Allelome.PRO, a pipeline to define allele-specific genomic features from high-throughput sequencing data

Detecting allelic biases from high-throughput sequencing data requires an approach that maximises sensitivity while minimizing false positives. Here, we present Allelome.PRO, an automated user-friendly bioinformatics pipeline, which uses high-throughput sequencing data from reciprocal crosses of two genetically distinct mouse strains to detect allele-specific expression and chromatin modifications. Allelome.PRO extends approaches used in previous studies that exclusively analyzed imprinted expression to give a complete picture of the ‘allelome’ by automatically categorising the allelic expression of all genes in a given cell type into imprinted, strain-biased, biallelic or non-informative. Allelome.PRO offers increased sensitivity to analyze lowly expressed transcripts, together with a robust false discovery rate empirically calculated from variation in the sequencing data. We used RNA-seq data from mouse embryonic fibroblasts from F1 reciprocal crosses to determine a biologically relevant allelic ratio cutoff, and define for the first time an entire allelome. Furthermore, we show that Allelome.PRO detects differential enrichment of H3K4me3 over promoters from ChIP-seq data validating the RNA-seq results. This approach can be easily extended to analyze histone marks of active enhancers, or transcription factor binding sites and therefore provides a powerful tool to identify candidate cis regulatory elements genome wide

Taurolidine: a novel anti-neoplastic agent induces apoptosis of osteosarcoma cell lines

Summary: Taurolidine, the active agent of Taurolin®, is a broad spectrum anti-biotic that has been used for over 15years for the treatment of severe surgical infections. Recently, taurolidine has been shown to possess anti-neoplastic properties in vitro and in vivo against a variety of cancers including ovarian, colon and prostate. In this study we assessed the cytotoxic activity of taurolidine against human osteosarcoma (OS) cell lines and normal human bone cells. Treatment with taurolidine inhibited the growth of all ten osteosarcoma cell lines tested and taurolidine was equally potent against cell lines with and without distinct genetic defects (i.e. p53, Rb). Moreover, taurolidine-induced growth inhibition was found to be associated with a dose dependent increase in the number of apoptotic cells and apoptosis was shown to be caspase-dependent. Taurolidine treatment was also found to inhibit adhesion of OS cell lines. Compared to OS cell lines, normal bone cells in primary culture were found to be less sensitive to the cytotoxic and anti-adhesive effects of taurolidine. These data indicate that taurolidine possesses potent anti-neoplastic activity against osteosarcoma cell lines and may have potential as a novel OS chemotherapeutic agen

A importância da educação no desenvolvimento de um processo de conscientização social sobre a separação e descarte de resíduos sólidos: um relato de experiência

A educação ambiental é um processo com grande impacto de transformação local que reflete tanto em mudanças sociais como ambientais.  Por conta disso, este trabalho foi desenvolvido a partir da disciplina Projeto Integrador: Ser Biológico e Social e trata sobre um relato de experiência acerca das atividades desenvolvidas em uma comunidade da região noroeste do estado do Rio Grande do Sul e seus resultados obtidos a partir dos momentos de conscientização com os moradores do local. A partir dos problemas ambientais encontrados na localidade, foram desenvolvidas atividades com o intuito de elucidar sobre a maneira correta de realizar o descarte do lixo gerado no local. Foram utilizados instrumentos como um jogo de perguntas e respostas na plataforma de aprendizado "Kahoot", folders informativos sobre as cores das lixeiras e seus respectivos significados, e uma confraternização acerca do tema com os moradores. Evidenciando-se por meio destas atividades o papel da comunidade na limpeza e melhora do ambiente em que estas famílias residem, ademais, discutiram-se estratégias para a intervenção efetiva destes problemas por meio do Poder Público municipal

Long non-coding RNAs display higher natural expression variation than protein-coding genes in healthy humans

Background: Long non-coding RNAs (lncRNAs) are increasingly implicated as gene regulators and may ultimately be more numerous than protein-coding genes in the human genome. Despite large numbers of reported lncRNAs, reference annotations are likely incomplete due to their lower and tighter tissue-specific expression compared to mRNAs. An unexplored factor potentially confounding lncRNA identification is inter-individual expression variability. Here, we characterize lncRNA natural expression variability in human primary granulocytes. Results: We annotate granulocyte lncRNAs and mRNAs in RNA-seq data from 10 healthy individuals, identifying multiple lncRNAs absent from reference annotations, and use this to investigate three known features (higher tissue-specificity, lower expression, and reduced splicing efficiency) of lncRNAs relative to mRNAs. Expression variability was examined in seven individuals sampled three times at 1- or more than 1-month intervals. We show that lncRNAs display significantly more inter-individual expression variability compared to mRNAs. We confirm this finding in two independent human datasets by analyzing multiple tissues from the GTEx project and lymphoblastoid cell lines from the GEUVADIS project. Using the latter dataset we also show that including more human donors into the transcriptome annotation pipeline allows identification of an increasing number of lncRNAs, but minimally affects mRNA gene number. Conclusions: A comprehensive annotation of lncRNAs is known to require an approach that is sensitive to low and tight tissue-specific expression. Here we show that increased inter-individual expression variability is an additional general lncRNA feature to consider when creating a comprehensive annotation of human lncRNAs or proposing their use as prognostic or disease markers

Multiclass cancer classification in fresh frozen and formalin-fixed paraffin-embedded tissue by DigiWest multiplex protein analysis

Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications

Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples

Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint.Peer Reviewe