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Zeitschriftenartikel zum Thema "RNAseq analysis"
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PAI, TUN-WEN, BO-HAN SU, PEI-CHIH WU, MARGARET DAH-TSYR CHANG, HAO-TENG CHANG, TAN-CHI FAN und SHI-HWEI LIU. „UNIQUE PEPTIDE IDENTIFICATION OF RNaseA SUPERFAMILY SEQUENCES BASED ON REINFORCED MERGING ALGORITHMS“. Journal of Bioinformatics and Computational Biology 04, Nr. 01 (Februar 2006): 75–92. http://dx.doi.org/10.1142/s0219720006001710.
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Human ribonuclease A (RNaseA) superfamily consists of eight RNases with high similarity in which RNase2 and RNase3 share 76.7% identity. The evolutionary variation of RNases results in differential structures and functions of the enzymes. To distinguish the characteristics of each RNase, we developed reinforced merging algorithms (RMA) to rapidly identify the unique peptide motifs for each member of the highly conserved human RNaseA superfamily. Many motifs in RNase3 identified by RMA correlated well with the antigenic regions predicted by DNAStar. Two unique peptide motifs were experimentally confirmed to contain epitopes for monoclonal antibodies (mAbs) specifically against RNase3. Further analysis of homologous RNases in different species revealed that the unique peptide motifs were located at the correspondent positions, and one of these motifs indeed matched the epitope for a specific anti-bovine pancreatic RNaseA (bpRNaseA) antibody. Our method provides a useful tool for identification of unique peptide motifs for further experimental design. The RMA system is available and free for academic use at and .
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Colombo, Anthony R., Timothy J. Triche Jr und Giridharan Ramsingh. „Arkas: Rapid reproducible RNAseq analysis“. F1000Research 6 (27.04.2017): 586. http://dx.doi.org/10.12688/f1000research.11355.1.
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The recently introduced Kallisto pseudoaligner has radically simplified the quantification of transcripts in RNA-sequencing experiments. We offer cloud-scale RNAseq pipelines Arkas-Quantification, which deploys Kallisto for parallel cloud computations, and Arkas-Analysis, which annotates the Kallisto results by extracting structured information directly from source FASTA files with per-contig metadata and calculates the differential expression and gene-set enrichment analysis on both coding genes and transcripts. The biologically informative downstream gene-set analysis maintains special focus on Reactome annotations while supporting ENSEMBL transcriptomes. The Arkas cloud quantification pipeline includes support for custom user-uploaded FASTA files, selection for bias correction and pseudoBAM output. The option to retain pseudoBAM output for structural variant detection and annotation provides a middle ground between de novo transcriptome assembly and routine quantification, while consuming a fraction of the resources used by popular fusion detection pipelines. Illumina's BaseSpace cloud computing environment, where these two applications are hosted, offers a massively parallel distributive quantification step for users where investigators are better served by cloud-based computing platforms due to inherent efficiencies of scale.
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Colombo, Anthony R., Timothy J. Triche Jr und Giridharan Ramsingh. „Arkas: Rapid reproducible RNAseq analysis“. F1000Research 6 (21.06.2017): 586. http://dx.doi.org/10.12688/f1000research.11355.2.
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The recently introduced Kallisto pseudoaligner has radically simplified the quantification of transcripts in RNA-sequencing experiments. We offer cloud-scale RNAseq pipelines Arkas-Quantification, and Arkas-Analysis available within Illumina’s BaseSpace cloud application platform which expedites Kallisto preparatory routines, reliably calculates differential expression, and performs gene-set enrichment of REACTOME pathways. Due to inherit inefficiencies of scale, Illumina's BaseSpace computing platform offers a massively parallel distributive environment improving data management services and data importing. Arkas-Quantification deploys Kallisto for parallel cloud computations and is conveniently integrated downstream from the BaseSpace Sequence Read Archive (SRA) import/conversion application titled SRA Import. Arkas-Analysis annotates the Kallisto results by extracting structured information directly from source FASTA files with per-contig metadata, calculates the differential expression and gene-set enrichment analysis on both coding genes and transcripts. The Arkas cloud pipeline supports ENSEMBL transcriptomes and can be used downstream from the SRA Import facilitating raw sequencing importing, SRA FASTQ conversion, RNA quantification and analysis steps.
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Lamping, Mario, Damian Tobias Rieke, Frederick Klauschen, Korinna Jöhrens, Ioannis Anagnostopoulos, Dido Lenze, Inge Tinhofer et al. „Clinical impact of comprehensive versus targeted genomic analysis for precision oncology.“ Journal of Clinical Oncology 37, Nr. 15_suppl (20.05.2019): e13033-e13033. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13033.
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e13033 Background: Panel sequencing (PS) has become a standard-of-care in cancer diagnostics. More comprehensive analyses such as whole-exome (WES) or RNA sequencing (RNAseq) allow for the detection of rare and unknown genetic aberrations that are not covered by predefined assays. The clinical impact of targeted versus comprehensive genomic assays were analyzed in patients presented at the Charité Molecular Tumor Board (MTB). Methods: Patients (pts) with advanced and/or metastatic cancer for whom no standard therapy was available were discussed in the MTB to allocate diagnostic profiling and guide biomarker-based treatment (BBT). Pts had to be < 50 years of age or diagnosed with a rare tumor entity to undergo WES/RNAseq, performed on fresh tissue. If ineligible, standard PS was performed on archival tissue. BBT recommendations, ranked by pre-specified evidence levels, were made by the MTB and pts were followed up. Results: 228 patients (median age 49 years, 108 female and 120 male) were discussed in the MTB between January 2016 and February 2019. We assigned 73 and 155 pts to PS and WES/RNAseq and results were obtained for 78.1% (n = 57/73) and 54.8% (n = 85/155) pts, respectively. Sequencing failed for 11 (PS; 15.1%) and 62 (WES/RNAseq; 40%) pts, most commonly due to insufficient tissue (n = 29). Sequencing was ongoing in 5 (PS) and 8 (WES/RNAseq) pts at the time of analysis. A median of 2 BBTs were recommended for 75.4% (43/57) of PS (range r: 1-3) and 90.6% (77/85) of WES/RNAseq pts (r: 1-6) each. 22% (n = 17/77) of WES/RNAseq pts had ≥4 BBTs made by the MTB. Treatment was initiated in 30.2% (n = 13/43) of PS and 40.2% (n = 31/77) of WES/RNAseq pts. Clinical benefit rates (CBRs) were 23.1% (2 PR, 1 SD) for PS and 45.2% (2 CR, 3 PR, 9 SD) for WES/RNAseq pts. Overall survival data was immature at the time of analysis. Conclusions: Utilizing WES/RNAseq is a feasible approach to perform tumor profiling in a heterogeneous cohort. We here show a higher rate of pts receiving confident evidence-based treatment recommendations in the WES/RNAseq group and a higher rate of treatment initiation. The CBR nearly doubled in the WES/RNAseq cohort when compared to standard PS pts, thus emphasizing the need for larger comparative analyses to guide diagnostic decision-making.
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Guo, Yan, Shilin Zhao, Chung-I. Li, Quanhu Sheng und Yu Shyr. „RNAseqPS: A Web Tool for Estimating Sample Size and Power for RNAseq Experiment“. Cancer Informatics 13s6 (Januar 2014): CIN.S17688. http://dx.doi.org/10.4137/cin.s17688.
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Sample size and power determination is the first step in the experimental design of a successful study. Sample size and power calculation is required for applications for National Institutes of Health (NIH) funding. Sample size and power calculation is well established for traditional biological studies such as mouse model, genome wide association study (GWAS), and microarray studies. Recent developments in high-throughput sequencing technology have allowed RNAseq to replace microarray as the technology of choice for high-throughput gene expression profiling. However, the sample size and power analysis of RNAseq technology is an underdeveloped area. Here, we present RNAseqPS, an advanced online RNAseq power and sample size calculation tool based on the Poisson and negative binomial distributions. RNAseqPS was built using the Shiny package in R. It provides an interactive graphical user interface that allows the users to easily conduct sample size and power analysis for RNAseq experimental design. RNAseqPS can be accessed directly at http://cqs.mc.vanderbilt.edu/shiny/RNAseqPS/ .
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Guo, Yan, Shilin Zhao, Fei Ye, Quanhu Sheng und Yu Shyr. „MultiRankSeq: Multiperspective Approach for RNAseq Differential Expression Analysis and Quality Control“. BioMed Research International 2014 (2014): 1–8. http://dx.doi.org/10.1155/2014/248090.
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Background. After a decade of microarray technology dominating the field of high-throughput gene expression profiling, the introduction of RNAseq has revolutionized gene expression research. While RNAseq provides more abundant information than microarray, its analysis has proved considerably more complicated. To date, no consensus has been reached on the best approach for RNAseq-based differential expression analysis. Not surprisingly, different studies have drawn different conclusions as to the best approach to identify differentially expressed genes based upon their own criteria and scenarios considered. Furthermore, the lack of effective quality control may lead to misleading results interpretation and erroneous conclusions. To solve these aforementioned problems, we propose a simple yet safe and practical rank-sum approach for RNAseq-based differential gene expression analysis named MultiRankSeq. MultiRankSeq first performs quality control assessment. For data meeting the quality control criteria, MultiRankSeq compares the study groups using several of the most commonly applied analytical methods and combines their results to generate a new rank-sum interpretation. MultiRankSeq provides a unique analysis approach to RNAseq differential expression analysis. MultiRankSeq is written in R, and it is easily applicable. Detailed graphical and tabular analysis reports can be generated with a single command line.
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Mora-Márquez, Fernando, José Luis Vázquez-Poletti und Unai López de Heredia. „NGScloud2: optimized bioinformatic analysis using Amazon Web Services“. PeerJ 9 (16.04.2021): e11237. http://dx.doi.org/10.7717/peerj.11237.
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Background NGScloud was a bioinformatic system developed to perform de novo RNAseq analysis of non-model species by exploiting the cloud computing capabilities of Amazon Web Services. The rapid changes undergone in the way this cloud computing service operates, along with the continuous release of novel bioinformatic applications to analyze next generation sequencing data, have made the software obsolete. NGScloud2 is an enhanced and expanded version of NGScloud that permits the access to ad hoc cloud computing infrastructure, scaled according to the complexity of each experiment. Methods NGScloud2 presents major technical improvements, such as the possibility of running spot instances and the most updated AWS instances types, that can lead to significant cost savings. As compared to its initial implementation, this improved version updates and includes common applications for de novo RNAseq analysis, and incorporates tools to operate workflows of bioinformatic analysis of reference-based RNAseq, RADseq and functional annotation. NGScloud2 optimizes the access to Amazon’s large computing infrastructures to easily run popular bioinformatic software applications, otherwise inaccessible to non-specialized users lacking suitable hardware infrastructures. Results The correct performance of the pipelines for de novo RNAseq, reference-based RNAseq, RADseq and functional annotation was tested with real experimental data, providing workflow performance estimates and tips to make optimal use of NGScloud2. Further, we provide a qualitative comparison of NGScloud2 vs. the Galaxy framework. NGScloud2 code, instructions for software installation and use are available at https://github.com/GGFHF/NGScloud2. NGScloud2 includes a companion package, NGShelper that contains Python utilities to post-process the output of the pipelines for downstream analysis at https://github.com/GGFHF/NGShelper.
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Kalinina, Alena, und Diane Lagace. „Single-Cell and Single-Nucleus RNAseq Analysis of Adult Neurogenesis“. Cells 11, Nr. 10 (13.05.2022): 1633. http://dx.doi.org/10.3390/cells11101633.
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The complexity of adult neurogenesis is becoming increasingly apparent as we learn more about cellular heterogeneity and diversity of the neurogenic lineages and stem cell niches within the adult brain. This complexity has been unraveled in part due to single-cell and single-nucleus RNA sequencing (sc-RNAseq and sn-RNAseq) studies that have focused on adult neurogenesis. This review summarizes 33 published studies in the field of adult neurogenesis that have used sc- or sn-RNAseq methods to answer questions about the three main regions that host adult neural stem cells (NSCs): the subventricular zone (SVZ), the dentate gyrus (DG) of the hippocampus, and the hypothalamus. The review explores the similarities and differences in methodology between these studies and provides an overview of how these studies have advanced the field and expanded possibilities for the future.
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Guo, Yan, Chung-I. Li, Fei Ye und Yu Shyr. „Evaluation of read count based RNAseq analysis methods“. BMC Genomics 14, Suppl 8 (2013): S2. http://dx.doi.org/10.1186/1471-2164-14-s8-s2.
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Penaherrera, Daniel, Sheri Skerget, Austin Christofferson, Jessica Aldrich, Sara Nasser, Christophe Legendre, Martin Boateng et al. „Development and Validation of a High Risk Multiple Myeloma Gene Expression Index from RNA Sequencing: An Mmrf Commpass Analysis“. Blood 132, Supplement 1 (29.11.2018): 1895. http://dx.doi.org/10.1182/blood-2018-99-119610.
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Abstract Multiple Myeloma (MM) is a genetically heterogeneous disease of plasma cells that generally exhibits chromosomal abnormalities and distinct gene expression signatures. Previous studies have sought to identify gene expression indices using microarray technology to discern genes associated with survival outcomes to predict whether a newly diagnosed patient has an aggressive form of the disease. One such MM-specific index is the UAMS 70 gene index, which is composed of 51 over- and 19 under-expressed genes. This index was developed using Affymetrix U133Plus2.0 microarray data from 532 MM patients at diagnosis by computing log-rank test statistics on gene expression quartiles. Despite consistently achieving a high performance across a variety of MM datasets, issues arise when applying this index to RNAseq data. Here we address those issues, deriving an independent index based on the RNAseq data from the Multiple Myeloma Research Foundation (MMRF) CoMMpass Study (NCT01454297), and benchmark its performance to an implementation of the UAMS 70 gene index. UAMS index scores are computed by taking the difference between the average log2-scale expression of the 51 over- and 19 under-expressed genes. We applied this calculation to RNAseq data analyzed using Sailfish, Salmon v7.2, and HTseq counts collected from 41 Multiple Myeloma Genomics Initiative samples and compared the results to scores from matching GCRMA, MAS5, RMA, and PLIER16 Affymetrix U133Plus2.0 microarray data. Differences in the distribution of index values across data types led to nonconforming classification of high-risk individuals. Additionally, when applied to RNAseq data, several Affymetrix probesets did not uniquely match to gene annotations from Ensembl-v74. This reduced the number of genes upon which our UAMS score was calculated to 61 genes. Of the original 51 over-expressed probes, only 44 uniquely mapped genes remained after 7 multi-mapped probes are removed and similarly, out of the 19 under-expressed genes only 17 were uniquely mapped. Given the complication of probe-gene mismatch and inconsistencies identifying high-risk individuals when applied to RNAseq data, we developed an independent index using the baseline RNAseq data from the MMRF CoMMpass Study IA13 dataset. From a training set (n=375) of RNAseq data measuring 56430 genes, we performed univariate log-rank tests on expression quartiles associated with disease-related survival while controlling for an FDR of 2.5%, resulting in 23 under- and 332 over-expressed genes. Subsequent multivariate Cox regression analysis and backward stepwise selection culminated in the identification of the CoMMpass RNAseq index, which is based on the ratio of mean expression values of 87 genes (19 under- and 68 over-expressed) predictive of high risk (hazard ratio [HR] = 8.7341, 95% CI = 5.615-13.58, p < 0.001). Validation on the test set (n=251) yielded a HR of 5.612 (95% CI = 3.066-10.27, p < 0.001) as compared to a HR of 4.753 (95% CI = 2.688-8.403, p < 0.001) achieved with the adapted UAMS index. Adjusting for a patient's International Staging System (ISS) stage revises these hazard ratios to 6.236 (95% CI = 3.345-11.627, p < 0.001) and 3.6420 (95% CI = 1.9726-6.724, p < 0.001) for the CoMMpass RNAseq and the adapted UAMS indices, respectively. Furthermore, the distribution of CoMMpass RNAseq index values across the training and test set show no observable bias with respect to three main therapy arms, suggesting it is predictive of high risk independent of treatment. Our newly derived CoMMpass RNAseq index shares one gene in common with the UAMS 61 gene index (CENPW) and recovers two over-expressed genes (FABP5, TAGLN2), which were removed from the UAMS 70 gene index due to probe multimapping. When the recovered genes are added back to the UAMS index, the unadjusted and adjusted hazard ratios measured for the test set are 5.173 (CI = 2.926-9.146, p < 0.001) and 4.022 (CI = 2.1840-7.408, p < 0.001), respectively. Of the original 70 genes in the UAMS index, 21 (30%) map to chromosome 1, which frequently exhibits copy number gains in MM. Only 11 of the 87 (13%) genes in our proposed index map to chr1, which indicates that, given its performance, the newly derived list of genes may represent a more diverse index to predict, and provide novel insights into, high risk MM. Altogether, the CoMMpass RNAseq index identifies a high risk signature in 13% of MM patients and outperforms the UAMS index. Disclosures Lonial: Amgen: Research Funding.
Dissertationen zum Thema "RNAseq analysis"
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Liu, Oscar H. „RNAseq Analysis of Gastric Bacteria in Helicobacter pylori-Associated Carcinogenesis“. Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-9937.
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Helicobacter pylori infects more than half of the world's population, and is known to be involved in several diseases including gastric cancer. Its close interactions with the stomach and host immune system serves as a good model to study the co-adaptation and co-evolution of the organisms in the stomach micro-environment. In this project, we utilized RNA-seq and data analysis tools to investigate differentially expressed genes by H. pylori in patients at different stages of early gastric cancer development. We also investigated the abundance and diversity of bacterial genera other than H. pylori, and looked for correlations with H. pylori presence and number. For differential gene expression of H. pylori, one gene was differentially expressed between samples of corpus atrophy without metaplasia vs. samples of antrum gastritis, and eight genes were found to be differentially expressed between samples of corpus atrophy with metaplasia vs. samples with pan-gastritis. When samples were clustered into different groups based on the expression data, 52 genes (shared or unique to the specific comparison groups) were found to be differentially expressed, but no apparent patterns were observed that could be explained by medical or sample collection data. For bacterial diversity and abundances, we found several genera colonizing the stomach, of which some have been previously identified. While most of these bacteria colonize regardless of the presence of H. pylori, the abundance of three genera, Wolinella, Campylobacter, and Veillonella, seem to be correlated with the presence of H. pylori.
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Simon, Svenja [Verfasser]. „Visual Analysis of RNAseq Data : Discovering Genes in Bacteria / Svenja Simon“. Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1114886580/34.
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Aksamit, Matthew Stephen. „Bioinformatic analysis of pea aphid salivary gland transcripts“. Thesis, Kansas State University, 2014. http://hdl.handle.net/2097/32836.
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Master of ScienceBiochemistry and Molecular Biophysics Interdepartmental Program
Gerald Reeck
Pea aphids (Acyrthosiphon pisum) are sap-sucking insects that feed on the phloem sap of some plants of the family Fabaceae (legumes). Aphids feed on host plants by inserting their stylets between plant cells to feed from phloem sap in sieve elements. Their feeding is of major agronomical importance, as aphids cause hundreds of millions of dollars in crop damage worldwide, annually. Salivary gland transcripts from plant-fed and diet-fed pea aphids were studied by RNASeq to analyze their expression. Most transcripts had higher expression in plant-fed pea aphids, likely due to the need for saliva protein in the aphid/plant interaction. Numerous salivary gland transcripts and saliva proteins have been identified in aphids, including a glutathione peroxidase. Glutathione peroxidases are a group of enzymes with the purpose of protecting organisms from oxidative damage. Here, I present a bioinformatic analysis of pea aphid expressed sequence tag libraries that identified four unique glutathione peroxidases in pea aphids. One glutathione peroxidase, ApGPx1 has high expression in the pea aphid salivary gland. Two glutathione peroxidase genes are present in the current annotation of the pea aphid genome. My work indicates that the two genes need to be revised.
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Ahmed, Firdous. „Identification of potential biomarkers in lung cancer as possible diagnostic agents using bioinformatics and molecular approaches“. University of the Western Cape, 2015. http://hdl.handle.net/11394/4862.
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>Magister Scientiae - MScLung cancer remains the leading cause of cancer deaths worldwide, with the majority of cases attributed to non-small cell lung carcinomas. At the time of diagnosis, a large percentage of patients present with advanced stage of disease, ultimately resulting in a poor prognosis. The identification circulatory markers, overexpressed by the tumour tissue, could facilitate the discovery of an early, specific, non-invasive diagnostic tool as well as improving prognosis and treatment protocols. The aim was to analyse gene expression data from both microarray and RNA sequencing platforms, using bioinformatics and statistical analysis tools. Enrichment analysis sought to identify genes, which were differentially expressed (p < 0.05, FC > 2) and had the potential to be secreted into the extracellular circulation, by using Gene Ontology terms of the Cellular Component. Results identified 1 657 statically significant genes between normal and early lung cancer tissue, with only 1 gene differentially expressed (DE) between the early and late stage disease. Following statistical analysis, 171 DE genes selected as potential early stage biomarkers. The overall sensitivity of RNAseq, in comparison to arrays enabled the identification of 57 potential serum markers. These genes of interest were all downregulated in the tumour tissue, and while they did not facilitate the discovery of an ideal diagnostic marker based on the set criteria in this study, their roles in disease initiation and progression require further analysis.
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Sadacca, Benjamin. „Pharmacogenomic and High-Throughput Data Analysis to Overcome Triple Negative Breast Cancers Drug Resistance“. Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS538/document.
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Devant le grand nombre de tumeurs du sein triple négatif résistant aux traitements, il est essentiel de comprendre les mécanismes de résistance et de trouver de nouvelles molécules efficaces. En premier lieu, nous analysons deux ensembles de données pharmacogénomiques à grande échelle. Nous proposons une nouvelle classification basée sur des profils transcriptomiques de lignées cellulaires, selon un processus de sélection de gènes basé sur des réseaux biologiques. Notre classification moléculaire montre une plus grande homogénéité dans la réponse aux médicaments que lorsque l’on regroupe les lignées cellulaires en fonction de leur tissu d'origine. Elle permet également d’identifier des profils similaires de réponse aux traitements. Dans un second travail, nous étudions une cohorte de patients atteints d’un cancer du sein triple négatif ayant résisté à la chimiothérapie néoadjuvante. Nous effectuons des analyses moléculaires complètes basées sur du RNAseq et WES. Nous constatons une forte hétérogénéité moléculaire des tumeurs avant et après traitement. Bien que nous observons une évolution clonale sous traitement, aucun mécanisme récurrent de résistance n’a pu être identifié. Nos résultats suggèrent fortement que chaque tumeur a un profil moléculaire unique et qu'il est important d'étudier de grandes séries de tumeurs. Enfin, nous améliorons une méthode pour tester la surreprésentation de motifs connus de protéines de liaison à l'ARN, dans un ensemble donné de séquences régulées. Cet outil utilise une approche innovante pour contrôler la proportion de faux positifs qui n'est pas réalisé par l'algorithme existant. Nous montrons l'efficacité de notre approche en utilisant deux séries de données différentesGiven the large number of treatment-resistant triple-negative breast cancers, it is essential to understand the mechanisms of resistance and to find new effective molecules. First, we analyze two large-scale pharmacogenomic datasets. We propose a novel classification based on transcriptomic profiles of cell lines, according to a biological network-driven gene selection process. Our molecular classification shows greater homogeneity in drug response than when cell lines are grouped according to their original tissue. It also helps identify similar patterns of treatment response. In a second analysis, we study a cohort of patients with triple-negative breast cancer who have resisted to neoadjuvant chemotherapy. We perform complete molecular analyzes based on RNAseq and WES. We observe a high molecular heterogeneity of tumors before and after treatment. Although we highlighted clonal evolution under treatment, no recurrent mechanism of resistance could be identified Our results strongly suggest that each tumor has a unique molecular profile and that that it is increasingly important to have large series of tumors. Finally, we are improving a method for testing the overrepresentation of known RNA binding protein motifs in a given set of regulated sequences. This tool uses an innovative approach to control the proportion of false positives that is not realized by the existing algorithm. We show the effectiveness of our approach using two different datasets
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Grosse-Holz, Friederike. „Proteases and inhibitors in the interaction between Nicotiana benthamiana and Agrobacterium tumefaciens : systematic analysis and emerging solutions for molecular farming“. Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:6146918c-3749-4604-88fa-01d426e4a817.
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Nicotiana benthamiana is now an established platform for molecular farming, the production of biopharmaceuticals in plants. Infiltration with Agrobacterium tumefaciens (agroinfiltration) is commonly used to transiently express one or multiple transgenes in N. benthamiana leaves. Agroinfiltrated N. benthamiana is a flexible and scalable recombinant protein (RP) production platform, but is impeded by low RP yields. Plant proteases can degrade RPs and thus limit RP accumulation. To inform, design and implement strategies for enhancing RP accumulation, I present four papers about proteases and protease inhibitors in agroinfiltrated N. benthamiana. First, I investigated the transcriptome, extracellular proteome and active secretome to understand the plant response to agroinfiltration and investigate the expressed proteases. I show that an extracellular immune response is mounted at the expense of photosynthesis. Comprehensive annotation and monitoring uncover a large, diverse repertoire of proteases in agroinfiltrated leaves, indicating that broad-range depletion of protease activity may be required to enhance RP accumulation. Second, I reviewed the literature on multifunctional plant protease inhibitors (PIs) and grouped them into three types of multifunctional PIs that evolved independently. Third, I screened candidate PIs and discovered that three new, unrelated PIs enhance RP accumulation. I present universal elements of the RP degradation machinery, uncovering new questions on our understanding of the protease network that degrades RPs. Fourth, I identified targets of SlCYS8, a PI that enhances RP accumulation. The target proteases of SlCYS8 are implicated in RP degradation and the high specificity of SlCYS8 can be used to study their role in other processes. By elucidating the immune response to agroinfiltration, by uncovering the N. benthamiana protease repertoire and by providing new tools to deplete the activity of specific proteases, this thesis makes a relevant contribution to both basic plant research and molecular farming.
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DAS, VIVEK. „LEVERAGING TRANSCRIPTOMIC ANALYSIS TO IDENTIFY TRANSCRIPTION FACTORS ORCHESTRATING CANCER PROGRESSION“. Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/559711.
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Next generation sequencing (NGS) technology is currently employed to explore the molecular profiles associated to different biological contexts. The application of this technology provides at same time a high-resolution and global view of the genome and epigenome phenomena, enabling us to study the molecular events underlying many human diseases, including cancer. Our lab tries to exploit the utility of high throughput sequencing technologies generating genomic, transcriptomic and epigenomic data from patient's cohort to study the underlying molecular mechanisms that characterize the specific diseases and map the key regulators that can be critical targets for relevant therapeutic measures. I take the advantage of this technology to mainly understand two aggressive cancers: Ovarian Cancer (OC) and Glioblastoma multiforme (GBM).
OC is a leading cause of cancer-related death for which no significant therapeutic progress has been made in the last decades. Also, in this case, despite multimodal treatment its prognosis remains extremely poor. This is due to the fact that the molecular mechanisms underlying OC tumorigenesis and progression are still poorly understood (Vaughan et al., 2011). GBM is the most common and aggressive primary brain malignancy with very poor prognosis (Frattini et al., 2013). The median survival rate is of 12-15 months (Singh et al., 2012) with 5-year survival that is less than 5% despite the multimodal treatment which include surgery, radiotherapy and chemotherapy. To this end, I will be integrating various genomic and transcriptomic analysis to define the key regulatory actors that characterize the disease progression paving. This integrated analysis has been devised in form of a computational workflow that gives way for a discovery pipeline for physiopathologically meaningful epigenetic targets that can lead to therapies.
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Couto, Joana Manuel Gonçalves Teixeira. „Transcriptomic analysis of Anopheles Stephensi salivary glands during the infection with Plasmodium Berghei“. Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/14639.
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Mestrado em Biologia Molecular e CelularMalaria remains the leading cause of morbidity and mortality in tropical and subtropical regions, contributing to the emergence of 198 million clinical cases in 2013. The mosquito Anopheles stephensi is one of the most prevalent malaria vectors in the Asian region having recently been implicated in malaria resurgence in Djibouti. Using techniques as RNA sequencing, differentially expressed genes in the salivary glands of the mosquito in response to infection by Plasmodium berghei were identified. Some of these genes can be selected to evaluate their potential as targets for malaria transmission blocking. Among the genes with differentially expression resulting from the analysis of RNA-seq results and confirmation by qPCR, a gene related transport of Cl- and HCO3 2-, prestin, was upregulated after infection with P. berghei. This gene plays a crucial role in parasite invasion in the midgut and the optimization of the environment in which the parasite develops. For this reason, the silencing of this transcript was made to evaluate the function of prestin in salivary glands. The gene silencing, using RNA interference technique, allow inferring about the role or function of prestin gene in a particular metabolic or physiological process. After prestin gene silencing, the number of viable mosquitoes had a significant decrease in comparison with the control (β2M). There was also a significant decrease in the number of mosquitoes before injection and at the last day after injection. The number of sporozoites were not generally affected by silencing of prestin when compared with the control. To clarify other results obtained during the study, as the influence of the silencing of prestin in the survival of mosquitoes and the presence and number of sporozoites in the salivary glands, will be essential to perform qPCR to determine differential expression of this gene after silencing. Furthermore, it is also important to examine differential expression of off-target (ASTE006714) after silencing prestin, since the sequence of this gene have a high percentage of identity with prestin.
A malária continua a ser a principal causa de morbilidade e mortalidade nas regiões tropicais e subtropicais, contribuindo para o surgimento de 198 milhões de casos clínicos no ano de 2013. O mosquito Anopheles stephensi é um dos vectores de malária mais prevalentes na região asiática, tendo sido recentemente implicado no ressurgimento de malária em Djibouti. Através de técnicas como sequenciação de RNA, genes diferenciadamente expressos nas glândulas salivares deste mosquito em resposta à infecção por Plasmodium berghei foram identificados. Alguns destes genes podem ser selecionados para avaliar a sua potencialidade como alvos para bloqueio da transmissão da malária. Entre os genes com expressão diferencial resultante da análise dos resultados de RNA-seq e confirmação por qPCR, um gene relacionado com o transporte de Cl- e HCO3 2-, prestin, estava sobrexpresso após infeção com P. berghei. Este gene tem um papel crucial na invasão do parasita no intestino médio e na optimização do meio em que o parasita se desenvolve. Por esse motivo, o silenciamento deste transcrito foi efectuado para averiguar o papel funcional nas glândulas salivares. O silenciamento de genes utilizando a técnica de RNA de interferência permite inferir sobre o seu papel ou função num dado processo metabólico ou fisiológico. Após o silenciamento do gene prestin, o número de mosquitos viáveis apresentou um decréscimo significativo em comparação com o controlo (β2M). Também houve uma queda significativa entre o número de mosquitos antes da injeção e no último dia após injecção. O número de esporozoítos em geral não foi afectado pelo silenciamento da prestin quando comparado com o controlo. Para esclarecer resultados obtidos durante o estudo, tais como a influência do silenciamento da prestin na sobrevivência dos mosquitos e a presença e número de esporozoítos na glândulas salivares, seria fundamental realizar ensaios de qPCR para determinar a expressão diferencial deste gene após silenciamento. Além disso, será também importante analisar a expressão diferencial do off-target (ASTE006714) após silenciamento da prestin, uma vez que a semelhança da sequência entre este e a prestin é elevada.
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Ahmed, Fathima Zuba. „Unravelling genes responsible for successful anthocyanin production in Nicotiana benthamiana“. Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/230763/1/Fathima%20Zuba_Ahmed_Thesis.pdf.
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This thesis utilised two approaches, forward genetics and comparative transcriptomic analysis, to investigate the contrasting response to anthocyanin production observed in two distinct Nicotiana benthamiana ecotypes, LAB and QLD. The thesis is a step forward in utilising N. benthamiana as a candidate in forward genetics, currently limited due to its large complex genome and polyploid nature. The study utilised a cross-population between LAB and QLD to investigate the nature of inheritance of the contrasting parental phenotypes in its progeny. Additionally, expression profiles of anthocyanin biosynthesis genes were analysed via differential expression analysis and a novel method of mathematical manifold analysis
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Meunier, Léa. „Analyse de signatures transcriptomiques et épigénétiques des carcinomes hépatocellulaires“. Thesis, Université de Paris (2019-....), 2020. http://www.theses.fr/2020UNIP7082.
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Élucider les processus transcriptionnels et épigénétiques dérégulés dans les cancers est fondamental pour mieux comprendre les voies biologiques impliquées et proposer une thérapie adaptée au phénotype moléculaire de chaque tumeur. Les approches classiques de classification non supervisée définissent des groupes moléculaires principaux pour chaque type tumoral. Cependant, ces méthodes, appliquées à des tumeurs complexes comme le carcinome hépatocellulaire (CHC), le 3ème cancer le plus mortel au monde, définissent des groupes qui restent relativement hétérogènes et ne reflètent qu’imparfaitement la diversité des mécanismes biologiques à l’œuvre dans ces tumeurs. Au cours de ma thèse, j’ai développé une stratégie d’analyses innovante, basée sur l’analyse en composantes indépendantes (ACI), pour extraire des signatures de processus biologiques précis à partir de grands jeux de données transcriptomiques et épigénétiques. Grace à cette nouvelle approche, j’ai identifié des groupes de gènes co-régulés, associés à des phénotypes ou altérations moléculaires précises. De même, l’analyse en composantes indépendantes du méthylome de 738 CHC m’a permis d’isoler 13 signatures épigénétiques stables, préférentiellement actives dans certaines tumeurs et certains sites CpG. Ces signatures incluent des signatures de méthylation précédemment associées au vieillissement et au cancer, mais aussi de nouvelles signatures d'hyper- et d'hypométhylation liées à des événements « drivers » et sous-groupes moléculaires spécifiques. Ces résultats nous éclairent sur la diversité des mécanismes moléculaires impliqués dans la carcinogenèse hépatique. Les outils d’analyse biostatistique innovants que j’ai développés ont été incorporés dans un package R librement utilisable par la communauté scientifiqueElucidating deregulated transcriptional and epigenetic processes in cancers is fundamental to better understand the biological pathways involved and to propose a therapy adapted to the molecular phenotype of each tumor. Classical unsupervised classification approaches define, for each tumor type, the main molecular groups. However, these methods, applied to complex tumors such as hepatocellular carcinoma (HCC), the 3rd cause of cancer-associated mortality worldwide, define groups that remain relatively heterogeneous and only imperfectly reflect the diversity of biological mechanisms at work in these tumors. During my PhD, I developed a, innovative strategy involving independent component analysis (ICA) to extract signatures of precise biological processes in large transcriptomic and epigenetic tumor data sets. This new approach allowed me to identify groups of co-regulated genes associated with specific phenotypes or molecular alterations. Similarly, independent component analysis of the methylomes of 738 HCC revealed 13 stable epigenetic signatures preferentially active in specific tumors and CpG sites. These signatures include signatures previously associated with ageing and cancer, but also new hyper- and hypomethylation signatures related to specific driver events and molecular subgroups. The work presented in this thesis sheds light on the diversity of molecular processes remodeling liver cancer transcriptomes and methylomes, improve the understanding of the molecular mechanisms involved in hepatic carcinogenesis and provides a statistical framework to unravel the signatures of these processes
Bücher zum Thema "RNAseq analysis"
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1925-, Cherayil J. D., Hrsg. Transfer RNAs and other soluble RNAs. Boca Raton: CRC Press, 1990.
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Cao, Haiming, Hrsg. Functional Analysis of Long Non-Coding RNAs. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1158-6.
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Rederstorff, Mathieu. Small non-coding RNAs: Methods and protocols. New York: Humana Press, 2015.
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R Bioinformatics Cookbook: Use R and Bioconductor to Perform RNAseq, Genomics, Data Visualization, and Bioinformatic Analysis. Packt Publishing, Limited, 2019.
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Nellen, Wolfgang, und Christian Hammann. Small RNAs : : Analysis and Regulatory Functions. Springer London, Limited, 2007.
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Cao, Haiming. Functional Analysis of Long Non-Coding RNAs: Methods and Protocols. Springer, 2020.
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Cao, Haiming. Functional Analysis of Long Non-Coding RNAs: Methods and Protocols. Springer, 2021.
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(Editor), Wolfgang Nellen, und Christian Hammann (Editor), Hrsg. Small RNAs:: Analysis and Regulatory Functions (Nucleic Acids and Molecular Biology). Springer, 2007.
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(Editor), Wolfgang Nellen, und Christian Hammann (Editor), Hrsg. Small RNAs:: Analysis and Regulatory Functions (Nucleic Acids and Molecular Biology). Springer, 2005.
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Rederstorff, Mathieu. Small Non-Coding RNAs: Methods and Protocols. Springer, 2022.
Den vollen Inhalt der Quelle findenBuchteile zum Thema "RNAseq analysis"
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Cagnin, Stefano, Enrico Alessio, Raphael Severino Bonadio und Gabriele Sales. „Single-Cell RNAseq Analysis of lncRNAs“. In Long Non-Coding RNAs in Cancer, 71–90. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1581-2_5.
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Sharma, Preeti, B. Sharan Sharma und Ramtej J. Verma. „A Guide to RNAseq Data Analysis Using Bioinformatics Approaches“. In Advances in Bioinformatics, 243–60. Singapore: Springer Singapore, 2021. http://dx.doi.org/10.1007/978-981-33-6191-1_12.
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Majerczyk, Charlotte D. „Global Expression Analysis of Quorum Sensing-Controlled Genes by RNAseq“. In Methods in Molecular Biology, 177–92. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7309-5_14.
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Croce, Olivier, und Eric Röttinger. „Creating a User-Friendly and Open-Access Gene Expression Database for Comparing Embryonic Development and Regeneration in Nematostella vectensis“. In Methods in Molecular Biology, 649–62. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2172-1_35.
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AbstractThe sea anemone Nematostella vectensis has emerged as a powerful research model to understand at the gene regulatory network level, to what extend regeneration recapitulates embryonic development. Such comparison involves massive transcriptomic analysis, a routine approach for identifying differential gene expression. Here we present a workflow to build a user-friendly, mineable, and open-access database providing access to the scientific community to various RNAseq datasets.
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Grassmann, Felix. „Conduct and Quality Control of Differential Gene Expression Analysis Using High-Throughput Transcriptome Sequencing (RNASeq)“. In Methods in Molecular Biology, 29–43. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-8669-9_2.
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Kopajtich, Robert, Johannes A. Mayr und Holger Prokisch. „Analysis of Mitochondrial RNA-Processing Defects in Patient-Derived Tissues by qRT-PCR and RNAseq“. In Methods in Molecular Biology, 379–90. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6824-4_23.
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Ocaña, Kary, Lucas Cruz, Micaella Coelho, Rafael Terra, Marcelo Galheigo, Andre Carneiro, Diego Carvalho et al. „ParslRNA-Seq: An Efficient and Scalable RNAseq Analysis Workflow for Studies of Differentiated Gene Expression“. In Communications in Computer and Information Science, 174–89. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-23821-5_13.
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Ender, Anna, Peter F. Stadler, Mario Mörl und Sven Findeiß. „RNA Design Principles for Riboswitches that Regulate RNase P-Mediated tRNA Processing“. In Riboregulator Design and Analysis, 179–202. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2421-0_11.
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Ender, Anna, Peter F. Stadler, Mario Mörl und Sven Findeiß. „RNA Design Principles for Riboswitches that Regulate RNase P-Mediated tRNA Processing“. In Riboregulator Design and Analysis, 179–202. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2421-0_11.
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Hallier, Marc, Svetlana Chabelskaya und Brice Felden. „Experimental Analyses of RNA-Based Regulations in Bacteria“. In Regulatory RNAs, 341–70. Berlin, Heidelberg: Springer Berlin Heidelberg, 2011. http://dx.doi.org/10.1007/978-3-662-45801-3_14.
Der volle Inhalt der QuelleKonferenzberichte zum Thema "RNAseq analysis"
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Oba-Shinjo, Sueli M., Lais C. Cardoso, Roseli da Silva, Antonio M. Lerario, Miyuki Uno und Suely S. K. Marie. „Abstract 66: CD99 functional analysis in glioblastoma by RNAseq“. In Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA. American Association for Cancer Research, 2015. http://dx.doi.org/10.1158/1538-7445.am2015-66.
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Legeai, Fabrice, Susete Alves-Carvalho, Kévin Gazengel, Anthony Bretaudeau, Stéphanie Robin und Stéphanie Daval. „AskoR, A R Package for Easy RNASeq Data Analysis“. In The 1st International Electronic Conference on Entomology. Basel, Switzerland: MDPI, 2021. http://dx.doi.org/10.3390/iece-10646.
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Bhuvaneshwar, Krithika, Coleman I. Smith, Alexander H. Kroemer, Aiwu Ruth He und Yuriy Gusev. „Abstract 548: RNAseq analysis of infiltrating immune cells in liver cancer“. In Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC. American Association for Cancer Research, 2017. http://dx.doi.org/10.1158/1538-7445.am2017-548.
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Ryan, Michael C., und John N. Weinstein. „Abstract 1796: Analysis of TCGA RNASeq data using SpliceSeq provides a survey of alternative splicing in cancer.“ In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-1796.
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Toccacieli, Ali, und Manuela Petti. „Identification of Cancer Biomarkers for Multi-class Diagnostics through Network Analysis of RNAseq Data of Tumor-Educated Platelets“. In 2022 IEEE International Conference on Bioinformatics and Biomedicine (BIBM). IEEE, 2022. http://dx.doi.org/10.1109/bibm55620.2022.9995086.
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Mavrommatis, Konstantinos, Lauren Intagliata, Garth McGrath, Daniel Civello und Maureen Cronin. „Abstract 3626: Establishing a robust NGS laboratory workflow and analysis pipeline for FFPE specimen RNAseq to support biopharmaceutical translational research“. In Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-3626.
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Wu, Xue, Yue Zhao, Xiaoyu Xie, Xiaoling Xuei, Yunlong Liu, Lijun Cheng und Lang Li. „Abstract 549: Genome-wide CRISPR-Cas9 screen and RNAseq analysis identify new candidate synthetic lethality partners to PARP inhibitor in triple-negative breast cancer“. In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-549.
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Pineda, R. H., N. Mitash, K. Kohler, M. C. Melo Narvaez, J. Sembrat, M. Fangping, M. Lehmann, N. Kaminski und M. Koenigshoff. „Differential RNAseq Analysis of an Ex-Vivo Human Fibrotic Tissue Slice Model Reveals Dysregulated Genes of Cellular Senescence and YAP/TAZ Signaling in Fibrosis“. In American Thoracic Society 2022 International Conference, May 13-18, 2022 - San Francisco, CA. American Thoracic Society, 2022. http://dx.doi.org/10.1164/ajrccm-conference.2022.205.1_meetingabstracts.a5229.
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Song, Hye-Won, Gretchen Y. Lam, Margaret Nakamoto, Punya Narayan, Ian Taylor und Stefanie Mortimer. „Abstract 1345: Advances in single cell whole transcriptome analysis: Single nucleus RNAseq and simultaneous protein and mRNA profiling using the BD RhapsodyTMSingle-Cell Analysis system and BD®AbSeq“. In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-1345.
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Zhang, Lan, Juan Zhang und Qian Shi. „Abstract A6: RNAseq and immune profiling analysis of syngeneic mouse models treated with immune checkpoint inhibitors enable biomarker discovery and model selection for cancer immunotherapy“. In Abstracts: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; November 5-9, 2015; Boston, MA. American Association for Cancer Research, 2015. http://dx.doi.org/10.1158/1535-7163.targ-15-a6.
Der volle Inhalt der QuelleBerichte der Organisationen zum Thema "RNAseq analysis"
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Lers, Amnon, E. Lomaniec, S. Burd, A. Khalchitski, L. Canetti und Pamela J. Green. Analysis of Senescence Inducible Ribonuclease in Tomato: Gene Regulation and Function. United States Department of Agriculture, Februar 2000. http://dx.doi.org/10.32747/2000.7570563.bard.
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Natural leaf senescence has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. Senescence is regulated by differential gene expression yet, functional characterization of the genes specifically induced and study of their expression control, is still in its infancy. Study of senescence-specific genes is required to allow identification of regulatory elements participating in senescence-induced expression and thus provide insights into the genetic regulation of senescence. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as RNases and proteases. This study was aimed a analysis of senescence-inducible RNases in tomato with the following objectives: Isolation of senescence-inducible RNase cDNA clones; Expression analyses of RNase genes during senescence; Identification of sequences required for senescence-induced gene expression; Functional analyses of senescence-inducible RNases. We narrowed our aims somewhat to focus on the first three objectives because the budget we were awarded was reduced from that requested. We have expanded our research for identification senescence-related RNase/nuclease activities as we thought it will direct us to new RNase/nuclease genes. We have also carried out research in Arabidopsis and parsley, which enabled us to draw mire general conclusions. We completed the first and second objectives and have made considerable progress on the remaining two. We have defined growth conditions suitable for this research and defined the physiological and biochemical parameters characteristic to the advance of leaf senescence. In tomato and arabidopsis we have focused on natural leaf senescence. Parsley was used mainly for study of postharvest senescence in detached leaves. We have identified a 41-kD a tomato nuclease, LeNUCI, specifically induced during senescence which can degrade both RNA and DNA. This activity could be induced by ethylene in young leaves and was subjected to detailed analysis, which enabled its classification as Nuclease I enzyme. LeNUCI may be involved in nucleic acid metabolism during tomato leaf senescence. In parsley senescing leaves we identified 2 main senescence-related nuclease activities of 41 and 39-kDa. These activities were induced in both naturally or artificially senescing leaves, could degrade both DNA and RNA and were very similar in their characteristics to the LeNUCI. Two senescence-induced RNase cDNAs were cloned from tomato. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both were demonstrated before to be induced following phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. LX gene expression was much more senescence specific and ethylene could activate it in detached young leaves. LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may be a defense-related protein. Transgenic plants were generated for altering LX gene expression. No major visible alterations in the phenotype were observed so far. Detailed analysis of senescence in these plants is performed currently. The LX promoter was cloned and its analysis is performed currently for identification of senescence-specific regulatory elements. In Arabidopsis we have identified and characterized a senescence-associated nuclease 1 gene, BFN1, which is highly expressed during leaf and stem senescence. BFN1, is the first example of a senescence- associated gene encoding a nuclease I enzyme as well as the first nuclease I cloned and characterized from Arabidopsis. Our progress should provide excellent tools for the continued analysis of regulation and function of senescence-inducible ribonucleases and nucleases in plants. The cloned genes can be used in reverse genetic approaches, already initiated, which can yield a more direct evidence for the function of these enzymes. Another contribution of this research will be in respect to the molecular mechanism, which controls senescence. We had already initiated in this project and will continue to identify and characterize regulatory elements involved in senescence-specific expression of the genes isolated in this work.
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Schuster, Gadi, und David Stern. Integrated Studies of Chloroplast Ribonucleases. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7697125.bard.
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Gene regulation at the RNA level encompasses multiple mechanisms in prokaryotes and eukaryotes, including splicing, editing, endo- and exonucleolytic cleavage, and various phenomena related to small or interfering RNAs. Ribonucleases are key players in nearly all of these post-transcriptional mechanisms, as the catalytic agents. This proposal continued BARD-funded research into ribonuclease activities in the chloroplast, where RNase mutation or deficiency can cause metabolic defects and is often associated with plant chlorosis, embryo or seedling lethality, and/or failure to tolerate nutrient stress. The first objective of this proposal was to examined a series of point mutations in the PNPase enzyme of Arabidopsis both in vivo and in vitro. This goal is related to structure-function analysis of an enzyme whose importance in many cellular processes in prokaryotes and eukaryotes has only begun to be uncovered. PNPase substrates are mostly generated by endonucleolytic cleavages for which the catalytic enzymes remain poorly described. The second objective of the proposal was to examine two candidate enzymes, RNase E and RNase J. RNase E is well-described in bacteria but its function in plants was still unknown. We hypothesized it catalyzes endonucleolytic cleavages in both RNA maturation and decay. RNase J was recently discovered in bacteria but like RNase E, its function in plants had yet to be explored. The results of this work are described in the scientific manuscripts attached to this report. We have completed the first objective of characterizing in detail TILLING mutants of PNPase Arabidopsis plants and in parallel introducing the same amino acids changes in the protein and characterize the properties of the modified proteins in vitro. This study defined the roles for both RNase PH core domains in polyadenylation, RNA 3’-end maturation and intron degradation. The results are described in the collaborative scientific manuscript (Germain et al 2011). The second part of the project aimed at the characterization of the two endoribonucleases, RNase E and RNase J, also in this case, in vivo and in vitro. Our results described the limited role of RNase E as compared to the pronounced one of RNase J in the elimination of antisense transcripts in the chloroplast (Schein et al 2008; Sharwood et al 2011). In addition, we characterized polyadenylation in the chloroplast of the green alga Chlamydomonas reinhardtii, and in Arabidopsis (Zimmer et al 2009). Our long term collaboration enabling in vivo and in vitro analysis, capturing the expertise of the two collaborating laboratories, has resulted in a biologically significant correlation of biochemical and in planta results for conserved and indispensable ribonucleases. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture.
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Glazer, Itamar, Alice Churchill, Galina Gindin und Michael Samish. Genomic and Organismal Studies to Elucidate the Mechanisms of Infectivity of Entomopathogenic Fungi to Ticks. United States Department of Agriculture, Januar 2013. http://dx.doi.org/10.32747/2013.7593382.bard.
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The overall goal of this research was to elucidate the factors affecting early development of Metarhizium spp. (previously named M. anisopliae) on ticks or tick cuticle extracts and the molecular basis of these early infection processes. The original objectives were: 1. Characterize the pre-penetration events (adhesion, germination and appressorium formation) of spores of M. anisopliae strains with high or low virulence during tick infection. 2. Create GFP-expressing strains of M. anisopliae tick pathogens having high and low virulence to compare their progress of infection by microscopy. 3. Use microarray analyses, primarily with existing M. anisopliae EST sequences in GenBank, to identify and characterize fungal genes whose expression is regulated in response to host cuticle extracts. Objective 3 was later modified (as approved by BARD) to use RNAseq to characterize the early stages of fungal gene expression during infection of intact host cuticles. This new method provides a massively larger and more informative dataset and allows us to take advantage of a) recently published genomes of Metarhizium robertsii and M. acridum for RNAseq data analysis, and b) newly developed and highly efficient cDNA sequencing technologies that are relatively low cost and, therefore, allow deep sequencing of multiple transcriptome samples. We examined pre-penetration and penetration events that differentiate high and low virulence strains of Metarhizium spp., focusing on spore adhesion, germination, appressorium formation, and penetration of tick integuments. Initiation of fungal infection was compared on susceptible and resistant tick species at different tick developmental stages. In vitro studies comparing the effects of protein and fatty acid profiles from tick cuticle extracts demonstrated that resistant tick cuticles contain higher concentrations of specific lipids that inhibit fungal development than do susceptible tick cuticles, suggesting one mechanism of Ixodidae resistance to fungal entomopathogens (Objective 1). We used molecular markers to determine that the three M. anisopliae strains from Israel that we studied actually were three distinct species. M. brunneum is highly virulent against the tick Rhipicephalus annulatus, M. pingshaense and M. robertsii are intermediate in virulence, and M. majus is of low virulence. We transformed all four Metarhizium species to express GFP and used them in pathogenicity assays against diverse tick species. Key findings were that a) resistant ticks inhibit Metarhizium infection prior to hemocoel invasion by reducing fungal viability on the cuticle surface (Objective 2), as was supported by the in vitro studies of Objective 1, and b) Metarhizium kills susceptible ticks after cuticle penetration but prior to hemocoel colonization. Transcriptome studies of the most virulent species, M. brunneum, are in progress and include analyses of ungerminated conidia and conidia germination and development on a low nutrient medium or on susceptible R. annulatus exoskeleton (Objective 3). We anticipate these studies will contribute to identifying fungal genetic factors that increase virulence and speed of kill and may help reveal tick chemistries that could be included in biocontrol formulations to increase efficacy. Methodologies developed to screen tick cuticle extracts for ability to support conidia germination and development may help in the selection of wild fungi with increased virulence against resistant ticks. The overall knowledge gained should contribute not only to the improvement of tick control but also to the control of other blood-sucking arthropods and related plant pests. Use of bio-based agents for controlling arthropods will contribute to a healthier, more sustainable environment and serve a growing number of organic food farmers.
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Ori, Naomi, und Mark Estelle. Specific mediators of auxin activity during tomato leaf and fruit development. United States Department of Agriculture, Januar 2012. http://dx.doi.org/10.32747/2012.7597921.bard.
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The plant hormone auxin is involved in numerous developmental processes, including leaf and fruit development. The tomato (Solanumlycopersicum) gene ENTIRE (E) encodes an auxin-response inhibitor from the Aux/IAA family. While most loss-offunction mutations in Aux/IAA genes are similar to the wild type due to genetic redundancy, entire (e) mutants show specific effects on leaf and fruit development. e mutants have simple leaves, in contrast to the compound leaves of wild type tomatoes. In addition, e plants produce parthenocarpic fruits, in which fruit set occurs independently of fertilization. The aim of this research program was to utilize the e mutation to identify and characterize genes that mediate the specific effect of auxin in leaf and fruit development. The specific objectives of the project were to: 1. Characterize and map modifiers of the e leaf phenotype. 2. Characterize and map suppressors of the e fruit phenotype. 3. Dissect the developmental specificity of the E gene. 4. Examine the effect of fruit-overexpression of identified genes on fruit set and seed production. To identify mediators of auxin in leaf development, we mainly focused on one mutant, crawling elephant (crel, previously called t282), which showed substantial suppression of the e phenotype and other auxin-relatedphenotypes. We have identified the CREL gene as a homolog of the Arabidopsis VRN5 gene, involved in recruiting polycomb silencing complexes to specific targets. We showed that CREL affects auxin sensitivity in tomato. Suppressors of the e fruit phenotype have been further characterized and selected for more profound effects. Expression profiling by RNAseq was used to analyze the effect of e as well as crel on gene expression in leaves and fruits. This analysis has identified putative E and CREL targets. We have initiated studies to assess the role of some of these targets in flower and fruit development. The research has identified potential mediators of auxin response in leaf, flower and fruit development.
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Lers, Amnon, und Pamela J. Green. LX Senescence-Induced Ribonuclease in Tomato: Function and Regulation. United States Department of Agriculture, September 2003. http://dx.doi.org/10.32747/2003.7586455.bard.
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Natural leaf senescence, which occurs even when growth conditions are near optimal, has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. However, the successful design of such strategies requires a better insight into the senescence machinery and control in higher plants. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as ribonucleases (RNases) and proteases. Previously we had identified and characterized the tomato LX RNase gene demonstrating its transcript to be highly and specifically induced during senescence. This reported study was focused on LX but also had broadened our research to other senescence-associated nucleic acids degrading enzymes to learn about their function and the regulation of their encoding genes. Beside tomato we used parsley and Arabidopsis for the study of: the bi-functional nuclease which has a role in senescence. The study of different senescence- associated nucleases in few plant systems will allow a more general view on function and regulation of these enzymes in senescence. The specific original proposed objectives included: 1. Study the consequences of alterations in LX RNase level on tomato leaf senescence and general development; 2. Analyze stimuli which may participate in senescence-specific activation of the LX gene; 3. Clone the senescence-associated BFNI nuclease gene homologue from tomato. 4. Further characterize the sequences required for senescence-specific gene expression. Homozygous transgenic plants in which LX gene was either inhibited or over-expressed were generated. In both of these LX mutated plants no major phenotypic consequences were observed, which may suggests that LX is not essential for plant growth under optimal growth conditions. Lack of any abnormalities in the LX over-expressing lines suggests that special system exist to allow function of the RNase only when needed. Detailed analyses of growth under stress and consequences to RNA metabolism are underway. We have analyzed LX expression on the protein level demonstrating that it is involved also in petal senescing. Our results suggest that LX is responding to complex regulation involving developmental, organ dependent factors and responds differently to hormonal or environmental stimuli in the different plant organs. The cloned 1.4 kb promoter was cloned and its analysis revealed that probably not all required elements for senescence induction are included. Biochemical analysis of senescence-associated be-functional nucleases in the different plants, tomato, parsley and Arabidopsis, suggests they belong to a sub-class within the type I plant nucleases. The parsley PcNUC1/2 nuclease protein was purified from senescing leaves its and activity was studied in vitro revealing endo-, double strand, nucleolytic activity and exo-nucleolytic activity. Its encoding gene was cloned and found to be induced on the mRNA level. The promoter of the related Arabidopsis BFNI nuclease was shown in both tomato and Arabidopsis to be able and direct senescence-specific expression suggesting that, at least part, the gene is regulated on the transcriptional level and that the mechanism for this senescence-specific regulation is conserved between different plants. Few plants in which the BFNI gene is mutated were identified which are subjected now to detailed analysis. Our results suggest that the senescence-related nucleic acid degrading enzymes share similarities in both function and regulation between different plants and possibly have important functions in processes un-related to senescence. Still, the function of these enzymes, at least in some cases is not essential to plant development under optimal growth conditions. We are now at the stage which permits in depth investigation of the specific functions and mode of molecular regulation of senescence-associated nucleases with the aid of the research tools developed. The isolated senescence-specific promoter, shown to be active in heterologous plant system, could be utilized in agricultural-related biotechnological applications for retardation of senescence.
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Eyal, Yoram, und Sheila McCormick. Molecular Mechanisms of Pollen-Pistil Interactions in Interspecific Crossing Barriers in the Tomato Family. United States Department of Agriculture, Mai 2000. http://dx.doi.org/10.32747/2000.7573076.bard.
