Extracellular vesicle-mediated export of fungal RNA

Extracellular vesicles (EVs) play an important role in the biology of various organisms, including fungi, in which they are required for the trafficking of molecules across the cell wall. Fungal EVs contain a complex combination of macromolecules, including proteins, lipids and glycans. in this work, we aimed to describe and characterize RNA in EV preparations from the human pathogens Cryptococcus neoformans, Paracoccidiodes brasiliensis and Candida albicans, and from the model yeast Saccharomyces cerevisiae. the EV RNA content consisted mostly of molecules less than 250 nt long and the reads obtained aligned with intergenic and intronic regions or specific positions within the mRNA. We identified 114 ncRNAs, among them, six small nucleolar (snoRNA), two small nuclear (snRNA), two ribosomal (rRNA) and one transfer (tRNA) common to all the species considered, together with 20 sequences with features consistent with miRNAs. We also observed some copurified mRNAs, as suggested by reads covering entire transcripts, including those involved in vesicle-mediated transport and metabolic pathways. We characterized for the first time RNA molecules present in EVs produced by fungi. Our results suggest that RNA-containing vesicles may be determinant for various biological processes, including cell communication and pathogenesis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Instituto Nacional de Ciencia e Tecnologia de Inovacao em Doencas Negligenciadas (INCT-IDN)Brazilian agency Fundacao Araucaria - PRONEXBrazilian agency Papes-FiocruzUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFundacao Oswaldo Cruz, CDTS, Rio de Janeiro, RJ, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Prof Paulo de Goes, BR-21941 Rio de Janeiro, RJ, BrazilFiocruz PR, Fundacao Oswaldo Cruz, Inst Carlos Chagas, Curitiba, PR, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Cytogenetic studies of Brazilian pediatric myelodysplastic syndrome cases: challenges and difficulties in a large and emerging country

Myelodysplastic syndromes (MDS) and juvenile myelomonocytic leukemia (JMML) are rare hematopoietic stem cell diseases affecting children. Cytogenetics plays an important role in the diagnosis of these diseases. We report here the experience of the Cytogenetic Subcommittee of the Brazilian Cooperative Group on Pediatric Myelodysplastic Syndromes (BCG-MDS-PED). We analyzed 168 cytogenetic studies performed in 23 different cytogenetic centers; 84 of these studies were performed in patients with confirmed MDS (primary MDS, secondary MDS, JMML, and acute myeloid leukemia/MDS+Down syndrome). Clonal abnormalities were found in 36.9% of the MDS cases and cytogenetic studies were important for the detection of constitutional diseases and for differential diagnosis with other myeloid neoplasms. These data show the importance of the Cooperative Group for continuing education in order to avoid a late or wrong diagnosis.Univ São Paulo, Fac Med, Dept Hematol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Hematol, São Paulo, BrazilUniv São Paulo, Fac Med, Dept Hematol, Lab Citogenet, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Oncol Pediat, São Paulo, BrazilUniv Estadual Paulista, Fac Med Botucatu, Dept Clin Med, Botucatu, SP, BrazilHosp Canc Barretos, Barretos, SP, BrazilCtr Tratamento Fabiana Macedo de Morais, Grp Assistencia Crianca Canc, Grp Cooperat Brasileiro Sindrome Mielodisplas Ped, Sao Jose Dos Campos, SP, BrazilUniversidade Federal de São Paulo, Dept Hematol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Oncol Pediat, São Paulo, BrazilWeb of Scienc

A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with alpha 1,3-glucans. in Cryptococcus neoformans, alpha 1,3-glucans interact with glucuronoxylomannan (GXM), a hetero-polysaccharide that is essential for fungal virulence. in this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. in addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. in comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. the P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis. (C) 2012 Elsevier Inc. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)NIHCenter for AIDS Research at EinsteinInterhemispheric Research Training Grant in Infectious Diseases, Fogarty International CenterDepartment of EnergyFiocruz MS, CDTS, BR-21040360 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Prof Paulo de Goes, BR-21941902 Rio de Janeiro, BrazilAlbert Einstein Coll Med, Dept Microbiol & Immunol, Bronx, NY 10461 USAUniversidade Federal de São Paulo, Disciplina Biol Celular, BR-04023062 São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Ultraestrutura Celular Hertha Meyer, BR-21941903 Rio de Janeiro, BrazilAlbert Einstein Coll Med, Div Infect Dis, Dept Med, Bronx, NY 10461 USAUniversidade Federal de São Paulo, Disciplina Biol Celular, BR-04023062 São Paulo, BrazilNIH: AI033142NIH: AI033774NIH: AI052733NIH: HL059842Interhemispheric Research Training Grant in Infectious Diseases, Fogarty International Center: NIH D43-TW007129Department of Energy: DE-FG-9-93ER-20097Web of Scienc

Motor recovery and synaptic preservation after ventral root avulsion and repair with a fibrin sealant derived from snake venom

Background: Ventral root avulsion is an experimental model of proximal axonal injury at the central/peripheral nervous system interface that results in paralysis and poor clinical outcome after restorative surgery. Root reimplantation may decrease neuronal degeneration in such cases. We describe the use of a snake venom-derived fibrin sealant during surgical reconnection of avulsed roots at the spinal cord surface. The present work investigates the effects of this fibrin sealant on functional recovery, neuronal survival, synaptic plasticity, and glial reaction in the spinal motoneuron microenvironment after ventral root reimplantation. Methodology/Principal Findings: Female Lewis rats (7 weeks old) were subjected to VRA and root replantation. The animals were divided into two groups: 1) avulsion only and 2) replanted roots with fibrin sealant derived from snake venom. Post-surgical motor performance was evaluated using the CatWalk system twice a week for 12 weeks. The rats were sacrificed 12 weeks after surgery, and their lumbar intumescences were processed for motoneuron counting and immunohistochemistry (GFAP, Iba-1 and synaptophysin antisera). Array based qRT-PCR was used to evaluate gene regulation of several neurotrophic factors and receptors as well as inflammatory related molecules. The results indicated that the root reimplantation with fibrin sealant enhanced motor recovery, preserved the synaptic covering of the motoneurons and improved neuronal survival. The replanted group did not show significant changes in microglial response compared to VRA-only. However, the astroglial reaction was significantly reduced in this group. Conclusions/Significance: In conclusion, the present data suggest that the repair of avulsed roots with snake venom fibrin glue at the exact point of detachment results in neuroprotection and preservation of the synaptic network at the microenvironment of the lesioned motoneurons. Also such procedure reduced the astroglial reaction and increased mRNA levels to neurotrophins and anti-inflammatory cytokines that may in turn, contribute to improving recovery of motor function

Avian influenza virus (H11N9) in migratory shorebirds wintering in the Amazon region, Brazil

Aquatic birds are the natural reservoir for avian influenza viruses (AIV). Habitats in Brazil provide stopover and wintering sites for water birds that migrate between North and South America. The current study was conducted to elucidate the possibility of the transport of influenza A viruses by birds that migrate annually between the Northern and Southern Hemispheres. In total, 556 orotracheal/cloacal swab samples were collected for influenza A virus screening using real-time RT-PCR (rRT-PCR). The influenza A virus-positive samples were subjected to viral isolation. Four samples were positive for the influenza A matrix gene by rRT-PCR. From these samples, three viruses were isolated, sequenced and characterized. All positive samples originated from a single bird species, the ruddy turnstone (Arenaria interpres), that was caught in the Amazon region at Caeté Bay, Northeast Pará, at Ilha de Canelas. To our knowledge, this is the first isolation of H11N9 in the ruddy turnstone in South America. (Résumé d'auteur

Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice

<p>Abstract</p> <p>Background</p> <p>Dengue, one of the most important arboviral diseases of humans, may cause severe systemic disease. Although dengue virus (DENV) has been considered to be a non-neurotropic virus, dengue infection has been associated recently with a series of neurological syndromes, including encephalitis. In this work, we evaluated behavioral changes and inflammatory parameters in C57BL/6 mice infected with non-adapted dengue virus 3 (DENV-3) genotype I.</p> <p>Methods</p> <p>C57BL/6 mice received 4 × 10³PFU of DENV-3 by an intracranial route. We evaluated the trafficking of leukocytes in brain microvasculature using intravital microscopy, and evaluated chemokine and cytokine profiling by an ELISA test at 3 and 6 days post infection (p.i.). Furthermore, we determined myeloperoxidase activity and immune cell populations, and also performed histopathological analysis and immunostaining for the virus in brain tissue.</p> <p>Results</p> <p>All animals developed signs of encephalitis and died by day 8 p.i. Motor behavior and muscle tone and strength parameters declined at day 7 p.i. We observed increased leukocyte rolling and adhesion in brain microvasculature of infected mice at days 3 and 6 p.i. The infection was followed by significant increases in IFN-γ, TNF-α, CCL2, CCL5, CXCL1, and CXCL2. Histological analysis showed evidence of meningoencephalitis and reactive gliosis. Increased numbers of neutrophils, CD4⁺and CD8⁺T cells were detected in brain of infected animals, notably at day 6 p.i. Cells immunoreactive for anti-NS-3 were visualized throughout the brain.</p> <p>Conclusion</p> <p>Intracerebral infection with non-adapted DENV-3 induces encephalitis and behavioral changes that precede lethality in mice.</p

Large emissions from floodplain trees close the Amazon methane budget

Wetlands are the largest global source of atmospheric methane (CH4), a potent greenhouse gas. However, methane emission inventories from the Amazon floodplain, the largest natural geographic source of CH4 in the tropics, consistently underestimate the atmospheric burden of CH4 determined via remote sensing and inversion modelling, pointing to a major gap in our understanding of the contribution of these ecosystems to CH4 emissions. Here we report CH4 fluxes from the stems of 2,357 individual Amazonian floodplain trees from 13 locations across the central Amazon basin. We find that escape of soil gas through wetland trees is the dominant source of regional CH4 emissions. Methane fluxes from Amazon tree stems were up to 200 times larger than emissions reported for temperate wet forests6 and tropical peat swamp forests, representing the largest non-ebullitive wetland fluxes observed. Emissions from trees had an average stable carbon isotope value (δ13C) of −66.2 ± 6.4 per mil, consistent with a soil biogenic origin. We estimate that floodplain trees emit 15.1 ± 1.8 to 21.2 ± 2.5 teragrams of CH4 a year, in addition to the 20.5 ± 5.3 teragrams a year emitted regionally from other sources. Furthermore, we provide a ‘top-down’ regional estimate of CH4 emissions of 42.7 ± 5.6 teragrams of CH4 a year for the Amazon basin, based on regular vertical lower-troposphere CH4 profiles covering the period 2010–2013. We find close agreement between our ‘top-down’ and combined ‘bottom-up’ estimates, indicating that large CH4 emissions from trees adapted to permanent or seasonal inundation can account for the emission source that is required to close the Amazon CH4 budget. Our findings demonstrate the importance of tree stem surfaces in mediating approximately half of all wetland CH4 emissions in the Amazon floodplain, a region that represents up to one-third of the global wetland CH4 source when trees are combined with other emission sources

Preparation of novel trifluoroacetylketene O,N-acetals and trifluoromethyl-containing S,S-sulfoximido N-substituted heterocycles

Two new trifluoroacetylketene O,N-acetals [CF3C(O)CH=C(OEt)(NS(O)R2), where R = CH3, Ph] derived from the reaction of 4,4-diethoxy-1,1,1-trifluorobut-3-en-2-one [CF3C(O)CH=C(OEt)2] with S,S-dimethyl- and S-methyl-S-phenyl-sulfoximide [HN=S(O)R2], in the presence of triethylamine, have been obtained, in 60-72% yields, and applied in the synthesis of S,S-dimethylsulfoximido-substituted pyrazoles, isoxazoles and pyrimidines, in 55-89% yields, from the reactions of 4-ethoxy-4-(S,S-dimethylsulfoximido)-1,1,1-trifluorobut-3-en-2-one with hydrazines, hydroxylamine hydrochloride and acetylguanidine