Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5

Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.     

Dark diversity in Amazonian stream fish communities: What factors determine species absence along environmental gradients?

1. Species distribution models often fail to predict observed patterns of species diversity, and this is because some species within a regional pool that are tolerant of conditions at a given location may nevertheless be absent from the local community. These missing species have been termed “dark diversity”. In the present study, we investigated which factors explain dark diversity among fish assemblages in Amazonian streams.
2. We sampled 71 streams in areas with different types of land use within two river basins and estimated dark diversity from patterns of species co-occurrence, using Beals’ index, along environmental gradients. From this procedure, taxa are designated as dark diversity components when they are absent from a given stream, but often co-occur with the local species at other streams, indicating similar ecological requirements. We used generalised linear models both to determine whether environmental or landscape variables, connectivity, instream environmental heterogeneity or some combination of these factors explained dark diversity of fishes, and to evaluate whether ecomorphology is associated with the extent to which a species contributes to dark diversity and which specific traits contribute the most to explaining variation in dark diversity.
3. Mean local diversity exceeded observed dark diversity. The magnitude of dark diversity was directly associated with the proportion of secondary forest in the immediate catchment and with the index of proximity to anthropogenic impact. Species that have high affinity for environments with higher current velocity, low swimming ability and that capture food mainly on the surface contributed more to dark diversity, which suggests that swimming ability, habitat preference and aspects related to diet are key predictors of the probability that a given species will be present at locations with suitable habitat.
4. Our findings reinforce the idea that dark diversity results from interactions between species traits and environmental factors, including anthropogenic impacts. Understanding the interplay among environmental factors and species traits that contribute to dark diversity provides targets for improved ecosystem restoration and sustainability of native species assemblages.

     

<Emphasis Type="Italic">Isospora lopesi</Emphasis> n. sp. (Apicomplexa: Eimeriidae) from the eastern white-throated spadebill <Emphasis Type="Italic">Platyrinchus mystaceus</Emphasis> Vieillot (Passeriformes: Tyranni: Tyrannidae) in South America

A species of Isospora Schneider, 1881 (Protozoa: Apicomplexa: Eimeriidae) considered as new to science is described and characterised molecularly from the eastern white-throated spadebill Platyrinchus mystaceus Vieillot in the Parque Nacional do Itatiaia, southeastern Brazil. Isospora lopesi n. sp. has oöcysts that are subspheroidal to ovoidal, 18–24 × 18–22 (20.6 × 19.7) µm, with smooth, bilayered wall, c.1.5 μm thick. Micropyle and oöcyst residuum are absent, but one polar granule is present. Sporocysts are ellipsoidal, 12–16 × 8–11 (14.4 × 8.6) µm. The Stieda body is flattened to half-moon-shaped and sub-Stieda body is rounded. Sporocyst residuum is present, consisting of numerous spherules of different sizes. Sporozoites are vermiform with anterior and posterior refractile bodies and nucleus. Molecular analysis was conducted at the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. This new isolate exhibited similarity greater than 98% with Isospora spp. isolates from spectacled warblers Sylvia conspicillata Temminck, 1820. This is the fourth isosporoid coccidian described from New World tyrannid birds, but is the first to have a complementary molecular characterisation.     

Nitrochalcones: Potential in vivo insulin secretagogues

In this study, the in vivo and in vitro anti-hyperglycemic activity of chalcone derivatives of 3,4-methylenedioxy, with a substituent electron-acceptor nitro group in the A or B ring, was investigated. As expected, the second generation sulfonylurea glipizide stimulated insulin secretion and reduced glycemia over the study period. Also, it was demonstrated for the first time that chalcones are able to increase insulin secretion and this event was coincident with serum glucose-lowering in the oral glucose tolerance test. Additionally, the chalcones studied had a similar effect on insulin secretion and serum glucose-lowering as glipizide. The effect of chalcones in terms of inducing insulin secretion was greater than that of glipizide after 30 min. Moreover, chalcones were not able to stimulate glucose uptake in soleus muscle, either in the presence of insulin or in the absence of this hormone. In addition, the oral treatment with chalcones did not alter glycemia in diabetic rats. These reports indicate that the effect of chalcones on serum glucose-lowering in hyperglycemic-normal rats is mainly a consequence of insulin secretion, highlighting these chalcones as novel compounds with strong anti-hyperglycemic properties.     

