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.     

Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

Angelman syndrome and severe infections in a patient with de novo 15q11.2–q13.1 deletion and maternally inherited 2q21.3 microdeletion

Angelman syndrome is a neurodevelopmental disorder characterized by mental retardation, severe speech disorder, facial dysmorphism, secondary microcephaly, ataxia, seizures, and abnormal behaviors such as easily provoked laughter. It is most frequently caused by a de novo maternal deletion of chromosome 15q11–q13 (about 70–90%), but can also be caused by paternal uniparental disomy of chromosome 15q11–q13 (3–7%), an imprinting defect (2–4%) or in mutations in the ubiquitin protein ligase E3A gene UBE3A mostly leading to frame shift mutation. In addition, for patients with overlapping clinical features (Angelman-like syndrome), mutations in methyl-CpG binding protein 2 gene MECP2 and cyclin-dependent kinase-like 5 gene CDKL5 as well as a microdeletion of 2q23.1 including the methyl-CpG binding domain protein 5 gene MBD5 have been described. Here, we describe a patient who carries a de novo 5 Mb-deletion of chromosome 15q11.2–q13.1 known to be associated with Angelman syndrome and a further, maternally inherited deletion 2q21.3 (~ 364 kb) of unknown significance. In addition to classic features of Angelman syndrome, she presented with severe infections in the first year of life, a symptom that has not been described in patients with Angelman syndrome. The 15q11.2–q13.1 deletion contains genes critical for Prader–Willi syndrome, the Angelman syndrome causing genes UBE3A and ATP10A/C, and several non-imprinted genes: GABRB3 and GABRA5 (both encoding subunits of GABA A receptor), GOLGA6L2, HERC2 and OCA2 (associated with oculocutaneous albinism II). The deletion 2q21.3 includes exons of the genes RAB3GAP1 (associated with Warburg Micro syndrome) and ZRANB3 (not disease-associated). Despite the normal phenotype of the mother, the relevance of the 2q21.3 microdeletion for the phenotype of the patient cannot be excluded, and further case reports will need to address this point.     

Microbial activity of suspended biomass from a nitritation–anammox SBR in dependence of operational condition and size fraction

Single-stage nitritation–anammox combines the growth of aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AnAOB) in one reactor. The necessary compromise of their milieu conditions often leads to the growth of nitrite-oxidizing bacteria (NOB). For this study, a sequencing batch reactor (SBR) for nitritation–anammox was operated for 180 days with sewage sludge reject water (removal capacity, 0.4 kg?N?m^?3?day^?1). The growth of NOB was favored by enhanced oxygen supply rather than extended aerobic phases. Suspended-type biomass from this SBR was taken regularly and sieved into three size fractions (all of them <1,000 μm). Batch experiments as well as fluorescence in situ hybridization were performed to study the distribution and activity of AnAOB, AOB, and NOB within those size fractions. Both the measured conversion rates and detected abundances decreased with increasing size fraction. The highest anammox conversion rates (15 g NH₄ ⁺–N per kilogram VSS per hour) and the highest abundances of Brocadia fulgida were found in the medium size fraction (100–315 μm). The batch experiments proved to be accurate tools for the monitoring of multiple processes in the reactor. The results were representative for reactor performance during the 6 months of reactor operation.     

Interactions between Fengycin and Model Bilayers Quantified by Coarse-Grained Molecular Dynamics

Bacteria, particularly of the genus Bacillus, produce a wide variety of antifungal compounds. They act by affecting the lipid bilayers of fungal membranes, causing curvature-induced strain and eventual permeabilization. One class of these, known as fengycins, has been commercialized for treating agricultural infections and shows some promise as a possible antifungal pharmaceutical. Understanding the mechanism by which fengycins damage lipid bilayers could prove useful to the future development of related antifungal treatments. In this work, we present multi-microsecond-long simulations of fengycin interacting with different lipid bilayer systems. We see fengycin aggregation and uncover a clear aggregation pattern that is partially influenced by bilayer composition. We also quantify some local bilayer perturbations caused by fengycin binding, including curvature of the lipid bilayer and local electrostatic-driven reorganization.     

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.     

DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain

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Highlights? DCC and netrin-1 are enriched at synapses in the adult mouse forebrain ? DCC is enriched in the PSD and regulates dendritic spine morphology ? LTP induction and memory formation require DCC expression by neurons ? DCC activation of Src is required for NMDAR-dependent LTP in adult CNS     

Microparticle-Induced Coagulation Relates to Coronary Artery Atherosclerosis in Severe Aortic Valve Stenosis

Background

Circulating microparticles (MPs) derived from endothelial cells and blood cells bear procoagulant activity and promote thrombin generation. Thrombin exerts proinflammatory effects mediating the progression of atherosclerosis. Aortic valve stenosis may represent an atherosclerosis-like process involving both the aortic valve and the vascular system. The aim of this study was to investigate whether MP-induced thrombin generation is related to coronary atherosclerosis and aortic valve calcification.

Methods

In a cross-sectional study of 55 patients with severe aortic valve stenosis, we assessed the coronary calcification score (CAC) as indicator of total coronary atherosclerosis burden, and aortic valve calcification (AVC) by computed tomography. Thrombin-antithrombin complex (TATc) levels were measured as a marker for thrombin formation. Circulating MPs were characterized by flow cytometry according to the expression of established surface antigens and by measuring MP-induced thrombin generation.

Results

Patients with CAC score below the median were classified as patients with low CAC, patients with CAC Score above the median as high CAC. In patients with high CAC compared to patients with low CAC we detected higher levels of TATc, platelet-derived MPs (PMPs), endothelial-derived MPs (EMPs) and MP-induced thrombin generation. Increased level of PMPs and MP-induced thrombin generation were independent predictors for the severity of CAC. In contrast, AVC Score did not differ between patients with high and low CAC and did neither correlate with MPs levels nor with MP-induced thrombin generation.

Conclusion

In patients with severe aortic valve stenosis MP-induced thrombin generation was independently associated with the severity of CAC but not AVC indicating different pathomechanisms involved in coronary artery and aortic valve calcification.