A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Enhancement of metabolic rates of yeast flocculent cells through the use of polymeric additives

The influence of several polymeric additives on specific glucose uptake rate of flocs of a S. cerevisiae strain — S. cerevisiae NRRLY 265 was studied. A special continuous membrane microreactor was used to measure glucose uptake on the presence of calcium and of the tested additives — two cationic polymers — bis(polyoxyethylene-bis(amine)) 20,000 and BPA 1,000 and one anionic polymer — Magna Floc LT25.An increase on glucose uptake rate was always observed when comparing with calcium bound flocs. For bis(polyoxyethylene-bis(amine)) 20,000 the increase was only 19% but for BPA 1,000 a value of more than 50% was observed. For Magna Floc LT25 a two fold increase was measured.The determination of floc size and porosity in the presence of the additives indicated that, on the basis of these parameters, it was not possible to explain the observed glucose uptake rates. The floc porosites in additive bound flocs were similar and 10% larger than for calcium bound flocs and glucose uptake rate was larger for the largest flocs — Magna Floc LT25 bound flocs were the largest followed by BPA 1,000, bis(polyoxyethylene-bis(amine)) 20,000 and calcium bound flocs. These values disagree with what should be expected in diffusion controlled processes.The calculation of intercellular floc distance indicated that polymeric additives act on the reduction of diffusional limitations by increasing the available flux area for glucose inside the flocs. By analysing different kinds of packings, it was also observed that the packing arrangement for yeast cells in flocs is close to the cubic packing. The simulation of this arrangement for the obtained floc sizes confirmed that the 10% increase in floc porosity is sufficient to explain the increase in the available flux area.     

Fruit infesting tephritids [Dipt.: Tephritidae] and associated parasitoids in Chiapas,Mexico

A total of 1,302 parasitoids representing 8 species and 4 families were recovered from 9,818 fruit fly host fruits sampled. The most common parasitoid species wasDiachasmimorpha longicaudata (Ashmead). Average percent parasitism ranged between 0.44 and 29.23%. Parasitoid emergence data indicate thatAnastrepha ludens (Loew),A. obliqua (Sein),A. serpentina (Wiedeman),A. striata (Schiner) andToxotrypana curvicauda (Gerstaecker) were subject to parasitism. We provide information on the population fluctuation ofAnastrepha ludens, A. obliqua, A. serpentina, A. distincta (Greene),A. striata, A. fraterculus (Wiedeman),A. chiclayae (Greene),A. montei (Costa Lima),A. leptozona (Hendel) andA. tripunctata (Wulp).Anastrepha ludens andA. obliqua were the most common species, representing 95.3% of all fruit fly species caught in McPhail traps.      

Dialysable leukocyte extracts modify the course of experimental paracoccidioidomycosis in the Syrian hamster

The effect of dialysable leukocyte extracts (DLE) obtained from hamsters immunized withParacoccidioides brasiliensis (immune DLE) and from non-immunized hamsters (non-immune DLE) was studied in hamsters inoculated withP. brasiliensis by the intratesticular route. Treatment with immune or non-immune DLE was started during the third week of infection and was repeated at 7, 11, 15 and 19 weeks. A group of untreated infected animals was used as control. Animals were submitted to the delayed hypersensitivity skin test toP. brasiliensis antigen (PbAg) in vivo and assayed in vitro by the macrophage migration inhibition test in the presence of Phytohemagglutinin (PHA) and PbAg and by immunodiffusion for specific antibody. The animals were sacrificed at 4, 8, 12, 16 and 20 weeks. The morphology and extension of the lesions were studied at the inoculation site, and in lymph nodes, lungs, liver, spleen and kidneys. In contrast to the controls, animals treated with both DLEs maintained a positive cell-mediated immune response throughout the experiment and developed less extensive infection with a significantly lower number of fungi in the lesions. The results suggest that immune and non-immune DLE preparations modified the evolution of experimental paracoccidioidomycosis with equal efficiency. This similarity may be explained by the immunoregulatory activities of both extracts.     

