hb) level in 199 non-dialyzed, non-transplanted patients with CKD stages 1–5. All had their glomerular filtration rate measured by ⁵¹Cr-EDTA renal clearance (mGFR), as well as measurements of iron markers including hepcidin and of erythropoietin (EPO). Hepcidin varied from 0.2 to 193 ng/mL. The median increased from 23.3 ng/mL [8.8–28.7] to 36.1 ng/mL [14.1–92.3] when mGFR decreased from ≥60 to <15 mL/min/1.73 m² (p = 0.02). Patients with absolute iron deficiency (transferrin saturation (TSAT) <20% and ferritin <40 ng/mL) had the lowest hepcidin levels (5.0 ng/mL [0.7–11.7]), and those with a normal iron profile (TSAT ≥20% and ferritin ≥40), the highest (34.5 ng/mL [23.7–51.6]). In multivariate analysis, absolute iron deficiency was associated with lower hepcidin values, and inflammation combined with a normal or functional iron profile with higher values, independent of other determinants of hepcidin concentration, including EPO, mGFR, and albuminemia. The hepcidin level, although it rose overall when mGFR declined, collapsed in patients with absolute iron deficiency. There was a significant interaction with iron status in the association between Hb and hepcidin. Except in absolute iron deficiency, hepcidin’s negative association with Hb level indicates that it is not down-regulated in CKD anemia.     

Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso

ABSTRACT: BACKGROUND: HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. METHODS: In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson's correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. RESULTS: After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the FcgammaRIIa H/R131 with IgG2 and IgG3 levels; iii) TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels. CONCLUSION: Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual's humoral response against malaria.     

Protein expression from synthetic genes: selection of clones using GFP

Construction of synthetic genes is today the most elegant way to optimize the heterologous expression of a recombinant protein. However, the selection of positive clones that incorporate the correct synthetic DNA fragments is a bottleneck as current methods of gene synthesis introduce 3.5 nucleotide deletions per kb. Furthermore, even when all predictable optimizations for protein production have been introduced into the synthetic gene, production of the protein is often disappointing: protein is produced in too low amounts or end up in inclusion bodies. We propose a strategy to overcome these two problems simultaneously by cloning the synthetic gene upstream of a reporter gene. This permits the selection of clones devoid of frame-shift mutations. In addition, beside nucleotide deletion, an average of three non-neutral mutations per kb are introduced during gene synthesis. Using a reporter protein downstream of the synthetic gene, allows the selection of clones with random mutations improving the expression or the folding of the protein of interest. The problem of errors found in synthetic genes is then turned into an advantage since it provides polymorphism useful for molecular evolution. The use of synthetic genes appears as an alternative to the error-prone PCR strategy to generate the variations necessary in protein engineering experiments.     

Distribution and Expression of Elicitin Genes in the Interspecific Hybrid Oomycete Phytophthora alni

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3′ untranslated region (3′UTR)-specific PCRs and sequencing. The occurrence of multiple 3′UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.     

Controlling Schistosomiasis: Significant Decrease of Anaemia Prevalence One Year after a Single Dose of Praziquantel in Nigerien Schoolchildren

Background

In the framework of the monitoring and evaluation of the Nigerien schistosomiasis and soil-transmitted helminth control programme, a follow-up of children took place in eight sentinel sites. The objective of the study was to assess the evolution of Schistosoma haematobium infection and anaemia in schoolchildren after a single administration of praziquantel (PZQ) and albendazole.

Methods/Principal Findings

Pre-treatment examination and follow-up at one year post-treatment of schoolchildren aged 7, 8, and 11 years, including interview, urine examination, ultrasound examination of the urinary tract, and measurement of haemoglobin. Before treatment, the overall prevalence of S. heamatobium infection was 75.4% of the 1,642 enrolled children, and 21.8% of children excreted more than 50 eggs/10 ml urine. Prevalence increased with age. The overall prevalence of anaemia (haemoglobin <11.5 g/dl) was 61.6%, decreasing significantly with increasing age. The mean haemoglobinemia was 11 g/dl. In bivariate analysis, anaemia was significantly more frequent in children infected with S. haematobium, although it was not correlated to the intensity of infection. Anaemia was also associated with micro-haematuria and to kidney distensions. In a sub-sample of 636 children tested for P. falciparum infection, anaemia was significantly more frequent in malaria-infected children. In multivariate analysis, significant predictors of anaemia were P. falciparum infection, kidney distension, and the village. One year after a single-dose praziquantel treatment (administered using the WHO PZQ dose pole) co-administered with albendazole (400 mg single dose) for de-worming, the prevalence of S. haematobium infection was 38%, while the prevalence of anaemia fell to 50.4%. The mean haemoglobinemia showed a statistically significant increase of 0.39 g/dl to reach 11.4 g/dl. Anaemia was no longer associated with S. haematobium or to P. falciparum infections, or to haematuria or ultrasound abnormalities of the urinary tract.

Conclusions

The high prevalence of anaemia in Nigerien children is clearly a result of many factors and not of schistosomiasis alone. Nevertheless, treatment of schistosomiasis and de-worming were followed by a partial, but significant, reduction of anaemia in schoolchildren, not explainable by any other obvious intervention.     

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.