Arkas: Rapid reproducible RNAseq analysis

PubMed Central

Colombo, Anthony R.; J. Triche Jr, Timothy; Ramsingh, Giridharan

2017-01-01

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. PMID:28868134

Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non-Small Cell Lung Cancer Tissue.

PubMed

Kristof, Jessica; Sakrison, Kellen; Jin, Xiaoping; Nakamaru, Kenji; Schneider, Matthias; Beckman, Robert A; Freeman, Daniel; Spittle, Cindy; Feng, Wenqin

2017-01-01

In preclinical studies, heregulin ( HRG ) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti-epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non-small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG -positive and HRG -negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8 , and EIF2B1 ), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.

PubMed

Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto

2012-07-11

Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

From the viral perspective: infectious salmon anemia virus (ISAV) transcriptome during the infective process in Atlantic salmon (Salmo salar).

PubMed

Valenzuela-Miranda, Diego; Cabrejos, María Eugenia; Yañez, José Manuel; Gallardo-Escárate, Cristian

2015-04-01

The infectious salmon anemia virus (ISAV) is a severe disease that mainly affects the Atlantic salmon (Salmo salar) aquaculture industry. Although several transcriptional studies have aimed to understand Salmon-ISAV interaction through the evaluation of host-gene transcription, none of them has focused their attention upon the viral transcriptional dynamics. For this purpose, RNA-Seq and RT-qPCR analyses were conducted in gills, liver and head-kidney of S. salar challenged by cohabitation with ISAV. Results evidence the time and tissue transcript patterns involved in the viral expression and how the transcription levels of ISAV segments are directly linked with the protein abundance found in other virus of the Orthomyxoviridae family. In addition, RT-qPCR result evidenced that quantification of ISAV through amplification of segment 3 would result in a more sensitive approach for detection and quantification of ISAV. This study offers a more comprehensive approach regarding the ISAV infective process and gives novel knowledge for its molecular detection. Copyright © 2014 Elsevier B.V. All rights reserved.

Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes.

PubMed

Mahakapuge, T A N; Scheerlinck, J-P Y; Rojas, C A Alvarez; Every, A L; Hagen, J

2016-03-01

With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization "geNorm" and "Norm Finder", PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep. Copyright © 2016. Published by Elsevier B.V.

Digital gene expression for non-model organisms

PubMed Central

Hong, Lewis Z.; Li, Jun; Schmidt-Küntzel, Anne; Warren, Wesley C.; Barsh, Gregory S.

2011-01-01

Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-high-throughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6–8 million reads. EDGE exhibits very little technical noise, reveals a large (106) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions. PMID:21844123

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.

PubMed

Auvré, Frédéric; Coutier, Julien; Martin, Michèle T; Fortunel, Nicolas O

2018-05-08

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references such as house-keeping gene expression, as it is initiated with defined cell numbers, precisely sorted by flow cytometry.

MITIE: Simultaneous RNA-Seq-based transcript identification and quantification in multiple samples.

PubMed

Behr, Jonas; Kahles, André; Zhong, Yi; Sreedharan, Vipin T; Drewe, Philipp; Rätsch, Gunnar

2013-10-15

High-throughput sequencing of mRNA (RNA-Seq) has led to tremendous improvements in the detection of expressed genes and reconstruction of RNA transcripts. However, the extensive dynamic range of gene expression, technical limitations and biases, as well as the observed complexity of the transcriptional landscape, pose profound computational challenges for transcriptome reconstruction. We present the novel framework MITIE (Mixed Integer Transcript IdEntification) for simultaneous transcript reconstruction and quantification. We define a likelihood function based on the negative binomial distribution, use a regularization approach to select a few transcripts collectively explaining the observed read data and show how to find the optimal solution using Mixed Integer Programming. MITIE can (i) take advantage of known transcripts, (ii) reconstruct and quantify transcripts simultaneously in multiple samples, and (iii) resolve the location of multi-mapping reads. It is designed for genome- and assembly-based transcriptome reconstruction. We present an extensive study based on realistic simulated RNA-Seq data. When compared with state-of-the-art approaches, MITIE proves to be significantly more sensitive and overall more accurate. Moreover, MITIE yields substantial performance gains when used with multiple samples. We applied our system to 38 Drosophila melanogaster modENCODE RNA-Seq libraries and estimated the sensitivity of reconstructing omitted transcript annotations and the specificity with respect to annotated transcripts. Our results corroborate that a well-motivated objective paired with appropriate optimization techniques lead to significant improvements over the state-of-the-art in transcriptome reconstruction. MITIE is implemented in C++ and is available from http://bioweb.me/mitie under the GPL license.

Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

PubMed Central

Straub, Cécile; Castellote, Jessie; Onesime, Djamila; Bonnarme, Pascal; Irlinger, Françoise

2013-01-01

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed. PMID:23124230

The Vigna unguiculata Gene Expression Atlas (VuGEA) from de novo assembly and quantification of RNA-seq data provides insights into seed maturation mechanisms.

PubMed

Yao, Shaolun; Jiang, Chuan; Huang, Ziyue; Torres-Jerez, Ivone; Chang, Junil; Zhang, Heng; Udvardi, Michael; Liu, Renyi; Verdier, Jerome

2016-10-01

Legume research and cultivar development are important for sustainable food production, especially of high-protein seed. Thanks to the development of deep-sequencing technologies, crop species have been taken to the front line, even without completion of their genome sequences. Black-eyed pea (Vigna unguiculata) is a legume species widely grown in semi-arid regions, which has high potential to provide stable seed protein production in a broad range of environments, including drought conditions. The black-eyed pea reference genotype has been used to generate a gene expression atlas of the major plant tissues (i.e. leaf, root, stem, flower, pod and seed), with a developmental time series for pods and seeds. From these various organs, 27 cDNA libraries were generated and sequenced, resulting in more than one billion reads. Following filtering, these reads were de novo assembled into 36 529 transcript sequences that were annotated and quantified across the different tissues. A set of 24 866 unique transcript sequences, called Unigenes, was identified. All the information related to transcript identification, annotation and quantification were stored into a gene expression atlas webserver (http://vugea.noble.org), providing a user-friendly interface and necessary tools to analyse transcript expression in black-eyed pea organs and to compare data with other legume species. Using this gene expression atlas, we inferred details of molecular processes that are active during seed development, and identified key putative regulators of seed maturation. Additionally, we found evidence for conservation of regulatory mechanisms involving miRNA in plant tissues subjected to drought and seeds undergoing desiccation. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Single cell genomic quantification by non-fluorescence nonlinear microscopy

NASA Astrophysics Data System (ADS)

Kota, Divya; Liu, Jing

2017-02-01

Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.

Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

PubMed

Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

2009-05-01

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

PubMed Central

2012-01-01

Background Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial. Methods Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females. Results The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers. Conclusions This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the two mechanisms are regulated independently. Moreover, neither the total DMD transcript level, nor the relative proportion of the wild-type transcript do correlate with the symptomatic phenotype. PMID:22894145

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

PubMed Central

Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

2013-01-01

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing. PMID:24236095

Mass Spectral Enhanced Detection of Ubls Using SWATH Acquisition: MEDUSA—Simultaneous Quantification of SUMO and Ubiquitin-Derived Isopeptides

NASA Astrophysics Data System (ADS)

Griffiths, John R.; Chicooree, Navin; Connolly, Yvonne; Neffling, Milla; Lane, Catherine S.; Knapman, Thomas; Smith, Duncan L.

2014-05-01

Protein modification by ubiquitination and SUMOylation occur throughout the cell and are responsible for numerous cellular functions such as apoptosis, DNA replication and repair, and gene transcription. Current methods for the identification of such modifications using mass spectrometry predominantly rely upon tryptic isopeptide tag generation followed by database searching with in vitro genetic mutation of SUMO routinely required. We have recently described a novel approach to ubiquitin and SUMO modification detection based upon the diagnostic a' and b' ions released from the isopeptide tags upon collision-induced dissociation of reductively methylated Ubl isopeptides (RUbI) using formaldehyde. Here, we significantly extend those studies by combining data-independent acquisition (DIA) with alternative labeling reagents to improve diagnostic ion coverage and enable relative quantification of modified peptides from both MS and MS/MS signals. Model synthetic ubiquitin and SUMO-derived isopeptides were labeled with mTRAQ reagents (Δ0, Δ4, and Δ8) and subjected to LC-MS/MS with SWATH acquisition. Novel diagnostic ions were generated upon CID, which facilitated the selective detection of these modified peptides. Simultaneous MS-based and MS/MS-based relative quantification was demonstrated for both Ub and SUMO-derived isopeptides across three channels in a background of mTRAQ-labeled Escherichia coli digest.

Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

PubMed

Cusick, Kathleen D; Fitzgerald, Lisa A; Pirlo, Russell K; Cockrell, Allison L; Petersen, Emily R; Biffinger, Justin C

2014-01-01

Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene expression changes during growth in bioreactors.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

PubMed

Shen, L P; Sheridan, P; Cao, W W; Dailey, P J; Salazar-Gonzalez, J F; Breen, E C; Fahey, J L; Urdea, M S; Kolberg, J A

1998-06-01

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Body image perceptions in women with pelvic organ prolapse: a qualitative study.

PubMed

Lowder, Jerry L; Ghetti, Chiara; Nikolajski, Cara; Oliphant, Sallie S; Zyczynski, Halina M

2011-05-01

To describe perceptions of prolapse-specific body image in women with symptomatic prolapse. Women with symptomatic pelvic organ prolapse quantification stage ≥ II prolapse participated in semistructured focus groups or self-report questionnaire. Transcripts were independently reviewed and body image themes were identified and confirmed by consensus. Twenty-five women participated in focus groups and 27 in online questionnaires. Transcript analysis revealed 3 central themes and 25 body-image related subthemes. Women living with prolapse were more likely to feel self-conscious, isolated, "different," less feminine, and less attractive. Women often changed sexual intimacy practices because of embarrassment or discomfort, and many avoided intimacy all together. Prolapse greatly affected women's personal and professional activities causing some women to adjust routines or stop activities. Women reported loss of interest in activities, distraction while performing daily/work-related tasks, and embarrassment when asking for help with activities. Themes identified in this qualitative study demonstrate the profound effect of prolapse on a woman's body image. Copyright © 2011 Mosby, Inc. All rights reserved.

Temporal Analysis of the Magnaporthe Oryzae Proteome During Conidial Germination and Cyclic AMP (cAMP)-mediated Appressorium Formation*

PubMed Central

Franck, William L.; Gokce, Emine; Oh, Yeonyee; Muddiman, David C.; Dean, Ralph A.

2013-01-01

Rice blast disease caused by Magnaporthe oryzae is one of the most serious threats to global rice production. During the earliest stages of rice infection, M. oryzae conidia germinate on the leaf surface and form a specialized infection structure termed the appressorium. The development of the appressorium represents the first critical stage of infectious development. A total of 3200 unique proteins were identified by nanoLC-MS/MS in a temporal study of conidial germination and cAMP-induced appressorium formation in M. oryzae. Using spectral counting based label free quantification, observed changes in relative protein abundance during the developmental process revealed changes in the cell wall biosynthetic machinery, transport functions, and production of extracellular proteins in developing appressoria. One hundred and sixty-six up-regulated and 208 down-regulated proteins were identified in response to cAMP treatment. Proteomic analysis of a cAMP-dependent protein kinase A mutant that is compromised in the ability to form appressoria identified proteins whose developmental regulation is dependent on cAMP signaling. Selected reaction monitoring was used for absolute quantification of four regulated proteins to validate the global proteomics data and confirmed the germination or appressorium specific regulation of these proteins. Finally, a comparison of the proteome and transcriptome was performed and revealed little correlation between transcript and protein regulation. A subset of regulated proteins were identified whose transcripts show similar regulation patterns and include many of the most strongly regulated proteins indicating a central role in appressorium formation. A temporal quantitative RT-PCR analysis confirmed a strong correlation between transcript and protein abundance for some but not all genes. Collectively, the data presented here provide the first comprehensive view of the M. oryzae proteome during early infection-related development and highlight biological processes important for pathogenicity. PMID:23665591

A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards.

PubMed

Kito, Keiji; Okada, Mitsuhiro; Ishibashi, Yuko; Okada, Satoshi; Ito, Takashi

2016-05-01

The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction.

PubMed

de Cremoux, P; Bieche, I; Tran-Perennou, C; Vignaud, S; Boudou, E; Asselain, B; Lidereau, R; Magdelénat, H; Becette, V; Sigal-Zafrani, B; Spyratos, F

2004-09-01

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure. We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene. A good correlation was observed for all target and reference genes in both centers using their own protocols and kits (P < 0.0001). The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERalpha and the PR transcripts (P < 0.001). A weak inverse correlation was observed in both centers between ERalpha and ERbeta levels, but only when TBP was the reference gene. No other correlation was observed with other parameters. Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of inter-laboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.

Quantification of functional genes from procaryotes in soil by PCR.

PubMed

Sharma, Shilpi; Radl, Viviane; Hai, Brigitte; Kloos, Karin; Fuka, Mirna Mrkonjic; Engel, Marion; Schauss, Kristina; Schloter, Michael

2007-03-01

Controlling turnover processes and fluxes in soils and other environments requires information about the gene pool and possibilities for its in situ induction. Therefore in the recent years there has been a growing interest in genes and transcripts coding for metabolic enzymes. Besides questions addressing redundancy and diversity, more and more attention is given on the abundance of specific DNA and mRNA in the different habitats. This review will describe several PCR techniques that are suitable for quantification of functional genes and transcripts such as MPN-PCR, competitive PCR and real-time PCR. The advantages and disadvantages of the mentioned methods are discussed. In addition, the problems of quantitative extraction of nucleic acid and substances that inhibit polymerase are described. Finally, some examples from recent papers are given to demonstrate the applicability and usefulness of the different approaches.

Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data

PubMed Central

Wood, David L. A.; Nones, Katia; Steptoe, Anita; Christ, Angelika; Harliwong, Ivon; Newell, Felicity; Bruxner, Timothy J. C.; Miller, David; Cloonan, Nicole; Grimmond, Sean M.

2015-01-01

Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual’s phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci. PMID:25965996

An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation.

PubMed

Rullan, Marc; Benzinger, Dirk; Schmidt, Gregor W; Milias-Argeitis, Andreas; Khammash, Mustafa

2018-05-17

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Information transduction capacity reduces the uncertainties in annotation-free isoform discovery and quantification

PubMed Central

Deng, Yue; Bao, Feng; Yang, Yang; Ji, Xiangyang; Du, Mulong; Zhang, Zhengdong

2017-01-01

Abstract The automated transcript discovery and quantification of high-throughput RNA sequencing (RNA-seq) data are important tasks of next-generation sequencing (NGS) research. However, these tasks are challenging due to the uncertainties that arise in the inference of complete splicing isoform variants from partially observed short reads. Here, we address this problem by explicitly reducing the inherent uncertainties in a biological system caused by missing information. In our approach, the RNA-seq procedure for transforming transcripts into short reads is considered an information transmission process. Consequently, the data uncertainties are substantially reduced by exploiting the information transduction capacity of information theory. The experimental results obtained from the analyses of simulated datasets and RNA-seq datasets from cell lines and tissues demonstrate the advantages of our method over state-of-the-art competitors. Our algorithm is an open-source implementation of MaxInfo. PMID:28911101

Characterization and improvement of RNA-Seq precision in quantitative transcript expression profiling.

PubMed

Łabaj, Paweł P; Leparc, Germán G; Linggi, Bryan E; Markillie, Lye Meng; Wiley, H Steven; Kreil, David P

2011-07-01

Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large-scale RNA-Seq datasets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. We report on a comprehensive study of target identification and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive recall of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, <30% of all transcripts could be quantified reliably with a relative error<20%. Based on established tools, we then introduce a new approach for mapping and analysing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision. rnaseq10@boku.ac.at

High flavonoid accompanied with high starch accumulation triggered by nutrient starvation in bioenergy crop duckweed (Landoltia punctata).

PubMed

Tao, Xiang; Fang, Yang; Huang, Meng-Jun; Xiao, Yao; Liu, Yang; Ma, Xin-Rong; Zhao, Hai

2017-02-15

As the fastest growing plant, duckweed can thrive on anthropogenic wastewater. The purple-backed duckweed, Landoltia punctata, is rich in starch and flavonoids. However, the molecular biological basis of high flavonoid and low lignin content remains largely unknown, as does the best method to combine nutrients removed from sewage and the utilization value improvement of duckweed biomass. A combined omics study was performed to investigate the biosynthesis of flavonoid and the metabolic flux changes in L. punctata grown in different culture medium. Phenylalanine metabolism related transcripts were identified and carefully analyzed. Expression quantification results showed that most of the flavonoid biosynthetic transcripts were relatively highly expressed, while most lignin-related transcripts were poorly expressed or failed to be detected by iTRAQ based proteomic analyses. This explains why duckweed has a much lower lignin percentage and higher flavonoid content than most other plants. Growing in distilled water, expression of most flavonoid-related transcripts were increased, while most were decreased in uniconazole treated L. punctata (1/6 × Hoagland + 800 mg•L -1 uniconazole). When L. punctata was cultivated in full nutrient medium (1/6 × Hoagland), more than half of these transcripts were increased, however others were suppressed. Metabolome results showed that a total of 20 flavonoid compounds were separated by HPLC in L. punctata grown in uniconazole and full nutrient medium. The quantities of all 20 compounds were decreased by uniconazole, while 11 were increased and 6 decreased when grown in full nutrient medium. Nutrient starvation resulted in an obvious purple accumulation on the underside of each frond. The high flavonoid and low lignin content of L. punctata appears to be predominantly caused by the flavonoid-directed metabolic flux. Nutrient starvation is the best option to obtain high starch and flavonoid accumulation simultaneously in a short time for biofuels fermentation and natural products isolation.

Defining the consequences of genetic variation on a proteome–wide scale

PubMed Central

Chick, Joel M.; Munger, Steven C.; Simecek, Petr; Huttlin, Edward L.; Choi, Kwangbom; Gatti, Daniel M.; Raghupathy, Narayanan; Svenson, Karen L.; Churchill, Gary A.; Gygi, Steven P.

2016-01-01

Genetic variation modulates protein expression through both transcriptional and post-transcriptional mechanisms. To characterize the consequences of natural genetic diversity on the proteome, here we combine a multiplexed, mass spectrometry-based method for protein quantification with an emerging outbred mouse model containing extensive genetic variation from eight inbred founder strains. By measuring genome-wide transcript and protein expression in livers from 192 Diversity outbred mice, we identify 2,866 protein quantitative trait loci (pQTL) with twice as many local as distant genetic variants. These data support distinct transcriptional and post-transcriptional models underlying the observed pQTL effects. Using a sensitive approach to mediation analysis, we often identified a second protein or transcript as the causal mediator of distant pQTL. Our analysis reveals an extensive network of direct protein–protein interactions. Finally, we show that local genotype can provide accurate predictions of protein abundance in an independent cohort of collaborative cross mice. PMID:27309819

RNA-Seq-Based Transcript Structure Analysis with TrBorderExt.

PubMed

Wang, Yejun; Sun, Ming-An; White, Aaron P

2018-01-01

RNA-Seq has become a routine strategy for genome-wide gene expression comparisons in bacteria. Despite lower resolution in transcript border parsing compared with dRNA-Seq, TSS-EMOTE, Cappable-seq, Term-seq, and others, directional RNA-Seq still illustrates its advantages: low cost, quantification and transcript border analysis with a medium resolution (±10-20 nt). To facilitate mining of directional RNA-Seq datasets especially with respect to transcript structure analysis, we developed a tool, TrBorderExt, which can parse transcript start sites and termination sites accurately in bacteria. A detailed protocol is described in this chapter for how to use the software package step by step to identify bacterial transcript borders from raw RNA-Seq data. The package was developed with Perl and R programming languages, and is accessible freely through the website: http://www.szu-bioinf.org/TrBorderExt .

Mechanistic evaluation of the pros and cons of digital RT-LAMP for HIV-1 viral load quantification on a microfluidic device and improved efficiency via a two-step digital protocol.

PubMed

Sun, Bing; Shen, Feng; McCalla, Stephanie E; Kreutz, Jason E; Karymov, Mikhail A; Ismagilov, Rustem F

2013-02-05

Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ∼2% to ∼23%, by (i) using a more efficient reverse transcriptase, (ii) introducing RNase H to break up the DNA:RNA hybrid, and (iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from four patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient's HIV RNA. We learned the following three lessons from this work: (i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful validation is essential before a method is considered to provide absolute quantification; (ii) the sensitivity of dLAMP to the sequence of the target nucleic acid necessitates additional validation with patient samples carrying the full spectrum of mutations; (iii) for multistep digital amplification chemistries, such as a combination of reverse transcription with amplification, microfluidic devices may be used to decouple these steps from one another and to perform them under different, individually optimized conditions for improved efficiency.

Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei

2008-12-01

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection ofmore » target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.« less

The MNS glycophorin variant GP.Mur affects differential erythroid expression of Rh/RhAG transcripts.

PubMed

Hsu, K; Kuo, M-S; Yao, C-C; Cheng, H-C; Lin, H-J; Chan, Y-S; Lin, M

2017-10-01

The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/RhAG subcomplex. Glycophorin B (GPB) is a component of the Rh/RhAG subcomplex that is also structurally associated with glycophorin A (GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (RhAG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/RhAG expression at the transcript level. GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for RhAG, RhD and RhCcEe. Quantification by real-time PCR revealed significantly fewer RhAG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both RhAG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of RhAG and RhD, but not between that of RhAG and RhCcEe. Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to RhAG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of RhAG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/RhAG expressions prior to protein translation. © 2017 International Society of Blood Transfusion.

Quantification of EVI1 transcript levels in acute myeloid leukemia by RT-qPCR analysis: A study by the ALFA Group.

PubMed

Smol, Thomas; Nibourel, Olivier; Marceau-Renaut, Alice; Celli-Lebras, Karine; Berthon, Céline; Quesnel, Bruno; Boissel, Nicolas; Terré, Christine; Thomas, Xavier; Castaigne, Sylvie; Dombret, Hervé; Preudhomme, Claude; Renneville, Aline

2015-12-01

EVI1 overexpression confers poor prognosis in acute myeloid leukemia (AML). Quantification of EVI1 expression has been mainly assessed by real-time quantitative PCR (RT-qPCR) based on relative quantification of EVI1-1D splice variant. In this study, we developed a RT-qPCR assay to perform quantification of EVI1 expression covering the different splice variants. A sequence localized in EVI1 exons 14 and 15 was cloned into plasmids that were used to establish RT-qPCR standard curves. Threshold values to define EVI1 overexpression were determined using 17 bone marrow (BM) and 31 peripheral blood (PB) control samples and were set at 1% in BM and 0.5% in PB. Samples from 64 AML patients overexpressing EVI1 included in the ALFA-0701 or -0702 trials were collected at diagnosis and during follow-up (n=152). Median EVI1 expression at AML diagnosis was 23.3% in BM and 3.6% in PB. EVI1 expression levels significantly decreased between diagnostic and post-induction samples, with an average variation from 21.6% to 3.56% in BM and from 4.0% to 0.22% in PB, but did not exceed 1 log10 reduction. Our study demonstrates that the magnitude of reduction in EVI1 expression levels between AML diagnosis and follow-up is not sufficient to allow sensitive detection of minimal residual disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Linear RNA amplification for the production of microarray hybridization probes.

PubMed

Klebes, Ansgar; Kornberg, Thomas B

2008-01-01

To understand Drosophila development and other genetically controlled processes, it is often desirable to identify differences in gene expression levels. An experimental approach to investigate these processes is to catalog the transcriptome by hybridization of mRNA to DNA microbar-rays. In these experiments mRNA-derived hybridization probes are produced and hybridized to an array of DNA spots on a solid support. The labeled cDNAs of the complex hybridization probe will bind to their complementary sequences and provide quantification of the relative concentration of the corresponding transcript in the starting material. However, such approaches are often limited by the scarcity of the experimental sample because standard methods of probe preparation require microgram quantities of mRNA template. Linear RNA amplification can alleviate such limitations to support the generation of microarray hybridization probes from a few 100 pg of mRNA. These smaller quantities can be isolated from a few 100 cells. Here, we present a linear amplification protocol designed to preserve both the relative abundance of transcripts as well as their sequence complexity.

Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia.

PubMed

Wattjes, M P; Krauter, J; Nagel, S; Heidenreich, O; Ganser, A; Heil, G

2000-02-01

The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leukemia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hematological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel competitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both techniques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages.

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring*

PubMed Central

Lawless, Craig; Holman, Stephen W.; Brownridge, Philip; Lanthaler, Karin; Harman, Victoria M.; Watkins, Rachel; Hammond, Dean E.; Miller, Rebecca L.; Sims, Paul F. G.; Grant, Christopher M.; Eyers, Claire E.; Beynon, Robert J.

2016-01-01

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. PMID:26750110

Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

PubMed

Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

2008-12-15

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)

PubMed Central

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults. PMID:26998411

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

PubMed

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been; Yang, Wei Cheng

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Digital Droplet PCR: CNV Analysis and Other Applications.

PubMed

Mazaika, Erica; Homsy, Jason

2014-07-14

Digital droplet PCR (ddPCR) is an assay that combines state-of-the-art microfluidics technology with TaqMan-based PCR to achieve precise target DNA quantification at high levels of sensitivity and specificity. Because quantification is achieved without the need for standard assays in an easy to interpret, unambiguous digital readout, ddPCR is far simpler, faster, and less error prone than real-time qPCR. The basic protocol can be modified with minor adjustments to suit a wide range of applications, such as CNV analysis, rare variant detection, SNP genotyping, and transcript quantification. This unit describes the ddPCR workflow in detail for the Bio-Rad QX100 system, but the theory and data interpretation are generalizable to any ddPCR system. Copyright © 2014 John Wiley & Sons, Inc.

Structure & Coupling of Semiotic Sets

NASA Astrophysics Data System (ADS)

Orsucci, Franco; Giuliani, Alessandro; Zbilut, Joseph

2004-12-01

We investigated the informational structure of written texts (also in the form of speech transcriptions) using Recurrence Quantification Analysis (RQA). RQA technique provides a quantitative description of text sequences at the orthographic level in terms of structuring, and may be useful for a variety of linguistics-related studies. We used RQA to measure differences in linguistic samples from different subjects. They were divided in subgroups based on personality and culture differences. We used RQA and KRQA (Cross Recurrence) to measure the coupling and synchronization during the conversation (semiotic interaction) of different subjects. We discuss results both for the improvement of methodology and some general implications for neurocognitive science.

Genome-wide Determinants of Proviral Targeting, Clonal Abundance and Expression in Natural HTLV-1 Infection

PubMed Central

Melamed, Anat; Laydon, Daniel J.; Gillet, Nicolas A.; Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

2013-01-01

The regulation of proviral latency is a central problem in retrovirology. We postulate that the genomic integration site of human T lymphotropic virus type 1 (HTLV-1) determines the pattern of expression of the provirus, which in turn determines the abundance and pathogenic potential of infected T cell clones in vivo. We recently developed a high-throughput method for the genome-wide amplification, identification and quantification of proviral integration sites. Here, we used this protocol to test two hypotheses. First, that binding sites for transcription factors and chromatin remodelling factors in the genome flanking the proviral integration site of HTLV-1 are associated with integration targeting, spontaneous proviral expression, and in vivo clonal abundance. Second, that the transcriptional orientation of the HTLV-1 provirus relative to that of the nearest host gene determines spontaneous proviral expression and in vivo clonal abundance. Integration targeting was strongly associated with the presence of a binding site for specific host transcription factors, especially STAT1 and p53. The presence of the chromatin remodelling factors BRG1 and INI1 and certain host transcription factors either upstream or downstream of the provirus was associated respectively with silencing or spontaneous expression of the provirus. Cells expressing HTLV-1 Tax protein were significantly more frequent in clones of low abundance in vivo. We conclude that transcriptional interference and chromatin remodelling are critical determinants of proviral latency in natural HTLV-1 infection. PMID:23555266

The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

DOE PAGES

Xiong, Yi; Coradetti, Samuel T.; Li, Xin; ...

2014-05-29

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggestsmore » that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.« less

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

PubMed Central

Masvidal, Laia; Igreja, Susana; Ramos, Maria D; Alvarez, Antoni; de Gracia, Javier; Ramalho, Anabela; Amaral, Margarida D; Larriba, Sara; Casals, Teresa

2014-01-01

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression. PMID:24129438

Edinburgh Working Papers in Applied Linguistics, 1998.

ERIC Educational Resources Information Center

Parkinson, Brian, Ed.

1998-01-01

Papers on applied linguistics and language pedagogy include: "Non-Exact Quantification in Slide Presentations of Medical Research" (Ron Howard); "Modality and Point of View: A Contrastive Analysis of Japanese Wartime and Peacetime Newspaper Discourse" (Noriko Iwamoto); "Classroom Transcripts and 'Noticing' in Teacher Education" (Tony Lynch);…

IAOseq: inferring abundance of overlapping genes using RNA-seq data.

PubMed

Sun, Hong; Yang, Shuang; Tun, Liangliang; Li, Yixue

2015-01-01

Overlapping transcription constitutes a common mechanism for regulating gene expression. A major limitation of the overlapping transcription assays is the lack of high throughput expression data. We developed a new tool (IAOseq) that is based on reads distributions along the transcribed regions to identify the expression levels of overlapping genes from standard RNA-seq data. Compared with five commonly used quantification methods, IAOseq showed better performance in the estimation accuracy of overlapping transcription levels. For the same strand overlapping transcription, currently existing high-throughput methods are rarely available to distinguish which strand was present in the original mRNA template. The IAOseq results showed that the commonly used methods gave an average of 1.6 fold overestimation of the expression levels of same strand overlapping genes. This work provides a useful tool for mining overlapping transcription levels from standard RNA-seq libraries. IAOseq could be used to help us understand the complex regulatory mechanism mediated by overlapping transcripts. IAOseq is freely available at http://lifecenter.sgst.cn/main/en/IAO_seq.jsp.

Gene expression profiling of human breast tissue samples using SAGE-Seq.

PubMed

Wu, Zhenhua Jeremy; Meyer, Clifford A; Choudhury, Sibgat; Shipitsin, Michail; Maruyama, Reo; Bessarabova, Marina; Nikolskaya, Tatiana; Sukumar, Saraswati; Schwartzman, Armin; Liu, Jun S; Polyak, Kornelia; Liu, X Shirley

2010-12-01

We present a powerful application of ultra high-throughput sequencing, SAGE-Seq, for the accurate quantification of normal and neoplastic mammary epithelial cell transcriptomes. We develop data analysis pipelines that allow the mapping of sense and antisense strands of mitochondrial and RefSeq genes, the normalization between libraries, and the identification of differentially expressed genes. We find that the diversity of cancer transcriptomes is significantly higher than that of normal cells. Our analysis indicates that transcript discovery plateaus at 10 million reads/sample, and suggests a minimum desired sequencing depth around five million reads. Comparison of SAGE-Seq and traditional SAGE on normal and cancerous breast tissues reveals higher sensitivity of SAGE-Seq to detect less-abundant genes, including those encoding for known breast cancer-related transcription factors and G protein-coupled receptors (GPCRs). SAGE-Seq is able to identify genes and pathways abnormally activated in breast cancer that traditional SAGE failed to call. SAGE-Seq is a powerful method for the identification of biomarkers and therapeutic targets in human disease.

