Evolutionary history of trypanosomes from South American caiman (Caiman yacare) and African crocodiles inferred by phylogenetic analyses using SSU rDNA and gGAPDH genes

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.     

Effect of the pH of growth on the survival of Lactobacillus delbrueckii subsp. bulgaricus to stress conditions during spray-drying

AIMS: The aim of this study was to optimize survival of Lactobacillus delbrueckii subsp. bulgaricus during spray-drying and subsequent storage through optimizing the pH of growth conditions. METHODS AND RESULTS: Cell concentrates previously grown without or with pH controlled were spray-dried and stored at 20 degrees C and heat treated at 57 degrees C. Cells grown under noncontrolled pH were more resistant to both drying and heating than cells grown under controlled pH but no significant differences were observed during storage. The intracellular proteins profile of cells grown under both conditions was studied by two-dimensional SDS-polyacrylamide gel electrophoresis. Eight proteins were identified using automated mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data acquisition. Of the identified proteins, only cochaperonin GroES corresponded to a known heat shock protein (HSP). The other proteins identified are proteins involved in glycolysis. For cells grown under noncontrolled pH the expression of the Hsp70, GroES and GroEL, measured by Western blotting, was enhanced. CONCLUSIONS: The higher resistance of cells grown under noncontrolled pH correlates with the enhanced production of heat shock proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth of L. bulgaricus under controlled pH (commonly used by the starter cultures production industry) results in cells more sensitive to stresses frequently encountered by the cells during starter cultures preparation/storage/utilization.     

Helianthus tuberosus agglutinin directly induces neutrophil migration, which can be modulated/inhibited by resident mast cells.

We investigated the effect of Helianthus tuberosus agglutinin (HTA) on neutrophil migration in vivo and in vitro. The role of resident cells in this effect was analyzed. Peritonitis was induced by injecting stimuli into rat (150-200 g) peritoneal cavities, and in vitro neutrophil chemotaxis was performed using a Boyden microchamber. HTA (80, 200, or 500 microg/mL per cavity) induced significant in vivo neutrophil migration (p < 0.05); in vitro assays showed that this lectin also induced neutrophil chemotaxis, an effect inhibited by the incubation of lectin associated with alpha-D(+)-mannose, its specific binding sugar. Depletion of the resident-cell population by peritoneal lavage did not alter HTA-induced neutrophil migration (200 microg/mL per cavity). The opposite strategy, increasing peritoneal macrophages by intraperitoneally injecting rats with thioglycollate, did not enhance the neutrophil migration produced by HTA (200 microg/mL per cavity). In addition, injection of supernatant from HTA-stimulated macrophage culture (300 microg/mL) into rat peritoneal cavities did not induce neutrophil migration. However, reduction of the peritoneal mast-cell population potentiated the neutrophil migration (p < 0.05) induced by HTA (200 microg/mL per cavity). Lectin from H. tuberosus has a direct neutrophil chemotatic effect that is modulated by mast cells.     

Life cycle of Amblyomma cooperi (Acari: Ixodidae) using capybaras (Hydrochaeris hydrochaeris) as hosts

The life cycle of Amblyomma cooperi was evaluated under laboratory conditions testing different host species. Larval infestations were performed on chickens (Gallus gallus) and capybaras (Hydrochaeris hydrochaeris). Nymphal infestations were performed on G. gallus, H. hydrochaeris, guinea pigs (Cavia porcellus) and wild mice (Calomys callosus). Infestations by adult ticks were performed only on capybaras. All free-living stages were observed in darkness at 27 degrees C and RH 85%. Capybaras were significantly (p < 0.05) the most suitable hosts for immature ticks, with the highest larval (63.6%) and nymphal (48%) recovery. Larval and nymphal feeding and premolt periods were significantly different (p < 0.05) between ticks fed on different host species. Male nymphs showed premolt period significantly shorter (p < 0.05) than female nymphs. The overall sex ratio of adult ticks was 0.92:1 (M:F). Infestations by adult ticks on capybaras yielded more than 76% of engorged female recovery. Only three out of 33 engorged females fed on capybaras did not lay fertile eggs. The life cycle of A. cooperi in laboratory, reported for the first time, was completed in an average period of 189.4 days. During the premolt period, all A. cooperi engorged nymphs secreted distinct blackish drops, which seem to be inherent to this species. Our results, associated data in the literature, confirm the high suitability of capybaras for the adult stage of A. cooperi and also indicate this animal species as a primary host for immature stages of A. cooperi in nature. On the other hand, the results of larval and nymphal infestation on chickens and guinea pigs suggest that birds and wild guinea pigs, which are also present in the distribution area of A. cooperi in South America, could be potentially infested by A. cooperi immature stages in nature.     

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.     

Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2

Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome.     

Long term effects of manure, charcoal and mineral fertilization on crop production and fertility on a highly weathered Central Amazonian upland soil

Application of organic fertilizers and charcoal increase nutrient stocks in the rooting zone of crops, reduce nutrient leaching and thus improve crop production on acid and highly weathered tropical soils. In a field trial near Manaus (Brazil) 15 different amendment combinations based on equal amounts of carbon (C) applied through chicken manure (CM), compost, charcoal, and forest litter were tested during four cropping cycles with rice (Oryza sativa L.) and sorghum (Sorghum bicolor L.) in five replicates. CM amendments resulted in the highest (P < 0.05) cumulative crop yield (12.4 Mg ha⁻¹) over four seasons. Most importantly, surface soil pH, phosphorus (P), calcium (Ca), and magnesium (Mg) were significantly enhanced by CM. A single compost application produced fourfold more grain yield (P < 0.05) than plots mineral fertilized in split applications. Charcoal significantly improved plant growth and doubled grain production if fertilized with NPK in comparison to the NPK-fertilizer without charcoal (P < 0.05). The higher yields caused a significantly greater nutrient export in charcoal-amended fields, but available nutrients did not decrease to the same extent as on just mineral fertilized plots. Exchangeable soil aluminum (Al) was further reduced if mineral fertilizer was applied with charcoal (from 4.7 to 0 mg kg⁻¹). The resilience of soil organic matter (SOM) in charcoal amended plots (8 and 4% soil C loss, mineral fertilized or not fertilized, respectively) indicates the refractory nature of charcoal in comparison to SOM losses over 20 months in CM (27%), compost amended (27%), and control plots (25% loss).     

Relationships between tree component structure,topography and soils of a riverside forest,Rio Botucaraí, Southern Brazil

The relationships among floristic, structural and physiognomic variables of the tree component, flooding regime variations and soil fertility were investigated in a riparian forest fragment (ca. 43 ha) in the Rio Botucaraí watershed near the confluence with the Rio Jacuí, southern Brazil. All the trees with a dbh ≥ 15 cm were surveyed in 100 contiguous 10 × 10 m plots and soil chemical and textural variables were obtained from the analyses of 15 topsoil samples (0–20 cm depth) collected at different locations. The sample sites encompass all the topographic variation of the area. We used canonical correspondence analysis (CCA) to seek correlations between environmental variables and tree component distribution. The 1,547 surveyed individuals belonged to 30 species and 16 botanical families. The Shannon diversity index (H′) and the Pielou equability (J′) were 1.995 and 0.586 nats ind.⁻¹, respectively. Sebastiania commersoniana, Casearia sylvestris, Eugenia uniflora and Eugenia hyemalis presented the highest importance values. Species’ densities chiefly correlated with site elevation, flooding regime and soil chemical fertility. The analyses of ecological categories (EC) revealed that most species are heliophylous and typical of early successional stages. Richness and diversity (P < 0.001) variations from different topographic positions reflect that at the local scale, flooding was a limiting factor for the establishment of certain species suggesting that local processes control species richness and diversity.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.     

Interaction between artemisinin and heme. A Density Functional Theory study of structures and interaction energies

Malaria is an infectious disease caused by the unicellular parasite Plasmodium sp. Currently, the malaria parasite is becoming resistant to the traditional pharmacological alternatives, which are ineffective. Artemisinin is the most recent advance in the chemotherapy of malaria. Since it has been proven that artemisinin may act on intracellular heme, we have undertaken a systematic study of several interactions and arrangements between artemisinin and heme. Density Functional Theory calculations were employed to calculate interaction energies, electronic states, and geometrical arrangements for the complex between the heme group and artemisinin. The results show that the interaction between the heme group and artemisinin at long distances occurs through a complex where the iron atom of the heme group retains its electronic features, leading to a quintet state as the most stable one. However, for interaction at short distances, due to artemisinin reduction by the heme group, the most stable complex has a septet spin state. These results suggest that a thermodynamically favorable interaction between artemisinin and heme may happen.