Experimental design and data analysis of Ago-RIP-Seq experiments for the identification of microRNA targets.

The identification of microRNA (miRNA) target genes is crucial for understanding miRNA function. Many methods for the genome-wide miRNA target identification have been developed in recent years; however, they have several limitations including the dependence on low-confident prediction programs and artificial miRNA manipulations. Ago-RNA immunoprecipitation combined with high-throughput sequencing (Ago-RIP-Seq) is a promising alternative. However, appropriate statistical data analysis algorithms taking into account the experimental design and the inherent noise of such experiments are largely lacking.Here, we investigate the experimental design for Ago-RIP-Seq and examine biostatistical methods to identify de novo miRNA target genes. Statistical approaches considered are either based on a negative binomial model fit to the read count data or applied to transformed data using a normal distribution-based generalized linear model. We compare them by a real data simulation study using plasmode data sets and evaluate the suitability of the approaches to detect true miRNA targets by sensitivity and false discovery rates. Our results suggest that simple approaches like linear regression models on (appropriately) transformed read count data are preferable.

Comparison of protein immunoprecipitation-multiple reaction monitoring with ELISA for assay of biomarker candidates in plasma.

Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978-0.995) between 10 and 640 ng/mL for the target proteins spiked into a "mock plasma" matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67-0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.

The diagnostic utility of anti-melanoma differentiation-associated gene 5 antibody testing for predicting the prognosis of Japanese patients with DM.

OBJECTIVE: Interstitial lung disease (ILD), especially rapidly progressive ILD (RPILD), is a major poor prognostic factor in patients with DM. We investigated the association of anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) with clinical characteristics and mortality in Japanese patients with DM. METHODS: Seventy-nine DM patients, comprising 58 classic DM and 21 clinically amyopathic DM (CADM) patients, were enrolled. Serum Abs were screened by immunoprecipitation assays, and an immunosorbent assay (ELISA) was used for MDA5. The relationships of clinical characteristics and mortality with each Ab were investigated. RESULTS: Anti-MDA5 Ab was detected in 17 patients. Anti-clinically amyopathic DM 140 kDa polypeptide Abs (anti-CADM-140 Abs) were found in 16 of the 17 anti-MDA5 Ab(+) patients. Skin ulcers, palmar papules, CADM, RPILD and mediastinal emphysema were widely distributed in anti-MDA5 Ab(+) patients. Mortality at 6 months as well as 5 years was also significantly higher in anti-MDA5 Ab(+) patients than in anti-MDA5 Ab(-) patients. In a multivariable Cox regression analysis, mortality was independently associated with anti-MDA5 Ab (relative hazard 6.33; 95% CI 1.43, 28.0). All of the deaths in anti-MDA5 Ab(+) patients were attributed to respiratory failure of RPILD; however, RPILD did not worsen in any of the anti-MDA5 Ab(+) patients who survived the first 6 months. CONCLUSION: The presence of anti-MDA5 Ab identifies the characteristic skin, musculoskeletal, pulmonary and prognostic features in patients with DM. In addition, anti-MDA5 Ab seems to predict a group of patients with CADM-complicated fatal RPILD.

Vulnerabilidades específicas de células malignas humanas dependentes de Ras oncogênico: FGF2 e PMA como supressores de tumor / Specific vulnerabilities of human malignant cells dependent on oncogenic Ras: FGF2 and PMA as tumor suppressors

