Expression profiling and cross-species RNA interference (RNAi) of desiccation-induced transcripts in the anhydrobiotic nematode Aphelenchus avenae

Abstract Background Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. Results To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. Conclusions This study has identified and characterised the expression profiles of members of the anhydrobiotic gene set in A. avenae. It also demonstrates the potential of RNAi for the analysis of anhydrobiosis and provides the first genetic data to underline the importance of effective antioxidant systems in metazoan desiccation tolerance.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Amycolatopsis thermophila sp nov and Amycolatopsis viridis sp nov., thermophilic actinomycetes isolated from arid soil

The taxonomic positions of two thermophilic actinomycetes isolated from an arid Australian soil sample were established based on an investigation using a polyphasic taxonomic approach. The organisms had chemical and morphological properties typical of members of the genus Amycolatopsis and formed distinct phyletic lines in the Amycolatopsis methanolica 16S rRNA subclade. The two organisms were distinguished from one another and from the type strains of related species of the genus Amycolatopsis using a range of phenotypic properties. Based on the combined genotypic and phenotypic data, it is proposed that the two isolates be classified in the genus Amycolatopsis as Amycolatopsis thermophila sp. nov. (type strain GY088(T)=NCIMB 14699(T)=NRRL B-24836(T)) and Amycolatopsis viridis sp. nov. (type strain GY115(T)=NCIMB 14700(T)= NRRL B-24837(T)).

Ensino de anestesiologia durante a graduação por meio de uma liga acadêmica: qual o impacto no aprendizado dos alunos?

Justificativa e objetivos: As ligas acadêmicas vêm se firmando como instrumentos de ensino médico e inserção dos estudantes de medicina na prática das especialidades, inclusive na anestesiologia. Como o papel das ligas no processo de desenvolvimento de competências e aprendizado de seus alunos não é bem conhecido, avaliou-se o aprendizado dos integrantes de uma liga acadêmica de anestesiologia após um ano de participação nas suas atividades. MÉTODO: Os alunos de uma liga acadêmica de anestesiologia foram acompanhados de março a dezembro de 2010 e avaliados por meio de testes cognitivos objetivos de múltipla escolha aplicados antes do início das atividades e após sua conclusão. A frequência nas atividades e o perfil epidemiológico dos alunos foram correlacionados aos resultados dos testes. RESULTADOS: Foram analisados 20 acadêmicos do 3º ao 6º ano, com média de idade de 22,8 (21-26) anos. A frequência média nas atividades propostas foi 10,4/13 (80%). A média de acertos no primeiro teste foi 8,1/17 (47,6%), sendo que os alunos do 3º ano apresentam notas menores (p < 0,02) em relação aos demais. No teste pós-liga, a média de acertos foi 11,9/17 (70%), mostrando melhora no desempenho dos alunos (p < 0,05), e não houve diferença entre as notas dos diferentes anos da graduação. Foi encontrada relação forte entre frequência nas atividades e melhora nas notas (r = 0,719; p < 0,001). CONCLUSÕES: Os alunos que participaram da liga apresentaram melhora nos testes de avaliação de conhecimento, sugerindo que a liga é útil instrumento de ensino e promove ganho de aprendizado em anestesiologia. As atividades desenvolvidas nas ligas podem ter papel positivo na formação acadêmica dos graduandos, destacando-se, neste artigo, a anestesiologia.

MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

Abstract Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.