Transcriptomic Studies of the Effect of nod Gene-Inducing Molecules in Rhizobia: Different Weapons, One Purpose

PubMed Central

Jiménez-Guerrero, Irene; Acosta-Jurado, Sebastián; Navarro-Gómez, Pilar; López-Baena, Francisco Javier; Ollero, Francisco Javier

2017-01-01

Simultaneous quantification of transcripts of the whole bacterial genome allows the analysis of the global transcriptional response under changing conditions. RNA-seq and microarrays are the most used techniques to measure these transcriptomic changes, and both complement each other in transcriptome profiling. In this review, we exhaustively compiled the symbiosis-related transcriptomic reports (microarrays and RNA sequencing) carried out hitherto in rhizobia. This review is specially focused on transcriptomic changes that takes place when five rhizobial species, Bradyrhizobium japonicum (=diazoefficiens) USDA 110, Rhizobium leguminosarum biovar viciae 3841, Rhizobium tropici CIAT 899, Sinorhizobium (=Ensifer) meliloti 1021 and S. fredii HH103, recognize inducing flavonoids, plant-exuded phenolic compounds that activate the biosynthesis and export of Nod factors (NF) in all analysed rhizobia. Interestingly, our global transcriptomic comparison also indicates that each rhizobial species possesses its own arsenal of molecular weapons accompanying the set of NF in order to establish a successful interaction with host legumes. PMID:29267254

QUANTIFICATION AND ASSOCIATED VARIABILITY OF INDUCED VITELLOGENIN GENE TRANSCRIPTS IN FATHEAD MINNOW (PIMEPHALES PROMELAS) BY QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION ASSAY

EPA Science Inventory

Ecological risk assessors have a growing need for sensitive and rapid indicators of environmental exposure in aquatic ecosystems resulting from natural and synthetic estrogen-like compounds. Investigators developing subcellular exposure markers in traditional sentinel organisms m...

A proteomic insight into vitellogenesis during tick ovary maturation.

PubMed

Xavier, Marina Amaral; Tirloni, Lucas; Pinto, Antônio F M; Diedrich, Jolene K; Yates, John R; Mulenga, Albert; Logullo, Carlos; da Silva Vaz, Itabajara; Seixas, Adriana; Termignoni, Carlos

2018-03-16

Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding of tick oogenesis and embryogenesis could contribute to the development of novel control methods. However, to date, studies on the temporal dynamics of proteins during ovary development were not reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of 3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the annotated proteins belong to categories related to basal metabolism, such as protein synthesis and modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/detoxification metabolism, signal transduction, and storage. The abundance of selected proteins involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide a framework for future studies to elucidate tick development and validate candidate targets for novel control methods.

Hepatitis E Virus (HEV) Detection and Quantification by a Real-Time Reverse Transcription-PCR Assay Calibrated to the World Health Organization Standard for HEV RNA

PubMed Central

Germer, Jeffrey J.; Ankoudinova, Irina; Belousov, Yevgeniy S.; Mahoney, Walt; Dong, Chen; Meng, Jihong; Mandrekar, Jayawant N.

2017-01-01

ABSTRACT Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA. PMID:28228493

Specific metabolic activity of ripening bacteria quantified by real-time reverse transcription PCR throughout Emmental cheese manufacture.

PubMed

Falentin, Hélène; Postollec, Florence; Parayre, Sandrine; Henaff, Nadine; Le Bivic, Pierre; Richoux, Romain; Thierry, Anne; Sohier, Danièle

2010-11-15

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 10(3) copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese. Copyright © 2010 Elsevier B.V. All rights reserved.

Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis*

PubMed Central

Wong, Ming-Kin; Guan, Daogang; Ng, Kaoru Hon Chun; Ho, Vincy Wing Sze; An, Xiaomeng; Li, Runsheng; Ren, Xiaoliang

2016-01-01

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development. PMID:27056332

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity

PubMed Central

Traverse, Charles C.

2017-01-01

ABSTRACT Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola, which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. PMID:28851848

Inference of alternative splicing from RNA-Seq data with probabilistic splice graphs

PubMed Central

LeGault, Laura H.; Dewey, Colin N.

2013-01-01

Motivation: Alternative splicing and other processes that allow for different transcripts to be derived from the same gene are significant forces in the eukaryotic cell. RNA-Seq is a promising technology for analyzing alternative transcripts, as it does not require prior knowledge of transcript structures or genome sequences. However, analysis of RNA-Seq data in the presence of genes with large numbers of alternative transcripts is currently challenging due to efficiency, identifiability and representation issues. Results: We present RNA-Seq models and associated inference algorithms based on the concept of probabilistic splice graphs, which alleviate these issues. We prove that our models are often identifiable and demonstrate that our inference methods for quantification and differential processing detection are efficient and accurate. Availability: Software implementing our methods is available at http://deweylab.biostat.wisc.edu/psginfer. Contact: cdewey@biostat.wisc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23846746

Detection and quantification of serum or plasma HCV RNA: mini review of commercially available assays.

PubMed

Le Guillou-Guillemette, Helene; Lunel-Fabiani, Francoise

2009-01-01

The treatment schedule (combination of compounds, doses, and duration) and the virological follow-up for management of antiviral treatment in patients chronically infected by HCV is now well standardized, but to ensure good monitoring of the treated patients, physicians need rapid, reproducible, and sensitive molecular virological tools with a wide range of detection and quantification of HCV RNA in blood samples. Several assays for detection and/or quantification of HCV RNA are currently commercially available. Here, all these assays are detailed, and a brief description of each step of the assay is provided. They are divided into two categories by method: those based on signal amplification and those based on target amplification. These two categories are then divided into qualitative, quantitative, and quantitative detection assays. The real-time reverse-transcription polymerase chain reaction (RT-PCR)-based assays are the most promising strategy in the HCV virological area.

Quantification of hsp70 mRNA from the Cryptosporidium parvum in soil by reverse transcription real-time PCR

EPA Science Inventory

As one of the leading causes of waterborne enteric disease, Cryptosporidium parvum poses significant threat to public health. Besides water, soil can also become an important environmental source of C. parvum once polluted. Detection of viable C. parvum in soil is a key issue whe...

In vitro quantitative and relative gene expression analysis of pancreatic transcription factors Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1 in trans-differentiated human hepatic progenitors.

PubMed

Vishwakarma, Sandeep Kumar; Rahamathulla, Syed; Bardia, Avinash; Tiwari, Santosh K; Srinivas, Gunda; Raj, Avinash; Tripura, Chaturvedula; Sandhya, Annamaneni; Habeeb, Mohammed Aejaz; Khan, Aleem A; Pande, Gopal; Reddy, K Pratap; Reddy, P Yugandhar

2014-09-01

Diabetes is a major health concern throughout the world because of its increasing prevalence in epidemic proportions. β-Cell deterioration in the pancreas is a crucial factor for the progression of diabetes mellitus. Therefore, the restoration of β-cell mass and its function is of vital importance for the development of effective therapeutic strategies and most accessible cell sources for the treatment of diabetes mellitus. Human fetuses (12-20 weeks gestation age) were used to isolate human hepatic progenitor cells (hHPCs) from fetal liver using a two-step collagenase digestion method. Epithelial cell adhesion molecule-positive (EpCAM+ve)-enriched hHPCs were cultured in vitro and induced with 5-30 mmol/L concentration of glucose for 0-32 h. Pdx-1 expression and insulin secretion was analyzed using immunophenotypic and chemifluorescence assays, respectively. Relative gene expression was quantified in induced hHPCs, and compared with uninduced and pancreatic cells to identify the activated transcription factors (Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1) involved in β-cell production. EpCAM+ve cells derived from human fetal liver showed high in vitro trans-differentiation potential towards the β-cell phenotype with 23 mmol/L glucose induction after 24 h. The transcription factors showed eminent expression in induced cells. The expression level of transcription factors was found significantly high in 23 mmol/L-induced hHPCs as compared with the uninduced cells. The present study has shown an exciting new insight into β-cell development from hHPCs trans-differentiation. Relative quantification of gene expression in trans-differentiated cells offers vast possibility for the production of a maximum number of functionally active pancreatic β-cells for a future cure of diabetes.

Evaluation of digital PCR for absolute RNA quantification.

PubMed

Sanders, Rebecca; Mason, Deborah J; Foy, Carole A; Huggett, Jim F

2013-01-01

Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.

A Java program for LRE-based real-time qPCR that enables large-scale absolute quantification.

PubMed

Rutledge, Robert G

2011-03-02

Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

PubMed Central

Rutledge, Robert G.

2011-01-01

Background Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples. PMID:21407812

Community structure of the metabolically active rumen bacterial and archaeal communities of dairy cows over the transition period

PubMed Central

Zhu, Zhigang; Noel, Samantha Joan; Difford, Gareth Frank; Al-Soud, Waleed Abu; Brejnrod, Asker; Sørensen, Søren Johannes; Lassen, Jan; Løvendahl, Peter; Højberg, Ole

2017-01-01

Dairy cows experience dramatic changes in host physiology from gestation to lactation period and dietary switch from high-forage prepartum diet to high-concentrate postpartum diet over the transition period (parturition +/- three weeks). Understanding the community structure and activity of the rumen microbiota and its associative patterns over the transition period may provide insight for e.g. improving animal health and production. In the present study, rumen samples from ten primiparous Holstein dairy cows were collected over seven weeks spanning the transition period. Total RNA was extracted from the rumen samples and cDNA thereof was subsequently used for characterizing the metabolically active bacterial (16S rRNA transcript amplicon sequencing) and archaeal (qPCR, T-RFLP and mcrA and 16S rRNA transcript amplicon sequencing) communities. The metabolically active bacterial community was dominated by three phyla, showing significant changes in relative abundance range over the transition period: Firmicutes (from prepartum 57% to postpartum 35%), Bacteroidetes (from prepartum 22% to postpartum 18%) and Proteobacteria (from prepartum 7% to postpartum 32%). For the archaea, qPCR analysis of 16S rRNA transcript number, revealed a significant prepartum to postpartum increase in Methanobacteriales, in accordance with an observed increase (from prepartum 80% to postpartum 89%) in relative abundance of 16S rRNA transcript amplicons allocated to this order. On the other hand, a significant prepartum to postpartum decrease (from 15% to 2%) was observed in relative abundance of Methanomassiliicoccales 16S rRNA transcripts. In contrast to qPCR analysis of the 16S rRNA transcripts, quantification of mcrA transcripts revealed no change in total abundance of metabolically active methanogens over the transition period. According to T-RFLP analysis of the mcrA transcripts, two Methanobacteriales genera, Methanobrevibacter and Methanosphaera (represented by the T-RFs 39 and 267 bp), represented more than 70% of the metabolically active methanogens, showing no significant changes over the transition period; minor T-RFs, likely to represent members of the order Methanomassiliicoccales and with a relative abundance below 5% in total, decreased significantly over the transition period. In accordance with the T-RFLP analysis, the mcrA transcript amplicon sequencing revealed Methanobacteriales to cover 99% of the total reads, dominated by the genera Methanobrevibacter (75%) and Methanosphaera (24%), whereas the Methanomassiliicoccales order covered only 0.2% of the total reads. In conclusion, the present study showed that the structure of the metabolically active bacterial and archaeal rumen communities changed over the transition period, likely in response to the dramatic changes in physiology and nutritional factors like dry matter intake and feed composition. It should be noted however that for the methanogens, the observed community changes were influenced by the analyzed gene (mcrA or 16S rRNA). PMID:29117259

Detection of mRNA by reverse transcription PCR as an indicator of viability in Phytophthora ramorum

Treesearch

Antonio Chimento; Santa Olga Cacciola; Matteo Garbelotto

2008-01-01

Real-Time PCR technologies offer increasing opportunities to detect and study phytopathogenic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing both real-time analysis of the reaction kinetics and quantification of specific DNA targets. Before the development of Real-Time PCR and...

Conserved and divergent rhythms of crassulacean acid metabolism-related and core clock gene expression in the cactus Opuntia ficus-indica.

PubMed

Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

2011-08-01

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional levels.

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

PubMed Central

Tiziano, Francesco Danilo; Pinto, Anna Maria; Fiori, Stefania; Lomastro, Rosa; Messina, Sonia; Bruno, Claudio; Pini, Antonella; Pane, Marika; D'Amico, Adele; Ghezzo, Alessandro; Bertini, Enrico; Mercuri, Eugenio; Neri, Giovanni; Brahe, Christina

2010-01-01

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I–III) on the basis of clinical severity. All patients have at least one or more (usually 2–4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II–III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials. PMID:19603064

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

PubMed

Muhar, Matthias; Ebert, Anja; Neumann, Tobias; Umkehrer, Christian; Jude, Julian; Wieshofer, Corinna; Rescheneder, Philipp; Lipp, Jesse J; Herzog, Veronika A; Reichholf, Brian; Cisneros, David A; Hoffmann, Thomas; Schlapansky, Moritz F; Bhat, Pooja; von Haeseler, Arndt; Köcher, Thomas; Obenauf, Anna C; Popow, Johannes; Ameres, Stefan L; Zuber, Johannes

2018-05-18

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Image-based quantification and mathematical modeling of spatial heterogeneity in ESC colonies.

PubMed

Herberg, Maria; Zerjatke, Thomas; de Back, Walter; Glauche, Ingmar; Roeder, Ingo

2015-06-01

Pluripotent embryonic stem cells (ESCs) have the potential to differentiate into cells of all three germ layers. This unique property has been extensively studied on the intracellular, transcriptional level. However, ESCs typically form clusters of cells with distinct size and shape, and establish spatial structures that are vital for the maintenance of pluripotency. Even though it is recognized that the cells' arrangement and local interactions play a role in fate decision processes, the relations between transcriptional and spatial patterns have not yet been studied. We present a systems biology approach which combines live-cell imaging, quantitative image analysis, and multiscale, mathematical modeling of ESC growth. In particular, we develop quantitative measures of the morphology and of the spatial clustering of ESCs with different expression levels and apply them to images of both in vitro and in silico cultures. Using the same measures, we are able to compare model scenarios with different assumptions on cell-cell adhesions and intercellular feedback mechanisms directly with experimental data. Applying our methodology to microscopy images of cultured ESCs, we demonstrate that the emerging colonies are highly variable regarding both morphological and spatial fluorescence patterns. Moreover, we can show that most ESC colonies contain only one cluster of cells with high self-renewing capacity. These cells are preferentially located in the interior of a colony structure. The integrated approach combining image analysis with mathematical modeling allows us to reveal potential transcription factor related cellular and intercellular mechanisms behind the emergence of observed patterns that cannot be derived from images directly. © 2015 International Society for Advancement of Cytometry.

Hepatitis E Virus (HEV) Detection and Quantification by a Real-Time Reverse Transcription-PCR Assay Calibrated to the World Health Organization Standard for HEV RNA.

PubMed

Germer, Jeffrey J; Ankoudinova, Irina; Belousov, Yevgeniy S; Mahoney, Walt; Dong, Chen; Meng, Jihong; Mandrekar, Jayawant N; Yao, Joseph D

2017-05-01

Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA. Copyright © 2017 American Society for Microbiology.

Adhesion signaling promotes protease‑driven polyploidization of glioblastoma cells.

PubMed

Mercapide, Javier; Lorico, Aurelio

2014-11-01

An increase in ploidy (polyploidization) causes genomic instability in cancer. However, the determinants for the increased DNA content of cancer cells have not yet been fully elucidated. In the present study, we investigated whether adhesion induces polyploidization in human U87MG glioblastoma cells. For this purpose, we employed expression vectors that reported transcriptional activation by signaling networks implicated in cancer. Signaling activation induced by intercellular integrin binding elicited both extracellular signal‑regulated kinase (ERK) and Notch target transcription. Upon the prolonged activation of both ERK and Notch target transcription induced by integrin binding to adhesion protein, cell cultures accumulated polyploid cells, as determined by cell DNA content distribution analysis and the quantification of polynucleated cells. This linked the transcriptional activation induced by integrin adhesion to the increased frequency of polyploidization. Accordingly, the inhibition of signaling decreased the extent of polyploidization mediated by protease‑driven intracellular invasion. Therefore, the findings of this study indicate that integrin adhesion induces polyploidization through the stimulation of glioblastoma cell invasiveness.

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification.

PubMed

Guan, Weihua; Chen, Liben; Rane, Tushar D; Wang, Tza-Huei

2015-09-03

We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

PubMed Central

Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

2015-01-01

We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples. PMID:26333806

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

NASA Astrophysics Data System (ADS)

Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

2015-09-01

We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

Detection of novel NF1 mutations and rapid mutation prescreening with Pyrosequencing.

PubMed

Brinckmann, Anja; Mischung, Claudia; Bässmann, Ingelore; Kühnisch, Jirko; Schuelke, Markus; Tinschert, Sigrid; Nürnberg, Peter

2007-12-01

Neurofibromatosis type 1 (NF1) is caused by mutations in the neurofibromin (NF1) gene. Mutation analysis of NF1 is complicated by its large size, the lack of mutation hotspots, pseudogenes and frequent de novo mutations. Additionally, the search for NF1 mutations on the mRNA level is often hampered by nonsense-mediated mRNA decay (NMD) of the mutant allele. In this study we searched for mutations in a cohort of 38 patients and investigated the relationship between mutation type and allele-specific transcription from the wild-type versus mutant alleles. Quantification of relative mRNA transcript numbers was done by Pyrosequencing, a novel real-time sequencing method whose signals can be quantified very accurately. We identified 21 novel mutations comprising various mutation types. Pyrosequencing detected a definite relationship between allelic NF1 transcript imbalance due to NMD and mutation type in 24 of 29 patients who all carried frame-shift or nonsense mutations. NMD was absent in 5 patients with missense and silent mutations, as well as in 4 patients with splice-site mutations that did not disrupt the reading frame. Pyrosequencing was capable of detecting NMD even when the effects were only moderate. Diagnostic laboratories could thus exploit this effect for rapid prescreening for NF1 mutations as more than 60% of the mutations in this gene disrupt the reading frame and are prone to NMD.

In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

PubMed

Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

2006-01-01

Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

PubMed Central

Wu, Jing Qin; Wang, Xi; Beveridge, Natalie J.; Tooney, Paul A.; Scott, Rodney J.; Carr, Vaughan J.; Cairns, Murray J.

2012-01-01

Background While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression. Methodology/Principal Findings The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05). Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1) gene. Conclusions This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia. PMID:22558445

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Tubulin C-terminal Post-translational Modifications Do Not Occur in Wood Forming Tissue of Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Hao; Gu, Xi; Xue, Liang-Jiao

Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem wheremore » high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.« less

Tubulin C-terminal Post-translational Modifications Do Not Occur in Wood Forming Tissue of Populus

DOE PAGES

Hu, Hao; Gu, Xi; Xue, Liang-Jiao; ...

2016-10-13

Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem wheremore » high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.« less

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1[w

PubMed Central

Czechowski, Tomasz; Stitt, Mark; Altmann, Thomas; Udvardi, Michael K.; Scheible, Wolf-Rüdiger

2005-01-01

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future. PMID:16166256

LocExpress: a web server for efficiently estimating expression of novel transcripts.

PubMed

Hou, Mei; Tian, Feng; Jiang, Shuai; Kong, Lei; Yang, Dechang; Gao, Ge

2016-12-22

The temporal and spatial-specific expression pattern of a transcript in multiple tissues and cell types can indicate key clues about its function. While several gene atlas available online as pre-computed databases for known gene models, it's still challenging to get expression profile for previously uncharacterized (i.e. novel) transcripts efficiently. Here we developed LocExpress, a web server for efficiently estimating expression of novel transcripts across multiple tissues and cell types in human (20 normal tissues/cells types and 14 cell lines) as well as in mouse (24 normal tissues/cell types and nine cell lines). As a wrapper to RNA-Seq quantification algorithm, LocExpress efficiently reduces the time cost by making abundance estimation calls increasingly within the minimum spanning bundle region of input transcripts. For a given novel gene model, such local context-oriented strategy allows LocExpress to estimate its FPKMs in hundreds of samples within minutes on a standard Linux box, making an online web server possible. To the best of our knowledge, LocExpress is the only web server to provide nearly real-time expression estimation for novel transcripts in common tissues and cell types. The server is publicly available at http://loc-express.cbi.pku.edu.cn .

Dehydration-induced WRKY genes from tobacco and soybean respond to jasmonic acid treatments in BY-2 cell culture.

PubMed

Rabara, Roel C; Tripathi, Prateek; Lin, Jun; Rushton, Paul J

2013-02-15

Drought is one of the important environmental factors affecting crop production worldwide and therefore understanding the molecular response of plant to stress is an important step in crop improvement. WRKY transcription factors are one of the 10 largest transcription factor families across the green lineage. In this study, highly upregulated dehydration-induced WRKY and enzyme-coding genes from tobacco and soybean were selected from microarray data for promoter analyses. Putative stress-related cis-regulatory elements such as TGACG motif, ABRE-like elements; W and G-like sequences were identified by an in silico analyses of promoter region of the selected genes. GFP quantification of transgenic BY-2 cell culture showed these promoters direct higher expression in-response to 100 μM JA treatment compared to 100 μM ABA, 10% PEG and 85 mM NaCl treatments. Thus promoter activity upon JA treatment and enrichment of MeJA-responsive elements in the promoter of the selected genes provides insights for these genes to be jasmonic acid responsive with potential of mediating cross-talk during dehydration responses. Copyright © 2013 Elsevier Inc. All rights reserved.

Target molecules in 3T3-L1 adipocytes differentiation are regulated by maslinic acid, a natural triterpene from Olea europaea.

PubMed

Pérez-Jiménez, Amalia; Rufino-Palomares, Eva E; Fernández-Gallego, Nieves; Ortuño-Costela, M Carmen; Reyes-Zurita, Fernando J; Peragón, Juan; García-Salguero, Leticia; Mokhtari, Khalida; Medina, Pedro P; Lupiáñez, José A

2016-11-15

Metabolic syndrome is a set of pathologies among which stand out the obesity, which is related to the lipid droplet accumulation and changes to cellular morphology regulated by several molecules and transcription factors. Maslinic acid (MA) is a natural product with demonstrated pharmacological functions including anti-inflammation, anti-tumor and anti-oxidation, among others. Here we report the effects of MA on the adipogenesis process in 3T3-L1 cells. Cell viability, glucose uptake, cytoplasmic triglyceride droplets, triglycerides quantification, gene transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte fatty acid-binding protein (aP2) and intracellular Ca 2+ levels were determined in pre-adipocytes and adipocytes of 3T3-L1 cells. MA increased glucose uptake. MA also decreased lipid droplets and triglyceride levels, which is in concordance with the down-regulation of PPARγ and aP2. Finally, MA increased the intracellular Ca 2+ concentration, which could also be involved in the demonstrated antiadipogenic effect of this triterpene. MA has been demonstrated as potential antiadipogenic compound in 3T3-L1 cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

Stemness-Related Transcriptional Factors and Homing Gene Expression Profiles in Hepatic Differentiation and Cancer

PubMed Central

Toraih, Eman A; Fawzy, Manal S; El-Falouji, Abdullah I; Hamed, Elham O; Nemr, Nader A; Hussein, Mohammad H; Fadeal, Noha M Abd El

2016-01-01

Stem cell transcriptional signature activation is an essential event in the development of cancer. This study aimed to investigate the differential expression profiles of three pluripotency-associated genes, OCT4, NANOG and SOX2, G-protein-coupled chemokine receptor 4 (CXCR4) and the ligand CXCL2, and alpha-fetoprotein (AFP) in hepatogenic differentiated stem cells and in sera of hepatitis C virus (HCV) and HCV-induced hepatocellular carcinoma (HCC) patients. Mesenchymal stem cells derived from umbilical cord blood were differentiated using hepatogenic differentiation media. Serum specimens were collected from 96 patients (32 cirrhotic HCV, 32 early HCC and 32 late HCC) and 96 controls. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for relative quantification of the six target genes using the Livak method. In silico network analysis was also executed to explore the pluripotency and tumorigenetic regulatory circuits in liver cancer. The expression levels of all genes declined gradually during the stages of stem cell differentiation. On univariate and multivariate analyses, NANOG, CXCR4 and AFP were significantly upregulated in late clinical stage HCC patients. In contrast, SOX2 and CXCL2 were markedly overexpressed in cirrhotic patients and could be used for clear demarcation between cirrhotic and HCC patients in our cases. In conclusion, our data highlight the potential role of the SOX2 stem cell marker and CXCL2 chemokine in liver cell degeneration and fibrogenesis in HCV-induced hepatic cirrhosis in our sample of the Egyptian population. In addition, the significant association of NANOG and CXCR4 high expression with late HCC could contribute to the acquisition of stem cell–like properties in hepatic cancer and dissemination in late stages, respectively. Taken together, our results could have potential application in HCC prognosis and treatment. PMID:27623812

Quantification of multiple gene expression in individual cells.

PubMed

Peixoto, António; Monteiro, Marta; Rocha, Benedita; Veiga-Fernandes, Henrique

2004-10-01

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

PubMed

El-Heliebi, Amin; Hille, Claudia; Laxman, Navya; Svedlund, Jessica; Haudum, Christoph; Ercan, Erkan; Kroneis, Thomas; Chen, Shukun; Smolle, Maria; Rossmann, Christopher; Krzywkowski, Tomasz; Ahlford, Annika; Darai, Evangelia; von Amsberg, Gunhild; Alsdorf, Winfried; König, Frank; Löhr, Matthias; de Kruijff, Inge; Riethdorf, Sabine; Gorges, Tobias M; Pantel, Klaus; Bauernhofer, Thomas; Nilsson, Mats; Sedlmayr, Peter

2018-03-01

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices. © 2017 American Association for Clinical Chemistry.

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

Evolutionary Convergence of Cell-Specific Gene Expression in Independent Lineages of C4 Grasses1[W][OPEN

PubMed Central

John, Christopher R.; Smith-Unna, Richard D.; Woodfield, Helen; Covshoff, Sarah; Hibberd, Julian M.

2014-01-01

Leaves of almost all C4 lineages separate the reactions of photosynthesis into the mesophyll (M) and bundle sheath (BS). The extent to which messenger RNA profiles of M and BS cells from independent C4 lineages resemble each other is not known. To address this, we conducted deep sequencing of RNA isolated from the M and BS of Setaria viridis and compared these data with publicly available information from maize (Zea mays). This revealed a high correlation (r = 0.89) between the relative abundance of transcripts encoding proteins of the core C4 pathway in M and BS cells in these species, indicating significant convergence in transcript accumulation in these evolutionarily independent C4 lineages. We also found that the vast majority of genes encoding proteins of the C4 cycle in S. viridis are syntenic to homologs used by maize. In both lineages, 122 and 212 homologous transcription factors were preferentially expressed in the M and BS, respectively. Sixteen shared regulators of chloroplast biogenesis were identified, 14 of which were syntenic homologs in maize and S. viridis. In sorghum (Sorghum bicolor), a third C4 grass, we found that 82% of these trans-factors were also differentially expressed in either M or BS cells. Taken together, these data provide, to our knowledge, the first quantification of convergence in transcript abundance in the M and BS cells from independent lineages of C4 grasses. Furthermore, the repeated recruitment of syntenic homologs from large gene families strongly implies that parallel evolution of both structural genes and trans-factors underpins the polyphyletic evolution of this highly complex trait in the monocotyledons. PMID:24676859

Recent advances in stable isotope labeling based techniques for proteome relative quantification.

PubMed

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2014-10-24

The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

Genome-Scale Transcriptome Analysis in Response to Nitric Oxide in Birch Cells: Implications of the Triterpene Biosynthetic Pathway

PubMed Central

Zeng, Fansuo; Sun, Fengkun; Li, Leilei; Liu, Kun; Zhan, Yaguang

2014-01-01

Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10−5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis. PMID:25551661

Elongated Hypocotyl 5-Homolog (HYH) Negatively Regulates Expression of the Ambient Temperature-Responsive MicroRNA Gene MIR169

PubMed Central

Serivichyaswat, Phanu T.; Susila, Hendry; Ahn, Ji Hoon

2017-01-01

Arabidopsis microRNA169 (miR169) is an ambient temperature-responsive microRNA that plays an important role in stress responses and the floral transition. However, the transcription factors that regulate the expression of MIR169 have remained unknown. In this study, we show that Elongated Hypocotyl 5-Homolog (HYH) directly binds to the promoter of MIR169a and negatively regulates its expression. Absolute quantification identified MIR169a as the major locus producing miR169. GUS reporter assays revealed that the deletion of a 498-bp fragment (–1,505 to –1,007, relative to the major transcriptional start site) of MIR169a abolished its ambient temperature-responsive expression. DNA-affinity chromatography followed by liquid chromatography-mass spectrometry analysis identified transcription factor HYH as a trans-acting factor that binds to the 498-bp promoter fragment of pri-miR169a. Electrophoretic mobility shift assays and chromatin immunoprecipitation–quantitative PCR demonstrated that the HYH.2 protein, a predominant isoform of HYH, directly associated with a G-box-like motif in the 498-bp fragment of pri-miR169a. Higher enrichment of HYH.2 protein on the promoter region of MIR169a was seen at 23°C, consistent with the presence of more HYH.2 protein in the cell at the temperature. Transcript levels of pri-miR169a increased in hyh mutants and decreased in transgenic plants overexpressing HYH. Consistent with the negative regulation of MIR169a by HYH, the diurnal levels of HYH mRNA and pri-miR169a showed opposite patterns. Taken together, our results suggest that HYH is a transcription factor that binds to a G-box-like motif in the MIR169a promoter and negatively regulates ambient temperature-responsive expression of MIR169a at higher temperatures in Arabidopsis. PMID:29270188

Conserved and Divergent Rhythms of Crassulacean Acid Metabolism-Related and Core Clock Gene Expression in the Cactus Opuntia ficus-indica1[C][W

PubMed Central

Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

2011-01-01

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional levels. PMID:21677095

De novo transcriptome assembly and quantification reveal differentially expressed genes between soft-seed and hard-seed pomegranate (Punica granatum L.).

PubMed

Xue, Hui; Cao, Shangyin; Li, Haoxian; Zhang, Jie; Niu, Juan; Chen, Lina; Zhang, Fuhong; Zhao, Diguang

2017-01-01

Pomegranate (Punica granatum L.) belongs to Punicaceae, and is valued for its social, ecological, economic, and aesthetic values, as well as more recently for its health benefits. The 'Tunisia' variety has softer seeds and big arils that are easily swallowed. It is a widely popular fruit; however, the molecular mechanisms of the formation of hard and soft seeds is not yet clear. We conducted a de novo assembly of the seed transcriptome in P. granatum L. and revealed differential gene expression between the soft-seed and hard-seed pomegranate varieties. A total of 35.1 Gb of data were acquired in this study, including 280,881,106 raw reads. Additionally, de novo transcriptome assembly generated 132,287 transcripts and 105,743 representative unigenes; approximately 13,805 unigenes (37.7%) were longer than 1,000 bp. Using bioinformatics annotation libraries, a total of 76,806 unigenes were annotated and, among the high-quality reads, 72.63% had at least one significant match to an existing gene model. Gene expression and differentially expressed genes were analyzed. The seed formation of the two pomegranate cultivars involves lignin biosynthesis and metabolism, including some genes encoding laccase and peroxidase, WRKY, MYB, and NAC transcription factors. In the hard-seed pomegranate, lignin-related genes and cellulose synthesis-related genes were highly expressed; in soft-seed pomegranates, expression of genes related to flavonoids and programmed cell death was slightly higher. We validated selection of the identified genes using qRT-PCR. This is the first transcriptome analysis of P. granatum L. This transcription sequencing greatly enriched the pomegranate molecular database, and the high-quality SSRs generated in this study will aid the gene cloning from pomegranate in the future. It provides important insights into the molecular mechanisms underlying the formation of soft seeds in pomegranate.

De novo transcriptome assembly and quantification reveal differentially expressed genes between soft-seed and hard-seed pomegranate (Punica granatum L.)