Um passo limitante no desenvolvimento de fármacos para terapias do câncer está na descoberta de vulnerabilidades específicas de células tumorais que sirvam à identificação de alvos moleculares apropriados à intervenção farmacológica. Esta é a motivação central desta tese, cuja abordagem experimental focaliza a ação oncogênica das proteínas Ras. Amplificação ou mutação ativadora nos proto-oncogenes ras estão entre as alterações genéticas mais frequentes em cânceres. Essas lesões genéticas aparecem na origem etiológica de múltiplas formas de fenótipos malignos. Mas, essas lesões oncogênicas também conferem susceptibilidades letais às células malignamente transformadas frente a determinados agentes que não interferem significativamente nas funções vitais de células normais. Nos últimos anos nosso laboratório vem estudando os mecanismos moleculares da ação antiproliferativa do fator de crescimento FGF2 (Fibroblast Growth Factor2) e do éster de forbol PMA (Phorbol-12-Myristate-13-Acetate) em linhagens de células murinas malignas dependentes de ras oncogênico. Nesta tese investigamos quanto de nossas observações anteriores com células murinas são aplicáveis a células humanas. Nesse sentido focalizamos a linhagem HaCaT de queratinócitos humanos imortalizados e seus subclones malignizados por expressão ectópica de H-RasV12; além disso, numa triagem inicial também examinamos treze linhagens celulares humanas derivadas de tumores naturais portadores de mutação ativadora em H-Ras, N-Ras ou K-Ras. Nossos resultados mostram que os queratinócitos da linhagem parental HaCaT expressam receptores de FGFs e respondem mitogenicamente tanto a FGF2 como a PMA; portanto, ambos FGF2 e PMA são benéficos aos queratinócitos HaCaT. Por outro lado, o FGF2 mostrou-se citotóxico para subclones HaCaT que expressam H-RasV12 induzível, mas sublinhagens HaCaT com expressão constitutiva de H-RasV12 mostraram-se resistentes à ação citotóxica de FGF2. Diferentemente de FGF2, PMA bloqueou a proliferação de sublinhagens clonais HaCaT-H-RasV12 em ambos substrato sólido e suspensão de agarose e, também, reduziu a estratificação dos queratinócitos HaCaT-H-RasV12 em culturas organotípicas. PMA foi citotóxico e não citostático, pois induziu morte apoptótica sem causar arresto em nenhuma fase específica do ciclo celular. Em HaCaT parental, PMA induziu aumento transitório dos níveis intracelulares de espécies reativas de oxigênio (ROS), mas nos queratinócitos HaCaT-H-RasV12, PMA causou aumentos mais altos e persistentes de ROS, o que promove forte estresse oxidativo, provavelmente responsável pela toxidez deste ester de forbol. Entre as treze linhagens celulares humanas malignas com H-Ras, N-Ras ou K-Ras mutados, onze foram vulneráveis à ação citotóxica de PMA; mas apenas uma delas, a linhagem de tumor urotelial UM-UC-3, foi sensível ao efeito anti-proliferativo de FGF2. Em conclusão, células malignas humanas com Ras mutado parecem superar rapidamente uma possível toxidez de FGF2, mas não ultrapassam a toxidez causada por PMA

A challenge in drug development for cancer therapy is the discovering of molecular targets suitable for pharmacological interference. This challenge was the main motivation of the present thesis. Amplification or activating mutation in ras proto-oncogenes are among the most frequent genetic lesions in human cancer. Actually, mutated Ras onco-proteins are in the etiological roots of multiple malignant phenotypes; however these onco-proteins also cause specific lethal vulnerabilities even in robust malignant cells. Recently, our laboratory reported that malignant murine cell lines dependent on oncogenic Ras are prone to toxicity initiated by FGF2 (Fibroblast Growth Factor 2) and PMA (Phorbol-12-Myristate-13-Acetate), which are not harmful to normal cells. This cytotoxicity of FGF2 and PMA very likely follows different molecular mechanisms, which, however, are not yet completely understood. The aim of this thesis was to investigate whether these vulnerabilities found in murine malignant cells were also valid for human malignant cell lines dependent on oncogenic Ras. To this end the experimental approach was focused on the HaCaT cell line of immortalized human keratinocytes and its sublines transformed by H-RasV12 ectopic expression. In addition thirteen human cell lines derived from natural tumor carrying mutated H-Ras, N-Ras or K-Ras oncogenes were also screened. The results showed that HaCaT keratinocytes express FGF receptors and respond mitogenically to both FGF2 and PMA. On the other hand, FGF2 was cytotoxic to HaCaT subclones expressing inducible H-RasV12. But, HaCaT sublines constitutively expressing H-RasV12 were resistant to FGF2 toxicity. However, PMA was toxic to all HaCaT-H-RasV12 sublines, inhibiting proliferation in both solid substrate and agarose suspension cultures and, also reducing stratification in organotypic cultures. Furthermore, in HaCaT-H-RasV12 sublines, but not in the parental HaCaT line, PMA caused a persistently high increase in intracellular levels of reactive oxygen species (ROS) and concomitantly induced apoptosis. Moreover, eleven of the thirteen human tumor cell lines with mutated H-Ras, N-Ras or K-Ras, were growth inhibited by PMA, whereas only one of them was inhibited by FGF2, the urothelial tumor cell line UM-UC-3. In conclusion, human malignant cells driven by Ras oncogenes very likely rapidly overcome FGF2 toxicity, whereas they remain stably vulnerable to PMA cytotoxicity

Mass spectrometry-based immuno-precipitation proteomics - the user's guide.

