High-Throughput最新文献_第3页

Investigation of RNA Editing Sites within Bound Regions of RNA-Binding Proteins RNA结合蛋白结合区内RNA编辑位点的研究

High-Throughput Pub Date : 2019-11-29 DOI: 10.3390/ht8040019

Tyler Weirick, G. Militello, M. R. Hosen, D. John, J. Moore, S. Uchida

{"title":"Investigation of RNA Editing Sites within Bound Regions of RNA-Binding Proteins","authors":"Tyler Weirick, G. Militello, M. R. Hosen, D. John, J. Moore, S. Uchida","doi":"10.3390/ht8040019","DOIUrl":"https://doi.org/10.3390/ht8040019","url":null,"abstract":"Studies in epitranscriptomics indicate that RNA is modified by a variety of enzymes. Among these RNA modifications, adenosine to inosine (A-to-I) RNA editing occurs frequently in the mammalian transcriptome. These RNA editing sites can be detected directly from RNA sequencing (RNA-seq) data by examining nucleotide changes from adenosine (A) to guanine (G), which substitutes for inosine (I). However, a careful investigation of such nucleotide changes must be conducted to distinguish sequencing errors and genomic mutations from the genuine editing sites. Building upon our recent introduction of an easy-to-use bioinformatics tool, RNA Editor, to detect RNA editing events from RNA-seq data, we examined the extent by which RNA editing events affect the binding of RNA-binding proteins (RBP). Through employing bioinformatic techniques, we uncovered that RNA editing sites occur frequently in RBP-bound regions. Moreover, the presence of RNA editing sites are more frequent when RNA editing islands were examined, which are regions in which RNA editing sites are present in clusters. When the binding of one RBP, human antigen R [HuR; encoded by ELAV-like protein 1 (ELAV1)], was quantified experimentally, its binding was reduced upon silencing of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) compared to the control—suggesting that the presence of RNA editing islands influence HuR binding to its target regions. These data indicate RNA editing as an important mediator of RBP–RNA interactions—a mechanism which likely constitutes an additional mode of post-transcription gene regulation in biological systems.","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht8040019","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49512814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Expression Profiling of Candidate Genes in Sugar Beet Leaves Treated with Leonardite-Based Biostimulant 基于Leonardite的生物刺激剂处理甜菜叶片中候选基因的表达谱

High-Throughput Pub Date : 2019-10-11 DOI: 10.3390/ht8040018

H. S. Hajizadeh, B. Heidari, G. Bertoldo, M. Della Lucia, Francesco Magro, C. Broccanello, A. Baglieri, I. Puglisi, A. Squartini, G. Campagna, G. Concheri, S. Nardi, P. Stevanato

{"title":"Expression Profiling of Candidate Genes in Sugar Beet Leaves Treated with Leonardite-Based Biostimulant","authors":"H. S. Hajizadeh, B. Heidari, G. Bertoldo, M. Della Lucia, Francesco Magro, C. Broccanello, A. Baglieri, I. Puglisi, A. Squartini, G. Campagna, G. Concheri, S. Nardi, P. Stevanato","doi":"10.3390/ht8040018","DOIUrl":"https://doi.org/10.3390/ht8040018","url":null,"abstract":"Leonardite-based biostimulants are a large class of compounds, including humic acid substances. Foliar application of biostimulants at field level improves plant growth, yield and quality through metabolic changes and stimulation of plant proton pumps. The present study aimed at identifying optimum dosage of BLACKJAK, a humic acid-based substance, which is able to modify genes involved in sugar beet growth. Thirty-three genes belonging to various biochemical pathway categories were tested in leaves of treated sugar beet (Beta vulgaris L.) samples to assess gene expression profiling in response to BLACKJAK. Seedlings of a diploid and multigerm variety were grown in plastic pots and sprayed with two dilutions of BLACKJAK (dilution 1:500–1.0 mg C L−1 and dilution 1:1000–0.5 mg C L−1). Leaf samples were collected after 24, 48, and 72 h treatment with BLACKJAK for each dilution. RNA was extracted and the quantification of gene expression was performed while using an OpenArray platform. Results of analysis of variance demonstrated that, 15 genes out of a total of 33 genes tested with OpenArray qPCR were significantly affected by treatment and exposure time. Analysis for annotation of gene products and pathways revealed that genes belonging to the mitochondrial respiratory pathways, nitrogen and hormone metabolisms, and nutrient uptake were up-regulated in the BLACKJAK treated samples. Among the up-regulated genes, Bv_PHT2;1 and Bv_GLN1 expression exerted a 2-fold change in 1:1000 and 1:500 BLACKJAK concentrations. Overall, the gene expression data in the BLACKJAK treated leaves demonstrated the induction of plant growth–related genes that were contributed almost to amino acid and nitrogen metabolism, plant defense system, and plant growth.","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht8040018","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42849635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Special Issue “Adductomics: Elucidating the Environmental Causes of Disease” 特刊“添加组学：阐明疾病的环境原因”