PubMed Central

Xue, Hui; Cao, Shangyin; Li, Haoxian; Zhang, Jie; Niu, Juan; Chen, Lina; Zhang, Fuhong; Zhao, Diguang

2017-01-01

Pomegranate (Punica granatum L.) belongs to Punicaceae, and is valued for its social, ecological, economic, and aesthetic values, as well as more recently for its health benefits. The ‘Tunisia’ variety has softer seeds and big arils that are easily swallowed. It is a widely popular fruit; however, the molecular mechanisms of the formation of hard and soft seeds is not yet clear. We conducted a de novo assembly of the seed transcriptome in P. granatum L. and revealed differential gene expression between the soft-seed and hard-seed pomegranate varieties. A total of 35.1 Gb of data were acquired in this study, including 280,881,106 raw reads. Additionally, de novo transcriptome assembly generated 132,287 transcripts and 105,743 representative unigenes; approximately 13,805 unigenes (37.7%) were longer than 1,000 bp. Using bioinformatics annotation libraries, a total of 76,806 unigenes were annotated and, among the high-quality reads, 72.63% had at least one significant match to an existing gene model. Gene expression and differentially expressed genes were analyzed. The seed formation of the two pomegranate cultivars involves lignin biosynthesis and metabolism, including some genes encoding laccase and peroxidase, WRKY, MYB, and NAC transcription factors. In the hard-seed pomegranate, lignin-related genes and cellulose synthesis-related genes were highly expressed; in soft-seed pomegranates, expression of genes related to flavonoids and programmed cell death was slightly higher. We validated selection of the identified genes using qRT-PCR. This is the first transcriptome analysis of P. granatum L. This transcription sequencing greatly enriched the pomegranate molecular database, and the high-quality SSRs generated in this study will aid the gene cloning from pomegranate in the future. It provides important insights into the molecular mechanisms underlying the formation of soft seeds in pomegranate. PMID:28594931

Optimal Information Processing in Biochemical Networks

NASA Astrophysics Data System (ADS)

Wiggins, Chris

2012-02-01

A variety of experimental results over the past decades provide examples of near-optimal information processing in biological networks, including in biochemical and transcriptional regulatory networks. Computing information-theoretic quantities requires first choosing or computing the joint probability distribution describing multiple nodes in such a network --- for example, representing the probability distribution of finding an integer copy number of each of two interacting reactants or gene products while respecting the `intrinsic' small copy number noise constraining information transmission at the scale of the cell. I'll given an overview of some recent analytic and numerical work facilitating calculation of such joint distributions and the associated information, which in turn makes possible numerical optimization of information flow in models of noisy regulatory and biochemical networks. Illustrating cases include quantification of form-function relations, ideal design of regulatory cascades, and response to oscillatory driving.

High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae.

PubMed

Wang, Renjie; Normand, Christophe; Gadal, Olivier

2016-01-01

Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell's nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.

Novel paramyxoviruses in Australian flying-fox populations support host-virus co-evolution.

PubMed

Vidgen, Miranda E; de Jong, Carol; Rose, Karrie; Hall, Jane; Field, Hume E; Smith, Craig S

2015-07-01

Understanding the diversity of henipaviruses and related viruses is important in determining the viral ecology within flying-fox populations and assessing the potential threat posed by these agents. This study sought to identify the abundance and diversity of previously unknown paramyxoviruses (UPVs) in Australian flying-fox species (Pteropus alecto, Pteropus scapulatus, Pteropus poliocephalus and Pteropus conspicillatus) and in the Christmas Island species Pteropus melanotus natalis. Using a degenerative reverse transcription-PCR specific for the L gene of known species of the genus Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus, we identified an abundance and diversity of previously UPVs, with a representative 31 UPVs clustering in eight distinct groups (100 UPVs/495 samples). No new henipaviruses were identified. The findings were consistent with a hypothesis of co-evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-speciation between host and virus (beyond the scope of this study) would strengthen this hypothesis.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

PubMed

Stein, Erica V; Duewer, David L; Farkas, Natalia; Romsos, Erica L; Wang, Lili; Cole, Kenneth D

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR

PubMed Central

Duewer, David L.; Farkas, Natalia; Romsos, Erica L.; Wang, Lili; Cole, Kenneth D.

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values. PMID:29145448

Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR.

PubMed

Theis, Torsten; Skurray, Ronald A; Brown, Melissa H

2007-08-01

Quantitative real-time PCR (qRT-PCR) has become a routine technique for gene expression analysis. Housekeeping genes are customarily used as endogenous references for the relative quantification of genes of interest. The aim of this study was to develop a quantitative real-time PCR assay to analyze gene expression in multidrug resistant Staphylococcus aureus in the presence of cationic lipophilic substrates of multidrug transport proteins. Eleven different housekeeping genes were analyzed for their expression stability in the presence of a range of concentrations of four structurally different antimicrobial compounds. This analysis demonstrated that the genes rho, pyk and proC were least affected by rhodamine 6G and crystal violet, whereas fabD, tpiA and gyrA or fabD, proC and pyk were stably expressed in cultures grown in the presence of ethidium or berberine, respectively. Subsequently, these housekeeping genes were used as internal controls to analyze expression of the multidrug transport protein QacA and its transcriptional regulator QacR in the presence of the aforementioned compounds. Expression of qacA was induced by all four compounds, whereas qacR expression was found to be unaffected, reduced or enhanced. This study demonstrates that staphylococcal gene expression, including housekeeping genes previously used to normalize qRT-PCR data, is affected by growth in the presence of different antimicrobial compounds. Thus, identification of suitable genes usable as a control set requires rigorous testing. Identification of a such a set enabled them to be utilized as internal standards for accurate quantification of transcripts of the qac multidrug resistance system from S. aureus grown under different inducing conditions. Moreover, the qRT-PCR assay presented in this study may also be applied to gene expression studies of other multidrug transporters from S. aureus.

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora

Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow thatmore » with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several clinical and biological applications.« less

Diversity and spatial distribution of hydrazine oxidoreductase (hzo) gene in the oxygen minimum zone off Costa Rica.

PubMed

Kong, Liangliang; Jing, Hongmei; Kataoka, Takafumi; Buchwald, Carolyn; Liu, Hongbin

2013-01-01

Anaerobic ammonia oxidation (anammox) as an important nitrogen loss pathway has been reported in marine oxygen minimum zones (OMZs), but the community composition and spatial distribution of anammox bacteria in the eastern tropical North Pacific (ETNP) OMZ are poorly determined. In this study, anammox bacterial communities in the OMZ off Costa Rica (CRD-OMZ) were analyzed based on both hydrazine oxidoreductase (hzo) genes and their transcripts assigned to cluster 1 and 2. The anammox communities revealed by hzo genes and proteins in CRD-OMZ showed a low diversity. Gene quantification results showed that hzo gene abundances peaked in the upper OMZs, associated with the peaks of nitrite concentration. Nitrite and oxygen concentrations may therefore colimit the distribution of anammox bacteria in this area. Furthermore, transcriptional activity of anammox bacteria was confirmed by obtaining abundant hzo mRNA transcripts through qRT-PCR. A novel hzo cluster 2x clade was identified by the phylogenetic analysis and these novel sequences were abundant and widely distributed in this environment. Our study demonstrated that both cluster 1 and 2 anammox bacteria play an active role in the CRD-OMZ, and the cluster 1 abundance and transcriptional activity were higher than cluster 2 in both free-living and particle-attached fractions at both gene and transcriptional levels.

Quorum Sensing Inhibitors for Staphylococcus aureus from Italian Medicinal Plants

PubMed Central

Quave, Cassandra L.; Plano, Lisa R.W.; Bennett, Bradley C.

2010-01-01

Morbidity and mortality estimates due to methicillin-resistant Staphylococcus aureus (MRSA) infections continue to rise. Therapeutic options are limited by antibiotic resistance. Anti-pathogenic compounds, which inhibit quorum sensing (QS) pathways, may be a useful alternative to antibiotics. Staphylococcal QS is encoded by the agr locus and is responsible for the production of δ-hemolysin. Quantification of δ-hemolysin found in culture supernatants permits the analysis of agr activity at the translational, rather than transcriptional, level. We employed RP-HPLC techniques to investigate the anti-QS activity of 168 extracts from 104 Italian plants through quantification of δ-hemolysin. Extracts from three medicinal plants (Ballota nigra, Castanea sativa, and Sambucus ebulus) exhibited a dose-dependent response in the production of δ-hemolysin, indicating strong anti-QS activity in a pathogenic MRSA isolate. PMID:20645243

DOE Office of Scientific and Technical Information (OSTI.GOV)

Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita

As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statisticalmore » inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.« less

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

PubMed

Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas

2013-05-21

Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR

PubMed Central

2013-01-01

Background Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects. PMID:23693071

Using Poisson mixed-effects model to quantify transcript-level gene expression in RNA-Seq.

PubMed

Hu, Ming; Zhu, Yu; Taylor, Jeremy M G; Liu, Jun S; Qin, Zhaohui S

2012-01-01

RNA sequencing (RNA-Seq) is a powerful new technology for mapping and quantifying transcriptomes using ultra high-throughput next-generation sequencing technologies. Using deep sequencing, gene expression levels of all transcripts including novel ones can be quantified digitally. Although extremely promising, the massive amounts of data generated by RNA-Seq, substantial biases and uncertainty in short read alignment pose challenges for data analysis. In particular, large base-specific variation and between-base dependence make simple approaches, such as those that use averaging to normalize RNA-Seq data and quantify gene expressions, ineffective. In this study, we propose a Poisson mixed-effects (POME) model to characterize base-level read coverage within each transcript. The underlying expression level is included as a key parameter in this model. Since the proposed model is capable of incorporating base-specific variation as well as between-base dependence that affect read coverage profile throughout the transcript, it can lead to improved quantification of the true underlying expression level. POME can be freely downloaded at http://www.stat.purdue.edu/~yuzhu/pome.html. yuzhu@purdue.edu; zhaohui.qin@emory.edu Supplementary data are available at Bioinformatics online.

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.

PubMed

Hoppe, Philipp S; Schwarzfischer, Michael; Loeffler, Dirk; Kokkaliaris, Konstantinos D; Hilsenbeck, Oliver; Moritz, Nadine; Endele, Max; Filipczyk, Adam; Gambardella, Adriana; Ahmed, Nouraiz; Etzrodt, Martin; Coutu, Daniel L; Rieger, Michael A; Marr, Carsten; Strasser, Michael K; Schauberger, Bernhard; Burtscher, Ingo; Ermakova, Olga; Bürger, Antje; Lickert, Heiko; Nerlov, Claus; Theis, Fabian J; Schroeder, Timm

2016-07-14

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

The pan-cancer pathological regulatory landscape

PubMed Central

Falco, Matias M.; Bleda, Marta; Carbonell-Caballero, José; Dopazo, Joaquín

2016-01-01

Dysregulation of the normal gene expression program is the cause of a broad range of diseases, including cancer. Detecting the specific perturbed regulators that have an effect on the generation and the development of the disease is crucial for understanding the disease mechanism and for taking decisions on efficient preventive and curative therapies. Moreover, detecting such perturbations at the patient level is even more important from the perspective of personalized medicine. We applied the Transcription Factor Target Enrichment Analysis, a method that detects the activity of transcription factors based on the quantification of the collective transcriptional activation of their targets, to a large collection of 5607 cancer samples covering eleven cancer types. We produced for the first time a comprehensive catalogue of altered transcription factor activities in cancer, a considerable number of them significantly associated to patient’s survival. Moreover, we described several interesting TFs whose activity do not change substantially in the cancer with respect to the normal tissue but ultimately play an important role in patient prognostic determination, which suggest they might be promising therapeutic targets. An additional advantage of this method is that it allows obtaining personalized TF activity estimations for individual patients. PMID:28000771

Cytokine and transcription factor expression by Aspergillus fumigatus-stimulated peripheral blood mononuclear cells in dogs with sino-nasal aspergillosis.

PubMed

Vanherberghen, M; Bureau, F; Peters, I R; Day, M J; Lynch, A; Fievez, L; Billen, F; Clercx, C; Peeters, D

2013-08-15

The causal agent of sino-nasal aspergillosis is usually Aspergillus fumigatus, which is a saprophytic and ubiquitous fungus that causes a severe rhinosinusitis in apparent healthy dogs. Affected dogs do not have systemic immuno-suppression. It has been shown previously that dogs affected by this disease have local over-expression of interleukin (IL)-10 and Th1 cytokines in nasal mucosal tissue. The aim of the present study was to assess the response of peripheral blood mononuclear cells (PBMC) from affected and unaffected dogs to antigen-specific stimulation with heat-inactivated Aspergillus spp. conidia, by quantifying gene expression for specific Th1, Th2, Th17 and Treg cytokines and their related transcription factors. Quantification of IL-4 and IFN-γ protein in culture supernatant was performed by enzyme-linked immunosorbent assay (ELISA). PBMC from dogs with SNA produced adequate mRNA encoding IFN-γ and IFN-γ protein. The expression of IL-17A mRNA was significantly greater in PBMC of affected compared with unaffected dogs. The amount of IL-10 mRNA in PBMC from affected dogs decreased after antigen-specific challenge. These results suggest that the incapacity of affected dogs to clear these fungal infections is not related to a defect in Th1 immunity or to an overwhelming regulatory reaction, but rather to an uncontrolled pro-inflammatory reaction driven by Th17 cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

PubMed

Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2013-10-10

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. Copyright © 2013 Elsevier B.V. All rights reserved.

Detection and Analysis of Circular RNAs by RT-PCR.

PubMed

Panda, Amaresh C; Gorospe, Myriam

2018-03-20

Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.

Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates.

PubMed

Kelnar, Kevin; Peltier, Heidi J; Leatherbury, Neil; Stoudemire, Jay; Bader, Andreas G

2014-02-04

MRX34, a microRNA (miRNA)-based therapy for cancer, has recently entered clinical trials as the first clinical candidate in its class. It is a liposomal nanoparticle loaded with a synthetic mimic of the tumor suppressor miRNA miR-34a as the active pharmaceutical ingredient. To understand the pharmacokinetic properties of the drug and to rationalize an optimal dosing regimen in the clinic, a method is needed to quantitatively detect the miRNA mimic. Here, we report the development and qualification of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay in support of pharmacokinetic and toxicokinetic assessments in the nonhuman primate. Detection and quantification were performed on total ribonucleic acid (RNA) isolated from whole blood. The qualified range of the standard curve spans 6 orders of magnitude from 2.5 × 10(-7) to 2.5 × 10(-1) ng per reverse transcription (RT) reaction, corresponding to an estimated blood concentration from 6.2 × 10(-5) to 6.2 × 10(1) ng/mL. Our results demonstrate that endogenous as well as the exogenous miR-34a can be accurately and precisely quantified. The assay was used to establish the pharmacokinetic profile of MRX34, showing a favorable residence time and exposure of the miRNA mimic in whole blood from nonhuman primates.

APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals

PubMed Central

You, Leiming; Wu, Jiexin; Feng, Yuchao; Fu, Yonggui; Guo, Yanan; Long, Liyuan; Zhang, Hui; Luan, Yijie; Tian, Peng; Chen, Liangfu; Huang, Guangrui; Huang, Shengfeng; Li, Yuxin; Li, Jie; Chen, Chengyong; Zhang, Yaqing; Chen, Shangwu; Xu, Anlong

2015-01-01

Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3′-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3′-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3′-untranslated regions (3′-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish. PMID:25378337

Quantification of Pea enation mosaic virus 1 and 2 during infection of Pisum sativum by one step real-time RT-PCR.

PubMed

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C; Miller, W Allen

2017-02-01

Pea enation mosaic virus 1 (PEMV1) and Pea enation mosaic virus 2 (PEMV2) are two viruses in an obligate symbiosis that cause pea enation mosaic disease mainly in plants in the Fabaceae family. This virus system is a valuable model to investigate plant virus replication, movement and vector transmission. Thus, here we describe growth conditions, virus detection methods, and virus accumulation behavior. To measure the accumulation and movement of PEMV1 and PEMV2 in plants during the course of infection, we developed a quantitative real-time one-step reverse transcription PCR procedure using the SYBR-green ® technology. Viral primers were designed that anneal to conserved but distinct regions in the RNA-dependent RNA polymerase gene of each virus. Moreover, the normalization of viral accumulation was performed to correct for sample-to-sample variation by designing primers to two different Pisum sativum housekeeping genes: actin and β-tubulin. Transcript levels for these housekeeping genes did not change significantly in response to PEMV infection. Conditions were established for maximum PCR efficiency for each gene, and quantification using QuBit ® technology. Both viruses reached maximum accumulation around 21days post-inoculation of pea plants. These results provide valuable tools and knowledge to allow reproducible studies of this emerging model virus system virus complex. Copyright © 2016 Elsevier B.V. All rights reserved.

Forensic molecular pathology of violent deaths.

PubMed

Maeda, Hitoshi; Zhu, Bao-li; Ishikawa, Takaki; Michiue, Tomomi

2010-12-15

In forensic pathology, while classical morphology remains a core procedure to investigate deaths, a spectrum of ancillary procedures has been developed and incorporated to detail the pathology. Among them, postmortem biochemistry is important to investigate the systemic pathophysiological changes involved in the dying process that cannot be detected by morphology. In addition, recent advances in molecular biology have provided a procedure to investigate genetic bases of diseases that might present with sudden death, which is called 'molecular autopsy'. Meanwhile, the practical application of RNA analyses to postmortem investigation has not been accepted due to rapid decay after death; however, recent experimental and practical studies using real-time reverse transcription-PCR have suggested that the relative quantification of mRNA transcripts can be applied in molecular pathology for postmortem investigation of deaths, which may be called 'advanced molecular autopsy'. In a broad sense, forensic molecular pathology implies applied medical sciences to investigate the genetic basis of diseases, and the pathophysiology of diseases and traumas leading to death at a biological molecular level in the context of forensic pathology. The possible applications include analyses of local pathology, including tissue injury, ischemia/hypoxia and inflammation at the site of insult or specific tissue damage from intoxication, systemic responses to violence or environmental hazards, disorders due to intoxication, and systemic pathophysiology of fatal process involving major life-support organs. A review of previous studies suggests that systematic postmortem quantitative analysis of mRNA transcripts can be established from multi-faceted aspects of molecular biology and incorporated into death investigations in forensic pathology, to support and reinforce morphological evidence. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Transcriptome Analysis of Salt Tolerant Common Bean (Phaseolus vulgaris L.) under Saline Conditions

PubMed Central

Hiz, Mahmut Can; Canher, Balkan; Niron, Harun; Turet, Muge

2014-01-01

Salinity is one of the important abiotic stress factors that limit crop production. Common bean, Phaseolus vulgaris L., a major protein source in developing countries, is highly affected by soil salinity and the information on genes that play a role in salt tolerance is scarce. We aimed to identify differentially expressed genes (DEGs) and related pathways by comprehensive analysis of transcriptomes of both root and leaf tissues of the tolerant genotype grown under saline and control conditions in hydroponic system. We have generated a total of 158 million high-quality reads which were assembled into 83,774 all-unigenes with a mean length of 813 bp and N50 of 1,449 bp. Among the all-unigenes, 58,171 were assigned with Nr annotations after homology analyses. It was revealed that 6,422 and 4,555 all-unigenes were differentially expressed upon salt stress in leaf and root tissues respectively. Validation of the RNA-seq quantifications (RPKM values) was performed by qRT-PCR (Quantitative Reverse Transcription PCR) analyses. Enrichment analyses of DEGs based on GO and KEGG databases have shown that both leaf and root tissues regulate energy metabolism, transmembrane transport activity, and secondary metabolites to cope with salinity. A total of 2,678 putative common bean transcription factors were identified and classified under 59 transcription factor families; among them 441 were salt responsive. The data generated in this study will help in understanding the fundamentals of salt tolerance in common bean and will provide resources for functional genomic studies. PMID:24651267

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Sodium selenite increases the transcript levels of iodothyronine deiodinases I and II in ovine and bovine fetal thyrocytes in vitro.

PubMed

Foroughi, Mohammad Ali; Dehghani, Hesam; Mahdavi-Shahri, Naser; Bassami, Mohammad Reza

2013-07-01

Selenium is essential for thyroid hormone homeostasis. Selenium is co-translationally incorporated into the protein backbone of 5' deiodinase enzymes, which are responsible for the intra- and extra-thyroidal activation of thyroid hormones. The objective of this study was to evaluate the effects of sodium selenite on the transcript levels of type I (DIO1) and II (DIO2) deiodinases in the primary culture of ovine and bovine fetal thyroid. By culture of fetal thyrocytes in the presence or absence of sodium selenite, and quantification of DIO1 and DIO2 transcripts using real-time reverse transcription polymerase chain reaction (RT-qPCR), we found that sodium selenite is able to increase the abundance of transcripts for DIO1 and DIO2 genes. We also found that cultured thyrocytes in the presence of sodium selenite compared to control cultured thyrocytes release more T3 into the culture medium. This indicates that in the presence of sodium selenite higher levels of DIO1 and DIO2 enzymes are produced, which are able to convert T4 to T3. In conclusion, we have shown that sodium selenite is increasing the abundance of DIO1 and DIO2 transcripts and increasing the production and release of T3 from cultured fetal thyrocytes. This finding emphasizes the role of selenium in transcriptional and expression processes during development and suggests that selenium deficiency during pregnancy in sheep and cattle may lead to the lower levels of DIO1 and DIO2 transcription in fetal thyroid, and thus, lower level of thyroidal T3 release into the fetal serum. Copyright © 2013 Elsevier GmbH. All rights reserved.

Integrating discrete stochastic models and single-cell experiments to infer predictive models of MAPK-induced transcription dynamics

NASA Astrophysics Data System (ADS)

Munsky, Brian

2015-03-01

MAPK signal-activated transcription plays central roles in myriad biological processes including stress adaptation responses and cell fate decisions. Recent single-cell and single-molecule experiments have advanced our ability to quantify the spatial, temporal, and stochastic fluctuations for such signals and their downstream effects on transcription regulation. This talk explores how integrating such experiments with discrete stochastic computational analyses can yield quantitative and predictive understanding of transcription regulation in both space and time. We use single-molecule mRNA fluorescence in situ hybridization (smFISH) experiments to reveal locations and numbers of multiple endogenous mRNA species in 100,000's of individual cells, at different times and under different genetic and environmental perturbations. We use finite state projection methods to precisely and efficiently compute the full joint probability distributions of these mRNA, which capture measured spatial, temporal and correlative fluctuations. By combining these experimental and computational tools with uncertainty quantification, we systematically compare models of varying complexity and select those which give optimally precise and accurate predictions in new situations. We use these tools to explore two MAPK-activated gene regulation pathways. In yeast adaptation to osmotic shock, we analyze Hog1 kinase activation of transcription for three different genes STL1 (osmotic stress), CTT1 (oxidative stress) and HSP12 (heat shock). In human osteosarcoma cells under serum induction, we analyze ERK activation of c-Fos transcription.

Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs

NASA Astrophysics Data System (ADS)

Che, You; Liang, Peixin; Gong, Ting; Cao, Xiangyu; Zhao, Ying; Yang, Chao; Song, Cunjiang

2017-03-01

We investigated nitrogen-cycle bacterial communities in activated sludge from 8 municipal wastewater treatment plants (WWTPs). Redundancy analyses (RDA) showed that temperature was the most significant driving force in shaping microbial community structure, followed by influent NH4+ and total nitrogen (TN). The diversity of ammonia oxidizing and nitrite reducing bacteria were investigated by the construction of amoA, nirS and nirK gene clone libraries. Phylogenetic analysis indicated that Thauera and Mesorhizobium were the predominant nitrite reducing bacteria, and Nitrosomonas was the only detected ammonia oxidizing bacteria in all samples. Quantification of transcription level of nirS and nirK genes indicated that nirS-type nitrite reducing bacteria played the dominant roles in nitrite reduction process. Transcription level of nirS gene positively correlated with influent NH4+ and TN significantly, whereas inversely linked with hydraulic retention time. Temperature had a strong positive correlation to transcription level of amoA gene. Overall, this study deepened our understanding of the major types of ammonia oxidizing and nitrite reducing bacteria in activated sludge of municipal WWTPs. The relationship between transcription level of nitrogen-cycle genes and operational or environmental variables of WWTPs revealed in this work could provide guidance for optimization of operating parameters and improving the performance of nitrogen removal.

Quantification of tomato and Arabidopsis mobile RNAs trafficking into the parasitic plant Cuscuta pentagona.

PubMed

LeBlanc, Megan; Kim, Gunjune; Patel, Beneeta; Stromberg, Verlyn; Westwood, James

2013-12-01

The cross-species movement of mRNA from hosts to the parasitic plant Cuscuta pentagona has been reported previously, but has not been characterized quantitatively or with attention to uptake patterns and the fate of specific mRNAs. Real-time PCR and RNA-Seq approaches were used to identify and characterize mobile transcripts from tomato and Arabidopsis hosts into C. pentagona. Tomato transcripts of Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Proteinase Inhibitor (SlPI) differed significantly in the rate of uptake into the parasite, but were then distributed over the length of the parasite shoot. When parasite shoots were detached from the hosts, the SlPI transcript concentrations in the parasite showed the greatest decrease within the first 8 h. Arabidopsis transcripts also varied in mobility into the parasite, and assay of specific regions of a Salt-inducible Zinc Finger Protein (AtSZF1) transcript revealed distinct patterns of abundance in the parasite. The uptake and distribution of host mRNAs into C. pentagona appears to vary among mRNAs, and perhaps even with the region of the mRNA under investigation. We propose that mRNAs traffic into the parasite via multiple routes, or that other mechanisms for selective uptake and mobility exist between host and parasite. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Diversity and Spatial Distribution of Hydrazine Oxidoreductase (hzo) Gene in the Oxygen Minimum Zone Off Costa Rica

PubMed Central

Kong, Liangliang; Jing, Hongmei; Kataoka, Takafumi; Buchwald, Carolyn; Liu, Hongbin

2013-01-01

Anaerobic ammonia oxidation (anammox) as an important nitrogen loss pathway has been reported in marine oxygen minimum zones (OMZs), but the community composition and spatial distribution of anammox bacteria in the eastern tropical North Pacific (ETNP) OMZ are poorly determined. In this study, anammox bacterial communities in the OMZ off Costa Rica (CRD-OMZ) were analyzed based on both hydrazine oxidoreductase (hzo) genes and their transcripts assigned to cluster 1 and 2. The anammox communities revealed by hzo genes and proteins in CRD-OMZ showed a low diversity. Gene quantification results showed that hzo gene abundances peaked in the upper OMZs, associated with the peaks of nitrite concentration. Nitrite and oxygen concentrations may therefore colimit the distribution of anammox bacteria in this area. Furthermore, transcriptional activity of anammox bacteria was confirmed by obtaining abundant hzo mRNA transcripts through qRT-PCR. A novel hzo cluster 2x clade was identified by the phylogenetic analysis and these novel sequences were abundant and widely distributed in this environment. Our study demonstrated that both cluster 1 and 2 anammox bacteria play an active role in the CRD-OMZ, and the cluster 1 abundance and transcriptional activity were higher than cluster 2 in both free-living and particle-attached fractions at both gene and transcriptional levels. PMID:24205176

Overcoming Heparin-Associated RT-qPCR Inhibition and Normalization Issues for microRNA Quantification in Patients with Acute Myocardial Infarction.

PubMed

Coelho-Lima, Jose; Mohammed, Ashfaq; Cormack, Suzanne; Jones, Samuel; Das, Rajiv; Egred, Mohaned; Panahi, Pedram; Ali, Simi; Spyridopoulos, Ioakim

2018-06-11

 Cardiac-enriched micro ribonucleic acids (miRNAs) are released into the circulation following ST-elevation myocardial infarction (STEMI). Lack of standardized approaches for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) data normalization and presence of RT-qPCR inhibitors (e.g. heparin) in patient blood samples have prevented reproducible miRNA quantification in this cohort and subsequent translation of these biomarkers to clinical practice.  Using a RT-qPCR miRNA screening platform, we identified and validated an endogenous circulating miRNA as a normalization control. In addition, we assessed the effects of in vivo and in vitro anticoagulant drugs administration (heparin and bivalirudin) on three RT-qPCR normalization strategies (global miRNA mean, exogenous spike-in control [cel-miR-39] and endogenous miRNA control). Finally, we evaluated the effect of heparin and its in vitro inhibition with heparinase on the quantification of cardiac-enriched miRNAs in STEMI patients.  miR-425-5p was validated as an endogenous miRNA control. Heparin administration in vitro and in vivo inhibited all RT-qPCR normalization strategies. In contrast, bivalirudin had no effects on cel-miR-39 or miR-425-5p quantification. In vitro RNA sample treatment with 0.3 U of heparinase overcame heparin-induced over-estimation of cardiac-enriched miRNA levels and improved their correlation with high-sensitivity troponin T.  miRNA quantification in STEMI patients receiving heparin is jeopardized by its effect on all RT-qPCR normalization approaches. Use of samples from bivalirudin-treated patients or in vitro treatment of heparin-contaminated samples with heparinase are suitable alternatives for miRNA quantification in this cohort. Finally, we reinforce the evidence that cardiac-enriched miRNAs early after myocardial reperfusion reflect the severity of cardiac injury. Schattauer GmbH Stuttgart.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

PubMed

Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped. Copyright © 2017 Traverse and Ochman.

Time Series Expression Analyses Using RNA-seq: A Statistical Approach

PubMed Central

Oh, Sunghee; Song, Seongho; Grabowski, Gregory; Zhao, Hongyu; Noonan, James P.

2013-01-01

RNA-seq is becoming the de facto standard approach for transcriptome analysis with ever-reducing cost. It has considerable advantages over conventional technologies (microarrays) because it allows for direct identification and quantification of transcripts. Many time series RNA-seq datasets have been collected to study the dynamic regulations of transcripts. However, statistically rigorous and computationally efficient methods are needed to explore the time-dependent changes of gene expression in biological systems. These methods should explicitly account for the dependencies of expression patterns across time points. Here, we discuss several methods that can be applied to model timecourse RNA-seq data, including statistical evolutionary trajectory index (SETI), autoregressive time-lagged regression (AR(1)), and hidden Markov model (HMM) approaches. We use three real datasets and simulation studies to demonstrate the utility of these dynamic methods in temporal analysis. PMID:23586021

Time series expression analyses using RNA-seq: a statistical approach.

PubMed

Oh, Sunghee; Song, Seongho; Grabowski, Gregory; Zhao, Hongyu; Noonan, James P

2013-01-01

RNA-seq is becoming the de facto standard approach for transcriptome analysis with ever-reducing cost. It has considerable advantages over conventional technologies (microarrays) because it allows for direct identification and quantification of transcripts. Many time series RNA-seq datasets have been collected to study the dynamic regulations of transcripts. However, statistically rigorous and computationally efficient methods are needed to explore the time-dependent changes of gene expression in biological systems. These methods should explicitly account for the dependencies of expression patterns across time points. Here, we discuss several methods that can be applied to model timecourse RNA-seq data, including statistical evolutionary trajectory index (SETI), autoregressive time-lagged regression (AR(1)), and hidden Markov model (HMM) approaches. We use three real datasets and simulation studies to demonstrate the utility of these dynamic methods in temporal analysis.

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus.

PubMed

Nombela, Ivan; Puente-Marin, Sara; Chico, Veronica; Villena, Alberto J; Carracedo, Begoña; Ciordia, Sergio; Mena, Maria Carmen; Mercado, Luis; Perez, Luis; Coll, Julio; Estepa, Amparo; Ortega-Villaizan, Maria Del Mar

2017-01-01

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1 ) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer

DOE PAGES

Wang, Sisi; Zhang, Hongyong; Scharadin, Tiffany M.; ...

2016-01-22

Here, we report the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formationmore » and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.« less

Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis.

