Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics

Metatranscriptomes generated by pyrosequencing hold significant potential for describing functional processes in complex microbial communities. Meeting this potential requires protocols that maximize mRNA recovery by reducing the relative abundance of ribosomal RNA, as well as systematic comparisons to identify methodological artifacts and test for reproducibility across data sets. Here, we implement a protocol for subtractive hybridization of bacterial rRNA (16S and 23S) that uses sample-specific probes and is applicable across diverse environmental samples. To test this method, rRNA-subtracted and unsubtracted transcriptomes were sequenced (454 FLX technology) from bacterioplankton communities at two depths in the oligotrophic open ocean, yielding 10 data sets representing ~350 Mbp. Subtractive hybridization reduced bacterial rRNA transcript abundance by 40–58%, increasing recovery of non-rRNA sequences up to fourfold (from 12% to 20% of total sequences to 40–49%). In testing this method, we established criteria for detecting sequences replicated artificially via pyrosequencing errors and identified such replicates as a significant component (6–39%) of total pyrosequencing reads. Following replicate removal, statistical comparisons of reference genes (identified via BLASTX to NCBI-nr) between technical replicates and between rRNA-subtracted and unsubtracted samples showed low levels of differential transcript abundance (<0.2% of reference genes). However, gene overlap between data sets was remarkably low, with no two data sets (including duplicate runs from the same pyrosequencing library template) sharing greater than 17% of unique reference genes. These results indicate that pyrosequencing captures a small subset of total mRNA diversity and underscores the importance of reliable rRNA subtraction procedures to enhance sequencing coverage across the functional transcript pool.Agouron InstituteGordon and Betty Moore FoundationUnited States. Dept. of Energy. Office of ScienceNational Science Foundation (U.S.) (NSF Science and Technology Center Award EF0424599

A defined mechanistic correlate of protection against Plasmodium falciparum malaria in non-human primates.

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection

Adrenaline Infusion and Adrenocorticotropin (acth) and Cortisol Release in Normotensive and Hypertensive Man

1. Adrenaline causes ACTH release from cultured rat pituitary corticotrophs (Vale et al. 1983) and there is evidence that it causes ACTH release in rats in vivo (Plotsky et al. 1985). 2. The present study examined the effects of intravenous adrenaline infusion with and without simultaneous administration of the known ACTH secretagog, arginine vasopressin, in normotensive and mild essential hypertensive men on their plasma ACTH and cortisol levels. 3. Low dose adrenaline infusion (0.013 μg/kg per min) does not cause ACTH or cortisol release, but appears to blunt the ACTH and cortisol rise caused by arginine vasopressin (0.14 pressor units/kg, i.m.)

Comparação dos resultados da fala após as cirurgias de retalho faríngeo e veloplastia intravelar para correção da disfunção velofaríngea Comparison of speech results following pharyngeal flap and intravelar veloplasty for correction of velopharyngeal dysfunction

OBJETIVO: Comparar a fala e o funcionamento velofaríngeo após as técnicas de retalho faríngeo e veloplastia intravelar para a correção da disfunção velofaríngea residual. MÉTODOS: Foi realizado um estudo retrospectivo com análise de 148 casos com fissura labiopalatina operada e submetidos à correção cirúrgica da disfunção velofaríngea, sendo 77 com retalho faríngeo (média de idade: 20,4 anos) e 71 com veloplastia intravelar (média de idade: 16,2 anos). Foram avaliadas a ressonância da fala, a presença de articulações compensatórias, a emissão de ar nasal e a extensão da falha no fechamento velofaríngeo antes e após as duas técnicas. RESULTADOS: Dos 77 casos submetidos ao retalho faríngeo 64 (83%) apresentaram melhora na ressonância, enquanto que dos 71 casos com veloplastia intravelar 48 (68%) revelaram melhora, havendo diferença significativa entre os grupos. No grupo com retalho faríngeo, seis (8%) apresentaram melhora na articulação compensatória e dois (3%) no grupo veloplastia intravelar, enquanto a emissão de ar nasal melhorou em 17 (22%) casos com retalho faríngeo e em 18 (26%) com veloplastia intravelar. Não houve diferença entre os grupos quanto à articulação compensatória e emissão de ar nasal. A falha no fechamento velofaríngeo reduziu em 75 (96%) casos com retalho faríngeo e 46 (66%) com a veloplastia intravelar, havendo diferença entre os grupos. CONCLUSÃO: A técnica de retalho faríngeo mostrou-se mais efetiva na melhora da ressonância e no fechamento velofaríngeo quando comparada à veloplastia intravelar.<br>PURPOSE: To compare speech and velopharyngeal function after pharyngeal flap and intravelar veloplasty techniques for the correction of residual velopharyngeal dysfunction. METHODS: It was carried out a retrospective study analyzing 148 cases of cleft lip and palate operated and submitted to velopharyngeal dysfunction surgical correction, 77 with pharyngeal flap (mean age: 20.4 years) and 71 with intravelar veloplasty (mean age: 16.2 years). Speech resonance, presence of compensatory articulations, nasal air emission, and velopharyngeal gap size were assessed before and after the use of both techniques. RESULTS: Sixty four (83%) of the 77 cases submitted to pharyngeal flap presented resonance improvement, while 48 (68%) of the 71 cases with intravelar veloplasty improved, with significant difference between the groups. Six (8%) subjects with pharyngeal flap, and two (3%) with intravelar veloplasty showed improvement in compensatory articulations, while nasal air emission improved in 17 (22%) cases with pharyngeal flap, and 18 (26%) with intravelar veloplasty. No significant differences were found between the groups regarding compensatory articulations and nasal air emission. The velopharyngeal gap size reduced in 75 (96%) cases with pharyngeal flap, and 46 (66%) with intravelar veloplasty, with a significant difference between the groups. CONCLUSION: The pharyngeal flap technique was more effective in improving resonance and velopharyngeal closure, when compared to intravelar veloplasty

Coral skeletons provide historical evidence of phosphorus runoff on the Great Barrier Reef

Recently, the inshore reefs of the Great Barrier Reef have declined rapidly because of deteriorating water quality. Increased catchment runoff is one potential culprit. The impacts of land-use on coral growth and reef health however are largely circumstantial due to limited long-term data on water quality and reef health. Here we use a 60 year coral core record to show that phosphorus contained in the skeletons (P/Ca) of long-lived, near-shore Porites corals on the Great Barrier Reef correlates with annual records of fertiliser application and particulate phosphorus loads in the adjacent catchment. Skeletal P/Ca also correlates with Ba/Ca, a proxy for fluvial sediment loading, again linking near-shore phosphorus records with river runoff. Coral core records suggest that phosphorus levels increased 8 fold between 1949 and 2008 with the greatest levels coinciding with periods of high fertiliser-phosphorus use. Periods of high P/Ca correspond with intense agricultural activity and increased fertiliser application in the river catchment following agricultural expansion and replanting after cyclone damage. Our results demonstrate how coral P/Ca records can be used to assess terrestrial nutrient loading of vulnerable near-shore reefs