High-Throughput Pub Date : 2019-07-31 DOI: 10.3390/ht8030017

S. Pereira, A. Antunes

引用次数: 2

Global Properties of Latent Virus Dynamics Models with Immune Impairment and Two Routes of Infection. 具有免疫损伤和两种感染途径的潜伏病毒动力学模型的全局性质。

High-Throughput Pub Date : 2019-06-03 DOI: 10.3390/ht8020016

Aeshah A Raezah, Ahmed M Elaiw, Badria S Alofi

引用次数: 3

Development and Optimization of a Miniaturized Western Blot-Based Screening Platform to Identify Regulators of Post-Translational Modifications. 小型Western blot筛选平台的开发和优化，以确定翻译后修饰的调节因子。

High-Throughput Pub Date : 2019-06-03 DOI: 10.3390/ht8020015

Florencia Villafañez, Vanesa Gottifredi, Gastón Soria

{"title":"Development and Optimization of a Miniaturized Western Blot-Based Screening Platform to Identify Regulators of Post-Translational Modifications.","authors":"Florencia Villafañez, Vanesa Gottifredi, Gastón Soria","doi":"10.3390/ht8020015","DOIUrl":"https://doi.org/10.3390/ht8020015","url":null,"abstract":"Post-translational modifications (PTMs) are fundamental traits of protein functionality and their study has been addressed using several approaches over the past years. However, screening methods developed to detect regulators of PTMs imply many challenges and are usually based on expensive techniques. Herein, we described the development and optimization of a western blot-based platform for identification of regulators of a specific PTM-mono-ubiquitylation of proliferating cell nuclear antigen (PCNA). This cell-based method does not require specific equipment, apart from the basic western blot (WB) devices and minor accessories, which are accessible for most research labs. The modifications introduced to the classical WB protocol allow the performance of PTM analysis from a single well of a 96-well plate with minimal sample manipulation and low intra- and inter-plate variability, making this method ideal to screen arrayed compound libraries in a 96-well format. As such, our experimental pipeline provides the proof of concept to design small screenings of PTM regulators by improving the quantitative accuracy and throughput capacity of classical western blots.","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":"8 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht8020015","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37307463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

A Multispecies Biofilm In Vitro Screening Model of Dental Caries for High-Throughput Susceptibility Testing. 用于高通量敏感性测试的多物种生物膜体外筛选龋齿模型。

High-Throughput Pub Date : 2019-05-30 DOI: 10.3390/ht8020014

Lara A Heersema, Hugh D C Smyth

{"title":"A Multispecies Biofilm In Vitro Screening Model of Dental Caries for High-Throughput Susceptibility Testing.","authors":"Lara A Heersema, Hugh D C Smyth","doi":"10.3390/ht8020014","DOIUrl":"10.3390/ht8020014","url":null,"abstract":"There is a current need to develop and optimize new therapeutics for the treatment of dental caries, but these efforts are limited by the relatively low throughput of relevant in vitro models. The aim of this work was to bridge the 96-well microtiter plate system with a relevant multispecies dental caries model that could be reproducibly grown to allow for the high-throughput screening of anti-biofilm therapies. Various media and inoculum concentrations were assessed using metabolic activity, biomass, viability, and acidity assays to determine the optimal laboratory-controlled conditions for a multispecies biofilm composed of Streptococcus gordonii, Streptococcus mutans, and Candida albicans. The selected model encompasses several of the known fundamental characteristics of dental caries-associated biofilms. The 1:1 RPMI:TSBYE 0.6% media supported the viability and biomass production of mono- and multispecies biofilms best. Kinetic studies over 48 h in 1:1 RPMI:TSBYE 0.6% demonstrated a stable biofilm phase between 10 and 48 h for all mono- and multispecies biofilms. The 1:1:0.1 S. gordonii: S. mutans: C. albicans multispecies biofilm in 1:1 RPMI:TSBYE 0.6% is an excellent choice for a high-throughput multispecies model of dental caries. This high-throughput multispecies model can be used for screening novel therapies and for better understanding the treatment effects on biofilm interactions and stability.","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":"8 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht8020014","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37296509","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Emerging Technologies in Mass Spectrometry-Based DNA Adductomics. 基于质谱的DNA内合组学的新兴技术。

High-Throughput Pub Date : 2019-05-14 DOI: 10.3390/ht8020013

Jingshu Guo, Robert J Turesky

{"title":"Emerging Technologies in Mass Spectrometry-Based DNA Adductomics.","authors":"Jingshu Guo, Robert J Turesky","doi":"10.3390/ht8020013","DOIUrl":"https://doi.org/10.3390/ht8020013","url":null,"abstract":"The measurement of DNA adducts, the covalent modifications of DNA upon the exposure to the environmental and dietary genotoxicants and endogenously produced electrophiles, provides molecular evidence for DNA damage. With the recent improvements in the sensitivity and scanning speed of mass spectrometry (MS) instrumentation, particularly high-resolution MS, it is now feasible to screen for the totality of DNA damage in the human genome through DNA adductomics approaches. Several MS platforms have been used in DNA adductomic analysis, each of which has its strengths and limitations. The loss of 2'-deoxyribose from the modified nucleoside upon collision-induced dissociation is the main transition feature utilized in the screening of DNA adducts. Several advanced data-dependent and data-independent scanning techniques originated from proteomics and metabolomics have been tailored for DNA adductomics. The field of DNA adductomics is an emerging technology in human exposure assessment. As the analytical technology matures and bioinformatics tools become available for analysis of the MS data, DNA adductomics can advance our understanding about the role of chemical exposures in DNA damage and disease risk.","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":"8 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht8020013","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37240851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 25

The Adductomics of Isolevuglandins: Oxidation of IsoLG Pyrrole Intermediates Generates Pyrrole⁻Pyrrole Crosslinks and Lactams. 等吡咯-吡咯中间体氧化生成吡咯-吡咯交联和内酰胺。

High-Throughput Pub Date : 2019-05-10 DOI: 10.3390/ht8020012

Wenzhao Bi, Geeng-Fu Jang, Lei Zhang, John W Crabb, James Laird, Mikhail Linetsky, Robert G Salomon

{"title":"The Adductomics of Isolevuglandins: Oxidation of IsoLG Pyrrole Intermediates Generates Pyrrole⁻Pyrrole Crosslinks and Lactams.","authors":"Wenzhao Bi, Geeng-Fu Jang, Lei Zhang, John W Crabb, James Laird, Mikhail Linetsky, Robert G Salomon","doi":"10.3390/ht8020012","DOIUrl":"https://doi.org/10.3390/ht8020012","url":null,"abstract":"Isoprostane endoperoxides generated by free radical-induced oxidation of arachidonates, and prostaglandin endoperoxides generated through enzymatic cyclooxygenation of arachidonate, rearrange nonenzymatically to isoprostanes and a family of stereo and structurally isomeric γ-ketoaldehyde seco-isoprostanes, collectively known as isolevuglandins (isoLGs). IsoLGs are stealthy toxins, and free isoLGs are not detected in vivo. Rather, covalent adducts are found to incorporate lysyl ε-amino residues of proteins or ethanolamino residues of phospholipids. In vitro studies have revealed that adduction occurs within seconds and is uniquely prone to cause protein-protein crosslinks. IsoLGs accelerate the formation of the type of amyloid beta oligomers that have been associated with neurotoxicity. Under air, isoLG-derived pyrroles generated initially are readily oxidized to lactams and undergo rapid oxidative coupling to pyrrole-pyrrole crosslinked dimers, and to more highly oxygenated derivatives of those dimers. We have now found that pure isoLG-derived pyrroles, which can be generated under anoxic conditions, do not readily undergo oxidative coupling. Rather, dimer formation only occurs after an induction period by an autocatalytic oxidative coupling. The stable free-radical TEMPO abolishes the induction period, catalyzing rapid oxidative coupling. The amine N-oxide TMAO is similarly effective in catalyzing the oxidative coupling of isoLG pyrroles. N-acetylcysteine abolishes the generation of pyrrole-pyrrole crosslinks. Instead pyrrole-cysteine adducts are produced. Two unified single-electron transfer mechanisms are proposed for crosslink and pyrrole-cysteine adduct formation from isoLG-pyrroles, as well as for their oxidation to lactams and hydroxylactams.","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":"8 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht8020012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37237841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides. 用常规玻片固定空间有序微球高效筛选组合肽库。

High-Throughput Pub Date : 2019-04-30 DOI: 10.3390/ht8020011

Timm Schwaar, Maike Lettow, Dario Remmler, Hans G Börner, Michael G Weller

{"title":"Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides.","authors":"Timm Schwaar, Maike Lettow, Dario Remmler, Hans G Börner, Michael G Weller","doi":"10.3390/ht8020011","DOIUrl":"https://doi.org/10.3390/ht8020011","url":null,"abstract":"Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput \"all-on-one chip\" system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":"8 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht8020011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37207500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

The mercapturomic profile of health and non-communicable diseases. 健康和非传染性疾病的医学概况。

High-Throughput Pub Date : 2019-04-23 DOI: 10.3390/ht8020010

Clara Gonçalves-Dias, Judit Morello, Valdir Semedo, M João Correia, Nuno R Coelho, Emilia C Monteiro, Alexandra M M Antunes, Sofia A Pereira

引用次数: 7