Immuno-precipitation (IP) experiments using MS provide a sensitive and accurate way of characterising protein complexes and their response to regulatory mechanisms. Differences in stoichiometry can be determined as well as the reliable identification of specific binding partners. The quality control of IP and protein interaction studies has its basis in the biology that is being observed. Is that unusual protein identification a genuine novelty, or an experimental irregularity? Antibodies and the solid matrices used in these techniques isolate not only the target protein and its specific interaction partners but also many non-specific 'contaminants' requiring a structured analysis strategy. These methodological developments and the speed and accuracy of MS machines, which has been increasing consistently in the last 5 years, have expanded the number of proteins identified and complexity of analysis. The European Science Foundation's Frontiers in Functional Genomics programme 'Quality Control in Proteomics' Workshop provided a forum for disseminating knowledge and experience on this subject. Our aim in this technical brief is to outline clearly, for the scientists wanting to carry out this kind of experiment, and recommend what, in our experience, are the best potential ways to design an IP experiment, to help identify possible pitfalls, discuss important controls and outline how to manage and analyse the large amount of data generated. Detailed experimental methodologies have been referenced but not described in the form of protocols.

Diagnosis of neuromyelitis spectrum disorders: comparative sensitivities and specificities of immunohistochemical and immunoprecipitation assays.

OBJECTIVE: To compare the sensitivity and specificity of immunofluorescence (IF) and immunoprecipitation (IP) assays using green fluorescent protein-tagged aquaporin-4 (AQP4) in 6335 patients for whom serological evaluation was requested on a service basis. DESIGN: Case-control study. SETTING: Mayo Clinic Neuroimmunology Laboratory (Rochester, Minnesota) and Departments of Neurology (Rochester, Minnesota; Scottsdale, Arizona; and Jacksonville, Florida). Patients Group 1, 835 Mayo Clinic patients, 100 with a neuromyelitis optica (NMO) spectrum disorder diagnosis and 735 without NMO spectrum disorder; group 2, 5500 non-Mayo Clinic patients. Main Outcome Measure Sensitivity and specificity of each assay for NMO or NMO spectrum disorder, individually and combined. RESULTS: In group 1, the sensitivity rates for NMO were IF, 58%; IP, 33%; and combined assays, 63%. The sensitivity rates for relapsing longitudinally extensive transverse myelitis were IF, 29%; IP, 6%; and combined assays, 29%. The specificity rates for NMO and relapsing longitudinally extensive transverse myelitis were IF, 99.6%; IP, 99.3%; and combined assays, 99.2%. In group 2, NMO-IgG was detected by IF in 498 of 5500 patients (9.1%) and by IP in 331 patients (6.0%); 76 of the 331 patients seropositive by IP (23%) were negative by IF. Clinical information was available for 124 patients (including 16 of those seropositive by IP only); 123 had a definite NMO spectrum disorder and 1 was at risk for NMO (monophasic optic neuritis). CONCLUSIONS: In this large, clinical practice-based study, NMO-IgG detected by IF or IP was highly specific for NMO spectrum disorders. The IP assay was significantly less sensitive than IF. Combined testing improved sensitivity by 5%.

Advances in RIP-chip analysis : RNA-binding protein immunoprecipitation-microarray profiling.

In eukaryotic organisms, gene regulatory networks require an additional level of coordination that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed as passive molecules in the pathway from transcription to translation. However, it is now clear that RNA-binding proteins (RBPs) play a major role in regulating multiple mRNAs to facilitate gene expression patterns. On this basis, post-transcriptional and transcriptional gene expression networks appear to be very analogous. Our previous research focused on targeting RBPs to develop a better understanding of post-transcriptional gene-expression processing and the regulation of mRNA networks. We developed technologies for purifying endogenously formed RBP-mRNA complexes from cellular extracts and identifying the associated messages using genome-scale, microarray technology, a method called ribonomics or RNA-binding protein immunoprecipitation-microarray (Chip) profiling or RIP-Chip. The use of the RIP-Chip methods has provided great insight into the infrastructure of coordinated eukaryotic post-transcriptional gene expression, insights which could not have been obtained using traditional RNA expression profiling approaches (1). This chapter describes the most current RIP-Chip techniques as we presently practice them. We also discuss some of the informatic aspects that are unique to analyzing RIP-Chip data.