Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-Trypanosoma cruzi activity without cytotoxicity to mammalian cells

Amphibian skin secretions are known as a rich source of biologically active molecules, most of which are alkaloids, biogenic amines, and peptides. Dermaseptins are a class of antimicrobial peptides present in tree frogs of the Phyllomedusa genus. They are cationic molecules of 28-34 residues that permeabilize the membrane of Gram-positive and Gram-negative bacteria, yeasts, and filamentous fungi, showing little or no hemolytic activity. This work reports the isolation, molecular mass analysis, primary structure determination, biological activities, and potential therapeutic applications of an antimicrobial peptide found in the skin secretion of Phyllomedusa oreades, which is a newly described amphibian species endemic of the Brazilian savanna. DS 01 is a 29-residue-long peptide with a molecular mass of 2793.39 Da showing antibacterial properties against Gram-positive and Gram-negative bacteria in the range of 3-25 microm. Anti-protozoan activity was investigated using T. cruzi in its trypomatigote and epimastigote forms cultivated in both cell culture and blood media. Within 2 h after incubation with DS 01 at a final concentration of approximately 6 microm, no protozoan cells were detected. Two synthetic dermaseptins, described previously by our group and named dermadistinctins K and L (DD K and DD L), also had their anti-Trypanosoma cruzi activity investigated and demonstrated similar properties. Toxicity of DS 01 to mouse erythrocytes and white blood cells was evaluated by means of atomic force microscopy and flow cytometry. No morphological alterations were observed at a lytic concentration of DS 01, suggesting its therapeutic value especially as an anti-T. cruzi agent to prevent infections during blood transfusion.     

Isolation and characterization of a novel bradykinin potentiating peptide (BPP) from the skin secretion of Phyllomedusa hypochondrialis

Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were first described in the middle of 1960s and were the first natural inhibitors of the angiotensin-converting enzyme (ACE). BPPs present a classical motif and can be recognized by their typical pyroglutamyl (Pyr)/proline rich sequences presenting, invariably, a proline residue at the C-terminus. In the present study, we describe the isolation and biological characterization of a novel BPP isolated from the skin secretion of the Brazilian tree-frog Phyllomedusa hypochondrialis. This new BPP, named Phypo Xa presents the sequence Pyr-Phe-Arg-Pro-Ser-Tyr-Gln-Ile-Pro-Pro and is able to potentiate bradykinin activities in vivo and in vitro, as well as efficiently and competitively inhibit ACE. This is the first canonical BPP (i.e. Pyr-Aaa(n)-Gln-Ile-Pro-Pro) to be found not only in the frog skin but also in any other natural source other than the snake venoms.     

Development of integument and cutaneous glands in larval,juvenile and adult toads (Rhinella granulosa): a morphological and morphometric study

In this study, the development of integument and cutaneous glands in the toad Rhinella granulosa (Bufonidae) at different larval stages and in postmetamorphic and adult forms was examined. The analyses were conducted using histological, ultrastructural and morphometric methods. The results showed that cellular aggregations of precursor epidermal glands start to appear in stage 31 of (Herpetologica, 16, 1960 and 183) and then proliferate and invade the dermis. After stage 41, granular and mucous glands are very similar to those found in adults. The granular glands are syncytial and are surrounded by a distinct layer of myoepithelial cells. In the region of parotoid macroglands, the granular glands accumulate and their alveoli progressively increase until they reach adult size. An analysis by scanning electron microscopy showed the inner distribution of the syncytial nuclei and the myoepithelial cells. The morphological changes observed in the integument of tadpoles are associated with the gradual adaptation to terrestrial environments by preparing the individual for future chemical defence against predators and micro‐organisms.     

Physiological basis for diurnal activity in dispersing juvenile Bufo granulosus in the Caatinga, a Brazilian semi-arid environment

Diurnal activity is characteristic of many toad species, including Bufo granulosus from the Brazilian semi-arid biome called the Caatinga. Because of their patterns of activity, juvenile toads are exposed to hot and dehydrating conditions. Our investigation focuses on temperature and water relationships, and is based on the prediction that anuran diurnal activity in a semi-arid environment must be associated with morphological, physiological and behavioral traits enhancing thermal tolerances, capacity for performance at high temperatures and water balance. To test specific hypothesis related with this prediction, we investigated postmetamorphic B. granulosus and collected data on thermal tolerances and preferences, thermal safety margins, thermal dependence of locomotor behavior, thermal and kinetic properties of citrate synthase (CS), and skin morphophysiology. This information was compared with additional data from adult conspecifics and adult toads from sympatric species or from species from more moderate environments. We found that juvenile B. granulosus exhibit the highest critical maximum temperature reported for toads (44.2 degrees C) and are well suited to move at high temperatures. However, and in contrast with juveniles of other Bufo species, they do not show thermal preferences in a gradient and appear to hydroregulate more than thermoregulate. The CS of adult and juvenile toads shows typical patterns of thermal sensibility, but the thermal stability of this enzyme is much higher in juveniles than in adult Bufo of any other species studied. The inguinal skin exhibits a complex folding pattern and seems highly specialized for capillary water uptake. Diurnal activity in juvenile B. granulosus is possible given high thermal tolerances, keen ability to detect and uptake water, and avoidance behaviors.     

A base substitution at IVS-19 3′-end splice junction causes exon 20 skipping in proα2(I) collagen mRNA and produces mild osteogenesis imperfecta

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the proband's fibroblasts showed the presence of two populations of 2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patient's pro2(I) mRNA:normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3 consensus splicing site. As a consequence pro2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported.     

Detailed structural characterization of succinoglycan, the major exopolysaccharide of Rhizobium meliloti Rm1021.

