Rapid and accurate quantification of isomiRs by RT-qPCR

Currently, microRNAs (miRs) are annotated as a single defined sequence (canonical), even though high-throughput small RNA sequencing has identified miR isoforms (isomiRs) that differ from their canonical counterparts in length, sequence, or both. Here we describe a simple reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)-based assay for quantification of the miR-100-5p_iso_3p:−2 variant. We chose miR-100-5p because the canonical sequence was underrepresented in our evaluation of human plasma. The quantification of miR-100-5p_iso_3 p:−2 from 57 plasma samples demonstrated high concordance between high-throughput RNA sequencing and RT-qPCR results (r = 0.55, p < 0.0001). Of note, we could not detect or quantify miR-100-5p in our plasma samples using a commercial TaqMan canonical miR-100-5p RT-qPCR kit. With these 57 samples, we also adapted this assay to specifically quantify the canonical sequences of miR-122-5p and miR-192-5p. Similar to the results obtained with miR-100-5p_iso_3p:−2, RT-qPCR results for miR-122-5p and miR-192-5p highly correlated with high-throughput RNA sequencing data (miR-122-5p: r = 0.44, p = 0.0005; miR-192-5p: r = 0.72, p < 0.0001). The assay described here can be easily adapted to many different identified isomiRs. Because of the high specificity of isomiRs, their reliable RT-qPCR-based quantification could provide greater resolution and higher accuracy than using canonical sequences

Draft genome sequences of Mycobacterium setense type strain DSM-45070 and the nonpathogenic strain Manresensis, isolated from the bank of the Cardener river in Manresa, Catalonia, Spain

This study was funded by the Spanish Government, Instituto de Salud Carlos III (ISCIII), through the CIBER CRP-TB and the FIS 011/01702 grants to PJC, and by Manresana de Micobacteriologia, s.l. Additional support came from IMPPC core funding from the Generalitat de Catalunya to LS. JV, GR, SS, were funded by grant ADE10/00026 from ISCIII. JV was also co-funded by ISIS contract II11/00014 from ISCIII. IC is supported by the Spanish Ministerio de Economia y Competitividad (MINECO) through a Ramón y Cajal contract RYC-2012-10627 and grant SAF2013-43521-R. E.J. was funded by Instituto de Salud Carlos III-PI1001438 and the European Regional Development Fund (FEDER), and CV by Miguel Servet Contract CP13/00174.We present here the draft genome sequences of two Mycobacterium setense strains. One of them corresponds to the M. setense type strain DSM-45070, originally isolated from a patient with a posttraumatic chronic skin abscess. The other one corresponds to the nonpathogenic M. setense strain Manresensis, isolated from the Cardener River crossing Manresa, Catalonia, Spain. A comparative genomic analysis shows a smaller genome size and fewer genes in M. setense strain Manresensis relative to those of the type strain, and it shows the genome segments unique to each strain

Draft genome sequences of Mycobacterium setense type strain DSM-45070 and the nonpathogenic strain Manresensis, isolated from the bank of the Cardener River in Manresa, Catalonia, Spain

We present here the draft genome sequences of two Mycobacterium setense strains. One of them corresponds to the M. setense type strain DSM-45070, originally isolated from a patient with a posttraumatic chronic skin abscess. The other one corresponds to the nonpathogenic M. setense strain Manresensis, isolated from the Cardener River crossing Manresa, Catalonia, Spain. A comparative genomic analysis shows a smaller genome size and fewer genes in M. setense strain Manresensis relative to those of the type strain, and it shows the genome segments unique to each strain

Curcumin mediates oxaliplatin-acquired resistance reversion in colorectal cancer cell lines through modulation of CXC-Chemokine/NF-κB signalling pathway

This study was funded by the ISCIII grant, project n° PI1202228 and Departament d'Innovació, Universitats i Empresa, Generalitat de Catalunya. SGR-PREDIVHICO. This work was done under the framework of the doctorate in Medicine from the Universitat Autònoma de Barcelona. We thank Dr. Lucía Sanjurjo (Innate Immunity Group, IGTP, Badalona, Spain) for her technical assistance and support and Dr. Verónica Guirao (Biobank research support unit, IGTP, Badalona, Spain) for her comments and editorial assistance.Resistance to oxaliplatin (OXA) is a complex process affecting the outcomes of metastatic colorectal cancer (CRC) patients treated with this drug. De-regulation of the NF-κB signalling pathway has been proposed as an important mechanism involved in this phenomenon. Here, we show that NF-κB was hyperactivated in in vitro models of OXA-acquired resistance but was attenuated by the addition of Curcumin, a non-toxic NF-κB inhibitor. The concomitant combination of Curcumin + OXA was more effective and synergistic in cell lines with acquired resistance to OXA, leading to the reversion of their resistant phenotype, through the inhibition of the NF-κB signalling cascade. Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells. Moreover, CXCL8 and CXCL1 gene silencing made resistant cells more sensitive to OXA through the inhibition of the Akt/NF-κB pathway. High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin. In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients

Curcumin mediates oxaliplatin-acquired resistance reversion in colorectal cancer cell lines through modulation of CXC-Chemokine/NF-κB signalling pathway

Resistance to oxaliplatin (OXA) is a complex process affecting the outcomes of metastatic colorectal cancer (CRC) patients treated with this drug. De-regulation of the NF-kappa B signalling pathway has been proposed as an important mechanism involved in this phenomenon. Here, we show that NF-kappa B was hyperactivated in in vitro models of OXA-acquired resistance but was attenuated by the addition of Curcumin, a non-toxic NF-kappa B inhibitor. The concomitant combination of Curcumin + OXA was more effective and synergistic in cell lines with acquired resistance to OXA, leading to the reversion of their resistant phenotype, through the inhibition of the NF-kappa B signalling cascade. Transcriptomic profiling revealed the up-regulation of three NF-kappa B-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells. Moreover, CXCL8 and CXCL1 gene silencing made resistant cells more sensitive to OXA through the inhibition of the Akt/NF-kappa B pathway. High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin. In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients

Multitrait genome association analysis identifies new susceptibility genes for human anthropometric variation in the GCAT cohort

Background Heritability estimates have revealed an important contribution of SNP variants for most common traits; however, SNP analysis by single-trait genome-wide association studies (GWAS) has failed to uncover their impact. In this study, we applied a multitrait GWAS approach to discover additional factor of the missing heritability of human anthropometric variation. Methods We analysed 205 traits, including diseases identified at baseline in the GCAT cohort (Genomes For Life- Cohort study of the Genomes of Catalonia) (n=4988), a Mediterranean adult population-based cohort study from the south of Europe. We estimated SNP heritability contribution and single-trait GWAS for all traits from 15 million SNP variants. Then, we applied a multitrait-related approach to study genome-wide association to anthropometric measures in a two-stage meta-analysis with the UK Biobank cohort (n=336 107). Results Heritability estimates (eg, skin colour, alcohol consumption, smoking habit, body mass index, educational level or height) revealed an important contribution of SNP variants, ranging from 18% to 77%. Single-trait analysis identified 1785 SNPs with genome-wide significance threshold. From these, several previously reported single-trait hits were confirmed in our sample with LINC01432 (p=1.9×10−9) variants associated with male baldness, LDLR variants with hyperlipidaemia (ICD-9:272) (p=9.4×10−10) and variants in IRF4 (p=2.8×10−57), SLC45A2 (p=2.2×10−130), HERC2 (p=2.8×10−176), OCA2 (p=2.4×10−121) and MC1R (p=7.7×10−22) associated with hair, eye and skin colour, freckling, tanning capacity and sun burning sensitivity and the Fitzpatrick phototype score, all highly correlated cross-phenotypes. Multitrait meta-analysis of anthropometric variation validated 27 loci in a two-stage meta-analysis with a large British ancestry cohort, six of which are newly reported here (p value threshold <5×10−9) at ZRANB2-AS2, PIK3R1, EPHA7, MAD1L1, CACUL1 and MAP3K9. Conclusion Considering multiple-related genetic phenotypes improve associated genome signal detection. These results indicate the potential value of data-driven multivariate phenotyping for genetic studies in large population-based cohorts to contribute to knowledge of complex traits.This work was supported in part by the Spanish Ministerio de Economía y Competitividad (MINECO) project ADE 10/00026, by the Catalan Departament de Salut and by the Departament d’Empresa i Coneixement de la Generalitat de Catalunya, the Agència de Gestió d’Estudis Universitaris i de Recerca (AGA UR) (SGR 1269, SGR 1589 and SGR 647). RdC is the recipient of a Ramon y Cajal grant (RYC-2011-07822). The Project GCAT is coordinated by the Germans Trias i Pujol Research Institute (IGTP), in collaboration with the Catalan Institute of Oncology (ICO), and in partnership with the Blood and Tissue Bank of Catalonia (BST). IGTP is part of the CERCA Programme/Generalitat de Catalunya.Peer ReviewedPostprint (published version

Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

<p>Abstract</p> <p>Background</p> <p>Epidermal Growth Factor (EGF) is a key regulatory growth factor activating many processes relevant to normal development and disease, affecting cell proliferation and survival. Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.</p> <p>Results</p> <p>By applying a procedure for cross-platform data meta-analysis based on RankProd and GlobalAncova tests, we establish a well validated gene set with transcript levels altered after EGF treatment. We use this robust gene list to build higher order networks of gene interaction by interconnecting associated networks, supporting and extending the important role of the EGF signaling pathway in cancer. In addition, we find an entirely new set of genes previously unrelated to the currently accepted EGF associated cellular functions.</p> <p>Conclusions</p> <p>We propose that the use of global genomic cross-validation derived from high content technologies (microarrays or deep sequencing) can be used to generate more reliable datasets. This approach should help to improve the confidence of downstream <it>in silico </it>functional inference analyses based on high content data.</p

Multitrait genome association analysis identifies new susceptibility genes for human anthropometric variation in the GCAT cohort

BACKGROUND: Heritability estimates have revealed an important contribution of SNP variants for most common traits; however, SNP analysis by single-trait genome-wide association studies (GWAS) has failed to uncover their impact. In this study, we applied a multitrait GWAS approach to discover additional factor of the missing heritability of human anthropometric variation. METHODS: We analysed 205 traits, including diseases identified at baseline in the GCAT cohort (Genomes For Life- Cohort study of the Genomes of Catalonia) (n=4988), a Mediterranean adult population-based cohort study from the south of Europe. We estimated SNP heritability contribution and single-trait GWAS for all traits from 15 million SNP variants. Then, we applied a multitrait-related approach to study genome-wide association to anthropometric measures in a two-stage meta-analysis with the UK Biobank cohort (n=336 107). RESULTS: Heritability estimates (eg, skin colour, alcohol consumption, smoking habit, body mass index, educational level or height) revealed an important contribution of SNP variants, ranging from 18% to 77%. Single-trait analysis identified 1785 SNPs with genome-wide significance threshold. From these, several previously reported single-trait hits were confirmed in our sample with LINC01432 (p=1.9×10-9) variants associated with male baldness, LDLR variants with hyperlipidaemia (ICD-9:272) (p=9.4×10-10) and variants in IRF4 (p=2.8×10-57), SLC45A2 (p=2.2×10-130), HERC2 (p=2.8×10-176), OCA2 (p=2.4×10-121) and MC1R (p=7.7×10-22) associated with hair, eye and skin colour, freckling, tanning capacity and sun burning sensitivity and the Fitzpatrick phototype score, all highly correlated cross-phenotypes. Multitrait meta-analysis of anthropometric variation validated 27 loci in a two-stage meta-analysis with a large British ancestry cohort, six of which are newly reported here (p value threshold <5×10-9) at ZRANB2-AS2, PIK3R1, EPHA7, MAD1L1, CACUL1 and MAP3K9. CONCLUSION: Considering multiple-related genetic phenotypes improve associated genome signal detection. These results indicate the potential value of data-driven multivariate phenotyping for genetic studies in large population-based cohorts to contribute to knowledge of complex traits

Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor

BACKGROUND: Epidermal Growth Factor (EGF) plays an important function in the regulation of cell growth, proliferation, and differentiation by binding to its receptor (EGFR) and providing cancer cells with increased survival responsiveness. Signal transduction carried out by EGF has been extensively studied at both transcriptional and post-transcriptional levels. Little is known about the involvement of microRNAs (miRNAs) in the EGF signaling pathway. miRNAs have emerged as major players in the complex networks of gene regulation, and cancer miRNA expression studies have evidenced a direct involvement of miRNAs in cancer progression. RESULTS: In this study, we have used an integrative high content analysis approach to identify the specific miRNAs implicated in EGF signaling in HeLa cells as potential mediators of cancer mediated functions. We have used microarray and deep-sequencing technologies in order to obtain a global view of the EGF miRNA transcriptome with a robust experimental cross-validation. By applying a procedure based on Rankprod tests, we have delimited a solid set of EGF-regulated miRNAs. After validating regulated miRNAs by reverse transcription quantitative PCR, we have derived protein networks and biological functions from the predicted targets of the regulated miRNAs to gain insight into the potential role of miRNAs in EGF-treated cells. In addition, we have analyzed sequence heterogeneity due to editing relative to the reference sequence (isomiRs) among regulated miRNAs. CONCLUSIONS: We propose that the use of global genomic miRNA cross-validation derived from high throughput technologies can be used to generate more reliable datasets inferring more robust networks of co-regulated predicted miRNA target genes

A comprehensive custom panel design for routine hereditary cancer testing: preserving control, improving diagnostics and revealing a complex variation landscape

We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk