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.     

Ants in their plants: Pseudomyrmex ants reduce primate,parrot and squirrel predation on Macrolobium acaciifolium (Fabaceae) seeds in Amazonian Brazil

Although plant‐inhabiting ants are known to act as effective deterrents to a variety of vertebrate and invertebrate herbivores, this has been reported only once before for primates, a group better known for their predation of ants. In the present study, we investigated the effects that colonies of Pseudomyrmex viduus ants living in individual Macrolobium acaciifolium (Fabaceae) trees have on the rates of visitation and fruit removal by four taxa of seed‐predating vertebrates: the primate Cacajao melanocephalus ouakary; macaws (Ara spp.); large parrots (Amazona spp.); and the Northern Amazonian red squirrel (Sciurus igniventris). We found that ant presence significantly reduced both rates of visitation and of fruit removal by C. m. ouakary. The same pattern of reduced fruit removal was also observed for other seed predators (parrots, macaws, and squirrels) but not for visitation rates (although this may be a result of the small sample size). This appears to be only the second‐known demonstration of the repellent effect of ants on primates and, indeed, the first for squirrels and psittacine birds. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 260–273.     

Association of Lung Inflammatory Cells with Small Airways Function and Exhaled Breath Markers in Smokers – Is There a Specific Role for Mast Cells?

Background

Smoking is associated with a mixed inflammatory infiltrate in the airways. We evaluated whether airway inflammation in smokers is related to lung function parameters and inflammatory markers in exhaled breath.

Methods

Thirty-seven smokers undergoing lung resection for primary lung cancer were assessed pre-operatively by lung function testing including single-breath-nitrogen washout test (sb-N2-test), measurement of fractional exhaled nitric oxide (FeNO) and pH/8-isoprostane in exhaled breath condensate (EBC). Lung tissue sections containing cancer-free large (LA) and small airways (SA) were stained for inflammatory cells. Mucosal (MC_T) respectively connective tissue mast cells (MC_TC) and interleukin-17A (IL-17A) expression by mast cells was analysed using a double-staining protocol.

Results

The median number of neutrophils, macrophages and mast cells infiltrating the lamina propria and adventitia of SA was higher than in LA. Both MCTC and MC_T were higher in the lamina propria of SA compared to LA (MC_TC: 49 vs. 27.4 cells/mm²; MC_T: 162.5 vs. 35.4 cells/mm²; P<0.005 for both instances). IL-17A expression was predominantly detected in MC_TC of LA. Significant correlations were found for the slope of phase III % pred. of the sb-N2-test (r_s= -0.39), for the FEV₁% pred. (r_s= 0.37) and for FEV₁/FVC ratio (r_s=0.38) with MC_T in SA (P<0.05 for all instances). 8-isoprostane concentration correlated with the mast cells in the SA (r_s=0.44), there was no correlation for pH or FeNO with cellular distribution in SA.

Conclusions

Neutrophils, macrophages and mast cells are more prominent in the SA indicating that these cells are involved in the development of small airway dysfunction in smokers. Among these cell types, the best correlation was found for mast cells with lung function parameters and inflammatory markers in exhaled breath. Furthermore, the observed predominant expression of IL-17A in mast cells warrants further investigation to elucidate their role in smoking-induced lung injury, despite the lack of correlation with lung function and exhaled breath parameters.     

Blood collection for biochemical analysis in adult zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies. There are relatively few publications related to adult zebrafish physiology of organs and systems, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. Recently, Gupta et al. described how to dissect adult zebrafish organs, Kinkle et al. described how to perform intraperitoneal injections, and Pugach et al. described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish. The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.     

Effect of cellulose physical characteristics, especially the water sorption value, on the efficiency of its hydrolysis catalyzed by free or immobilized cellulase

