Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein

The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing. Mapping of the sequenced arms to a reference transcriptome identifies the exact locations of duplexes. hiCLIP data can directly identify all types of RNA duplexes bound by RBPs, including those that are challenging to predict computationally, such as intermolecular and long-range intramolecular duplexes. Moreover, the use of an adaptor that links the two arms of the RNA duplex permits hiCLIP to unambiguously identify the duplexes. Here we describe in detail the procedure for a hiCLIP experiment and the subsequent streamlined data analysis with an R package, 'hiclipr' (https://github.com/luslab/hiclipr/). Preparation of the library for high-throughput DNA sequencing takes ∼7 d and the basic bioinformatic pipeline takes 1 d

Estudo histológico e ultra-estrutural da mucosa do seio maxilar em pacientes com rinossinusite crônica e polipose nasossinusal Histology and ultrastructural study of the mucosa of the maxillary sinus in patients with chronic rhinosinusitis and nasosinusal polyposis

Na rinossinusite crônica, a inflamação da mucosa nasossinusal provoca alterações qualitativas e quantitativas do epitélio respiratório que recobre toda a cavidade nasossinusal, levando à manutenção do quadro inflamatório. FORMA DE ESTUDO: Caso-controle. MATERIAL E MÉTODO: Foram avaliados histopatologicamente dez pacientes com rinossinusite crônica (RC) e polipose nasossinusal (PN) por meio da história clínica e alérgica, estudo microbiológico, microscopia óptica, eletrônica de transmissão e varredura. RESULTADO: A diminuição do número de células colunares ciliadas, o aumento das células caliciformes, a diminuição do número de cílios por célula afetada e a metaplasia escamosa foram alterações freqüentemente encontradas nos casos de rinossinusite, explicando a persistência do quadro pela destruição no epitélio e quebra do sistema mucociliar.<br>In chronic rhinosinusitis, inflammation of the rhinosinusal mucosa provokes qualitative and quantitative changes in the respiratory epithelium that lines the entire rhinosinusal cavity, leading to the maintenance of an inflammatory picture. STUDY DESIGN: Case-control. MATERIAL AND METHOD: In the present study we evaluated histopathologically ten patients with chronic rhinosinusitis on the basis of clinical and allergic history, microbiological study, and light, electron and scanning electron microscopy. RESULTS: A reduced number of ciliated columnar cells, an increase in goblet-like cells, a reduction in the number of cilia per affected cell and squamous metaplasia were changes frequently detected in the cases of rhinosinusitis, explaining the persistence of the signs and symptoms due to the destruction of the epithelium and to the breakdown of the mucociliary system