PubMed

Ciura, Sorana; Lattante, Serena; Le Ber, Isabelle; Latouche, Morwena; Tostivint, Hervé; Brice, Alexis; Kabashi, Edor

2013-08-01

To define the role that repeat expansions of a GGGGCC hexanucleotide sequence of the C9orf72 gene play in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A genetic model for ALS was developed to determine whether loss of function of the zebrafish orthologue of C9orf72 (zC9orf72) leads to abnormalities in neuronal development. C9orf72 mRNA levels were quantified in brain and lymphoblasts derived from FTLD and ALS/FTLD patients and in zebrafish. Knockdown of the zC9orf72 was performed using 2 specific antisense morpholino oligonucleotides to block transcription. Quantifications of spontaneous swimming and tactile escape response, as well as measurements of axonal projections from the spinal cord, were performed. Significantly decreased expression of C9orf72 transcripts in brain and lymphoblasts was found in sporadic FTLD and ALS/FTLD patients with normal-size or expanded hexanucleotide repeats. The zC9orf72 is selectively expressed in the developing nervous system at developmental stages. Loss of function of the zC9orf72 transcripts causes both behavioral and cellular deficits related to locomotion without major morphological abnormalities. These deficits were rescued upon overexpression of human C9orf72 mRNA transcripts. Our results indicate C9orf72 haploinsufficiency could be a contributing factor in the spectrum of ALS/FTLD neurodegenerative disorders. Loss of function of the zebrafish orthologue of zC9orf72 expression in zebrafish is associated with axonal degeneration of motor neurons that can be rescued by expressing human C9orf72 mRNA, highlighting the specificity of the induced phenotype. These results reveal a pathogenic consequence of decreased C9orf72 levels, supporting a loss of function mechanism of disease. © 2013 American Neurological Association.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Sisi; Zhang, Hongyong; Scharadin, Tiffany M.

Here, we report the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formationmore » and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.« less

A novel quantitative reverse-transcription PCR (qRT-PCR) for the enumeration of total bacteria, using meat micro-flora as a model.

PubMed

Dolan, Anthony; Burgess, Catherine M; Barry, Thomas B; Fanning, Seamus; Duffy, Geraldine

2009-04-01

A sensitive quantitative reverse-transcription PCR (qRT-PCR) method was developed for enumeration of total bacteria. Using two sets of primers separately to target the ribonuclease-P (RNase P) RNA transcripts of gram positive and gram negative bacteria. Standard curves were generated using SYBR Green I kits for the LightCycler 2.0 instrument (Roche Diagnostics) to allow quantification of mixed microflora in liquid media. RNA standards were used and extracted from known cell equivalents and subsequently converted to cDNA for the construction of standard curves. The number of mixed bacteria in culture was determined by qRT-PCR, and the results correlated (r(2)=0.88, rsd=0.466) with the total viable count over the range from approx. Log(10) 3 to approx. Log(10) 7 CFU ml(-1). The rapid nature of this assay (8 h) and its potential as an alternative method to the standard plate count method to predict total viable counts and shelf life are discussed.

Analytical workflow profiling gene expression in murine macrophages

PubMed Central

Nixon, Scott E.; González-Peña, Dianelys; Lawson, Marcus A.; McCusker, Robert H.; Hernandez, Alvaro G.; O’Connor, Jason C.; Dantzer, Robert; Kelley, Keith W.

2015-01-01

Comprehensive and simultaneous analysis of all genes in a biological sample is a capability of RNA-Seq technology. Analysis of the entire transcriptome benefits from summarization of genes at the functional level. As a cellular response of interest not previously explored with RNA-Seq, peritoneal macrophages from mice under two conditions (control and immunologically challenged) were analyzed for gene expression differences. Quantification of individual transcripts modeled RNA-Seq read distribution and uncertainty (using a Beta Negative Binomial distribution), then tested for differential transcript expression (False Discovery Rate-adjusted p-value < 0.05). Enrichment of functional categories utilized the list of differentially expressed genes. A total of 2079 differentially expressed transcripts representing 1884 genes were detected. Enrichment of 92 categories from Gene Ontology Biological Processes and Molecular Functions, and KEGG pathways were grouped into 6 clusters. Clusters included defense and inflammatory response (Enrichment Score = 11.24) and ribosomal activity (Enrichment Score = 17.89). Our work provides a context to the fine detail of individual gene expression differences in murine peritoneal macrophages during immunological challenge with high throughput RNA-Seq. PMID:25708305

Single-molecule quantification of lipotoxic expression of activating transcription factor 3

PubMed Central

Wilson, Dennis W.; Rutledge, John C.

2014-01-01

Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500/h. After three hours we detected 33,090 ± 3,491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at ~3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipolysis products occurs in large aggregates. PMID:25189785

Data-Independent MS/MS Quantification of Neuropeptides for Determination of Putative Feeding-Related Neurohormones in Microdialysate

PubMed Central

2015-01-01

Food consumption is an important behavior that is regulated by an intricate array of neuropeptides (NPs). Although many feeding-related NPs have been identified in mammals, precise mechanisms are unclear and difficult to study in mammals, as current methods are not highly multiplexed and require extensive a priori knowledge about analytes. New advances in data-independent acquisition (DIA) MS/MS and the open-source quantification software Skyline have opened up the possibility to identify hundreds of compounds and quantify them from a single DIA MS/MS run. An untargeted DIA MSE quantification method using Skyline software for multiplexed, discovery-driven quantification was developed and found to produce linear calibration curves for peptides at physiologically relevant concentrations using a protein digest as internal standard. By using this method, preliminary relative quantification of the crab Cancer borealis neuropeptidome (<2 kDa, 137 peptides from 18 families) was possible in microdialysates from 8 replicate feeding experiments. Of these NPs, 55 were detected with an average mass error below 10 ppm. The time-resolved profiles of relative concentration changes for 6 are shown, and there is great potential for the use of this method in future experiments to aid in correlation of NP changes with behavior. This work presents an unbiased approach to winnowing candidate NPs related to a behavior of interest in a functionally relevant manner, and demonstrates the success of such a UPLC-MSE quantification method using the open source software Skyline. PMID:25552291

Data-independent MS/MS quantification of neuropeptides for determination of putative feeding-related neurohormones in microdialysate.

PubMed

Schmerberg, Claire M; Liang, Zhidan; Li, Lingjun

2015-01-21

Food consumption is an important behavior that is regulated by an intricate array of neuropeptides (NPs). Although many feeding-related NPs have been identified in mammals, precise mechanisms are unclear and difficult to study in mammals, as current methods are not highly multiplexed and require extensive a priori knowledge about analytes. New advances in data-independent acquisition (DIA) MS/MS and the open-source quantification software Skyline have opened up the possibility to identify hundreds of compounds and quantify them from a single DIA MS/MS run. An untargeted DIA MS(E) quantification method using Skyline software for multiplexed, discovery-driven quantification was developed and found to produce linear calibration curves for peptides at physiologically relevant concentrations using a protein digest as internal standard. By using this method, preliminary relative quantification of the crab Cancer borealis neuropeptidome (<2 kDa, 137 peptides from 18 families) was possible in microdialysates from 8 replicate feeding experiments. Of these NPs, 55 were detected with an average mass error below 10 ppm. The time-resolved profiles of relative concentration changes for 6 are shown, and there is great potential for the use of this method in future experiments to aid in correlation of NP changes with behavior. This work presents an unbiased approach to winnowing candidate NPs related to a behavior of interest in a functionally relevant manner, and demonstrates the success of such a UPLC-MS(E) quantification method using the open source software Skyline.

Dynamic Regulation of Tgf-B Signaling by Tif1γ: A Computational Approach

PubMed Central

Andrieux, Geoffroy; Fattet, Laurent; Le Borgne, Michel; Rimokh, Ruth; Théret, Nathalie

2012-01-01

TIF1γ (Transcriptional Intermediary Factor 1 γ) has been implicated in Smad-dependent signaling by Transforming Growth Factor beta (TGF-β). Paradoxically, TIF1γ functions both as a transcriptional repressor or as an alternative transcription factor that promotes TGF-β signaling. Using ordinary differential-equation models, we have investigated the effect of TIF1γ on the dynamics of TGF-β signaling. An integrative model that includes the formation of transient TIF1γ-Smad2-Smad4 ternary complexes is the only one that can account for TGF-β signaling compatible with the different observations reported for TIF1γ. In addition, our model predicts that varying TIF1γ/Smad4 ratios play a critical role in the modulation of the transcriptional signal induced by TGF-β, especially for short stimulation times that mediate higher threshold responses. Chromatin immunoprecipitation analyses and quantification of the expression of TGF-β target genes as a function TIF1γ/Smad4 ratios fully validate this hypothesis. Our integrative model, which successfully unifies the seemingly opposite roles of TIF1γ, also reveals how changing TIF1γ/Smad4 ratios affect the cellular response to stimulation by TGF-β, accounting for a highly graded determination of cell fate. PMID:22461896

5S rRNA and accompanying proteins in gonads: powerful markers to identify sex and reproductive endocrine disruption in fish.

PubMed

Diaz de Cerio, Oihane; Rojo-Bartolomé, Iratxe; Bizarro, Cristina; Ortiz-Zarragoitia, Maren; Cancio, Ibon

2012-07-17

In anuran ovaries, 5S rDNA is regulated transcriptionally by transcription factor IIIA (TFIIIA), which upon transcription, binds 5S rRNA, forming 7S RNP. 5S rRNA can be stockpiled also in the form of 42S RNP bound to 42sp43. The aim of the present study was to assess the differential transcriptional regulation of 5S rRNA and associated proteins in thicklip gray mullet (Chelon labrosus) gonads. Up to 75% of the total RNA from mullet ovaries was 5S rRNA. qPCR quantification of 5S rRNA expression, in gonads of histologically sexed individuals from different geographical areas, successfully sexed animals. All males had expression levels that were orders of magnitude below expression levels in females, throughout an annual reproductive cycle, with the exception of two individuals: one in November and one in December. Moreover, intersex mullets from a polluted harbor had expression levels between both sexes. TFIIIA and 42sp43 were also very active transcriptionally in gonads of female and intersex mullets, in comparison to males. Nucleocytoplasmatic transport is important in this context and we also analyzed transcriptional levels of importins-α1, -α2, and -β2 and different exportins. Importin-αs behaved similarly to 5S rRNA. Thus, 5S rRNA and associated proteins constitute very powerful molecular markers of sex and effects of xenosterogens in fish gonads, with potential technological applications in the analysis of fish stock dynamics and reproduction as well as in environmental health assessment.

Proteomic Analysis of Arginine Methylation Sites in Human Cells Reveals Dynamic Regulation During Transcriptional Arrest*

PubMed Central

Sylvestersen, Kathrine B.; Horn, Heiko; Jungmichel, Stephanie; Jensen, Lars J.; Nielsen, Michael L.

2014-01-01

The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function, and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared with the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression levels are not altered, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA data set in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase. PMID:24563534

Epithelial response to a high-protein diet in rat colon.

PubMed

Beaumont, Martin; Andriamihaja, Mireille; Armand, Lucie; Grauso, Marta; Jaffrézic, Florence; Laloë, Denis; Moroldo, Marco; Davila, Anne-Marie; Tomé, Daniel; Blachier, François; Lan, Annaïg

2017-01-31

High-protein diets (HPD) alter the large intestine microbiota composition in association with a metabolic shift towards protein degradation. Some amino acid-derived metabolites produced by the colon bacteria are beneficial for the mucosa while others are deleterious at high concentrations. The aim of the present work was to define the colonic epithelial response to an HPD. Transcriptome profiling was performed on colonocytes of rats fed an HPD or an isocaloric normal-protein diet (NPD) for 2 weeks. The HPD downregulated the expression of genes notably implicated in pathways related to cellular metabolism, NF-κB signaling, DNA repair, glutathione metabolism and cellular adhesion in colonocytes. In contrast, the HPD upregulated the expression of genes related to cell proliferation and chemical barrier function. These changes at the mRNA level in colonocytes were not associated with detrimental effects of the HPD on DNA integrity (comet assay), epithelium renewal (quantification of proliferation and apoptosis markers by immunohistochemistry and western blot) and colonic barrier integrity (Ussing chamber experiments). The modifications of the luminal environment after an HPD were associated with maintenance of the colonic homeostasis that might be the result of adaptive processes in the epithelium related to the observed transcriptional regulations.

Estrogenic Effects of Several BPA Analogs in the Developing Zebrafish Brain

PubMed Central

Cano-Nicolau, Joel; Vaillant, Colette; Pellegrini, Elisabeth; Charlier, Thierry D.; Kah, Olivier; Coumailleau, Pascal

2016-01-01

Important set of studies have demonstrated the endocrine disrupting activity of Bisphenol A (BPA). The present work aimed at defining estrogenic-like activity of several BPA structural analogs, including BPS, BPF, BPAF, and BPAP, on 4- or 7-day post-fertilization (dpf) zebrafish larva as an in vivo model. We measured the induction level of the estrogen-sensitive marker cyp19a1b gene (Aromatase B), expressed in the brain, using three different in situ/in vivo strategies: (1) Quantification of cyp19a1b transcripts using RT-qPCR in wild type 7-dpf larva brains exposed to bisphenols; (2) Detection and distribution of cyp19a1b transcripts using in situ hybridization on 7-dpf brain sections (hypothalamus); and (3) Quantification of the cyp19a1b promoter activity in live cyp19a1b-GFP transgenic zebrafish (EASZY assay) at 4-dpf larval stage. These three different experimental approaches demonstrated that BPS, BPF, or BPAF exposure, similarly to BPA, significantly activates the expression of the estrogenic marker in the brain of developing zebrafish. In vitro experiments using both reporter gene assay in a glial cell context and competitive ligand binding assays strongly suggested that up-regulation of cyp19a1b is largely mediated by the zebrafish estrogen nuclear receptor alpha (zfERα). Importantly, and in contrast to other tested bisphenol A analogs, the bisphenol AP (BPAP) did not show estrogenic activity in our model. PMID:27047331

Simultaneous isoform discovery and quantification from RNA-seq.

PubMed

Hiller, David; Wong, Wing Hung

2013-05-01

RNA sequencing is a recent technology which has seen an explosion of methods addressing all levels of analysis, from read mapping to transcript assembly to differential expression modeling. In particular the discovery of isoforms at the transcript assembly stage is a complex problem and current approaches suffer from various limitations. For instance, many approaches use graphs to construct a minimal set of isoforms which covers the observed reads, then perform a separate algorithm to quantify the isoforms, which can result in a loss of power. Current methods also use ad-hoc solutions to deal with the vast number of possible isoforms which can be constructed from a given set of reads. Finally, while the need of taking into account features such as read pairing and sampling rate of reads has been acknowledged, most existing methods do not seamlessly integrate these features as part of the model. We present Montebello, an integrated statistical approach which performs simultaneous isoform discovery and quantification by using a Monte Carlo simulation to find the most likely isoform composition leading to a set of observed reads. We compare Montebello to Cufflinks, a popular isoform discovery approach, on a simulated data set and on 46.3 million brain reads from an Illumina tissue panel. On this data set Montebello appears to offer a modest improvement over Cufflinks when considering discovery and parsimony metrics. In addition Montebello mitigates specific difficulties inherent in the Cufflinks approach. Finally, Montebello can be fine-tuned depending on the type of solution desired.

Stress induction in the bacteria Shewanella oneidensis and Deinococcus radiodurans in response to below-background ionizing radiation.

PubMed

Castillo, Hugo; Schoderbek, Donald; Dulal, Santosh; Escobar, Gabriela; Wood, Jeffrey; Nelson, Roger; Smith, Geoffrey

2015-01-01

The 'Linear no-threshold' (LNT) model predicts that any amount of radiation increases the risk of organisms to accumulate negative effects. Several studies at below background radiation levels (4.5-11.4 nGy h(-1)) show decreased growth rates and an increased susceptibility to oxidative stress. The purpose of our study is to obtain molecular evidence of a stress response in Shewanella oneidensis and Deinococcus radiodurans grown at a gamma dose rate of 0.16 nGy h(-1), about 400 times less than normal background radiation. Bacteria cultures were grown at a dose rate of 0.16 or 71.3 nGy h(-1) gamma irradiation. Total RNA was extracted from samples at early-exponential and stationary phases for the rt-PCR relative quantification (radiation-deprived treatment/background radiation control) of the stress-related genes katB (catalase), recA (recombinase), oxyR (oxidative stress transcriptional regulator), lexA (SOS regulon transcriptional repressor), dnaK (heat shock protein 70) and SOA0154 (putative heavy metal efflux pump). Deprivation of normal levels of radiation caused a reduction in growth of both bacterial species, accompanied by the upregulation of katB, recA, SOA0154 genes in S. oneidensis and the upregulation of dnaK in D. radiodurans. When cells were returned to background radiation levels, growth rates recovered and the stress response dissipated. Our results indicate that below-background levels of radiation inhibited growth and elicited a stress response in two species of bacteria, contrary to the LNT model prediction.

The Role of CYP3A4 mRNA Transcript with Shortened 3′-Untranslated Region in Hepatocyte Differentiation, Liver Development, and Response to Drug InductionS⃞

PubMed Central

Li, Dan; Gaedigk, Roger; Hart, Steven N.; Leeder, J. Steven

2012-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3′-untranslated region (UTR), one had a shorter 3′-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3′-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3′-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3′-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3′-UTR. We conclude that the 3′-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction. PMID:21998292

Maternal metabolism affects endometrial expression of oxidative stress and FOXL2 genes in cattle

PubMed Central

Forde, Niamh; Poirée, Mélanie; Healey, Gareth D.; Giraud-Delville, Corinne; Reinaud, Pierrette; Eozenou, Caroline; Vitorino Carvalho, Anaïs; Galio, Laurent; Raliou, Mariam; Oudin, Jean-François; Richard, Christophe; Sheldon, I. Martin; Charpigny, Gilles; Lonergan, Pat; Sandra, Olivier

2017-01-01

Intensive selection for milk production has led to reduced reproductive efficiency in high-producing dairy cattle. The impact of intensive milk production on oocyte quality as well as early embryo development has been established but few analyses have addressed this question at the initiation of implantation, a critical milestone ensuring a successful pregnancy and normal post-natal development. Our study aimed to determine if contrasted maternal metabolism affects the previously described sensory properties of the endometrium to the conceptus in cattle. Following embryo transfer at Day 7 post-oestrus, endometrial caruncular (CAR) and intercaruncular (ICAR) areas were collected at Day 19 from primiparous postpartum Holstein-Friesian cows that were dried-off immediately after parturition (i.e., never milked; DRY) or milked twice daily (LACT). Gene quantification indicated no significant impact of lactation on endometrial expression of transcripts previously reported as conceptus-regulated (PLET1, PTGS2, SOCS6) and interferon-tau stimulated (RSAD2, SOCS1, SOCS3, STAT1) factors or known as female hormone-regulated genes (FOXL2, SCARA5, PTGS2). Compared with LACT cows, DRY cows exhibited mRNA levels with increased expression for FOXL2 transcription factor and decreased expression for oxidative stress-related genes (CAT, SOD1, SOD2). In vivo and in vitro experiments highlighted that neither interferon-tau nor FOXL2 were involved in transcriptional regulation of CAT, SOD1 and SOD2. In addition, our data showed that variations in maternal metabolism had a higher impact on gene expression in ICAR areas. Collectively, our findings prompt the need to fully understand the extent to which modifications in endometrial physiology drive the trajectory of conceptus development from implantation onwards when maternal metabolism is altered. PMID:29281695

Maternal metabolism affects endometrial expression of oxidative stress and FOXL2 genes in cattle.

PubMed

Lesage-Padilla, Audrey; Forde, Niamh; Poirée, Mélanie; Healey, Gareth D; Giraud-Delville, Corinne; Reinaud, Pierrette; Eozenou, Caroline; Vitorino Carvalho, Anaïs; Galio, Laurent; Raliou, Mariam; Oudin, Jean-François; Richard, Christophe; Sheldon, I Martin; Charpigny, Gilles; Lonergan, Pat; Sandra, Olivier

2017-01-01

Intensive selection for milk production has led to reduced reproductive efficiency in high-producing dairy cattle. The impact of intensive milk production on oocyte quality as well as early embryo development has been established but few analyses have addressed this question at the initiation of implantation, a critical milestone ensuring a successful pregnancy and normal post-natal development. Our study aimed to determine if contrasted maternal metabolism affects the previously described sensory properties of the endometrium to the conceptus in cattle. Following embryo transfer at Day 7 post-oestrus, endometrial caruncular (CAR) and intercaruncular (ICAR) areas were collected at Day 19 from primiparous postpartum Holstein-Friesian cows that were dried-off immediately after parturition (i.e., never milked; DRY) or milked twice daily (LACT). Gene quantification indicated no significant impact of lactation on endometrial expression of transcripts previously reported as conceptus-regulated (PLET1, PTGS2, SOCS6) and interferon-tau stimulated (RSAD2, SOCS1, SOCS3, STAT1) factors or known as female hormone-regulated genes (FOXL2, SCARA5, PTGS2). Compared with LACT cows, DRY cows exhibited mRNA levels with increased expression for FOXL2 transcription factor and decreased expression for oxidative stress-related genes (CAT, SOD1, SOD2). In vivo and in vitro experiments highlighted that neither interferon-tau nor FOXL2 were involved in transcriptional regulation of CAT, SOD1 and SOD2. In addition, our data showed that variations in maternal metabolism had a higher impact on gene expression in ICAR areas. Collectively, our findings prompt the need to fully understand the extent to which modifications in endometrial physiology drive the trajectory of conceptus development from implantation onwards when maternal metabolism is altered.

viGEN: An Open Source Pipeline for the Detection and Quantification of Viral RNA in Human Tumors.

PubMed

Bhuvaneshwar, Krithika; Song, Lei; Madhavan, Subha; Gusev, Yuriy

2018-01-01

An estimated 17% of cancers worldwide are associated with infectious causes. The extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using human transcriptome (RNA-seq) data from tumor samples. We present an open source bioinformatics pipeline viGEN, which allows for not only the detection and quantification of viral RNA, but also variants in the viral transcripts. The pipeline includes 4 major modules: The first module aligns and filter out human RNA sequences; the second module maps and count (remaining un-aligned) reads against reference genomes of all known and sequenced human viruses; the third module quantifies read counts at the individual viral-gene level thus allowing for downstream differential expression analysis of viral genes between case and controls groups. The fourth module calls variants in these viruses. To the best of our knowledge, there are no publicly available pipelines or packages that would provide this type of complete analysis in one open source package. In this paper, we applied the viGEN pipeline to two case studies. We first demonstrate the working of our pipeline on a large public dataset, the TCGA cervical cancer cohort. In the second case study, we performed an in-depth analysis on a small focused study of TCGA liver cancer patients. In the latter cohort, we performed viral-gene quantification, viral-variant extraction and survival analysis. This allowed us to find differentially expressed viral-transcripts and viral-variants between the groups of patients, and connect them to clinical outcome. From our analyses, we show that we were able to successfully detect the human papilloma virus among the TCGA cervical cancer patients. We compared the viGEN pipeline with two metagenomics tools and demonstrate similar sensitivity/specificity. We were also able to quantify viral-transcripts and extract viral-variants using the liver cancer dataset. The results presented corresponded with published literature in terms of rate of detection, and impact of several known variants of HBV genome. This pipeline is generalizable, and can be used to provide novel biological insights into microbial infections in complex diseases and tumorigeneses. Our viral pipeline could be used in conjunction with additional type of immuno-oncology analysis based on RNA-seq data of host RNA for cancer immunology applications. The source code, with example data and tutorial is available at: https://github.com/ICBI/viGEN/.

Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair

PubMed Central

Wienholz, Franziska; Vermeulen, Wim

2017-01-01

Abstract Nucleotide excision repair (NER) comprises two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which remove a wide variety of helix-distorting lesions including UV-induced damage. During NER, a short stretch of single-stranded DNA containing damage is excised and the resulting gap is filled by DNA synthesis in a process called unscheduled DNA synthesis (UDS). UDS is measured by quantifying the incorporation of nucleotide analogues into repair patches to provide a measure of NER activity. However, this assay is unable to quantitatively determine TC-NER activity due to the low contribution of TC-NER to the overall NER activity. Therefore, we developed a user-friendly, fluorescence-based single-cell assay to measure TC-NER activity. We combined the UDS assay with tyramide-based signal amplification to greatly increase the UDS signal, thereby allowing UDS to be quantified at low UV doses, as well as DNA-repair synthesis of other excision-based repair mechanisms such as base excision repair and mismatch repair. Importantly, we demonstrated that the amplified UDS is sufficiently sensitive to quantify TC-NER-derived repair synthesis in GG-NER-deficient cells. This assay is important as a diagnostic tool for NER-related disorders and as a research tool for obtaining new insights into the mechanism and regulation of excision repair. PMID:28088761

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

PubMed

Zhu, Jie; Feng, Xiaolu; Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Detection and quantification of RNA 2′-O-methylation and pseudouridylation

PubMed Central

Karijolich, John

2016-01-01

RNA-guided RNA modification is a naturally occurring process that introduces 2′-O-methylation and pseudouridylation into rRNA, spliceosomal snRNA and several other types of RNA. The Box C/D ribonucleoproteins (RNP) and Box H/ACA RNP, each containing one unique guide RNA (Box C/D RNA or Box H/ACA RNA) and a set of core proteins, are responsible for 2′-O-methylation and pseudouridylation respectively. Box C/D RNA and Box H/ACA RNA provide the modification specificity through base pairing with their RNA substrate. These post-transcriptional modifications could profoundly alter the properties and functions of substrate RNAs. Thus it is desirable to establish reliable and standardized modification methods to study biological functions of modified nucleotides in RNAs. Here, we present several sensitive and efficient methods and protocols for detecting and quantifying post-transcriptional 2′-O-methylation and pseudouridylation. PMID:26853326

Use of multiple competitors for quantification of human immunodeficiency virus type 1 RNA in plasma.

PubMed

Vener, T; Nygren, M; Andersson, A; Uhlén, M; Albert, J; Lundeberg, J

1998-07-01

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.

18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

PubMed

Kim, Jong-Seo; Fillmore, Thomas L; Liu, Tao; Robinson, Errol; Hossain, Mahmud; Champion, Boyd L; Moore, Ronald J; Camp, David G; Smith, Richard D; Qian, Wei-Jun

2011-12-01

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

A review of the application of inflammatory biomarkers in epidemiologic cancer research

PubMed Central

Brenner, Darren R.; Scherer, Dominique; Muir, Kenneth; Schildkraut, Joellen; Boffetta, Paolo; Spitz, Margaret R.; LeMarchand, Loic; Chan, Andrew T.; Goode, Ellen L.; Ulrich, Cornelia M.; Hung, Rayjean J.

2014-01-01

Inflammation is a facilitating process for multiple cancer types. It is believed to affect cancer development and progression through several etiologic pathways including increased levels of DNA adduct formation, increased angiogenesis and altered anti-apoptotic signaling. This review highlights the application of inflammatory biomarkers in epidemiologic studies and discusses the various cellular mediators of inflammation characterizing the innate immune system response to infection and chronic insult from environmental factors. Included is a review of six classes of inflammation-related biomarkers: cytokines/chemokines, immune-related effectors, acute phase proteins, reactive oxygen and nitrogen species, prostaglandins and cyclooxygenase-related factors, and mediators such as transcription factors and growth factors. For each of these biomarkers we provide a brief overview of the etiologic role in the inflammation response and how they have been related to cancer etiology and progression within the literature. We provide a discussion of the common techniques available for quantification of each marker including strengths, weaknesses and potential pitfalls. Subsequently, we highlight a few under-studied measures to characterize the inflammatory response and their potential utility in epidemiologic studies of cancer. Finally, we suggest integrative methods for future studies to apply multi-faceted approaches to examine the relationship between inflammatory markers and their roles in cancer development. PMID:24962838

TTF-1 and napsin A on cell blocks and supernatants of pleural fluids for labeling malignant effusions.

PubMed

Porcel, José M; Palma, Rosa; Bielsa, Silvia; Esquerda, Aureli; Gatius, Sonia; Matias-Guiu, Xavier; Salud, Antonieta

2015-07-01

In this retrospective study of 80 pleural effusions, the combination of thyroid transcription factor 1 (TTF-1) and napsin A immunostaining on fluid cell blocks was positive in 80% of lung adenocarcinomas. Although measuring TTF-1 pleural fluid concentrations was of no value, quantification of napsin A levels allowed the identification of one third of the double-negative stained lung adenocarcinomas, with an overall accuracy similar to classical tumour markers for malignant-benign discrimination (sensitivity 40%, specificity 100%). © 2015 Asian Pacific Society of Respirology.

Circulating metastasis associated in colon cancer 1 transcripts in gastric cancer patient plasma as diagnostic and prognostic biomarker

PubMed Central

Burock, Susen; Herrmann, Pia; Wendler, Ina; Niederstrasser, Markus; Wernecke, Klaus-Dieter; Stein, Ulrike

2015-01-01

AIM: To evaluate the diagnostic and prognostic value of circulating Metastasis Associated in Colon Cancer 1 (MACC1) transcripts in plasma of gastric cancer patients. METHODS: We provide for the first time a blood-based assay for transcript quantification of the metastasis inducer MACC1 in a prospective study of gastric cancer patient plasma. MACC1 is a strong prognostic biomarker for tumor progression and metastasis in a variety of solid cancers. We conducted a study to define the diagnostic and prognostic power of MACC1 transcripts using 76 plasma samples from gastric cancer patients, either newly diagnosed with gastric cancer, newly diagnosed with metachronous metastasis of gastric cancer, as well as follow-up patients. Findings were controlled by using plasma samples from 54 tumor-free volunteers. Plasma was separated, RNA was isolated, and levels of MACC1 as well as S100A4 transcripts were determined by quantitative RT-PCR. RESULTS: Based on the levels of circulating MACC1 transcripts in plasma we significantly discriminated tumor-free volunteers and gastric cancer patients (P < 0.001). Levels of circulating MACC1 transcripts were increased in gastric cancer patients of each disease stage, compared to tumor-free volunteers: patients with tumors without metastasis (P = 0.005), with synchronous metastasis (P = 0.002), with metachronous metastasis (P = 0.005), and patients during follow-up (P = 0.021). Sensitivity was 0.68 (95%CI: 0.45-0.85) and specificity was 0.89 (95%CI: 0.77-0.95), respectively. Importantly, gastric cancer patients with high circulating MACC1 transcript levels in plasma demonstrated significantly shorter survival when compared with patients demonstrating low MACC1 levels (P = 0.0015). Furthermore, gastric cancer patients with high circulating transcript levels of MACC1 as well as of S100A4 in plasma demonstrated significantly shorter survival when compared with patients demonstrating low levels of both biomarkers or with only one biomarker elevated (P = 0.001). CONCLUSION: Levels of circulating MACC1 transcripts in plasma of gastric cancer patients are of diagnostic value and are prognostic for patient survival in a prospective study. PMID:25574109

Some novel insights on HPV16 related cervical cancer pathogenesis based on analyses of LCR methylation, viral load, E7 and E2/E4 expressions.

PubMed

Das Ghosh, Damayanti; Bhattacharjee, Bornali; Sen, Shrinka; Premi, Laikangbam; Mukhopadhyay, Indranil; Chowdhury, Rahul Roy; Roy, Sudipta; Sengupta, Sharmila

2012-01-01

This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1/E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1/E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 C(T)/E4 C(T). Relative quantification based on comparative C(T) (threshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 C(T) (p = 0.007) and E2 C(T) (p<0.0001), respectively, each normalized with ACTB C(T), among episomal cases only. The k-means clustering analysis considering E7 C(T) from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

ORMAN: optimal resolution of ambiguous RNA-Seq multimappings in the presence of novel isoforms.

PubMed

Dao, Phuong; Numanagić, Ibrahim; Lin, Yen-Yi; Hach, Faraz; Karakoc, Emre; Donmez, Nilgun; Collins, Colin; Eichler, Evan E; Sahinalp, S Cenk

2014-03-01

RNA-Seq technology is promising to uncover many novel alternative splicing events, gene fusions and other variations in RNA transcripts. For an accurate detection and quantification of transcripts, it is important to resolve the mapping ambiguity for those RNA-Seq reads that can be mapped to multiple loci: >17% of the reads from mouse RNA-Seq data and 50% of the reads from some plant RNA-Seq data have multiple mapping loci. In this study, we show how to resolve the mapping ambiguity in the presence of novel transcriptomic events such as exon skipping and novel indels towards accurate downstream analysis. We introduce ORMAN ( O ptimal R esolution of M ultimapping A mbiguity of R N A-Seq Reads), which aims to compute the minimum number of potential transcript products for each gene and to assign each multimapping read to one of these transcripts based on the estimated distribution of the region covering the read. ORMAN achieves this objective through a combinatorial optimization formulation, which is solved through well-known approximation algorithms, integer linear programs and heuristics. On a simulated RNA-Seq dataset including a random subset of transcripts from the UCSC database, the performance of several state-of-the-art methods for identifying and quantifying novel transcripts, such as Cufflinks, IsoLasso and CLIIQ, is significantly improved through the use of ORMAN. Furthermore, in an experiment using real RNA-Seq reads, we show that ORMAN is able to resolve multimapping to produce coverage values that are similar to the original distribution, even in genes with highly non-uniform coverage. ORMAN is available at http://orman.sf.net

21 CFR 530.24 - Procedure for announcing analytical methods for drug residue quantification.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS EXTRALABEL DRUG USE IN ANIMALS Specific Provisions Relating to Extralabel Use of Animal and Human Drugs in Food-Producing Animals § 530.24 Procedure for announcing analytical methods for drug residue quantification. (a...

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma.

PubMed

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman

2014-02-05

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*10(5) MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts.

High serum levels of pregenomic RNA reflect frequently failing reverse transcription in hepatitis B virus particles.

PubMed

Prakash, Kasthuri; Rydell, Gustaf E; Larsson, Simon B; Andersson, Maria; Norkrans, Gunnar; Norder, Heléne; Lindh, Magnus

2018-05-15

Hepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Particles containing HBV RNA are present in serum of infected individuals, and quantification of this HBV RNA could be clinically useful. In a retrospective study of 95 patients with chronic HBV infection, we characterised HBV RNA in serum in terms of concentration, particle association and sequence. HBV RNA was detected by real-time PCR at levels almost as high as HBV DNA. The HBV RNA was protected from RNase and it was found in particles of similar density as particles containing HBV DNA after fractionation on a Nycodenz gradient. Sequencing the epsilon region of the RNA did not reveal mutations that would preclude its binding to the viral polymerase before encapsidation. Specific quantification of precore RNA and pgRNA by digital PCR showed almost seven times lower ratio of precore RNA/pgRNA in serum than in liver tissue, which corresponds to poorer encapsidation of this RNA as compared with pgRNA. The serum ratio between HBV DNA and HBV RNA was higher in genotype D as compared with other genotypes. The results suggest that HBV RNA in serum is present in viral particles with failing reverse transcription activity, which are produced at almost as high rates as viral particles containing DNA. The results encourage further studies of the mechanisms by which these particles are produced, the impact of genotype, and the potential clinical utility of quantifying HBV RNA in serum.

A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

PubMed Central

Kang, Kang; Zhang, Xiaoying; Liu, Hongtao; Wang, Zhiwei; Zhong, Jiasheng; Huang, Zhenting; Peng, Xiao; Zeng, Yan; Wang, Yuna; Yang, Yi; Luo, Jun; Gou, Deming

2012-01-01

Background MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. Methodology/Principal Findings A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. Conclusions/Significance With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers. PMID:23152780

Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

PubMed Central

Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

2015-01-01

Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

Effect of doxorubicin, oxaliplatin, and methotrexate administration on the transcriptional activity of BCL-2 family gene members in stomach cancer cells.

PubMed

Florou, Dimitra; Patsis, Christos; Ardavanis, Alexandros; Scorilas, Andreas

2013-07-01

Defective apoptosis comprises the main reason for tumor aggressiveness and chemotherapy tolerance in solid neoplasias. Among the BCL-2 family members, whose mRNA or protein expression varies considerably in different human malignancies, BCL2L12 is the one for which we have recently shown its propitious prognostic value in gastric cancer. The purpose of the current work was to investigate the expression behavior of BCL2L12, BAX, and BCL-2 in human stomach adenocarcinoma cells following their exposure to anti-tumor substances. The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue methods assessed the impact of doxorubicin, oxaliplatin and methotrexate on AGS cells' viability and growth. Following isolation from cells, total RNA was reverse-transcribed to cDNA. Quantification of target genes' expression was performed with real-time PCR using SYBR Green detection system. The relative changes in their mRNA levels between drug-exposed and untreated cells were calculated with the comparative Ct method (2(-ddCt)). All three drugs, as a result of their administration to AGS cancer cells for particular time intervals, provoked substantial fluctuations in the transcriptional levels of the apoptosis-related genes studied. While BAX was principally upregulated, striking similar were the notable changes regarding BCL-2 and BCL2L12 expression in our cellular system. Our findings indicate the growth suppressive effects of doxorubicin, oxaliplatin and methotrexate treatment on stomach carcinoma cells and the implication of BCL2L12, BAX, and BCL-2 expression profiles in the molecular signaling pathways triggered by chemotherapy.

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

PubMed

Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

2018-03-19

To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

Quaternary ammonium isobaric tag for a relative and absolute quantification of peptides.

PubMed

Setner, Bartosz; Stefanowicz, Piotr; Szewczuk, Zbigniew

2018-02-01

Isobaric labeling quantification of peptides has become a method of choice for mass spectrometry-based proteomics studies. However, despite of wide variety of commercially available isobaric tags, none of the currently available methods offers significant improvement of sensitivity of detection during MS experiment. Recently, many strategies were applied to increase the ionization efficiency of peptides involving chemical modifications introducing quaternary ammonium fixed charge. Here, we present a novel quaternary ammonium-based isobaric tag for relative and absolute quantification of peptides (QAS-iTRAQ 2-plex). Upon collisional activation, the new stable benzylic-type cationic reporter ion is liberated from the tag. Deuterium atoms were used to offset the differential masses of a reporter group. We tested the applicability of QAS-iTRAQ 2-plex reagent on a series of model peptides as well as bovine serum albumin tryptic digest. Obtained results suggest usefulness of this isobaric ionization tag for relative and absolute quantification of peptides. Copyright © 2017 John Wiley & Sons, Ltd.

Use of Multiple Competitors for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma

PubMed Central

Vener, Tanya; Nygren, Malin; Andersson, AnnaLena; Uhlén, Mathias; Albert, Jan; Lundeberg, Joakim

1998-01-01

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens. PMID:9650926

DiffSplice: the genome-wide detection of differential splicing events with RNA-seq

PubMed Central

Hu, Yin; Huang, Yan; Du, Ying; Orellana, Christian F.; Singh, Darshan; Johnson, Amy R.; Monroy, Anaïs; Kuan, Pei-Fen; Hammond, Scott M.; Makowski, Liza; Randell, Scott H.; Chiang, Derek Y.; Hayes, D. Neil; Jones, Corbin; Liu, Yufeng; Prins, Jan F.; Liu, Jinze

2013-01-01

The RNA transcriptome varies in response to cellular differentiation as well as environmental factors, and can be characterized by the diversity and abundance of transcript isoforms. Differential transcription analysis, the detection of differences between the transcriptomes of different cells, may improve understanding of cell differentiation and development and enable the identification of biomarkers that classify disease types. The availability of high-throughput short-read RNA sequencing technologies provides in-depth sampling of the transcriptome, making it possible to accurately detect the differences between transcriptomes. In this article, we present a new method for the detection and visualization of differential transcription. Our approach does not depend on transcript or gene annotations. It also circumvents the need for full transcript inference and quantification, which is a challenging problem because of short read lengths, as well as various sampling biases. Instead, our method takes a divide-and-conquer approach to localize the difference between transcriptomes in the form of alternative splicing modules (ASMs), where transcript isoforms diverge. Our approach starts with the identification of ASMs from the splice graph, constructed directly from the exons and introns predicted from RNA-seq read alignments. The abundance of alternative splicing isoforms residing in each ASM is estimated for each sample and is compared across sample groups. A non-parametric statistical test is applied to each ASM to detect significant differential transcription with a controlled false discovery rate. The sensitivity and specificity of the method have been assessed using simulated data sets and compared with other state-of-the-art approaches. Experimental validation using qRT-PCR confirmed a selected set of genes that are differentially expressed in a lung differentiation study and a breast cancer data set, demonstrating the utility of the approach applied on experimental biological data sets. The software of DiffSplice is available at http://www.netlab.uky.edu/p/bioinfo/DiffSplice. PMID:23155066

Comparison of non-invasive assessment of liver fibrosis in patients with alpha1-antitrypsin deficiency using magnetic resonance elastography (MRE), acoustic radiation force impulse (ARFI) Quantification, and 2D-shear wave elastography (2D-SWE).

PubMed

Reiter, Rolf; Wetzel, Martin; Hamesch, Karim; Strnad, Pavel; Asbach, Patrick; Haas, Matthias; Siegmund, Britta; Trautwein, Christian; Hamm, Bernd; Klatt, Dieter; Braun, Jürgen; Sack, Ingolf; Tzschätzsch, Heiko

2018-01-01

Although it has been known for decades that patients with alpha1-antitrypsin deficiency (AATD) have an increased risk of cirrhosis and hepatocellular carcinoma, limited data exist on non-invasive imaging-based methods for assessing liver fibrosis such as magnetic resonance elastography (MRE) and acoustic radiation force impulse (ARFI) quantification, and no data exist on 2D-shear wave elastography (2D-SWE). Therefore, the purpose of this study is to evaluate and compare the applicability of different elastography methods for the assessment of AATD-related liver fibrosis. Fifteen clinically asymptomatic AATD patients (11 homozygous PiZZ, 4 heterozygous PiMZ) and 16 matched healthy volunteers were examined using MRE and ARFI quantification. Additionally, patients were examined with 2D-SWE. A high correlation is evident for the shear wave speed (SWS) determined with different elastography methods in AATD patients: 2D-SWE/MRE, ARFI quantification/2D-SWE, and ARFI quantification/MRE (R = 0.8587, 0.7425, and 0.6914, respectively; P≤0.0089). Four AATD patients with pathologically increased SWS were consistently identified with all three methods-MRE, ARFI quantification, and 2D-SWE. The high correlation and consistent identification of patients with pathologically increased SWS using MRE, ARFI quantification, and 2D-SWE suggest that elastography has the potential to become a suitable imaging tool for the assessment of AATD-related liver fibrosis. These promising results provide motivation for further investigation of non-invasive assessment of AATD-related liver fibrosis using elastography.

Multidimensional protein identification technology (MudPIT): technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue.

PubMed

Kislinger, Thomas; Gramolini, Anthony O; MacLennan, David H; Emili, Andrew

2005-08-01

An optimized analytical expression profiling strategy based on gel-free multidimensional protein identification technology (MudPIT) is reported for the systematic investigation of biochemical (mal)-adaptations associated with healthy and diseased heart tissue. Enhanced shotgun proteomic detection coverage and improved biological inference is achieved by pre-fractionation of excised mouse cardiac muscle into subcellular components, with each organellar fraction investigated exhaustively using multiple repeat MudPIT analyses. Functional-enrichment, high-confidence identification, and relative quantification of hundreds of organelle- and tissue-specific proteins are achieved readily, including detection of low abundance transcriptional regulators, signaling factors, and proteins linked to cardiac disease. Important technical issues relating to data validation, including minimization of artifacts stemming from biased under-sampling and spurious false discovery, together with suggestions for further fine-tuning of sample preparation, are discussed. A framework for follow-up bioinformatic examination, pattern recognition, and data mining is also presented in the context of a stringent application of MudPIT for probing fundamental aspects of heart muscle physiology as well as the discovery of perturbations associated with heart failure.

Comparative proteomic and bioinformatic analysis of Theileria luwenshuni and Theileria uilenbergi.

PubMed

Zhang, Xiao; Li, Youquan; Chen, Ze; Liu, Zhijie; Ren, Qiaoyun; Yang, Jifei; Zhu, Xinquan; Guan, Guiquan; Liu, Aihong; Luo, Jianxun; Yin, Hong

2016-07-01

Theileria is an obligatory intraerythrocytic protozoan parasite that causes economic losses to the cattle, sheep and goats industry. However, very little information is available on the genomes, transcriptomes, and proteomes of the ovine parasites, Theileria luwenshuni and Theileria uilenbergi. Differences in protein expression between these species were investigated to better understand their biology. Parasites were digested with trypsin, and the resulting peptides labeled with isobaric tags for relative and absolute quantification, followed by LC-MS/MS. More than 670 proteins, classified into categories primarily related to cellular process (29.78%), metabolic process (28.80%), localization (5.22%) and biological regulation (5.00%), were identified. Seventy-one proteins were differentially expressed; T. luwenshuni had 39 proteins more highly expressed than in T. uilenbergi, whereas T. uilenbergi had 32 that were more highly expressed. Several proteins related to parasite virulence and invasion (cysteine proteinase, histone deacetylase, pyruvate kinase, small nuclear ribonucleoprotein and orotate phosphoribosyltransferase) were differentially expressed. Real-time quantitative PCR validated protein expression changes at the transcript level. This is the first report on protein expression for the two most economically important Theileria species in China, and our findings may provide novel opportunities for ovine and caprine theileriosis control. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene Expression in Mammalian Cells After Exposure to 95 MeV Argon Ions

NASA Astrophysics Data System (ADS)

Arenz, A.; Hellweg, C. E.; Baumstark-Khan, C.

Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). The unique feature of the space radiation environment is the dominance of high-energy charged particles (HZE or high LET radiation) which present a significant hazard to space flight crews, and accelerator-based experiments are underway to quantify the health risks due to unavoidable radiation exposure. High linear energy transfer (LET) radiation has an increased relative biological effectiveness (RBE) as compared to X-rays for cell death induction, gene mutation, genomic instability, and carcinogenesis. The tumour suppressor gene p53 plays a crucial role in maintaining the integrity of the genome. The p53 protein acts as a transcription factor that mediates cell cycle arrest and apoptosis by binding to DNA and activating transcription of specific genes. It is also though to be involved in damage repair by transcriptional activation of the newly identified p53 dependent ribonuclease subunit R2 (p53R2) that is directly involved in the p53 cell cycle checkpoint for repair of damaged DNA. In that case it is responsible for nucleotide delivery for DNA repair synthesis. DNA damages of cultured human cells (e.g. MCF-7, AGS, A549) exposed to accelerated argon ions at the French heavy ion facility GANIL were analysed for expression levels of certain damage- and apoptosis-relevant genes. RNA was extracted from cells exposed to different particle fluences after various recovery times. A real-time QRT-PCR assay was applied, which employs both relative and absolute quantification of a candidate mRNA biomarker. The expressions of different DNA damage inducible genes (e.g. p53R2, GADD45, p21) were analysed. A reproducible up-regulation representing a twofold to fourfold change in p53R2 gene expression level was confirmed for X-irradiated and Ar-ion exposed cells dependent on dose. Kinetics of p53R2 gene expression modulations shows a response lasting up to 24 hours after irradiation.

Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

PubMed Central

Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

Hepatitis E virus RNA in Australian blood donations.

PubMed

Shrestha, Ashish C; Flower, Robert L P; Seed, Clive R; Keller, Anthony J; Harley, Robert; Chan, Hiu-Tat; Hoad, Veronica; Warrilow, David; Northill, Judith; Holmberg, Jerry A; Faddy, Helen M

2016-12-01

Hepatitis E virus (HEV) poses a risk to transfusion safety. In Australia, locally acquired HEV is rare and cases are mainly reported in travelers returning from countries endemic for HEV. The risk posed by HEV to transfusion safety in Australia is unknown; therefore, we aimed to measure the rate of current HEV infection in Australian blood donations. A total of 14,799 blood donations were tested for HEV RNA by transcription-mediated amplification, with confirmatory testing by reverse transcription-polymerase chain reaction. Viral load quantification and phylogenetic analysis was performed on HEV RNA-positive samples. One (0.0068%; 95% confidence interval [CI], 0.0002%-0.0376%) sample was confirmed positive for HEV RNA, resulting in a risk of collecting a HEV-viremic donation of 1 in 14,799 (95% CI, 1 in 584,530 to 1 in 2,657). The viral load in this sample was approximately 15,000 IU/mL, and it was determined to be Genotype 3. Our finding of 1 in 14,799 Australian donations positive for HEV RNA is lower than that from many other developed countries; this is consistent with the relatively low seroprevalence in Australia. As this HEV RNA-positive sample was Genotype 3, it seems likely that this infection was acquired through zoonotic transmission, either within Australia or overseas in a developed nation. HEV has the potential to pose a risk to transfusion safety in Australia; however, additional, larger studies are required to quantify the magnitude of this risk. © 2016 AABB.

APAtrap: identification and quantification of alternative polyadenylation sites from RNA-seq data.

PubMed

Ye, Congting; Long, Yuqi; Ji, Guoli; Li, Qingshun Quinn; Wu, Xiaohui

2018-06-01

Alternative polyadenylation (APA) has been increasingly recognized as a crucial mechanism that contributes to transcriptome diversity and gene expression regulation. As RNA-seq has become a routine protocol for transcriptome analysis, it is of great interest to leverage such unprecedented collection of RNA-seq data by new computational methods to extract and quantify APA dynamics in these transcriptomes. However, research progress in this area has been relatively limited. Conventional methods rely on either transcript assembly to determine transcript 3' ends or annotated poly(A) sites. Moreover, they can neither identify more than two poly(A) sites in a gene nor detect dynamic APA site usage considering more than two poly(A) sites. We developed an approach called APAtrap based on the mean squared error model to identify and quantify APA sites from RNA-seq data. APAtrap is capable of identifying novel 3' UTRs and 3' UTR extensions, which contributes to locating potential poly(A) sites in previously overlooked regions and improving genome annotations. APAtrap also aims to tally all potential poly(A) sites and detect genes with differential APA site usages between conditions. Extensive comparisons of APAtrap with two other latest methods, ChangePoint and DaPars, using various RNA-seq datasets from simulation studies, human and Arabidopsis demonstrate the efficacy and flexibility of APAtrap for any organisms with an annotated genome. Freely available for download at https://apatrap.sourceforge.io. liqq@xmu.edu.cn or xhuister@xmu.edu.cn. Supplementary data are available at Bioinformatics online.

Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

PubMed Central

Jiwaji, Meesbah; Sandison, Mairi E.; Reboud, Julien; Stevenson, Ross; Daly, Rónán; Barkess, Gráinne; Faulds, Karen; Kolch, Walter; Graham, Duncan; Girolami, Mark A.; Cooper, Jonathan M.; Pitt, Andrew R.

2014-01-01

Introduction Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. PMID:24926959

Curcumin is a potent modulator of microglial gene expression and migration

PubMed Central

2011-01-01

Background Microglial cells are important effectors of the neuronal innate immune system with a major role in chronic neurodegenerative diseases. Curcumin, a major component of tumeric, alleviates pro-inflammatory activities of these cells by inhibiting nuclear factor kappa B (NFkB) signaling. To study the immuno-modulatory effects of curcumin on a transcriptomic level, DNA-microarray analyses were performed with resting and LPS-challenged microglial cells after short-term treatment with curcumin. Methods Resting and LPS-activated BV-2 cells were stimulated with curcumin and genome-wide mRNA expression patterns were determined using DNA-microarrays. Selected qRT-PCR analyses were performed to confirm newly identified curcumin-regulated genes. The migration potential of microglial cells was determined with wound healing assays and transwell migration assays. Microglial neurotoxicity was estimated by morphological analyses and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium. Results Curcumin treatment markedly changed the microglial transcriptome with 49 differentially expressed transcripts in a combined analysis of resting and activated microglial cells. Curcumin effectively triggered anti-inflammatory signals as shown by induced expression of Interleukin 4 and Peroxisome proliferator activated receptor α. Several novel curcumin-induced genes including Netrin G1, Delta-like 1, Platelet endothelial cell adhesion molecule 1, and Plasma cell endoplasmic reticulum protein 1, have been previously associated with adhesion and cell migration. Consequently, curcumin treatment significantly inhibited basal and activation-induced migration of BV-2 microglia. Curcumin also potently blocked gene expression related to pro-inflammatory activation of resting cells including Toll-like receptor 2 and Prostaglandin-endoperoxide synthase 2. Moreover, transcription of NO synthase 2 and Signal transducer and activator of transcription 1 was reduced in LPS-triggered microglia. These transcriptional changes in curcumin-treated LPS-primed microglia also lead to decreased neurotoxicity with reduced apoptosis of 661W photoreceptor cultures. Conclusions Collectively, our results suggest that curcumin is a potent modulator of the microglial transcriptome. Curcumin attenuates microglial migration and triggers a phenotype with anti-inflammatory and neuroprotective properties. Thus, curcumin could be a nutraceutical compound to develop immuno-modulatory and neuroprotective therapies for the treatment of various neurodegenerative disorders. PMID:21958395

Normal Databases for the Relative Quantification of Myocardial Perfusion

PubMed Central

Rubeaux, Mathieu; Xu, Yuan; Germano, Guido; Berman, Daniel S.; Slomka, Piotr J.

2016-01-01

Purpose of review Myocardial perfusion imaging (MPI) with SPECT is performed clinically worldwide to detect and monitor coronary artery disease (CAD). MPI allows an objective quantification of myocardial perfusion at stress and rest. This established technique relies on normal databases to compare patient scans against reference normal limits. In this review, we aim to introduce the process of MPI quantification with normal databases and describe the associated perfusion quantitative measures that are used. Recent findings New equipment and new software reconstruction algorithms have been introduced which require the development of new normal limits. The appearance and regional count variations of normal MPI scan may differ between these new scanners and standard Anger cameras. Therefore, these new systems may require the determination of new normal limits to achieve optimal accuracy in relative myocardial perfusion quantification. Accurate diagnostic and prognostic results rivaling those obtained by expert readers can be obtained by this widely used technique. Summary Throughout this review, we emphasize the importance of the different normal databases and the need for specific databases relative to distinct imaging procedures. use of appropriate normal limits allows optimal quantification of MPI by taking into account subtle image differences due to the hardware and software used, and the population studied. PMID:28138354

Determination of Oversulphated Chondroitin Sulphate and Dermatan Sulphate in unfractionated heparin by (1)H-NMR - Collaborative study for quantification and analytical determination of LoD.

PubMed

McEwen, I; Mulloy, B; Hellwig, E; Kozerski, L; Beyer, T; Holzgrabe, U; Wanko, R; Spieser, J-M; Rodomonte, A

2008-12-01

Oversulphated Chondroitin Sulphate (OSCS) and Dermatan Sulphate (DS) in unfractionated heparins can be identified by nuclear magnetic resonance spectrometry (NMR). The limit of detection (LoD) of OSCS is 0.1% relative to the heparin content. This LoD is obtained at a signal-to-noise ratio (S/N) of 2000:1 of the heparin methyl signal. Quantification is best obtained by comparing peak heights of the OSCS and heparin methyl signals. Reproducibility of less than 10% relative standard deviation (RSD) has been obtained. The accuracy of quantification was good.

Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans

PubMed Central

Watson, Emma; Olin-Sandoval, Viridiana; Hoy, Michael J; Li, Chi-Hua; Louisse, Timo; Yao, Victoria; Mori, Akihiro; Holdorf, Amy D; Troyanskaya, Olga G; Ralser, Markus; Walhout, Albertha JM

2016-01-01

Metabolic network rewiring is the rerouting of metabolism through the use of alternate enzymes to adjust pathway flux and accomplish specific anabolic or catabolic objectives. Here, we report the first characterization of two parallel pathways for the breakdown of the short chain fatty acid propionate in Caenorhabditis elegans. Using genetic interaction mapping, gene co-expression analysis, pathway intermediate quantification and carbon tracing, we uncover a vitamin B12-independent propionate breakdown shunt that is transcriptionally activated on vitamin B12 deficient diets, or under genetic conditions mimicking the human diseases propionic- and methylmalonic acidemia, in which the canonical B12-dependent propionate breakdown pathway is blocked. Our study presents the first example of transcriptional vitamin-directed metabolic network rewiring to promote survival under vitamin deficiency. The ability to reroute propionate breakdown according to B12 availability may provide C. elegans with metabolic plasticity and thus a selective advantage on different diets in the wild. DOI: http://dx.doi.org/10.7554/eLife.17670.001 PMID:27383050

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

NASA Technical Reports Server (NTRS)

Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

1998-01-01

A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

Network analysis for the visualization and analysis of qualitative data.

PubMed

Pokorny, Jennifer J; Norman, Alex; Zanesco, Anthony P; Bauer-Wu, Susan; Sahdra, Baljinder K; Saron, Clifford D

2018-03-01

We present a novel manner in which to visualize the coding of qualitative data that enables representation and analysis of connections between codes using graph theory and network analysis. Network graphs are created from codes applied to a transcript or audio file using the code names and their chronological location. The resulting network is a representation of the coding data that characterizes the interrelations of codes. This approach enables quantification of qualitative codes using network analysis and facilitates examination of associations of network indices with other quantitative variables using common statistical procedures. Here, as a proof of concept, we applied this method to a set of interview transcripts that had been coded in 2 different ways and the resultant network graphs were examined. The creation of network graphs allows researchers an opportunity to view and share their qualitative data in an innovative way that may provide new insights and enhance transparency of the analytical process by which they reach their conclusions. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus

PubMed Central

Nombela, Ivan; Puente-Marin, Sara; Chico, Veronica; Villena, Alberto J.; Carracedo, Begoña; Ciordia, Sergio; Mena, Maria Carmen; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria del Mar

2018-01-01

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. PMID:29527292

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study[W][OA

PubMed Central

Graeber, Kai; Linkies, Ada; Wood, Andrew T.A.; Leubner-Metzger, Gerhard

2011-01-01

Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR. PMID:21666000

Establishing Ion Ratio Thresholds Based on Absolute Peak Area for Absolute Protein Quantification using Protein Cleavage Isotope Dilution Mass Spectrometry

PubMed Central

Loziuk, Philip L.; Sederoff, Ronald R.; Chiang, Vincent L.; Muddiman, David C.

2014-01-01

Quantitative mass spectrometry has become central to the field of proteomics and metabolomics. Selected reaction monitoring is a widely used method for the absolute quantification of proteins and metabolites. This method renders high specificity using several product ions measured simultaneously. With growing interest in quantification of molecular species in complex biological samples, confident identification and quantitation has been of particular concern. A method to confirm purity or contamination of product ion spectra has become necessary for achieving accurate and precise quantification. Ion abundance ratio assessments were introduced to alleviate some of these issues. Ion abundance ratios are based on the consistent relative abundance (RA) of specific product ions with respect to the total abundance of all product ions. To date, no standardized method of implementing ion abundance ratios has been established. Thresholds by which product ion contamination is confirmed vary widely and are often arbitrary. This study sought to establish criteria by which the relative abundance of product ions can be evaluated in an absolute quantification experiment. These findings suggest that evaluation of the absolute ion abundance for any given transition is necessary in order to effectively implement RA thresholds. Overall, the variation of the RA value was observed to be relatively constant beyond an absolute threshold ion abundance. Finally, these RA values were observed to fluctuate significantly over a 3 year period, suggesting that these values should be assessed as close as possible to the time at which data is collected for quantification. PMID:25154770

A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.

2013-02-01

Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred frommore » one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.« less

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

A novel method for room temperature distribution and conservation of RNA and DNA reference materials for guaranteeing performance of molecular diagnostics in onco-hematology: A GBMHM study.

PubMed

Cayuela, Jean-Michel; Mauté, Carole; Fabre, Anne-Lise; Nibourel, Olivier; Dulucq, Stéphanie; Delabesse, Eric; Villarèse, Patrick; Hayette, Sandrine; Mozziconacci, Marie-Joelle; Macintyre, Elizabeth

2015-10-01

Performance of methods used for molecular diagnostics must be closely controlled by regular analysis of internal quality controls. However, conditioning, shipping and long lasting storage of nucleic acid controls remain problematic. Therefore, we evaluated the minicapsule-based innovative process developed by Imagene (Evry, France) for implementing DNA and RNA controls designed for clonality assessment of lymphoproliferations and BCR-ABL1 mRNA quantification, respectively. DNA samples were extracted from 12 cell lines selected for giving specific amplifications with most BIOMED-2 PCR tubes. RNA samples were extracted from 8 cell line mixtures expressing various BCR-ABL1 transcript levels. DNA and RNA were encapsulated by Imagene and shipped at room temperature to participating laboratories. Biologists were asked to report quality data of recovered nucleic acids as well as PCR results. Encapsulated nucleic acids samples were easily and efficiently recovered from minicapsules. The expected rearrangements at immunoglobulin, T-cell receptor and BCL2 loci were detected in DNA samples by all laboratories. Quality of RNA was consistent between laboratories and met the criteria requested for quantification of BCR-ABL1 transcripts. Expression levels measured by the 5 laboratories were within ±2 fold interval from the corresponding pre-encapsulation reference value. Moreover aging studies of encapsulated RNA simulating up to 100 years storage at room temperature show no bias in quantitative outcome. Therefore, Imagene minicapsules are suitable for storage and distribution at room temperature of genetic material designed for proficiency control of molecular diagnostic methods based on end point or real-time quantitative PCR. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application.

PubMed

D'Antonio, Mattia; D'Onorio De Meo, Paolo; Pallocca, Matteo; Picardi, Ernesto; D'Erchia, Anna Maria; Calogero, Raffaele A; Castrignanò, Tiziana; Pesole, Graziano

2015-01-01

The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs.

Predicting conformational ensembles and genome-wide transcription factor binding sites from DNA sequences.

PubMed

Andrabi, Munazah; Hutchins, Andrew Paul; Miranda-Saavedra, Diego; Kono, Hidetoshi; Nussinov, Ruth; Mizuguchi, Kenji; Ahmad, Shandar

2017-06-22

DNA shape is emerging as an important determinant of transcription factor binding beyond just the DNA sequence. The only tool for large scale DNA shape estimates, DNAshape was derived from Monte-Carlo simulations and predicts four broad and static DNA shape features, Propeller twist, Helical twist, Minor groove width and Roll. The contributions of other shape features e.g. Shift, Slide and Opening cannot be evaluated using DNAshape. Here, we report a novel method DynaSeq, which predicts molecular dynamics-derived ensembles of a more exhaustive set of DNA shape features. We compared the DNAshape and DynaSeq predictions for the common features and applied both to predict the genome-wide binding sites of 1312 TFs available from protein interaction quantification (PIQ) data. The results indicate a good agreement between the two methods for the common shape features and point to advantages in using DynaSeq. Predictive models employing ensembles from individual conformational parameters revealed that base-pair opening - known to be important in strand separation - was the best predictor of transcription factor-binding sites (TFBS) followed by features employed by DNAshape. Of note, TFBS could be predicted not only from the features at the target motif sites, but also from those as far as 200 nucleotides away from the motif.

Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

PubMed

Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

2017-04-01

Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10 2 to 10 3 copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.).

PubMed

Mariot, Roberta Fogliatto; de Oliveira, Luisa Abruzzi; Voorhuijzen, Marleen M; Staats, Martijn; Hutten, Ronald C B; Van Dijk, Jeroen P; Kok, Esther; Frazzon, Jeverson

2015-01-01

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.

Archaeal RNA polymerase and transcription regulation

PubMed Central

Jun, Sung-Hoon; Reichlen, Matthew J.; Tajiri, Momoko; Murakami, Katsuhiko S.