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During the evolutionary process of speciation in plants, naturally occurring barriers to reproduction have developed that affect the transfer of genes within and between related species. These barriers can occur at several different levels beginning with pollination-barriers and ending with hybrid-breakdown. The interaction between pollen and pistils presents one of the major barriers to intra- and inter-specific crosses and is the focus of this research project. Our long-term goal in this research proposal was defined to resolve questions on recognition and communication during pollen-pistil interactions in the extended tomato family. In this context, this work was initiated and planned to study the potential involvement of tomato pollen-specific receptor-like kinases (RLK's) in the interaction between pollen and pistils. By special permission from BARD the objectives of this research were extended to include studies on pollen-pistil interactions and pollination barriers in horticultural crops with an emphasis on citrus. Functional characterization of 2 pollen-specific RLK's from tomato was carried out. The data shows that both encode functional kinases that were active as recombinant proteins. One of the kinases was shown to accumulate mainly after pollen germination and to be phosphorylated in-vitro in pollen membranes as well as in-vivo. The presence of style extract resulted in dephosphorylation of the RLK, although no species specificity was observed. This data implies a role for at least one RLK in pollination events following pollen germination. However, a transgenic plant analysis of the RLK's comprising overexpression, dominant-negative and anti-sense constructs failed to provide answers on their role in pollination. While genetic effects on some of the plants were observed in both the Israeli and American labs, no clear functional answers were obtained. An alternative approach to addressing function was pursued by screening for an artificial ligand for the receptor domain using a peptide phage display library. An enriched peptide sequence was obtained and will be used to design a peptide-ligand to be tested for its effect o pollen germination and tube growth. Self-incompatibility (SI) in citrus was studied on 3 varieties of pummelo. SI was observed using fluorescence microscopy in each of the 3 varieties and compatibility relations between varieties was determined. An initial screen for an S-RNase SI mechanism yielded only a cDNA homologous to the group of S-like RNases, suggesting that SI results from an as yet unknown mechanism. 2D gel electrophoresis was applied to compare pollen and style profiles of different compatibility groups. A "polymorphic" protein band from style extracts was observed, isolated and micro-sequenced. Degenerate primers designed based on the peptide sequence date will be used to isolate the relevant genes i order to study their potential involvement in SI. A study on SI in the apple cultivar Top red was initiated. SI was found, as previously shown, to be complete thus requiring a compatible pollinator variety. A new S-RNase allele was discovered fro Top red styles and was found to be highly homologous to pear S-RNases, suggesting that evolution of these genes pre-dated speciation into apples and pears but not to other Rosaceae species. The new allele provides molecular-genetic tools to determine potential pollinators for the variety Top red as well as a tool to break-down SI in this important variety.
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Lers, Amnon, und Pamela J. Green. Analysis of Small RNAs Associated with Plant Senescence. United States Department of Agriculture, März 2013. http://dx.doi.org/10.32747/2013.7593393.bard.
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Senescence is an agriculturally significant process due to its negative impact to crop yield and postharvest quality. The genetic regulatory systems controlling senescence induction and progress respond to both developmental and environmental stress signals and involve numerous gene expression changes. Knowledge about the key molecular factors which control senescence is very limited. MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, responses to environmental stresses, and senescence. The long-term goal of this work is to elucidate roles of small RNAs associated with plant senescence. The hypothesis underlying this research is that miRNA-mediated regulation makes important contributions to the senescence process in plants. Specific, original research objectives included: 1) Profiling of small RNAs from senescing plants; 2) Data Analysis and public access via a user-friendly web interface; 3) Validation of senescence-associated miRNAs and target RNAs; 4) Development of transgenic plants for functional analysis of miRNAs in Arabidopsis. Major revisions made in the research compared to the original work plan included 1) Exclusion of the planned work with tomato as recommended by the BARD review panel; 2) Performing miRNA study also in senescing Arabidopsis siliques, in addition to senescing leaves. To identify senescenceregulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel Analysis of RNA Ends (PARE) libraries, which enable the large-scale examination of miRNA-guided cleavage products, were also constructed and sequenced, resulting in over 750 million genome-matched sequences. These massive datasets lead to the identification of new miRNAs, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence. Experiments were initiated for functional analysis of specific senescence-associated miRNAs and respective target genes. Transgenic Arabidopsis plants were generated in which miR408, found in this study to be significantly induced in leaf senescence, was over-expressed either constitutively or under a senescence-specific promoter. These plants are currently being characterized for any altered phenotypes. In addition T-DNA knock out mutants for various target genes identified in this research are being analyzed. This work provides insights about specific miRNAs that contribute to leaf and silique senescence. The knowledge generated may suggest new strategies to monitor and alter the progression of senescence in crops for agricultural improvement.
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Splitter, Gary A., Menachem Banai und Jerome S. Harms. Brucella second messenger coordinates stages of infection. United States Department of Agriculture, Januar 2011. http://dx.doi.org/10.32747/2011.7699864.bard.
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Aim 1: To determine levels of this second messenger in: a) B. melitensiscyclic-dimericguanosinemonophosphate-regulating mutants (BMEI1448, BMEI1453, and BMEI1520), and b) B. melitensis16M (wild type) and mutant infections of macrophages and immune competent mice. (US lab primary) Aim 2: To determine proteomic differences between Brucelladeletion mutants BMEI1453 (high cyclic-dimericguanosinemonophosphate, chronic persistent state) and BMEI1520 (low cyclicdimericguanosinemonophosphate, acute virulent state) compared to wild type B. melitensisto identify the role of this second messenger in establishing the two polar states of brucellosis. (US lab primary with synergistic assistance from the Israel lab Aim 3: Determine the level of Brucellacyclic-dimericguanosinemonophosphate and transcriptional expression from naturally infected placenta. (Israel lab primary with synergistic assistance from the US lab). B. Background Brucellaspecies are Gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, the most prevalent zoonosis worldwide. Brucellosis is characterized by increased abortion, weak offspring, and decreased milk production in animals. Humans are infected with Brucellaby consuming contaminated milk products or via inhalation of aerosolized bacteria from occupational hazards. Chronic human infections can result in complications such as liver damage, orchitis, endocarditis, and arthritis. Brucellaspp. have the ability to infect both professional and non-professional phagocytes. Because of this, Brucellaencounter varied environments both throughout the body and within a cell and must adapt accordingly. To date, few virulence factors have been identified in B. melitensisand even less is known about how these virulence factors are regulated. Subsequently, little is known about how Brucellaadapt to its rapidly changing environments, and how it alternates between acute and chronic virulence. Our studies suggest that decreased concentrations of cyclic dimericguanosinemonophosphate (c-di-GMP) lead to an acute virulent state and increased concentrations of c-di-GMP lead to persistent, chronic state of B. melitensisin a mouse model of infection. We hypothesize that B. melitensisuses c-di-GMP to transition from the chronic state of an infected host to the acute, virulent stage of infection in the placenta where the bacteria prepare to infect a new host. Studies on environmental pathogens such as Vibrio choleraeand Pseudomonas aeruginosasupport a mechanism where changes in c-di-GMP levels cause the bacterium to alternate between virulent and chronic states. Little work exists on understanding the role of c-di-GMP in dangerous intracellular pathogens, like Brucellathat is a frequent pathogen in Israeli domestic animals and U.S. elk and bison. Brucellamust carefully regulate virulence factors during infection of a host to ensure proper expression at appropriate times in response to host cues. Recently, the novel secondary signaling molecule c-di-GMP has been identified as a major component of bacterial regulation and we have identified c-di-GMP as an important signaling factor in B. melitensishost adaptation. C. Major conclusions, solutions, achievements 1. The B. melitensis1453 deletion mutant has increased c-di-GMP, while the 1520 deletion mutant has decreased c-di-GMP. 2. Both mutants grow similarly in in vitro cultures; however, the 1453 mutant has a microcolony phenotype both in vitro and in vivo 3. The 1453 mutant has increased crystal violet staining suggesting biofilm formation. 4. Scanning electron microscopy revealed an abnormal coccus appearance with in increased cell area. 5. Proteomic analysis revealed the 1453 mutant possessed increased production of proteins involved in cell wall processes, cell division, and the Type IV secretion system, and a decrease in proteins involved in amino acid transport/metabolism, carbohydrate metabolism, fatty acid production, and iron acquisition suggesting less preparedness for intracellular survival. 6. RNAseq analysis of bone marrow derived macrophages infected with the mutants revealed the host immune response is greatly reduced with the 1453 mutant infection. These findings support that microlocalization of proteins involved in c-di-GMP homeostasis serve a second messenger to B. melitensisregulating functions of the bacteria during infection of the host.
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Zhong, xiaoling. Diagnostic Significance of Noncoding RNAs in Kawasaki Disease: A Systematic Review and Meta-Analysis. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, Oktober 2022. http://dx.doi.org/10.37766/inplasy2022.10.0035.
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Xu, Jianhao, Fang Cao, Yongwei Hu und Zaichang Chen. Circulating long noncoding RNAs as potential biomarkers for stomach cancer: A systematic review and meta-analysis. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, Februar 2021. http://dx.doi.org/10.37766/inplasy2021.2.0079.
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