The detailed structure of the symbiotically important exopolysaccharide succinoglycan from Rhizobium meliloti Rm1021 was determined by mass spectrometry with electrospray ionization and collision-induced dissociation of the octameric oligosaccharide repeating unit. Previously undetermined locations of the succinyl and acetyl modifications were determined, in respect to both residue locations within the octamer and the carbon positions within the pyranose ring. Glycosidic linkages determined previously by methylation analysis were also verified.     

Diurnal foraging ant–tree co-occurrence networks are similar between canopy and understorey in a Neotropical rain forest

Discussion of the vertical stratification of organisms in tropical forests has traditionally focused on species distribution. Most studies have shown that, due to differences in abiotic conditions and resource distribution, species can be distributed along the vertical gradient according to their ecophysiological needs. However, the network structure between distinct vertical strata remains little-explored. To fill this gap in knowledge, we used baits to sample ants in the canopy and understorey trees of a Mexican tropical rain forest to record the ant–tree co-occurrences. We examined the ant–tree co-occurrences in the canopy and understorey using complementary network metrics (i.e., specialization, interaction diversity, modularity, and nestedness). In addition, we evaluated co-occurrence patterns between ant species on trees, using C-score analysis. In general, we found no differences in the network structure, although the interaction diversity was greater in the understorey than in the canopy networks. We also observed that co-occurrence networks of each vertical stratum featured four ant species in the central core of highly co-occurring species, with three species unique to each stratum. Moreover, we found a similar trend toward ant species segregation in the both strata. These findings reveal a similar pattern of ant–ant co-occurrences in both vertical strata, probably due to the presence of arboreal-nesting ants in the understorey. Overall, we showed that despite the marked differences in species composition and environmental conditions between understorey and canopy strata, ant–tree co-occurrences in these habitats could be governed by similar mechanisms, related to dominance and resource monopolization by ants.     

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.     

Acute Disseminated Encephalomyelitis Onset: Evaluation Based on Vaccine Adverse Events Reporting Systems

Objective

To evaluate epidemiological features of post vaccine acute disseminated encephalomyelitis (ADEM) by considering data from different pharmacovigilance surveillance systems.

Methods

The Vaccine Adverse Event Reporting System (VAERS) database and the EudraVigilance post-authorisation module (EVPM) were searched to identify post vaccine ADEM cases. Epidemiological features including sex and related vaccines were analysed.

Results

We retrieved 205 and 236 ADEM cases from the EVPM and VAERS databases, respectively, of which 404 were considered for epidemiological analysis following verification and causality assessment. Half of the patients had less than 18 years and with a slight male predominance. The time interval from vaccination to ADEM onset was 2-30 days in 61% of the cases. Vaccine against seasonal flu and human papilloma virus vaccine were those most frequently associated with ADEM, accounting for almost 30% of the total cases. Mean number of reports per year between 2005 and 2012 in VAERS database was 40±21.7, decreasing after 2010 mainly because of a reduction of reports associated with human papilloma virus and Diphtheria, Pertussis, Tetanus, Polio and Haemophilus Influentiae type B vaccines.

Conclusions

This study has a high epidemiological power as it is based on information on adverse events having occurred in over one billion people. It suffers from lack of rigorous case verification due to the weakness intrinsic to the surveillance databases used. At variance with previous reports on a prevalence of ADEM in childhood we demonstrate that it may occur at any age when post vaccination. This study also shows that the diminishing trend in post vaccine ADEM reporting related to Diphtheria, Pertussis, Tetanus, Polio and Haemophilus Influentiae type B and human papilloma virus vaccine groups is most likely due to a decline in vaccine coverage indicative of a reduced attention to this adverse drug reaction.     

The phylogeny of the Casque‐headed Treefrogs (Hylidae: Hylinae: Lophyohylini)

The South American and West Indian Casque‐headed Treefrogs (Hylidae: Hylinae: Lophyohylini) include 85 species. These are notably diverse in morphology (e.g. disparate levels of cranial hyperossification) and life history (e.g. different reproductive modes, chemical defences), have a wide distribution, and occupy habitats from the tropical rainforests to semiarid scrubland. In this paper, we present a phylogenetic analysis of this hylid tribe based on sequence fragments of up to five mitochondrial (12S, 16S, ND1, COI, Cytb) and six nuclear genes (POMC, RAG‐1, RHOD, SIAH, TNS3, TYR). We included most of its species (> 96%), in addition to a number of new species. Our results indicate: (i) the paraphyly of Trachycephalus with respect to Aparasphenodon venezolanus; (ii) the nonmonophyly of Aparasphenodon, with Argenteohyla siemersi, Corythomantis galeata and Nyctimantis rugiceps nested within it, and Ap. venezolanus nested within Trachycephalus; (iii) the polyphyly of Corythomantis; (iv) the nonmonophyly of the recognized species groups of Phyllodytes; and (v) a pervasive low support for the deep relationships among the major clades of Lophyohylini, including C. greeningi and the monotypic genera Itapotihyla and Phytotriades. To remedy the nonmonophyly of Aparasphenodon, Corythomantis, and Trachycephalus, we redefined Nyctimantis to include Aparasphenodon (with the exception of Ap. venezolanus, which we transferred to Trachycephalus), Argenteohyla, and C. galeata. Additionally, our results indicate the need for taxonomic work in the following clades: (i) Trachycephalus dibernardoi and Tr. imitatrix; (ii) Tr. atlas, Tr. mambaiensis and Tr. nigromaculatus; and (iii) Phyllodytes. On the basis of our phylogenetic results, we analyzed the evolution of skull hyperossification and reproductive biology, with emphasis on the multiple independent origins of phytotelm breeding, in the context of Anura. We also analyzed the inter‐related aspects of chemical defences, venom delivery, phragmotic behaviour, co‐ossification, and prevention of evaporative water loss.