Determination of Parameters for the Supercritical Extraction of Antioxidant Compounds from Green Propolis Using Carbon Dioxide and Ethanol as Co-Solvent

The aim of this study was to determine the best processing conditions to extract Brazilian green propolis using a supercritical extraction technology. For this purpose, the influence of different parameters was evaluated such as S/F (solvent mass in relation to solute mass), percentage of co-solvent (1 and 2% ethanol), temperature (40 and 50°C) and pressure (250, 350 and 400 bar) using supercritical carbon dioxide. The Global Yield Isotherms (GYIs) were obtained through the evaluation of the yield, and the chemical composition of the extracts was also obtained in relation to the total phenolic compounds, flavonoids, antioxidant activity and 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C) and acid 4-hydroxycinnamic (p-coumaric acid). The best results were identified at 50°C, 350 bar, 1% ethanol (co-solvent) and S/F of 110. These conditions, a content of 8.93±0.01 and 0.40±0.05 g/100 g of Artepillin C and p-coumaric acid, respectively, were identified indicating the efficiency of the extraction process. Despite of low yield of the process, the extracts obtained had high contents of relevant compounds, proving the viability of the process to obtain green propolis extracts with important biological applications due to the extracts composition.     

Genomic Ancestry,Self-Rated Health and Its Association with Mortality in an Admixed Population: 10 Year Follow-Up of the Bambui-Epigen (Brazil) Cohort Study of Ageing

Background

Self-rated health (SRH) has strong predictive value for mortality in different contexts and cultures, but there is inconsistent evidence on ethnoracial disparities in SRH in Latin America, possibly due to the complexity surrounding ethnoracial self-classification.

Materials/Methods

We used 370,539 Single Nucleotide Polymorphisms (SNPs) to examine the association between individual genomic proportions of African, European and Native American ancestry, and ethnoracial self-classification, with baseline and 10-year SRH trajectories in 1,311 community dwelling older Brazilians. We also examined whether genomic ancestry and ethnoracial self-classification affect the predictive value of SRH for subsequent mortality.

Results

European ancestry predominated among participants, followed by African and Native American (median = 84.0%, 9.6% and 5.3%, respectively); the prevalence of Non-White (Mixed and Black) was 39.8%. Persons at higher levels of African and Native American genomic ancestry, and those self-identified as Non-White, were more likely to report poor health than other groups, even after controlling for socioeconomic conditions and an array of self-reported and objective physical health measures. Increased risks for mortality associated with worse SRH trajectories were strong and remarkably similar (hazard ratio ~3) across all genomic ancestry and ethno-racial groups.

Conclusions

Our results demonstrated for the first time that higher levels of African and Native American genomic ancestry—and the inverse for European ancestry—were strongly correlated with worse SRH in a Latin American admixed population. Both genomic ancestry and ethnoracial self-classification did not modify the strong association between baseline SRH or SRH trajectory, and subsequent mortality.     

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

Thyroid Hormones Reorganize the Cytoskeleton of Glial Cells Through Gfap Phosphorylation and Rhoa-Dependent Mechanisms

Thyroid hormones (3,5,3′-triiodo-l-thyronine, T₃; 3,5,3′,5′-l-tetraiodothyronine, T₄; TH) play crucial roles in the growth and differentiation of the central nervous system. In this study, we investigated the actions of TH on proliferation, viability, cell morphology, in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and actin reorganization in C6 glioma cells. We first observe that long-term exposure to TH stimulates cell proliferation without induce cell death. We also demonstrate that after 3, 6, 12, 18, and 24 h treatment with TH, C6 cells and cortical astrocytes show a process-bearing shape. Furthermore, immunocytochemistry with anti-actin and anti-GFAP antibodies reveals that TH induces reorganization of actin and GFAP cytoskeleton. We also observe an increased in vitro ³²P incorporation into GFAP recovered into the high-salt Triton insoluble cytoskeletal fraction after 3 and 24 h exposure to 5×10⁻⁸ and 10⁻⁶ M T₃, and only after 24 h exposure to 10⁻⁹ M T₄. These results show a T₃ action on the phosphorylating system associated to GFAP and suggest a T₃-independent effect of T₄ on this cytoskeletal protein. In addition, C6 cells and astrocytes treated with lysophosphatidic acid, an upstream activator of the RhoA GTPase pathway, totally prevented the morphological alterations induced by TH, indicating that this effect could be mediated by the RhoA signaling pathway. Considering that IF network can be regulated by phosphorylation leading to reorganization of IF filamentous structure and that alterations of the microfilament organization may have important implications in glial functions, the effects of TH on glial cell cytoskeleton could be implicated in essential neural events such as brain development.