Cellulase, an enzymatic complex that synergically promotes the degradation of cellulose to glucose and cellobiose, free or adsorbed onto Si/SiO₂ wafers at 60 °C has been employed as catalyst in the hydrolysis of microcrystalline cellulose (Avicel), microcrystalline cellulose pre-treated with hot phosphoric acid (CP), cotton cellulose (CC) and eucalyptus cellulose (EC). The physical characteristics such as index of crystallinity (I_C), degree of polymerization (DP) and water sorption values were determined for all samples. The largest conversion rates of cellulose into the above-mentioned products using free cellulase were observed for samples with the largest water sorption values; conversion rates showed no correlation with either I_C or DP of the biopolymer. Cellulose with large water sorption value possesses large pore volumes, hence higher accessibility. The catalytic efficiency of immobilized cellulase could not be correlated with the physical characteristics of cellulose samples. The hydrolysis rates of the same cellulose samples with immobilized cellulase were lower than those by the free enzyme, due to the diffusion barrier (biopolymer chains approaching to the immobilized enzyme) and less effective contact between the enzyme active site and its substrate. Immobilized cellulase, unlike its free counterpart, can be recycled at least six times without loss of catalytic activity, leading to higher overall cellulose conversion.     

Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai

The secreted form of mouse meprin A is a homooligomer of meprin alpha subunits that contain a prosequence, a catalytic domain, and three domains designated as MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu), MATH (meprin and TRAF homology), and AM (AfterMath). Previous studies indicated that wild-type mouse meprin alpha is predominantly a secreted protein, while the MAM deletion mutant (DeltaMAM) is degraded intracellularly. The work herein indicates that the DeltaMAM mutant is ubiquitinated and degraded via the proteasomal pathway. Both wild-type meprin alpha and the DeltaMAM mutant interact with the molecular chaperones calnexin and calreticulin in the endoplasmic reticulum. The interactions of the chaperones with the DeltaMAM mutant were significantly prolonged in the presence of lactacystin, a specific inhibitor of the proteasome, whereas those with the wild type were not affected by this inhibitor. Trimming of the Asn-linked core oligosaccharides of meprin subunits was required for interactions with the chaperones. The data indicated that folding of the wild-type protein was accelerated by chaperones, whereas the rate of dimerization was unaffected. Thus, calnexin and calreticulin are intimately involved in the correct folding and transport of meprin to the plasma membrane, as well as in retrograde transport of the DeltaMAM mutant to the ubiquitin-dependent proteasomal degradative pathway in the cytosol.     

Chemical Composition and Cytotoxic Evaluation of the Essential Oil of Phyllogonium viride (Phyllogoniaceae,Bryophyta)

The present study aimed to determine the chemical composition and biological activity of the essential oil obtained from Phyllogonium viride Brid. (Phyllogoniaceae, Bryophyta), whose samples were collected in southern Brazil. For the first time, the cytotoxic activity of the essential oil of P. viride in breast and colorectal tumor cells (MCF-7 and HCT-116) was evaluated, as well as the cytotoxic potential of this oil in non-tumoral cells of human immortalized keratinocytes (HaCaT) via MTT assay. The compounds majorly found in P. viride essential oil were β-bazzanene (20.30 %), β-caryophyllene (17.06 %), β-chamigrene (14.02), and germacrene B (11.72 %). Treatment with P. viride essential oil in the different tested cell lines did not induce any toxicity in most of the tested concentrations. These data contribute to generating new scientific information about this understudied plant species. Furthermore, the chemical characterization of the compounds present in the essential oil of P. viride can lead to greater elucidation of its biotechnological potential.     

Water conditions influence the detectability of Crossodactylus gaudichaudii (Anura,Hylodidae) in streams of the Atlantic Forest

Stream-dwelling amphibians' occurrence, behaviour and reproductive success are strongly influenced by dynamic abiotic factors, for example, water flow and spray. These factors can disproportionately affect these frogs due to their dependency on specific favourable conditions for development and incapacity to disperse from unfavourable environments. We analysed the influence of environmental covariates on the detectability of Crossodactylus gaudichaudii, an amphibian species endemic to streams in the Brazilian Atlantic Forest. We conducted sampling in the streams of the Duas Bocas Biological Reserve, Brazil, and we measured air and water temperature, air humidity and pH of water on each sampling occasion. We estimated the effects of variables on the detectability of the species using single-season occupancy models. Our results indicated that the detectability of the species increases on occasions with higher water temperatures (24–26°C) and lower pH (5.0–5.5). We investigated the influence of these covariates only on the detectability of adult frogs, but it is likely that the physiochemical properties of stream water are more important to aquatic larvae, and hence, the probability of their detection. Given this, further studies should examine the relevance of covariates on the detectability of adult frogs as well as larvae.     

Genomic Characterization of Novel Circular ssDNA Viruses from Insectivorous Bats in Southern Brazil

Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.