Glucosylenamines as glycosyl acceptors: synthesis of gentiobiosylenamines.

The preparation of 2,3,4-tri-O-benzyl- (3), 2,3,4-tri-O-acetyl- (4), and 2,3,4-tri-O-benzoyl-N-(2,2-diethoxycarbonylvinyl)-6-O-trityl-beta- D-glucopyranosylamine (5) is described. The reaction of 3-5 with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide yields 2,3,4-tri-O-benzyl- (9), 2,3,4-tri-O-acetyl- (10), and 2,3,4-tri-O-benzoyl-N-(2,2-diethoxycarbonylvinyl)-6-O-(2,3,4,6-tet ra-O- acetyl-beta-D-glucopyranosyl)-beta-D-glucopyranosylamine (11), respectively. 2,3,4-Tri-O-benzyl- (6), 2,3,4-tri-O-acetyl- (7), and 2,3,4-tri-O- benzoyl-N-(2,2-diethoxycarbonylvinyl)-beta-D-glucopyranosylamine (8) are also described.     

Inflammatory intestinal damage induced by 5-fluorouracil requires IL-4

Background5-Fluorouracil (5-FU) induces intestinal mucositis, which is characterized by epithelial ulcerations in the mucosa and clinical manifestations, such as pain and dyspeptic symptoms. Cytokines participate in the inflammatory and functional events of intestinal mucositis. IL-4 is an important mediator of intestinal inflammation, with either anti-inflammatory or pro-inflammatory functions, depending on the model of intestinal inflammation. This study aimed to evaluate the role of IL-4 in 5-FU-induced intestinal mucositis.MethodsIL-4^+/+ or IL-4^?/? mice (25–30 g) were intraperitoneally injected with 5-FU (450 mg/Kg) or saline (C). After 3 days, the mice were sacrificed and the duodenum was evaluated for epithelial damage, MPO activity and cytokine concentration.Results5-FU induced significant damage in the intestinal epithelium of IL-4^+/+ mice (reduction in the villus/crypt ratio: control = 3.31 ± 0.21 μm, 5-FU = 0.99 ± 0.10 μm). However, the same treatment did not induce significant damage in IL-4^?/? mice (5-FU = 2.87 ± 0.19 μm) compared to wild-type mice. 5-FU-induced epithelial damage increased the MPO activity (neutrophil number) and the level of pro-inflammatory cytokines (IL-4, TNF-α, IL-1β and CXCL-8) in the duodenum. These results were not observed in IL-4^?/? mice treated with 5-FU.ConclusionOur data suggest that IL-4 participates as a pro-inflammatory cytokine in a 5-FU-induced intestinal damage model and suggests that IL-4 antagonists may be novel therapeutics for this condition.     

Independent evolution of song diversity and song motor performance in canaries,goldfinches and allies indicates clade-specific trade-offs in birdsong

The diversity and the motor performance of birdsongs can both be sexually selected. In wood warblers, most species with high motor performance sing a greater proportion of trills, presumably to advertise performance, and thus have lower syllable diversity. We tested if this trade-off between motor performance and syllable diversity extends to canaries, goldfinches and allies, a clade with much longer and more varied songs. We assembled a molecular phylogeny and inferred song motor performance based on the speed of frequency modulation either in trills or in within-song intervals. The two metrics of performance were positively, but only mildly, related across species. While performance evaluated in intervals had high phylogenetic signal, performance evaluated in trills changed independently of phylogeny and was constrained by body size. Species in densely vegetated habitats sang fewer trills, but did not differ in motor performance. Contrary to wood warblers, song motor performance did not predict the proportion of trilled syllables nor within-song syllable diversity, perhaps because large differences in the song duration of canaries, goldfinches and allies prevent trills from severely compromising syllable diversity. Opposed results in wood warblers and in these finches indicate the existence of clade-specific trade-offs in the evolution of birdsong.