2010-01-01

To elucidate the mechanism of transcription by cellular RNA polymerases (RNAPs), high resolution X-ray crystal structures together with structure-guided biochemical, biophysical and genetics studies are essential. The recently-solved X-ray crystal structures of archaeal RNA polymerase (RNAP) allow a structural comparison of the transcription machinery among all three domains of life. The archaea were once thought of closely related to bacteria, but they are now considered to be more closely related to the eukaryote at the molecular level than bacteria. According to these structures, the archaeal transcription apparatus, which includes RNAP and general transcription factors, is similar to the eukaryotic transcription machinery. Yet, the transcription regulators, activators and repressors, encoded by archaeal genomes are closely related to bacterial factors. Therefore, archaeal transcription appears to possess an intriguing hybrid of eukaryotic-type transcription apparatus and bacterial-like regulatory mechanisms. Elucidating the transcription mechanism in archaea, which possesses a combination of bacterial and eukaryotic transcription mechanisms that are commonly regarded as separate and mutually exclusive, can provide data that will bring basic transcription mechanisms across all three domains of life. PMID:21250781

On the Confounding Effect of Temperature on Chemical Shift-Encoded Fat Quantification

PubMed Central

Hernando, Diego; Sharma, Samir D.; Kramer, Harald; Reeder, Scott B.

2014-01-01

Purpose To characterize the confounding effect of temperature on chemical shift-encoded (CSE) fat quantification. Methods The proton resonance frequency of water, unlike triglycerides, depends on temperature. This leads to a temperature dependence of the spectral models of fat (relative to water) that are commonly used by CSE-MRI methods. Simulation analysis was performed for 1.5 Tesla CSE fat–water signals at various temperatures and echo time combinations. Oil–water phantoms were constructed and scanned at temperatures between 0 and 40°C using spectroscopy and CSE imaging at three echo time combinations. An explanted human liver, rejected for transplantation due to steatosis, was scanned using spectroscopy and CSE imaging. Fat–water reconstructions were performed using four different techniques: magnitude and complex fitting, with standard or temperature-corrected signal modeling. Results In all experiments, magnitude fitting with standard signal modeling resulted in large fat quantification errors. Errors were largest for echo time combinations near TEinit ≈ 1.3 ms, ΔTE ≈ 2.2 ms. Errors in fat quantification caused by temperature-related frequency shifts were smaller with complex fitting, and were avoided using a temperature-corrected signal model. Conclusion Temperature is a confounding factor for fat quantification. If not accounted for, it can result in large errors in fat quantifications in phantom and ex vivo acquisitions. PMID:24123362

Pesticide Side Effects in an Agricultural Soil Ecosystem as Measured by amoA Expression Quantification and Bacterial Diversity Changes

PubMed Central

Feld, Louise; Hjelmsø, Mathis Hjort; Nielsen, Morten Schostag; Jacobsen, Anne Dorthe; Rønn, Regin; Ekelund, Flemming; Krogh, Paul Henning; Strobel, Bjarne Westergaard; Jacobsen, Carsten Suhr

2015-01-01

Background and Methods Assessing the effects of pesticide hazards on microbiological processes in the soil is currently based on analyses that provide limited insight into the ongoing processes. This study proposes a more comprehensive approach. The side effects of pesticides may appear as changes in the expression of specific microbial genes or as changes in diversity. To assess the impact of pesticides on gene expression, we focused on the amoA gene, which is involved in ammonia oxidation. We prepared soil microcosms and exposed them to dazomet, mancozeb or no pesticide. We hypothesized that the amount of amoA transcript decreases upon pesticide application, and to test this hypothesis, we used reverse-transcription qPCR. We also hypothesized that bacterial diversity is affected by pesticides. This hypothesis was investigated via 454 sequencing and diversity analysis of the 16S ribosomal RNA and RNA genes, representing the active and total soil bacterial communities, respectively. Results and Conclusion Treatment with dazomet reduced both the bacterial and archaeal amoA transcript numbers by more than two log units and produced long-term effects for more than 28 days. Mancozeb also inhibited the numbers of amoA transcripts, but only transiently. The bacterial and archaeal amoA transcripts were both sensitive bioindicators of pesticide side effects. Additionally, the numbers of bacterial amoA transcripts correlated with nitrate production in N-amended microcosms. Dazomet reduced the total bacterial numbers by one log unit, but the population size was restored after twelve days. The diversity of the active soil bacteria also seemed to be re-established after twelve days. However, the total bacterial diversity as reflected in the 16S ribosomal RNA gene sequences was largely dominated by Firmicutes and Proteobacteria at day twelve, likely reflecting a halt in the growth of early opportunists and the re-establishment of a more diverse population. We observed no effects of mancozeb on diversity. PMID:25938467

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

PubMed

Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

2017-08-30

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these findings suggest that RbSTAT2 is involved in various biologically crucial mechanisms, and provides immune protection to the rock bream. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.

PubMed

Vendrame, Marco; Manzano, Marisa; Comi, Giuseppe; Bertrand, Julien; Iacumin, Lucilla

2014-09-01

Brettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B. bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B. bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B. bruxellensis DSMZ 70726. The obtained detection limits were 0.83 log CFU/mL in red wine, 0.63 log CFU/mL in white wine and 0.23 log CFU/mL in beer. Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials. Copyright © 2014 Elsevier Ltd. All rights reserved.

An in-advance stable isotope labeling strategy for relative analysis of multiple acidic plant hormones in sub-milligram Arabidopsis thaliana seedling and a single seed.

PubMed

Sun, Xiaohong; Ouyang, Yue; Chu, Jinfang; Yan, Jing; Yu, Yan; Li, Xiaoqiang; Yang, Jun; Yan, Cunyu

2014-04-18

A sensitive and reliable in-advance stable isotope labeling strategy was developed for simultaneous relative quantification of 8 acidic plant hormones in sub-milligram amount of plant materials. Bromocholine bromide (BETA) and its deuterated counterpart D9-BETA were used to in-advance derivatize control and sample extracts individually, which were then combined and subjected to solid-phase extraction (SPE) purification followed by UPLC-MS/MS analysis. Relative quantification of target compounds was obtained by calculation of the peak area ratios of BETA/D9-BETA labeled plant hormones. The in-advance stable isotope labeling strategy realized internal standard-based relative quantification of multiple kinds of plant hormones independent of availability of internal standard of every analyte with enhanced sensitivity of 1-3 orders of magnitude. Meanwhile, the in-advance labeling contributes to higher sample throughput and more reliability. The method was successfully applied to determine 8 plant hormones in 0.8mg DW (dry weight) of seedlings and 4 plant hormones from single seed of Arabidopsis thaliana. The results show the potential of the method in relative quantification of multiple plant hormones in tiny plant tissues or organs, which will advance the knowledge of the crosstalk mechanism of plant hormones. Copyright © 2014 Elsevier B.V. All rights reserved.

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

PubMed

Tian, Hui; Sun, Yuanyuan; Liu, Chenghui; Duan, Xinrui; Tang, Wei; Li, Zhengping

2016-12-06

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

Dietary cholecalciferol regulates the recruitment and growth of skeletal muscle fibers and the expressions of myogenic regulatory factors and the myosin heavy chain in European sea bass larvae.

PubMed

Alami-Durante, Hélène; Cluzeaud, Marianne; Bazin, Didier; Mazurais, David; Zambonino-Infante, José L

2011-12-01

The aim of this study was to determine whether dietary cholecalciferol affects the recruitment and growth of axial skeletal muscle fibers in first-feeding European sea bass. Larvae were fed diets containing 0.28 (VD-L, low dose), 0.69 (VD-C, control dose), or 3.00 (VD-H, high dose) mg cholecalciferol/kg from 9 to 44 d posthatching (dph). Larvae were sampled at 44 dph for quantification of somatic growth, muscle growth, and muscle growth dynamics and at 22 and 44 dph for the relative quantification of transcripts encoded by genes involved in myogenesis, cell proliferation, and muscle structure. The weight increase of the VD-L-fed larvae was less than that of the VD-H-fed group, whereas that of VD-C-fed larvae was intermediate. The level of expression of genes involved in cell proliferation (PCNA) and early myogenesis (Myf5) decreased between 22 and 44 dph, whereas that of the myogenic determination factor MyoD1 and that of genes involved in muscle structure and function (myosin heavy chain, myosin light chains 2 and 3) increased. Dietary cholecalciferol regulated Myf5, MyoD1, myogenin, and myosin heavy chain gene expression, with a gene-specific shape of response. The maximum hypertrophy of white muscle fibers was higher in larvae fed the VD-C and VD-H diets than in larvae fed the VD-L diet. White muscle hyperplasia was highly stimulated in VD-H-fed larvae compared to VD-L- and VD-C-fed ones. These findings demonstrate a dietary cholecalciferol effect on skeletal muscle growth mechanisms of a Teleost species.

41 CFR 60-30.22 - Official transcript.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 41 Public Contracts and Property Management 1 2011-07-01 2009-07-01 true Official transcript. 60-30.22 Section 60-30.22 Public Contracts and Property Management Other Provisions Relating to Public... ORDER 11246 Hearings and Related Matters § 60-30.22 Official transcript. The official transcripts of...

41 CFR 60-30.22 - Official transcript.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Official transcript. 60-30.22 Section 60-30.22 Public Contracts and Property Management Other Provisions Relating to Public... ORDER 11246 Hearings and Related Matters § 60-30.22 Official transcript. The official transcripts of...

22 CFR 92.61 - Transcription and signing of record of examination.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Transcription and signing of record of... RELATED SERVICES Depositions and Letters Rogatory § 92.61 Transcription and signing of record of... be fully transcribed and the transcription attached securely to any document or documents to which...

22 CFR 92.61 - Transcription and signing of record of examination.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Transcription and signing of record of... RELATED SERVICES Depositions and Letters Rogatory § 92.61 Transcription and signing of record of... be fully transcribed and the transcription attached securely to any document or documents to which...

Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

PubMed Central

Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

1994-01-01

Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

Molecular cloning and transcriptional analysis of a NPY receptor-like in common Chinese cuttlefish Sepiella japonica

NASA Astrophysics Data System (ADS)

Yang, Jingwen; Xu, Yuchao; Xu, Ke; Ping, Hongling; Shi, Huilai; Lü, Zhenming; Wu, Changwen; Wang, Tianming

2017-08-01

Neuropeptide Y (NPY) has a pivotal role in the regulation of many physiological processes. In this study, the gene encoding a NPY receptor-like from the common Chinese cuttlefish Sepiella japonica (SjNPYR-like) was identified and characterized. The full-length SjNPYR-like cDNA was cloned containing a 492-bp of 5' untranslated region (UTR), 1 182 bp open reading frame (ORF) encoding a protein of 393 amino acid residues, and 228 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 45.54 kDa and an isoelectric point (pI) of 8.13. By informatic analyses, SjNPYR-like was identified as belonging to the class A G protein coupled receptor (GPCR) family (the rhodopsin-type). The amino acid sequence contained 12 potential phosphorylation sites and five predicted N-linked glycosylation sites. Multiple sequence alignment and 3D structure modeling were conducted to clarify SjNPYR bioinformatics characteristics. Phylogenetic analysis identifies it as an NPYR with identity of 33% to Lymnaea stagnalis NPFR. Transmembrane properties of SjNPYR-like were demonstrated in vitro using HEK293 cells and the pEGFP-N1 plasmid. Relative quantification of SjNPYR-like mRNA level confirmed a high level expression and broad distribution of SjNPYR - like in various tissues of female S. japonica. In addition, the transcriptional profile of SjNPYR - like in the brain, liver, and ovary during gonadal development was analyzed. The results provide basic understanding on the molecular characteristics of SjNPYR-like and its potentially physical functions.

Forensic molecular pathology: its impacts on routine work, education and training.

PubMed

Maeda, Hitoshi; Ishikawa, Takaki; Michiue, Tomomi

2014-03-01

The major role of forensic pathology is the investigation of human death in relevance to social risk management to determine the cause and process of death, especially in violent and unexpected sudden deaths, which involve social and medicolegal issues of ultimate, personal and public concerns. In addition to the identification of victims and biological materials, forensic molecular pathology contributes to general explanation of the human death process and assessment of individual death on the basis of biological molecular evidence, visualizing dynamic functional changes involved in the dying process that cannot be detected by morphology (pathophysiological or molecular biological vital reactions); the genetic background (genomics), dynamics of gene expression (up-/down-regulation: transcriptomics) and vital phenomena, involving activated biological mediators and degenerative products (proteomics) as well as metabolic deterioration (metabolomics), are detected by DNA analysis, relative quantification of mRNA transcripts using real-time reverse transcription-PCR (RT-PCR), and immunohisto-/immunocytochemistry combined with biochemistry, respectively. Thus, forensic molecular pathology involves the application of omic medical sciences to investigate the genetic basis, and cause and process of death at the biological molecular level in the context of forensic pathology, that is, 'advanced molecular autopsy'. These procedures can be incorporated into routine death investigations as well as guidance, education and training programs in forensic pathology for 'dynamic assessment of the cause and process of death' on the basis of autopsy and laboratory data. Postmortem human data can also contribute to understanding patients' critical conditions in clinical management. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

TransAtlasDB: an integrated database connecting expression data, metadata and variants

PubMed Central

Adetunji, Modupeore O; Lamont, Susan J; Schmidt, Carl J

2018-01-01

Abstract High-throughput transcriptome sequencing (RNAseq) is the universally applied method for target-free transcript identification and gene expression quantification, generating huge amounts of data. The constraint of accessing such data and interpreting results can be a major impediment in postulating suitable hypothesis, thus an innovative storage solution that addresses these limitations, such as hard disk storage requirements, efficiency and reproducibility are paramount. By offering a uniform data storage and retrieval mechanism, various data can be compared and easily investigated. We present a sophisticated system, TransAtlasDB, which incorporates a hybrid architecture of both relational and NoSQL databases for fast and efficient data storage, processing and querying of large datasets from transcript expression analysis with corresponding metadata, as well as gene-associated variants (such as SNPs) and their predicted gene effects. TransAtlasDB provides the data model of accurate storage of the large amount of data derived from RNAseq analysis and also methods of interacting with the database, either via the command-line data management workflows, written in Perl, with useful functionalities that simplifies the complexity of data storage and possibly manipulation of the massive amounts of data generated from RNAseq analysis or through the web interface. The database application is currently modeled to handle analyses data from agricultural species, and will be expanded to include more species groups. Overall TransAtlasDB aims to serve as an accessible repository for the large complex results data files derived from RNAseq gene expression profiling and variant analysis. Database URL: https://modupeore.github.io/TransAtlasDB/ PMID:29688361

Identification of Proteins Involved in Carbohydrate Metabolism and Energy Metabolism Pathways and Their Regulation of Cytoplasmic Male Sterility in Wheat.

PubMed

Geng, Xingxia; Ye, Jiali; Yang, Xuetong; Li, Sha; Zhang, Lingli; Song, Xiyue

2018-01-23

Cytoplasmic male sterility (CMS) where no functional pollen is produced has important roles in wheat breeding. The anther is a unique organ for male gametogenesis and its abnormal development can cause male sterility. However, the mechanisms and regulatory networks related to plant male sterility are poorly understood. In this study, we conducted comparative analyses using isobaric tags for relative and absolute quantification (iTRAQ) of the pollen proteins in a CMS line and its wheat maintainer. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology classifications, metabolic pathways and transcriptional regulation networks using Blast2GO. We identified 5570 proteins based on 23,277 peptides, which matched with 73,688 spectra, including proteins in key pathways such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and 6-phosphofructokinase 1 in the glycolysis pathway, isocitrate dehydrogenase and citrate synthase in the tricarboxylic acid cycle and nicotinamide adenine dinucleotide (NADH)-dehydrogenase and adenosine-triphosphate (ATP) synthases in the oxidative phosphorylation pathway. These proteins may comprise a network that regulates male sterility in wheat. Quantitative real time polymerase chain reaction (qRT-PCR) analysis, ATP assays and total sugar assays validated the iTRAQ results. These DAPs could be associated with abnormal pollen grain formation and male sterility. Our findings provide insights into the molecular mechanism related to male sterility in wheat.

Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs

NASA Astrophysics Data System (ADS)

Wetzel, Collin; Li, Siwei; Limbach, Patrick A.

2014-07-01

Mapping, sequencing, and quantifying individual noncoding ribonucleic acids (ncRNAs), including post-transcriptionally modified nucleosides, by mass spectrometry is a challenge that often requires rigorous sample preparation prior to analysis. Previously, we have described a simplified method for the comparative analysis of RNA digests (CARD) that is applicable to relatively complex mixtures of ncRNAs. In the CARD approach for transfer RNA (tRNA) analysis, two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. This approach allows one to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging because of convoluted mass spectra arising from the natural 13C and 15 N isotopes present in the ribonuclease-digested tRNA samples. Here, we demonstrate that culturing in 12C-enriched/13C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality yield a 35 % improvement in detection of tRNA digestion products that can be uniquely assigned to particular tRNAs. These mass spectral improvements lead to a significant reduction in data processing attributable to the ease of spectral identification of labeled digestion products and will enable improvements in the relative quantification of modified RNAs by the 16O/18O differential labeling approach.

Design of RNA splicing analysis null models for post hoc filtering of Drosophila head RNA-Seq data with the splicing analysis kit (Spanki)

PubMed Central

2013-01-01

Background The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment. Results We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb.umd.edu/software/spanki. Conclusions Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools. PMID:24209455

Design of RNA splicing analysis null models for post hoc filtering of Drosophila head RNA-Seq data with the splicing analysis kit (Spanki).

PubMed

Sturgill, David; Malone, John H; Sun, Xia; Smith, Harold E; Rabinow, Leonard; Samson, Marie-Laure; Oliver, Brian

2013-11-09

The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment. We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb.umd.edu/software/spanki. Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools.

Complementary regulation of four Eucalyptus CBF genes under various cold conditions

PubMed Central

Navarro, M.; Marque, G.; Ayax, C.; Keller, G.; Borges, J. P.; Marque, C.; Teulières, C.

2009-01-01

CBF transcription factors play central roles in the control of freezing tolerance in plants. The isolation of two additional CBF genes, EguCBF1c and EguCBF1d, from E. gunnii, one of the cold-hardiest Eucalyptus species, is described. While the EguCBF1D protein sequence is very similar to the previously characterized EguCBF1A and EguCBF1B sequences, EguCBF1C is more distinctive, in particular in the AP2-DBD (AP2-DNA binding domain). The expression analysis of the four genes by RT-qPCR reveals that none of them is specific to one stress but they are all preferentially induced by cold, except for the EguCBF1c gene which is more responsive to salt. The calculation of the transcript copy number enables the quantification of constitutive CBF gene expression. This basal level, significant for the four genes, greatly influences the final EguCBF1 transcript level in the cold. A cold shock at 4 °C, as well as a progressive freezing which mimics a natural frost episode, trigger a fast and strong response of the EguCBF1 genes, while growth at acclimating temperatures results in a lower but more durable induction. The differential expression of the four EguCBF1 genes under these cold regimes suggests that there is a complementary regulation. The high accumulation of the CBF transcript, observed in response to the different types of cold conditions, might be a key for the winter survival of this evergreen broad-leaved tree. PMID:19457981

A phase quantification method based on EBSD data for a continuously cooled microalloyed steel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, H.; Wynne, B.P.; Palmiere, E.J., E-mail: e.j

2017-01-15

Mechanical properties of steels depend on the phase constitutions of the final microstructures which can be related to the processing parameters. Therefore, accurate quantification of different phases is necessary to investigate the relationships between processing parameters, final microstructures and mechanical properties. Point counting on micrographs observed by optical or scanning electron microscopy is widely used as a phase quantification method, and different phases are discriminated according to their morphological characteristics. However, it is difficult to differentiate some of the phase constituents with similar morphology. Differently, for EBSD based phase quantification methods, besides morphological characteristics, other parameters derived from the orientationmore » information can also be used for discrimination. In this research, a phase quantification method based on EBSD data in the unit of grains was proposed to identify and quantify the complex phase constitutions of a microalloyed steel subjected to accelerated coolings. Characteristics of polygonal ferrite/quasi-polygonal ferrite, acicular ferrite and bainitic ferrite on grain averaged misorientation angles, aspect ratios, high angle grain boundary fractions and grain sizes were analysed and used to develop the identification criteria for each phase. Comparing the results obtained by this EBSD based method and point counting, it was found that this EBSD based method can provide accurate and reliable phase quantification results for microstructures with relatively slow cooling rates. - Highlights: •A phase quantification method based on EBSD data in the unit of grains was proposed. •The critical grain area above which GAM angles are valid parameters was obtained. •Grain size and grain boundary misorientation were used to identify acicular ferrite. •High cooling rates deteriorate the accuracy of this EBSD based method.« less

Hyperplex-MRM: a hybrid multiple reaction monitoring method using mTRAQ/iTRAQ labeling for multiplex absolute quantification of human colorectal cancer biomarker.

PubMed

Yin, Hong-Rui; Zhang, Lei; Xie, Li-Qi; Huang, Li-Yong; Xu, Ye; Cai, San-Jun; Yang, Peng-Yuan; Lu, Hao-Jie

2013-09-06

Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.

Constitutive Transcription and Stable RNA Accumulation in Plastids during the Conversion of Chloroplasts to Chromoplasts in Ripening Tomato Fruits 1

PubMed Central

Marano, María Rosa; Carrillo, Néstor

1992-01-01

The size distribution of plastid transcripts during chromoplast differentiation in ripening tomato (Lycopersicon esculentum L.) fruits was determined using northern blot analysis. Hybridization of total cellular RNA from leaves and fruits with several tobacco chloroplast DNA probes showed distinct transcript patterns in chloroplasts and chromoplasts. We also compared transcriptional rates by probing immobilized DNA fragments of small size (representing about 85% of the plastid genome) with run-on transcripts from tomato plastids. The relative rates of transcription of the various DNA regions were very similar in chloro- and chromoplasts. Parallel determination of the steady-state levels of plastid RNA showed no strict correlation between synthesis rate and RNA accumulation. Differences in the relative abundance of transcripts between chloro- and chromoplasts were not very pronounced and were limited to a small number of genes. The results indicate that the conversion of chloroplasts to chromoplasts at the onset of tomato fruit ripening proceeds with no important variations in the relative transcription rates and with only moderate changes in the relative stability of plastid-encoded transcripts. Images Figure 1 Figure 4 PMID:16653091

Identification and Quantification Soil Redoximorphic Features by Digital Image Processing

USDA-ARS?s Scientific Manuscript database

Soil redoximorphic features (SRFs) have provided scientists and land managers with insight into relative soil moisture for approximately 60 years. The overall objective of this study was to develop a new method of SRF identification and quantification from soil cores using a digital camera and imag...

Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR.

PubMed

Witte, Anna Kristina; Fister, Susanne; Mester, Patrick; Schoder, Dagmar; Rossmanith, Peter

2016-11-01

Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA.

Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

PubMed

Mehta, Nickita; Porterfield, Mindy; Struwe, Weston B; Heiss, Christian; Azadi, Parastoo; Rudd, Pauline M; Tiemeyer, Michael; Aoki, Kazuhiro

2016-09-02

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A Novel Glycan Relative Quantification Strategy

PubMed Central

Walker, S. Hunter; Taylor, Amber D.; Muddiman, David C.

2013-01-01

The INLIGHT strategy for the sample preparation, data analysis, and relative quantification of N-linked glycans is presented. Glycans are derivatized with either natural (L) or stable-isotope labeled (H) hydrazide reagents and analyzed using reversed phase liquid chromatography coupled online to a Q Exactive mass spectrometer. A simple glycan ladder, maltodextrin, is first used to demonstrate the relative quantification strategy in samples with negligible analytical and biological variability. It is shown that after a molecular weight correction due to isotopic overlap and a post-acquisition normalization of the data to account for both the systematic variability, a plot of the experimental H:L ratio vs. the calculated H:L ratio exhibits a correlation of unity for maltodextrin samples mixed in different ratios. We also demonstrate that the INLIGHT approach can quantify species over four orders of magnitude in ion abundance. The INLIGHT strategy is further demonstrated in pooled human plasma, where it is shown that the post-acquisition normalization is more effective than using a single spiked-in internal standard. Finally, changes in glycosylation are able to be detected in complex biological matrices, when spiked with a glycoprotein. The ability to spike in a glycoprotein and detect change at the glycan level validates both the sample preparation and data analysis strategy, making INLIGHT an invaluable relative quantification strategy for the field of glycomics. PMID:23860851

Quantitative Proteomics via High Resolution MS Quantification: Capabilities and Limitations

PubMed Central

Higgs, Richard E.; Butler, Jon P.; Han, Bomie; Knierman, Michael D.

2013-01-01

Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference. PMID:23710359

Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR

PubMed Central

Kembou Tsofack, Japhette Esther; Zamostiano, Rachel; Watted, Salsabeel; Berkowitz, Asaf; Rosenbluth, Ezra; Mishra, Nischay; Briese, Thomas; Lipkin, W. Ian; Kabuusu, Richard M.; Ferguson, Hugh; del Pozo, Jorge

2016-01-01

ABSTRACT Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen. PMID:27974544

Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

PubMed

Kembou Tsofack, Japhette Esther; Zamostiano, Rachel; Watted, Salsabeel; Berkowitz, Asaf; Rosenbluth, Ezra; Mishra, Nischay; Briese, Thomas; Lipkin, W Ian; Kabuusu, Richard M; Ferguson, Hugh; Del Pozo, Jorge; Eldar, Avi; Bacharach, Eran

2017-03-01

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen. Copyright © 2017 American Society for Microbiology.

Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

PubMed Central

Dressman, Devin; Yan, Hai; Traverso, Giovanni; Kinzler, Kenneth W.; Vogelstein, Bert

2003-01-01

Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single magnetic particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently labeled particles via flow cytometry. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and used for further experimentation. BEAMing can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues. PMID:12857956

Alternative splicing of natriuretic peptide A and B receptor transcripts in the rat brain.

PubMed

Francoeur, F; Gossard, F; Hamet, P; Tremblay, J

1995-12-01

1. In the present study we searched for variants of alternative splicing of guanylyl cyclase A and B mRNA in rats in vivo. 2. Guanylyl cyclase A2 and guanylyl cyclase B2 isoforms of guanylyl cyclase produced by alternative splicing leading to the deletion of exon 9 of both transcripts were quantified in several rat organs. 3. Only one alternative splicing was found in the regulatory domain, encoded by exons 8-15. 4. Quantification of the guanylyl cyclase B2 isoform in different rat organs and in cultured aortic smooth muscle cells showed that this alternative splicing was tissue-specific and occurred predominantly in the central nervous system where the alternatively spliced variant represented more than 50% of the guanylyl cyclase B mRNA. 5. The same alternative splicing existed for guanylyl cyclase A mRNA but at very low levels in the organs studied. 6. Alternative splicing of guanylyl cyclase B exon 9 in the brain may play an important role in signal transduction, since the expressed protein possesses a constitutionally active guanylyl cyclase acting independently of C-type natriuretic peptide regulation.

CPTAC Evaluates Long-Term Reproducibility of Quantitative Proteomics Using Breast Cancer Xenografts | Office of Cancer Clinical Proteomics Research

Cancer.gov

Liquid chromatography tandem-mass spectrometry (LC-MS/MS)- based methods such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT) have been shown to provide overall better quantification accuracy and reproducibility over other LC-MS/MS techniques. However, large scale projects like the Clinical Proteomic Tumor Analysis Consortium (CPTAC) require comparisons across many genomically characterized clinical specimens in a single study and often exceed the capability of traditional iTRAQ-based quantification.

RNA-FISH to Study Regulatory RNA at the Site of Transcription.

PubMed

Soler, Marta; Boque-Sastre, Raquel; Guil, Sonia

2017-01-01

The increasing role of all types of regulatory RNAs in the orchestration of cellular programs has enhanced the development of a variety of techniques that allow its precise detection, quantification, and functional scrutiny. Recent advances in imaging and fluoresecent in situ hybridization (FISH) methods have enabled the utilization of user-friendly protocols that provide highly sensitive and accurate detection of ribonucleic acid molecules at both the single cell and subcellular levels. We herein describe the approach originally developed by Stellaris ® , in which the target RNA molecule is fluoresecently labeled with multiple tiled complementary probes each carrying a fluorophore, thus improving sensitivity and reducing the chance of false positives. We have applied this method to the detection of nascent RNAs that partake of special regulatory structures called R loops. Their growing role in active gene expression regulation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012; Ginno et al., Mol Cell 45:814-825, 2012; Sun et al., Science 340:619-621, 2013; Bhatia et al., Nature 511:362-365, 2014) imposes the use of a combination of in vivo and in vitro techniques for the detailed analysis of the transcripts involved. Therefore, their study is a good example to illustrate how RNA FISH, combined with transcriptional arrest and/or cell synchronization, permits localization and temporal characterization of potentially regulatory RNA sequences.

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

PubMed Central

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman

2014-01-01

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*105 MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC – circulating tumour cells, CxCa – cervical cancer, DD-RT-PCR – Digital-Direct Reverse Transcriptase PCR, HPV – Human Papilloma Virus, MNBC – mononuclear blood cells, ICC – immunocytochemistry. PMID:24496006

Digital quantification of gene expression using emulsion PCR.

PubMed

Shi, Xiaolong; Tang, Chao; Wang, Wei; Zhou, Dequan; Lu, Zuhong

2010-01-01

Here we describe a single-molecule quantitative assay of mRNA levels based on mRNA mediate-ligation and BEAMing (beads, emulsion, amplification, and magnetics) technique, which allows accurate and parallel measurement of multiple genes from a small amount of cells. In this method, a pair of oligos complementary target mRNA was used to probe transcripts for each gene of interest. The ligated products of oligos pair were clonally amplified on beads in millions of parallel compartmentalized droplets in a water-in-oil emulsion. The levels of each transcript within a sample were measured by counting the number of the correspondingly amplified beads which were immobilized on a glass surface. To demonstrate its utility, this method has been applied to the quantitation of the mRNA levels for two transcription factors, Klf4 and Sox5, and a housekeeping gene, Gapdh, in human leukemia K562 cells before and after induction with phorbol 12-myristate 13-acetate. Interestingly, we found a significant downregulation of the mRNA level of Sox5 after phorbol 12-myristate 13-acetate treatment. The mRNA mediate-ligation and BEAMing technique provides an accurate and sensitive way to quantify the amount of multiple specific mRNA in a very small number of cells, which may be valuable in the studies requiring precise and parallel quantization of multiple mRNA in the defined cell populations.

Detection of cyprinid herpesvirus 2 in peripheral blood cells of silver crucian carp, Carassius auratus gibelio (Bloch), suggests its potential in viral diagnosis.

PubMed

Wang, H; Xu, Lj; Lu, Lq

2016-02-01

Epidemics caused by cyprinid herpesvirus 2 (CyHV-2) in domestic cyprinid species have been reported in both European and Asian countries. Although the mechanisms remain unknown, acute CyHV-2 infections generally result in high mortality, and the surviving carps become chronic carriers displaying no external clinical signs. In this study, in situ hybridization analysis showed that CyHV-2 tended to infect peripheral blood cells during either acute or chronic infections in silver crucian carp, Carassius auratus gibelio (Bloch). Laboratory challenge experiments coupled with real-time PCR quantification assays further indicated that steady-state levels of the viral genomic copy number in fish serum exhibited a typical 'one-step' growth curve post-viral challenge. Transcriptional expression of open reading frames (ORF) 121, which was selected due to its highest transcriptional levels in almost all tested tissues, was monitored to represent the replication kinetics of CyHV-2 in peripheral blood cells. Similar kinetic curve of active viral gene transcription in blood cells was obtained as that of serum viral load, indicating that CyHV-2 replicated in peripheral blood cells as well as in other well-characterized tissues. This study should pave the way for designing non-invasive and cost-effective serum diagnostic methods for quick detection of CyHV-2 infection. © 2015 John Wiley & Sons Ltd.

ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data.

PubMed

Perkins, James R; Dawes, John M; McMahon, Steve B; Bennett, David L H; Orengo, Christine; Kohl, Matthias

2012-07-02

Measuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR) technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s). We have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.html These packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things) read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

Transcriptional Analysis of Tendril and Inflorescence Development in Grapevine (Vitis vinifera L.)

PubMed Central

Díaz-Riquelme, José; Martínez-Zapater, José M.; Carmona, María J.

2014-01-01

In grapevine (Vitis vinifera L.), the lateral meristem can give rise to either tendrils or inflorescences which are determined organs. To get insights into the processes of tendril and inflorescence development, we characterized the transcriptional variation taking place in both organs. The results of the global transcriptional analyses along tendril and inflorescence development suggested that these two homologous organs initially share a common transcriptional program related to cell proliferation and growth functions. In later developmental stages they showed organ specific gene expression programs related to the particular differentiation processes taking place in each organ. In this way, tendrils showed higher transcription of genes related to photosynthesis, hormone signaling and secondary metabolism than inflorescences, while inflorescences displayed higher transcriptional activity for genes encoding transcription factors, mainly those belonging to the MADS-box gene family. The expression profiles of selected transcription factors related with inflorescence and flower meristem identity and with flower organogenesis were generally conserved with respect to their homologs in model species. Regarding tendrils, it was interesting to find that genes related with reproductive development in other species were also recruited for grapevine tendril development. These results suggest a role for those genes in the regulation of basic cellular mechanisms common to both developmental processes. PMID:24637773

IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System

PubMed Central

Aboulnasr, Fatma; Hazari, Sidhartha; Nayak, Satyam; Chandra, Partha K.; Panigrahi, Rajesh; Ferraris, Pauline; Chava, Srinivas; Kurt, Ramazan; Song, Kyongsub; Dash, Asha; Balart, Luis A.; Garry, Robert F.; Wu, Tong; Dash, Srikanta

2015-01-01

Background HCV replication in persistently infected cell culture remains resistant to IFN-α/RBV combination treatment, whereas IFN-λ1 induces viral clearance. The antiviral mechanisms by which IFN-λ1 induces sustained HCV clearance have not been determined. Aim To investigate the mechanisms by which IFN-λ clears HCV replication in an HCV cell culture model. Methods IFN-α sensitive (S3-GFP) and resistant (R4-GFP) cells were treated with equivalent concentrations of either IFN-α or IFN-λ. The relative antiviral effects of IFN-α and IFN-λ1 were compared by measuring the HCV replication, quantification of HCV-GFP expression by flow cytometry, and viral RNA levels by real time RT-PCR. Activation of Jak-Stat signaling, interferon stimulated gene (ISG) expression, and miRNA-122 transcription in S3-GFP and R4-GFP cells were examined. Results We have shown that IFN-λ1 induces HCV clearance in IFN-α resistant and sensitive replicon cell lines in a dose dependent manner through Jak-Stat signaling, and induces STAT 1 and STAT 2 activation, ISRE-luciferase promoter activation and ISG expression. Stat 3 activation is also involved in IFN-λ1 induced antiviral activity in HCV cell culture. IFN-λ1 induced Stat 3 phosphorylation reduces the expression of hepatocyte nuclear factor 4 alpha (HNF4α) through miR-24 in R4-GFP cells. Reduced expression of HNF4α is associated with decreased expression of miR-122 resulting in an anti-HCV effect. Northern blot analysis confirms that IFN-λ1 reduces miR-122 levels in R4-GFP cells. Our results indicate that IFN-λ1 activates the Stat 3-HNF4α feedback inflammatory loop to inhibit miR-122 transcription in HCV cell culture. Conclusions In addition to the classical Jak–Stat antiviral signaling pathway, IFN-λ1 inhibits HCV replication through the suppression of miRNA-122 transcription via an inflammatory Stat 3–HNF4α feedback loop. Inflammatory feedback circuits activated by IFNs during chronic inflammation expose non-responders to the risk of hepatocellular carcinoma. PMID:26657215

Vitamin D Receptor gene (VDR) transcripts in bone, cartilage, muscles and blood and microarray analysis of vitamin D responsive genes expression in paravertebral muscles of Juvenile and Adolescent Idiopathic Scoliosis patients

PubMed Central

2012-01-01

Background VDR may be considered as a candidate gene potentially related to Idiopathic Scoliosis susceptibility and natural history. Transcriptional profile of VDR mRNA isoforms might be changed in the structural tissues of the scoliotic spine and potentially influence the expression of VDR responsive genes. The purpose of the study was to determine differences in mRNA abundance of VDR isoforms in bone, cartilage and paravertebral muscles between tissues from curve concavity and convexity, between JIS and AIS and to identify VDR responsive genes differentiating Juvenile and Adolescent Idiopathic Scoliosis in paravertebral muscles. Methods In a group of 29 patients with JIS and AIS, specimens of bone, cartilage, paravertebral muscles were harvested at the both sides of the curve apex together with peripheral blood samples. Extracted total RNA served as a matrix for VDRs and VDRl mRNA quantification by QRT PCR. Subsequent microarray analysis of paravertebral muscular tissue samples was performed with HG U133A chips (Affymetrix). Quantitative data were compared by a nonparametric Mann Whitney U test. Microarray results were analyzed with GeneSpring 11GX application. Matrix plot of normalized log-intensities visualized the degree of differentiation between muscular tissue transcriptomes of JIS and AIS group. Fold Change Analysis with cutoff of Fold Change ≥2 identified differentially expressed VDR responsive genes in paravertebral muscles of JIS and AIS. Results No significant differences in transcript abundance of VDR isoforms between tissues of the curve concavity and convexity were found. Statistically significant difference between JIS and AIS group in mRNA abundance of VDRl isoform was found in paravertebral muscles of curve concavity. Higher degree of muscular transcriptome differentiation between curve concavity and convexity was visualized in JIS group. In paravertebral muscles Tob2 and MED13 were selected as genes differentially expressed in JIS and AIS group. Conclusions In Idiopathic Scolioses transcriptional activity and alternative splicing of VDR mRNA in osseous, cartilaginous, and paravertebral muscular tissues are tissue specific and equal on both sides of the curve. The number of mRNA copies of VDRl izoform in concave paravertebral muscles might be one of the factors differentiating JIS and AIS. In paravertebral muscles Tob2 and Med13 genes differentiate Adolescent and Juvenile type of Idiopathic Scoliosis. PMID:23259508

A general approach to quantification of hydroxycinnamic acid derivatives and flavones, flavonols, and their glycosides by UV spectrophotometry

USDA-ARS?s Scientific Manuscript database

A general method was developed for the quantification of hydroxycinnamic acid derivatives and flavones, flavonols, and their glycosides based on the UV molar relative response factors (MRRF) of the standards. Each of these phenolic compounds contains a cinnamoyl structure and has a maximum absorban...

Quantitative Single-Cell mRNA Analysis in Hydrogel Beads.

PubMed

Rakszewska, Agata; Stolper, Rosa J; Kolasa, Anna B; Piruska, Aigars; Huck, Wilhelm T S

2016-06-01

In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

PubMed Central

Schmidt, Carla; Grønborg, Mads; Deckert, Jochen; Bessonov, Sergey; Conrad, Thomas; Lührmann, Reinhard; Urlaub, Henning

2014-01-01

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique. PMID:24448447

Large scale systematic proteomic quantification from non-metastatic to metastatic colorectal cancer

NASA Astrophysics Data System (ADS)

Yin, Xuefei; Zhang, Yang; Guo, Shaowen; Jin, Hong; Wang, Wenhai; Yang, Pengyuan

2015-07-01

A systematic proteomic quantification of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues from stage I to stage IIIC was performed in large scale. 1017 proteins were identified with 338 proteins in quantitative changes by label free method, while 341 proteins were quantified with significant expression changes among 6294 proteins by iTRAQ method. We found that proteins related to migration expression increased and those for binding and adherent decreased during the colorectal cancer development according to the gene ontology (GO) annotation and ingenuity pathway analysis (IPA). The integrin alpha 5 (ITA5) in integrin family was focused, which was consistent with the metastasis related pathway. The expression level of ITA5 decreased in metastasis tissues and the result has been further verified by Western blotting. Another two cell migration related proteins vitronectin (VTN) and actin-related protein (ARP3) were also proved to be up-regulated by both mass spectrometry (MS) based quantification results and Western blotting. Up to now, our result shows one of the largest dataset in colorectal cancer proteomics research. Our strategy reveals a disease driven omics-pattern for the metastasis colorectal cancer.

Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast

PubMed Central

Di Talia, Stefano; Wang, Hongyin; Skotheim, Jan M.; Rosebrock, Adam P.; Futcher, Bruce; Cross, Frederick R.

2009-01-01

In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle. PMID:19841732

Unexpected distribution of CA19.9 and other type 1 chain Lewis antigens in normal and cancer tissues of colon and pancreas: Importance of the detection method and role of glycosyltransferase regulation.

PubMed

Aronica, Adele; Avagliano, Laura; Caretti, Anna; Tosi, Delfina; Bulfamante, Gaetano Pietro; Trinchera, Marco

2017-01-01

CA19.9 antigen has been assumed as an abundant product of cancer cells, due to the reactivity found by immunohistochemical staining of cancer tissues with anti-CA19.9 antibody. Expression and biosynthesis of type 1 chain Lewis antigens in the colon and the pancreas were studied by immunodetection in tissue sections and lysates, quantification of glycosyltransferase transcripts, bisulfite sequencing, and chromatin immunoprecipitation assays. CA19.9 was poorly detectable in normal colon mucosa and almost undetectable in colon cancer, while it was easily detected in the pancreatic ducts, together with Lewis b antigen, under both normal and cancer conditions. B3GALT5 transcripts were down-regulated in colon cancer, while they remained expressed in pancreatic cancer. Even ST3GAL3 transcript appeared well expressed in the pancreas but poorly in the colon, irrespective of normal or cancer conditions. CpG islands flanking B3GALT5 native promoter presented an extremely low degree of methylation in pancreatic cancer with respect to colon cancer. In a DNA region about 1kb away from the B3GALT5 retroviral promoter, a stretch of CG dinucleotides presented a methylation pattern potentially associated with transcription. Such a DNA region and the transcription factor binding site provided overlapping results by chromatin immunoprecipitation assays, corroborating the hypothesis. CA19.9 appears as a physiological product whose synthesis strongly depends on the tissue specific and epigenetically-regulated expression of B3GALT5 and ST3GAL3. CA19.9 and other Lewis antigens acquire tumor marker properties in the pancreas due to mechanisms giving rise to reabsorption into vessels and elevation in circulating levels. Copyright © 2016 Elsevier B.V. All rights reserved.

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application

PubMed Central

2015-01-01

Background The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). Moreover, the huge volume of data generated by NGS platforms introduces unprecedented computational and technological challenges to efficiently analyze and store sequence data and results. Methods In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Results Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs. PMID:26046471

Cues, quantification, and agreement in language comprehension.

PubMed

Tanner, Darren; Bulkes, Nyssa Z

2015-12-01

We investigated factors that affect the comprehension of subject-verb agreement in English, using quantification as a window into the relationship between morphosyntactic processes in language production and comprehension. Event-related brain potentials (ERPs) were recorded while participants read sentences with grammatical and ungrammatical verbs, in which the plurality of the subject noun phrase was either doubly marked (via overt plural quantification and morphological marking on the noun) or singly marked (via only plural morphology on the noun). Both acceptability judgments and the ERP data showed heightened sensitivity to agreement violations when quantification provided an additional cue to the grammatical number of the subject noun phrase, over and above plural morphology. This is consistent with models of grammatical comprehension that emphasize feature prediction in tandem with cue-based memory retrieval. Our results additionally contrast with those of prior studies that showed no effects of plural quantification on agreement in language production. These findings therefore highlight some nontrivial divergences in the cues and mechanisms supporting morphosyntactic processing in language production and comprehension.

A quantification model for the structure of clay materials.

PubMed

Tang, Liansheng; Sang, Haitao; Chen, Haokun; Sun, Yinlei; Zhang, Longjian

2016-07-04

In this paper, the quantification for clay structure is explicitly explained, and the approach and goals of quantification are also discussed. The authors consider that the purpose of the quantification for clay structure is to determine some parameters that can be used to quantitatively characterize the impact of clay structure on the macro-mechanical behaviour. According to the system theory and the law of energy conservation, a quantification model for the structure characteristics of clay materials is established and three quantitative parameters (i.e., deformation structure potential, strength structure potential and comprehensive structure potential) are proposed. And the corresponding tests are conducted. The experimental results show that these quantitative parameters can accurately reflect the influence of clay structure on the deformation behaviour, strength behaviour and the relative magnitude of structural influence on the above two quantitative parameters, respectively. These quantitative parameters have explicit mechanical meanings, and can be used to characterize the structural influences of clay on its mechanical behaviour.

Bayesian forecasting and uncertainty quantifying of stream flows using Metropolis-Hastings Markov Chain Monte Carlo algorithm

NASA Astrophysics Data System (ADS)

Wang, Hongrui; Wang, Cheng; Wang, Ying; Gao, Xiong; Yu, Chen

2017-06-01

This paper presents a Bayesian approach using Metropolis-Hastings Markov Chain Monte Carlo algorithm and applies this method for daily river flow rate forecast and uncertainty quantification for Zhujiachuan River using data collected from Qiaotoubao Gage Station and other 13 gage stations in Zhujiachuan watershed in China. The proposed method is also compared with the conventional maximum likelihood estimation (MLE) for parameter estimation and quantification of associated uncertainties. While the Bayesian method performs similarly in estimating the mean value of daily flow rate, it performs over the conventional MLE method on uncertainty quantification, providing relatively narrower reliable interval than the MLE confidence interval and thus more precise estimation by using the related information from regional gage stations. The Bayesian MCMC method might be more favorable in the uncertainty analysis and risk management.

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop

PubMed Central

Ligerot, Yasmine; de Saint Germain, Alexandre; Troadec, Christelle; Citerne, Sylvie; Pillot, Jean-Paul; Prigge, Michael; Aubert, Grégoire; Bendahmane, Abdelhafid; Estelle, Mark; Debellé, Frédéric

2017-01-01

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner. PMID:29220348

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

PubMed

Ligerot, Yasmine; de Saint Germain, Alexandre; Waldie, Tanya; Troadec, Christelle; Citerne, Sylvie; Kadakia, Nikita; Pillot, Jean-Paul; Prigge, Michael; Aubert, Grégoire; Bendahmane, Abdelhafid; Leyser, Ottoline; Estelle, Mark; Debellé, Frédéric; Rameau, Catherine

2017-12-01

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry.

PubMed

Li, Ming; Josephs, Ralf D; Daireaux, Adeline; Choteau, Tiphaine; Westwood, Steven; Wielgosz, Robert I; Li, Hongmei

2018-06-04

Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.

Evaluation of a mass-balance approach to determine consumptive water use in northeastern Illinois

USGS Publications Warehouse

Mills, Patrick C.; Duncker, James J.; Over, Thomas M.; Marian Domanski,; ,; Engel, Frank

2014-01-01

Under ideal conditions, accurate quantification of consumptive use at the sewershed scale by the described mass-balance approach might be possible. Under most prevailing conditions, quantification likely would be more costly and time consuming than that of the present study, given the freely contributed technical support of the host community and relatively appropriate conditions of the study area. Essentials to quantification of consumptive use are a fully cooperative community, storm and sanitary sewers that are separate, and newer sewer infrastructure and (or) a robust program for limiting infiltration, exfiltration, and inflow.

Embryonal Control of Yellow Seed Coat Locus ECY1 Is Related to Alanine and Phenylalanine Metabolism in the Seed Embryo of Brassica napus

PubMed Central

Wang, Fulin; He, Jiewang; Shi, Jianghua; Zheng, Tao; Xu, Fei; Wu, Guanting; Liu, Renhu; Liu, Shengyi

2016-01-01

Seed coat color is determined by the type of pigment deposited in the seed coat cells. It is related to important agronomic traits of seeds such as seed dormancy, longevity, oil content, protein content and fiber content. In Brassica napus, inheritance of seed coat color is related to maternal effects and pollen effects (xenia effects). In this research we isolated a mutation of yellow seeded B. napus controlled by a single Mendelian locus, which is named Embryonal Control of Yellow seed coat 1 (Ecy1). Microscopy of transverse sections of the mature seed show that pigment is deposited only in the outer layer of the seed coat. Using Illumina Hisequation 2000 sequencing technology, a total of 12 GB clean data, 116× coverage of coding sequences of B. napus, was achieved from seeds 26 d after pollination (DAP). It was assembled into 172,238 independent transcripts, and 55,637 unigenes. A total of 139 orthologous genes of Arabidopsis transparent testa (TT) genes were mapped in silico to 19 chromosomes of B. napus. Only 49 of the TT orthologous genes are transcribed in seeds. However transcription of all orthologs was independent of embryonal control of seed coat color. Only 55 genes were found to be differentially expressed between brown seeds and the yellow mutant. Of these 55, 50 were upregulated and five were downregulated in yellow seeds as compared to their brown counterparts. By KEGG classification, 14 metabolic pathways were significantly enriched. Of these, five pathways: phenylpropanoid biosynthesis, cyanoamino acid metabolism, plant hormone signal transduction, metabolic pathways, and biosynthesis of secondary metabolites, were related with seed coat pigmentation. Free amino acid quantification showed that Ala and Phe were present at higher levels in the embryos of yellow seeds as compared to those of brown seeds. This increase was not observed in the seed coat. Moreover, the excess amount of free Ala was exactly twice that of Phe in the embryo. The pigment substrate chalcone is synthesized from two molecules of Ala and one molecule of Phe. The correlation between accumulation of Ala and Phe, and disappearance of pigment in the yellow seeded mutant, suggests that embryonal control of seed coat color is related with Phe and Ala metabolism in the embryo of B. napus. PMID:26896439

[Transcriptome analysis of Dunaliella viridis].

PubMed

Zhu, Shuai-qi; Gong, Yi-fu; Hang, Yu-qing; Liu, Hao; Wang, He-yu

2015-08-01

In order to understand the gene information, function, haloduric pathway (glycerolipid metabolism) and related key genes for Dunaliella viridis, we used Illumina HiSeqTM 2000 high-throughput sequencing technology to sequence its transcriptome. Trinity soft was used to assemble the data to form transcripts. Based on the Clusters of Orthologous Groups (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG ) databases, we carried out functional annotation and classification, pathway annotation, and the opening reading fragment (ORF) sequence prediction of transcripts. The key genes in the glycerolipid metabolism were analyzed. The results suggested that 81,593 transcripts were found, and 77,117 ORF sequences were predicted, accounting for 94.50% of all transcripts. COG classification results showed that 16,569 transcripts were assigned to 24 categories. GO classification annotated 76,436 transcripts. The number of transcripts for biologcial processes was 30,678, accounting for 40.14% of all transcripts. KEGG pathway analysis showed that 26,428 transcripts were annotated to 317 pathways, and 131 pathways were related to metabolism, accounting for 41.32% of all annotated pathways. Only one transcript was annotated as coding the key enzyme dihydroxyacetone kinase involved in the glycerolipid pathway. This enzyme could be related to glycerol biosynthesis under salt stress. This study further improved the gene information and laid the foundation of metabolic pathway research for Dunaliella viridis.

Development of a targeted method for twenty-three metabolites related to polyphenol gut microbial metabolism in biological samples, using SPE and UHPLC-ESI-MS/MS.

PubMed

Gasperotti, Mattia; Masuero, Domenico; Guella, Graziano; Mattivi, Fulvio; Vrhovsek, Urska

2014-10-01

An increasing number of studies have concerned the profiling of polyphenol microbial metabolites, especially in urine or plasma, but only a few have regarded their accurate quantification. This study reports on a new ultra-performance liquid chromatography tandem mass spectrometry method with electrospray ionisation (UHPLC-ESI-MS/MS) using a simple clean-up step with solid phase extraction (SPE) and validation on different biological matrices. The method was tested with spiked samples of liver, heart, kidneys, brain, blood and urine. The purification procedure, after the evaluation of three different cartridges, makes it possible to obtain cleaner samples and better quantification of putative trace metabolites, especially related to dietary studies, with concentrations below ng/g in tissue and for urine and blood, starting from ng/ml. Limits of detection and linear range were also assessed using mixed polyphenol metabolite standards. Short chromatographic separation was carried out for 23 target compounds related to the polyphenol microbial metabolism, coupled with a triple quadrupole mass spectrometer for their accurate quantification. By analysing different spiked biological samples we were able to test metabolite detection in the matrix and validate the overall recovery of the method, from purification to quantification. The method developed can be successfully applied and is suitable for high-throughput targeted metabolomics analysis related to nutritional intervention, or the study of the metabolic mechanism in response to a polyphenol-rich diet. Copyright © 2014 Elsevier B.V. All rights reserved.

Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quina, Ana Sofia; Instituto Gulbenkian de Ciencia, 2781-901 Oeiras; Parreira, Leonor

2005-07-01

Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters uponmore » activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments.« less

Gene transcript profiling in sea otters post-Exxon Valdez oil spill: A tool for marine ecosystem health assessment

USGS Publications Warehouse

Bowen, Lizabeth; Miles, A. Keith; Ballachey, Brenda E.; Waters, Shannon C.; Bodkin, James L.

2016-01-01

Using a panel of genes stimulated by oil exposure in a laboratory study, we evaluated gene transcription in blood leukocytes sampled from sea otters captured from 2006–2012 in western Prince William Sound (WPWS), Alaska, 17–23 years after the 1989 Exxon Valdez oil spill (EVOS). We compared WPWS sea otters to reference populations (not affected by the EVOS) from the Alaska Peninsula (2009), Katmai National Park and Preserve (2009), Clam Lagoon at Adak Island (2012), Kodiak Island (2005) and captive sea otters in aquaria. Statistically, sea otter gene transcript profiles separated into three distinct clusters: Cluster 1, Kodiak and WPWS 2006–2008 (higher relative transcription); Cluster 2, Clam Lagoon and WPWS 2010–2012 (lower relative transcription); and Cluster 3, Alaska Peninsula, Katmai and captive sea otters (intermediate relative transcription). The lower transcription of the aryl hydrocarbon receptor (AHR), an established biomarker for hydrocarbon exposure, in WPWS 2010–2012 compared to earlier samples from WPWS is consistent with declining hydrocarbon exposure, but the pattern of overall low levels of transcription seen in WPWS 2010–2012 could be related to other factors, such as food limitation, pathogens or injury, and may indicate an inability to mount effective responses to stressors. Decreased transcriptional response across the entire gene panel precludes the evaluation of whether or not individual sea otters show signs of exposure to lingering oil. However, related studies on sea otter demographics indicate that by 2012, the sea otter population in WPWS had recovered, which indicates diminishing oil exposure.

Detection and quantification of infectious avian influenza A (H5N1) virus in environmental water by using real-time reverse transcription-PCR.

PubMed

Dovas, C I; Papanastassopoulou, M; Georgiadis, M P; Chatzinasiou, E; Maliogka, V I; Georgiades, G K

2010-04-01

Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.

A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

PubMed Central

Richardson, Keith; Denny, Richard; Hughes, Chris; Skilling, John; Sikora, Jacek; Dadlez, Michał; Manteca, Angel; Jung, Hye Ryung; Jensen, Ole Nørregaard; Redeker, Virginie; Melki, Ronald; Langridge, James I.; Vissers, Johannes P.C.

2013-01-01

A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods. PMID:22871168

Quantification of lithium at ppm level in geological samples using nuclear reaction analysis.

PubMed

De La Rosa, Nathaly; Kristiansson, Per; Nilsson, E J Charlotta; Ros, Linus; Pallon, Jan; Skogby, Henrik

2018-01-01

Proton-induced reaction (p,α) is one type of nuclear reaction analysis (NRA) suitable especially for light element quantification. In the case of lithium quantification presented in this work, accelerated protons with an energy about of 850 keV were used to induce the 7 Li(p,α) 4 He reaction in standard reference and geological samples such as tourmaline and other Li-minerals. It is shown that this technique for lithium quantification allowed for measurement of concentrations down below one ppm. The possibility to relate the lithium content with the boron content in a single analysis was also demonstrated using tourmaline samples, both in absolute concentration and in lateral distribution. In addition, Particle induced X-ray emission (PIXE) was utilized as a complementary IBA technique for simultaneous mapping of elements heavier than sodium.

22 CFR 1004.8 - Transcripts, recording of closed meetings.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 22 Foreign Relations 2 2013-04-01 2009-04-01 true Transcripts, recording of closed meetings. 1004.8 Section 1004.8 Foreign Relations INTER-AMERICAN FOUNDATION RULES FOR IMPLEMENTING OPEN MEETINGS WITHIN THE INTER-AMERICAN FOUNDATION § 1004.8 Transcripts, recording of closed meetings. (a) The IAF...

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

PubMed

Sjödin, Marcus O D; Wetterhall, Magnus; Kultima, Kim; Artemenko, Konstantin

2013-06-01

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar. Copyright © 2013 Elsevier B.V. All rights reserved.

Mitochondrial dysfunction, oxidative stress and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson’s disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chin, Mark H.; Qian, Weijun; Wang, Haixing

2008-02-10

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson disease (PD) are not completely understood. Here we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 85 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA following neurotoxin treatment. The combined protein and gene list providesmore » a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response and apoptosis. Additionally, codon usage and miRNAs may play an important role in translational control in the striatum. These results constitute one of the largest datasets integrating protein and transcript changes for these neurotoxin models with many similar endpoint phenotypes but distinct mechanisms.« less

Real-time PCR quantification of gene expression in embryonic mouse tissue.

PubMed

Villalon, Eric; Schulz, David J; Waters, Samuel T

2014-01-01

The Gbx family of transcription factors consists of two closely related proteins GBX1 and GBX2. A defining feature of the GBX family is a highly conserved 60 amino acid DNA-binding domain, which differs by just two amino acids. Gbx1 and Gbx2 are co-expressed in several areas of the developing central nervous system including the forebrain, anterior hindbrain, and spinal cord, suggesting the potential for genetic redundancy. However, there is a spatiotemporal difference in expression of Gbx1 and Gbx2 in the forebrain and spinal cord. Gbx2 has been shown to play a critical role in positioning the midbrain/hindbrain boundary and developing anterior hindbrain, whereas gene-targeting experiments in mice have revealed an essential function for Gbx1 in the spinal cord for normal locomotion. To determine if Gbx2 could potentially compensate for a loss of Gbx1 in the developing spinal cord, we performed real-time PCR to examine levels of Gbx2 expression in Gbx1(-/-) spinal cord at embryonic day (E) 13.5, a developmental stage when Gbx2 is rapidly downregulated. We demonstrate that Gbx2 expression is elevated in the spinal cord of Gbx1(-/-) embryos.

Advances in Quantitative Proteomics of Microbes and Microbial Communities

NASA Astrophysics Data System (ADS)

Waldbauer, J.; Zhang, L.; Rizzo, A. I.

2015-12-01

Quantitative measurements of gene expression are key to developing a mechanistic, predictive understanding of how microbial metabolism drives many biogeochemical fluxes and responds to environmental change. High-throughput RNA-sequencing can afford a wealth of information about transcript-level expression patterns, but it is becoming clear that expression dynamics are often very different at the protein level where biochemistry actually occurs. These divergent dynamics between levels of biological organization necessitate quantitative proteomic measurements to address many biogeochemical questions. The protein-level expression changes that underlie shifts in the magnitude, or even the direction, of metabolic and biogeochemical fluxes can be quite subtle and test the limits of current quantitative proteomics techniques. Here we describe methodologies for high-precision, whole-proteome quantification that are applicable to both model organisms of biogeochemical interest that may not be genetically tractable, and to complex community samples from natural environments. Employing chemical derivatization of peptides with multiple isotopically-coded tags, this strategy is rapid and inexpensive, can be implemented on a wide range of mass spectrometric instrumentation, and is relatively insensitive to chromatographic variability. We demonstrate the utility of this quantitative proteomics approach in application to both isolates and natural communities of sulfur-metabolizing and photosynthetic microbes.

Bayesian forecasting and uncertainty quantifying of stream flows using Metropolis–Hastings Markov Chain Monte Carlo algorithm

DOE PAGES

Wang, Hongrui; Wang, Cheng; Wang, Ying; ...

2017-04-05

This paper presents a Bayesian approach using Metropolis-Hastings Markov Chain Monte Carlo algorithm and applies this method for daily river flow rate forecast and uncertainty quantification for Zhujiachuan River using data collected from Qiaotoubao Gage Station and other 13 gage stations in Zhujiachuan watershed in China. The proposed method is also compared with the conventional maximum likelihood estimation (MLE) for parameter estimation and quantification of associated uncertainties. While the Bayesian method performs similarly in estimating the mean value of daily flow rate, it performs over the conventional MLE method on uncertainty quantification, providing relatively narrower reliable interval than the MLEmore » confidence interval and thus more precise estimation by using the related information from regional gage stations. As a result, the Bayesian MCMC method might be more favorable in the uncertainty analysis and risk management.« less

Surface Enhanced Raman Spectroscopy (SERS) methods for endpoint and real-time quantification of miRNA assays

NASA Astrophysics Data System (ADS)

Restaino, Stephen M.; White, Ian M.

2017-03-01

Surface Enhanced Raman spectroscopy (SERS) provides significant improvements over conventional methods for single and multianalyte quantification. Specifically, the spectroscopic fingerprint provided by Raman scattering allows for a direct multiplexing potential far beyond that of fluorescence and colorimetry. Additionally, SERS generates a comparatively low financial and spatial footprint compared with common fluorescence based systems. Despite the advantages of SERS, it has remained largely an academic pursuit. In the field of biosensing, techniques to apply SERS to molecular diagnostics are constantly under development but, most often, assay protocols are redesigned around the use of SERS as a quantification method and ultimately complicate existing protocols. Our group has sought to rethink common SERS methodologies in order to produce translational technologies capable of allowing SERS to compete in the evolving, yet often inflexible biosensing field. This work will discuss the development of two techniques for quantification of microRNA, a promising biomarker for homeostatic and disease conditions ranging from cancer to HIV. First, an inkjet-printed paper SERS sensor has been developed to allow on-demand production of a customizable and multiplexable single-step lateral flow assay for miRNA quantification. Second, as miRNA concentrations commonly exist in relatively low concentrations, amplification methods (e.g. PCR) are therefore required to facilitate quantification. This work presents a novel miRNA assay alongside a novel technique for quantification of nuclease driven nucleic acid amplification strategies that will allow SERS to be used directly with common amplification strategies for quantification of miRNA and other nucleic acid biomarkers.

Basic fibroblastic growth factor affects the osteogenic differentiation of dental pulp stem cells in a treatment-dependent manner.

PubMed

Qian, J; Jiayuan, W; Wenkai, J; Peina, W; Ansheng, Z; Shukai, S; Shafei, Z; Jun, L; Longxing, N

2015-07-01

To determine how basic fibroblastic growth factor (bFGF) affected the osteogenic differentiation of human dental pulp stem cells (DPSCs) in vitro and in vivo. Basic fibroblastic growth factor stimulation of DPSCs was divided into a pre-treatment period and an osteogenic differentiation period. Alizarin red quantification experiments and alkaline phosphatase activity quantification assay were performed to examine the osteogenic differentiation of DPSCs after different bFGF stimulation. Quantification reverse transcription polymerase chain reaction was used to analyze the osteogenic gene expression of DPSCs after different bFGF stimulation. In addition, DPSCs that received the 1 and 2 weeks bFGF pre-treatments as in the in vitro experiments were mineralized for 1 week and seeded into hydroxyapatite/tricalcium phosphate (HA/TCP) pills and subcutaneously transplanted into naked mice for 2 or 3 months. The transplants were removed, sliced and stained using Modified Ponceau Trichrome Stain to observe the formation of mineralized tissue. Basic fibroblastic growth factor stimulation in the osteogenic differentiation period decreased the in vitro osteogenic differentiation ability of DPSCs. One week pre-treatment with bFGF increased the in vitro osteogenic differentiation ability of DPSCs, whereas 2 weeks pre-treatment with bFGF decreased the in vitro osteogenic differentiation ability of DPSCs. The pre-treatment period was vital for the osteogenic differentiation of DPSCs in vitro. The in vivo results were similar to the in vitro results. Basic fibroblastic growth factor affected the osteogenic differentiation of DPSCs in a treatment-dependent manner both in vitro and in vivo. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

PubMed

Marcolino-Gomes, Juliana; Rodrigues, Fabiana Aparecida; Fuganti-Pagliarini, Renata; Nakayama, Thiago Jonas; Ribeiro Reis, Rafaela; Bouças Farias, Jose Renato; Harmon, Frank G; Correa Molinari, Hugo Bruno; Correa Molinari, Mayla Daiane; Nepomuceno, Alexandre

2015-01-01

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-β and β-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.

Differential expression analysis of the broiler tracheal proteins responsible for the immune response and muscle contraction induced by high concentration of ammonia using iTRAQ-coupled 2D LC-MS/MS.

PubMed

Xiong, Yan; Tang, Xiangfang; Meng, Qingshi; Zhang, Hongfu

2016-11-01

Ammonia has been considered the contaminant primarily responsible for respiratory disease in poultry. Even though it can cause tracheal lesions, its adverse effects on the trachea have not been sufficiently studied. The present study investigated tracheal changes in Arbor Acres broilers (Gallus gallus) induced by high concentration of ammonia using isobaric tag for relative and absolute quantification (iTRAQ)-based proteome analysis. In total, 3,706 proteins within false discovery rate of 1% were identified, including 119 significantly differentially expressed proteins. Functional analysis revealed that proteins related to immune response and muscle contraction were significantly enriched. With respect to the immune response, up-regulated proteins (like FGA) were pro-inflammatory, while down-regulated proteins participated in antigen processing and antigen presenting (like MYO1G), immunoglobulin and cathelicidin production (like fowlicidin-2), and immunodeficiency (like PTPRC). Regarding muscle contraction, all differentially expressed proteins (like TPM1) were up-regulated. An over-expression of mucin, which is a common feature of airway disease, was also observed. Additionally, the transcriptional alterations of 6 selected proteins were analyzed by quantitative RT-PCR. Overall, proteomic changes suggested the onset of airway obstruction and diminished host defense in trachea after ammonia exposure. These results may serve as a valuable reference for future interventions against ammonia toxicity.

Non-Lethal Heat Shock Increased Hsp70 and Immune Protein Transcripts but Not Vibrio Tolerance in the White-Leg Shrimp

PubMed Central

Loc, Nguyen Hong; MacRae, Thomas H.; Musa, Najiah; Bin Abdullah, Muhd Danish Daniel; Abdul Wahid, Mohd. Effendy; Sung, Yeong Yik

2013-01-01

Non-lethal heat shock boosts bacterial and viral disease tolerance in shrimp, possibly due to increases in endogenous heat shock protein 70 (Hsp70) and/or immune proteins. To further understand the mechanisms protecting shrimp against infection, Hsp70 and the mRNAs encoding the immune-related proteins prophenoloxidase (proPO), peroxinectin, penaeidin, crustin and hemocyanin were studied in post-larvae of the white-leg shrimp Litopenaeus vannamei, following a non-lethal heat shock. As indicated by RT-qPCR, a 30 min abrupt heat shock increased Hsp70 mRNA in comparison to non-heated animals. Immunoprobing of western blots and quantification by ELISA revealed that Hsp70 production after heat shock was correlated with enhanced Hsp70 mRNA. proPO and hemocyanin mRNA levels were augmented, whereas peroxinectin and crustin mRNA levels were unchanged following non-lethal heat shock. Penaeidin mRNA was decreased by all heat shock treatments. Thirty min abrupt heat shock failed to improve survival of post-larvae in a standardized challenge test with Vibrio harveyi, indicating that under the conditions of this study, L. vannamei tolerance to Vibrio infection was influenced neither by Hsp70 accumulation nor the changes in the immune-related proteins, observations dissimilar to other shrimp species examined. PMID:24039886

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

PubMed Central

Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

2005-01-01

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated. PMID:16145106

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

PubMed Central

Hashemipetroudi, Seyyed Hamidreza; Nematzadeh, Ghorbanali; Ahmadian, Gholamreza; Yamchi, Ahad; Kuhlmann, Markus

2018-01-01

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species PMID:29443017

Proteomic profiling of early degenerative retina of RCS rats.

PubMed

Zhu, Zhi-Hong; Fu, Yan; Weng, Chuan-Huang; Zhao, Cong-Jian; Yin, Zheng-Qin

2017-01-01

To identify the underlying cellular and molecular changes in retinitis pigmentosa (RP). Label-free quantification-based proteomics analysis, with its advantages of being more economic and consisting of simpler procedures, has been used with increasing frequency in modern biological research. Dystrophic RCS rats, the first laboratory animal model for the study of RP, possess a similar pathological course as human beings with the diseases. Thus, we employed a comparative proteomics analysis approach for in-depth proteome profiling of retinas from dystrophic RCS rats and non-dystrophic congenic controls through Linear Trap Quadrupole - orbitrap MS/MS, to identify the significant differentially expressed proteins (DEPs). Bioinformatics analyses, including Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and upstream regulatory analysis, were then performed on these retina proteins. Finally, a Western blotting experiment was carried out to verify the difference in the abundance of transcript factor E2F1. In this study, we identified a total of 2375 protein groups from the retinal protein samples of RCS rats and non-dystrophic congenic controls. Four hundred thirty-four significantly DEPs were selected by Student's t -test. Based on the results of the bioinformatics analysis, we identified mitochondrial dysfunction and transcription factor E2F1 as the key initiation factors in early retinal degenerative process. We showed that the mitochondrial dysfunction and the transcription factor E2F1 substantially contribute to the disease etiology of RP. The results provide a new potential therapeutic approach for this retinal degenerative disease.

ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data

PubMed Central

2012-01-01

Background Measuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR) technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s). Results We have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.html Conclusions These packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things) read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples. PMID:22748112

[Detection of recombinant-DNA in foods from stacked genetically modified plants].

PubMed

Sorokina, E Iu; Chernyshova, O N

2012-01-01

A quantitative real-time multiplex polymerase chain reaction method was applied to the detection and quantification of MON863 and MON810 in stacked genetically modified maize MON 810xMON 863. The limit of detection was approximately 0,1%. The accuracy of the quantification, measured as bias from the accepted value and the relative repeatability standard deviation, which measures the intra-laboratory variability, were within 25% at each GM-level. A method verification has demonstrated that the MON 863 and the MON810 methods can be equally applied in quantification of the respective events in stacked MON810xMON 863.

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

PubMed

Pezzoli, N; Silvy, M; Woronko, A; Le Treut, T; Lévy-Mozziconacci, A; Reviron, D; Gabert, J; Picard, C

2007-09-01

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

Quantification is Neither Necessary Nor Sufficient for Measurement

NASA Astrophysics Data System (ADS)

Mari, Luca; Maul, Andrew; Torres Irribarra, David; Wilson, Mark

2013-09-01

Being an infrastructural, widespread activity, measurement is laden with stereotypes. Some of these concern the role of measurement in the relation between quality and quantity. In particular, it is sometimes argued or assumed that quantification is necessary for measurement; it is also sometimes argued or assumed that quantification is sufficient for or synonymous with measurement. To assess the validity of these positions the concepts of measurement and quantitative evaluation should be independently defined and their relationship analyzed. We contend that the defining characteristic of measurement should be the structure of the process, not a feature of its results. Under this perspective, quantitative evaluation is neither sufficient nor necessary for measurement.

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

PubMed

Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

2013-01-01

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

22 CFR 51.72 - Transcript and record of the hearing.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Transcript and record of the hearing. 51.72 Section 51.72 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS Procedures for Review of Certain Denials and Revocations § 51.72 Transcript and record of the hearing. A qualified...

22 CFR 51.72 - Transcript and record of the hearing.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 22 Foreign Relations 1 2013-04-01 2013-04-01 false Transcript and record of the hearing. 51.72 Section 51.72 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS Procedures for Review of Certain Denials and Revocations § 51.72 Transcript and record of the hearing. A qualified...

22 CFR 51.72 - Transcript and record of the hearing.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Transcript and record of the hearing. 51.72 Section 51.72 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS Procedures for Review of Certain Denials and Revocations § 51.72 Transcript and record of the hearing. A qualified...

22 CFR 51.72 - Transcript and record of the hearing.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Transcript and record of the hearing. 51.72 Section 51.72 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS Procedures for Review of Certain Denials and Revocations § 51.72 Transcript and record of the hearing. A qualified...

22 CFR 51.72 - Transcript and record of the hearing.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 22 Foreign Relations 1 2014-04-01 2014-04-01 false Transcript and record of the hearing. 51.72 Section 51.72 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS Procedures for Review of Certain Denials and Revocations § 51.72 Transcript and record of the hearing. A qualified...

Successful treatment by double-filtration plasmapheresis of a patient with bullous pemphigoid: effects in vivo on transcripts of several genes for chemokines and cytokines in peripheral blood mononuclear cells.

PubMed

Hatano, Y; Katagiri, K; Arakawa, S; Umeki, T; Takayasu, S; Fujiwara, S

2003-03-01

The involvement of various cytokines and chemokines has been reported in the pathogenesis of bullous pemphigoid (BP). Double-filtration plasmapheresis (DFPP) is an effective treatment for BP but the mechanism of action remains unclear. Using semiquantitative reverse transcription-polymerase chain reaction, we examined levels of transcripts for various cytokines and chemokines in freshly isolated peripheral blood mononuclear cells in a patient with BP before and after DFPP treatment. DFPP was performed four times. Relative levels of transcripts for interleukin (IL)-8, macrophage inflammatory protein (MIP)-1alpha and IL-5, and the ratio of relative levels of transcripts for IL-4 and interferon (IFN)-gamma, were higher, before treatment, than in healthy controls, and decreased when the extent of the lesions was reduced. Relative levels of transcripts for tumour necrosis factor (TNF)-alpha and IL-4 also decreased with regression of lesions, although they were similar to or lower than the corresponding levels in healthy individuals. When eruptions recurred, relative levels of transcripts for IL-8, MIP-1alpha, RANTES (regulated upon activation normal T cell expressed and secreted), IL-2, IFN-gamma and TNF-alpha were very much higher than those prior to the recurrence, while relative levels of mRNAs for IL-4 and IL-5 did not increase. Relative levels of transcripts for IL-8, MIP-1alpha, TNF-alpha and IL-2 were lower at the end of each individual DFPP and after the four treatments than at the beginning of treatment. Our observations suggest that cytokines and chemokines produced in mononuclear cells play important roles in the pathogenesis of BP and that regulation of their expression might be involved in the therapeutic effects of DFPP in BP.

Housekeeping genes as internal standards: use and limits.

PubMed

Thellin, O; Zorzi, W; Lakaye, B; De Borman, B; Coumans, B; Hennen, G; Grisar, T; Igout, A; Heinen, E

1999-10-08

Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.

Colour thresholding and objective quantification in bioimaging

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Gerber, M. A.; Torre-Bueno, J. R.

1992-01-01

Computer imaging is rapidly becoming an indispensable tool for the quantification of variables in research and medicine. Whilst its use in medicine has largely been limited to qualitative observations, imaging in applied basic sciences, medical research and biotechnology demands objective quantification of the variables in question. In black and white densitometry (0-256 levels of intensity) the separation of subtle differences between closely related hues from stains is sometimes very difficult. True-colour and real-time video microscopy analysis offer choices not previously available with monochrome systems. In this paper we demonstrate the usefulness of colour thresholding, which has so far proven indispensable for proper objective quantification of the products of histochemical reactions and/or subtle differences in tissue and cells. In addition, we provide interested, but untrained readers with basic information that may assist decisions regarding the most suitable set-up for a project under consideration. Data from projects in progress at Tulane are shown to illustrate the advantage of colour thresholding over monochrome densitometry and for objective quantification of subtle colour differences between experimental and control samples.

Microarray Analysis of Differential Gene Expression Profile Between Human Fetal and Adult Heart.

PubMed

Geng, Zhimin; Wang, Jue; Pan, Lulu; Li, Ming; Zhang, Jitai; Cai, Xueli; Chu, Maoping

2017-04-01

Although many changes have been discovered during heart maturation, the genetic mechanisms involved in the changes between immature and mature myocardium have only been partially elucidated. Here, gene expression profile changed between the human fetal and adult heart was characterized. A human microarray was applied to define the gene expression signatures of the fetal (13-17 weeks of gestation, n = 4) and adult hearts (30-40 years old, n = 4). Gene ontology analyses, pathway analyses, gene set enrichment analyses, and signal transduction network were performed to predict the function of the differentially expressed genes. Ten mRNAs were confirmed by quantificational real-time polymerase chain reaction. 5547 mRNAs were found to be significantly differentially expressed. "Cell cycle" was the most enriched pathway in the down-regulated genes. EFGR, IGF1R, and ITGB1 play a central role in the regulation of heart development. EGFR, IGF1R, and FGFR2 were the core genes regulating cardiac cell proliferation. The quantificational real-time polymerase chain reaction results were concordant with the microarray data. Our data identified the transcriptional regulation of heart development in the second trimester and the potential regulators that play a prominent role in the regulation of heart development and cardiac cells proliferation.

The Effects of Statistical Multiplicity of Infection on Virus Quantification and Infectivity Assays.

PubMed

Mistry, Bhaven A; D'Orsogna, Maria R; Chou, Tom

2018-06-19

Many biological assays are employed in virology to quantify parameters of interest. Two such classes of assays, virus quantification assays (VQAs) and infectivity assays (IAs), aim to estimate the number of viruses present in a solution and the ability of a viral strain to successfully infect a host cell, respectively. VQAs operate at extremely dilute concentrations, and results can be subject to stochastic variability in virus-cell interactions. At the other extreme, high viral-particle concentrations are used in IAs, resulting in large numbers of viruses infecting each cell, enough for measurable change in total transcription activity. Furthermore, host cells can be infected at any concentration regime by multiple particles, resulting in a statistical multiplicity of infection and yielding potentially significant variability in the assay signal and parameter estimates. We develop probabilistic models for statistical multiplicity of infection at low and high viral-particle-concentration limits and apply them to the plaque (VQA), endpoint dilution (VQA), and luciferase reporter (IA) assays. A web-based tool implementing our models and analysis is also developed and presented. We test our proposed new methods for inferring experimental parameters from data using numerical simulations and show improvement on existing procedures in all limits. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Application of targeted proteomics to metabolically engineered Escherichia coli.

PubMed

Singh, Pragya; Batth, Tanveer S; Juminaga, Darmawi; Dahl, Robert H; Keasling, Jay D; Adams, Paul D; Petzold, Christopher J

2012-04-01

As synthetic biology matures to compete with chemical transformation of commodity and high-value compounds, a wide variety of well-characterized biological parts are needed to facilitate system design. Protein quantification based on selected-reaction monitoring (SRM) mass spectrometry compliments metabolite and transcript analysis for system characterization and optimizing flux through engineered pathways. By using SRM quantification, we assayed red fluorescent protein (RFP) expressed from plasmids containing several inducible and constitutive promoters and subsequently assessed protein production from the same promoters driving expression of eight mevalonate pathway proteins in Escherichia coli. For each of the promoter systems, the protein level for the first gene in the operon followed that of RFP, however, the levels of proteins produced from genes farther from the promoter were much less consistent. Second, we used targeted proteomics to characterize tyrosine biosynthesis pathway proteins after removal of native regulation. The changes were not expected to cause significant impact on protein levels, yet significant variation in protein abundance was observed and tyrosine production for these strains spanned a range from less than 1 mg/L to greater than 250 mg/L. Overall, our results underscore the importance of targeted proteomics for determining accurate protein levels in engineered systems and fine-tuning metabolic pathways. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Next generation sequencing and de novo transcriptome analysis of Costus pictus D. Don, a non-model plant with potent anti-diabetic properties

PubMed Central

2012-01-01

Background Phyto-remedies for diabetic control are popular among patients with Type II Diabetes mellitus (DM), in addition to other diabetic control measures. A number of plant species are known to possess diabetic control properties. Costus pictus D. Don is popularly known as “Insulin Plant” in Southern India whose leaves have been reported to increase insulin pools in blood plasma. Next Generation Sequencing is employed as a powerful tool for identifying molecular signatures in the transcriptome related to physiological functions of plant tissues. We sequenced the leaf transcriptome of C. pictus using Illumina reversible dye terminator sequencing technology and used combination of bioinformatics tools for identifying transcripts related to anti-diabetic properties of C. pictus. Results A total of 55,006 transcripts were identified, of which 69.15% transcripts could be annotated. We identified transcripts related to pathways of bixin biosynthesis and geraniol and geranial biosynthesis as major transcripts from the class of isoprenoid secondary metabolites and validated the presence of putative norbixin methyltransferase, a precursor of Bixin. The transcripts encoding these terpenoids are known to be Peroxisome Proliferator-Activated Receptor (PPAR) agonists and anti-glycation agents. Sequential extraction and High Performance Liquid Chromatography (HPLC) confirmed the presence of bixin in C. pictus methanolic extracts. Another significant transcript identified in relation to anti-diabetic, anti-obesity and immuno-modulation is of Abscisic Acid biosynthetic pathway. We also report many other transcripts for the biosynthesis of antitumor, anti-oxidant and antimicrobial metabolites of C. pictus leaves. Conclusion Solid molecular signatures (transcripts related to bixin, abscisic acid, and geranial and geraniol biosynthesis) for the anti-diabetic properties of C. pictus leaves and vital clues related to the other phytochemical functions like antitumor, anti-oxidant, immuno-modulatory, anti-microbial and anti-malarial properties through the secondary metabolite pathway annotations are reported. The data provided will be of immense help to researchers working in the treatment of DM using herbal therapies. PMID:23176672

Microarray Analyses of Gene Expression during Adventitious Root Development in Pinus contorta1[w

PubMed Central

Brinker, Monika; van Zyl, Leonel; Liu, Wenbin; Craig, Deborah; Sederoff, Ronald R.; Clapham, David H.; von Arnold, Sara

2004-01-01

In order to investigate the gene expression pattern during adventitious root development, RNA of Pinus contorta hypocotyls, pulse-treated with the auxin indole-3-butyric acid and harvested at distinct developmental time points of root development, was hybridized to microarrays containing 2,178 cDNAs from Pinus taeda. Over the period of observation of root development, the transcript levels of 220 genes changed significantly. During the root initiation phase, genes involved in cell replication and cell wall weakening and a transcript encoding a PINHEAD/ZWILLE-like protein were up-regulated, while genes related to auxin transport, photosynthesis, and cell wall synthesis were down-regulated. In addition, there were changes in transcript abundance of genes related to water stress. During the root meristem formation phase the transcript abundances of genes involved in auxin transport, auxin responsive transcription, and cell wall synthesis, and of a gene encoding a B-box zinc finger-like protein, increased, while those encoding proteins involved in cell wall weakening decreased. Changes of transcript abundance of genes related to water stress during the root meristem formation and root formation phase indicate that the plant roots had become functional in water transport. Simultaneously, genes involved in auxin transport were up-regulated, while genes related to cell wall modification were down-regulated. Finally, during the root elongation phase down-regulation of transcripts encoding proteins involved in cell replication and stress occurred. Based on the observed changes in transcript abundances, we suggest hypotheses about the relative importance of various physiological processes during the auxin-induced development of roots in P. contorta. PMID:15247392

pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

PubMed

Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

2009-11-23

Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

Quantification of transcription factor-DNA binding affinity in a living cell

PubMed Central

Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

PubMed

Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

The transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women.

PubMed

Moore, A G; Brown, D A; Fairlie, W D; Bauskin, A R; Brown, P K; Munier, M L; Russell, P K; Salamonsen, L A; Wallace, E M; Breit, S N

2000-12-01

Macrophage inhibitory cytokine-1 (MIC-1) is a recently described divergent member of the transforming growth factor-ss superfamily. MIC-1 transcription up-regulation is associated with macrophage activation, and this observation led to its cloning. Northern blots indicate that MIC-1 is also present in human placenta. A sensitive sandwich enzyme-linked immunosorbent assay for the quantification of MIC-1 was developed and used to examine the role of this cytokine in pregnancy. High levels of MIC-1 are present in the sera of pregnant women. The level rises substantially with progress of gestation. MIC-1 can also be detected, in large amounts, in amniotic fluid and placental extracts. In addition, the BeWo placental trophoblastic cell line was found to constitutively express the MIC-1 transcript and secrete large amounts of MIC-1. These findings suggest that the placental trophoblast is a major source of the MIC-1 present in maternal serum and amniotic fluid. We suggest that MIC-1 may promote fetal survival by suppressing the production of maternally derived proinflammatory cytokines within the uterus.

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.

PubMed

Weighardt, Florian; Barbati, Cristina; Paoletti, Claudia; Querci, Maddalena; Kay, Simon; De Beuckeleer, Marc; Van den Eede, Guy

2004-01-01

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr) values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.

Microarray Analyses and Comparisons of Upper or Lower Flanks of Rice Shoot Base Preceding Gravitropic Bending

PubMed Central

Zang, Aiping; Chen, Haiying; Dou, Xianying; Jin, Jing; Cai, Weiming

2013-01-01

Gravitropism is a complex process involving a series of physiological pathways. Despite ongoing research, gravitropism sensing and response mechanisms are not well understood. To identify the key transcripts and corresponding pathways in gravitropism, a whole-genome microarray approach was used to analyze transcript abundance in the shoot base of rice (Oryza sativa sp. japonica) at 0.5 h and 6 h after gravistimulation by horizontal reorientation. Between upper and lower flanks of the shoot base, 167 transcripts at 0.5 h and 1202 transcripts at 6 h were discovered to be significantly different in abundance by 2-fold. Among these transcripts, 48 were found to be changed both at 0.5 h and 6 h, while 119 transcripts were only changed at 0.5 h and 1154 transcripts were changed at 6 h in association with gravitropism. MapMan and PageMan analyses were used to identify transcripts significantly changed in abundance. The asymmetric regulation of transcripts related to phytohormones, signaling, RNA transcription, metabolism and cell wall-related categories between upper and lower flanks were demonstrated. Potential roles of the identified transcripts in gravitropism are discussed. Our results suggest that the induction of asymmetrical transcription, likely as a consequence of gravitropic reorientation, precedes gravitropic bending in the rice shoot base. PMID:24040303

Inhabitancy of active Nitrosopumilus-like ammonia-oxidizing archaea and Nitrospira nitrite-oxidizing bacteria in the sponge Theonella swinhoei

PubMed Central

Feng, Guofang; Sun, Wei; Zhang, Fengli; Karthik, Loganathan; Li, Zhiyong

2016-01-01

Nitrification directly contributes to the ammonia removal in sponges, and it plays an indispensable role in sponge-mediated nitrogen cycle. Previous studies have demonstrated genomic evidences of nitrifying lineages in the sponge Theonella swinhoei. However, little is known about the transcriptional activity of nitrifying community in this sponge. In this study, combined DNA- and transcript-based analyses were performed to reveal the composition and transcriptional activity of the nitrifiers in T. swinhoei from the South China Sea. Transcriptional activity of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in this sponge were confirmed by targeting their nitrifying genes,16S rRNA genes and their transcripts. Phylogenetic analysis coupled with RDP rRNA classification indicated that archaeal 16S rRNA genes, amoA (the subunit of ammonia monooxygenase) genes and their transcripts were closely related to Nitrosopumilus-like AOA; whereas nitrifying bacterial 16S rRNA genes, nxrB (the subunit of nitrite oxidoreductase) genes and their transcripts were closely related to Nitrospira NOB. Quantitative assessment demonstrated relative higher abundances of nitrifying genes and transcripts of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. This study illustrated the transcriptional potentials of Nitrosopumilus-like archaea and Nitrospira bacteria that would predominantly contribute to the nitrification functionality in the South China Sea T. swinhoei. PMID:27113140

Novel isotopic N, N-Dimethyl Leucine (iDiLeu) Reagents Enable Absolute Quantification of Peptides and Proteins Using a Standard Curve Approach

NASA Astrophysics Data System (ADS)

Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

2015-01-01

Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N, N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis.

PubMed

Smith, Benjamin; Li, Jianfang; Metruccio, Matteo; Wan, Stephanie; Evans, David; Fleiszig, Suzanne

2018-04-20

A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples.

Concordant gene regulation related to perturbations of three GDP-mannose-related genes.

PubMed

Törmä, Anssi; Pitkänen, Juha-Pekka; Huopaniemi, Laura; Mattila, Pirkko; Renkonen, Risto

2009-02-01

Glycosylation of proteins is one of the most crucial post-translational modifications. In order to access system-level and state-dependent data related to the regulation of glycosylation events, we cultivated yeast cell strains each harboring a selected conditional knockdown construct for a gene (either SEC53, VRG4 or DPM1) related to GDP-mannose synthesis or its utilization in glycan biosynthesis. In order to carry this out efficiently, we developed automated sampling from bioreactor cultivations, a collection of in silico workflows for data analysis as well as their integration into a large data warehouse. Using the above-mentioned approaches, we could show that conditional knocking down of transcripts related to GDP-mannose synthesis or transportation led to altered levels of over 300 transcripts. These transcripts and their corresponding proteins were characterized by their gene ontology (GO) annotations, and their putative transcriptional regulation was analyzed. Furthermore, novel pathways were generated indicating interactions between GO categories with common proteins, putative transcriptional regulators of such induced GO categories, and the large protein-protein interaction network among the proteins whose transcripts indicated altered expression levels. When these results are always added to an ever-expanding data warehouse as annotations, they will incrementally increase the knowledge of biological systems.

Rapid quantification of iodopropynyl butylcarbamate as the preservative in cosmetic formulations using high-performance liquid chromatography-electrospray mass spectrometry.

PubMed

Frauen, M; Steinhart, H; Rapp, C; Hintze, U

2001-07-01

A simple, rapid and reproducible method for identification and quantification of iodopropynyl butylcarbamate (IPBC) in different cosmetic formulations is presented. The determination was carried out using a high-performance liquid chromatography (HPLC) procedure on a reversed phase column coupled to a single quadrupole mass spectrometer (MS) via an electrospray ionization (ESI) interface. Detection was performed in the positive selected ion-monitoring mode. In methanol/water extracts from different cosmetic formulations a detection limit between 50 and 100 ng/g could be achieved. A routine analytical procedure could be set up with good quantification reliability (relative standard deviation between 0.9 and 2.9%).

Maize Iranian mosaic virus shows a descending transcript accumulation order in plant and insect hosts.

PubMed

Hortamani, Mozhgan; Massah, Amir; Izadpanah, Keramat

2018-04-01

Maize Iranian mosaic virus (MIMV) is a distinct member of the genus Nucleorhabdovirus. In this study, expression of all MIMV genes in maize for four weeks after inoculation and in inoculative planthoppers was examined using a quantitative RT-PCR (RT-qPCR) assay. Accumulation of MIMV P, gene 3, M, G and L transcripts relative to N transcripts was measured and normalized to 18S rRNA in maize plants and to the ribosomal protein S13 gene (RPS13) in planthoppers using the comparative C T method. In plants, higher levels of MIMV N transcripts were found relative to other transcripts, while MIMV L transcripts were at the lowest levels. The highest accumulation of MIMV transcripts was found at 14 days postinoculation (dpi). At 21 dpi, we found the lowest transcript levels for all genes, which increased again at 28 dpi, although in lower amounts than at 14 dpi. In Laodelphax striatellus, MIMV M, G and L transcripts accumulated at lower levels than other transcripts. The gene 3 transcript level was high in both plants and planthoppers. Our results showed that transcript accumulation for the MIMV genes was similar in both hosts and followed the pattern of sequential transcriptional attenuation from the 3' to the 5' end of the genome, similar to vertebrate rhabdoviruses. These results indicate that the regulation of virus gene transcription for this plant-infecting rhabdovirus is similar to that of some vertebrate-infecting rhabdoviruses.

Medically important carotenoids from Momordica charantia and their gene expressions in different organs.

PubMed

Cuong, Do Manh; Arasu, Mariadhas Valan; Jeon, Jin; Park, Yun Ji; Kwon, Soon-Jae; Al-Dhabi, Naif Abdullah; Park, Sang Un

2017-12-01

Carotenoids, found in the fruit and different organs of bitter melon ( Momordica charantia ), have attracted great attention for their potential health benefits in treating several major chronic diseases. Therefore, study related to the identification and quantification of the medically important carotenoid metabolites is highly important for the treatment of various disorderes. The present study involved in the identification and quantification of the various carotenoids present in the different organs of M. charantia and the identification of the genes responsible for the accumulation of the carotenoids with respect to the transcriptome levels were investigated. In this study, using the transcriptome database of bitter melon, a partial-length cDNA clone encoding geranylgeranyl pyrophosphate synthase ( McGGPPS2 ), and several full-length cDNA clones encoding geranylgeranyl pyrophosphate synthase ( McGGPPS1 ), zeta-carotene desaturase ( McZDS ), lycopene beta-cyclase ( McLCYB ), lycopene epsilon cyclases ( McLCYE1 and McLCYE2 ), beta-carotene hydroxylase ( McCHXB ), and zeaxanthin epoxidase ( McZEP ) were identified in bitter melon . The expression levels of the mRNAs encoding these eight putative biosynthetic enzymes, as well as the accumulation of lycopene, α-carotene, lutein, 13Z-β-carotene, E-β-carotene, 9Z-β-carotene, β-cryptoxanthin, zeaxanthin, antheraxanthin, and violaxanthin were investigated in different organs from M. charantia as well as in the four different stages of its fruit maturation. Transcripts were found to be constitutively expressed at high levels in the leaves where carotenoids were also found at the highest levels . Collectively, these results indicate that the putative McGGPPS2, McZDS, McLCYB, McLCYE1, McLCYE2, and McCHXB enzymes might be key factors in controlling carotenoid content in bitter melon . In conclusion, the over expression of the carotenoid biosynthetic genes from M. charantia crops to increase the yield of these medically important carotenoids.

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

PubMed Central

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus. PMID:25014071

The α2β2 isoform combination dominates the astrocytic Na+ /K+ -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation.

PubMed

Stoica, Anca; Larsen, Brian Roland; Assentoft, Mette; Holm, Rikke; Holt, Leanne Melissa; Vilhardt, Frederik; Vilsen, Bente; Lykke-Hartmann, Karin; Olsen, Michelle Lynne; MacAulay, Nanna

2017-11-01

Synaptic activity results in transient elevations in extracellular K + , clearance of which is critical for sustained function of the nervous system. The K + clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na + /K + -ATPase. The Na + /K + -ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K + clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na + /K + -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na + /K + -ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2 +/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K + dynamics. © 2017 Wiley Periodicals, Inc.

Morphological observation, RNA-Seq quantification, and expression profiling: novel insight into grafting-responsive carotenoid biosynthesis in watermelon grafted onto pumpkin rootstock.

PubMed

Liu, Guang; Yang, Xingping; Xu, Jinhua; Zhang, Man; Hou, Qian; Zhu, Lingli; Huang, Ying; Xiong, Aisheng

2017-03-01

Watermelon is an important and economical horticultural crop in China, where ~20% of the plants are grafted. The development of grafted watermelon fruit involves a diverse range of gene interactions that results in dynamic changes in fruit. However, the molecular mechanisms underlying grafting-induced fruit quality change are unclear. In the present study, we measured the lycopene content by high-performance liquid chromatography and used RNA-Seq (quantification) to perform a genome-wide transcript analysis of fruits from watermelon grafted onto pumpkin rootstock (pumpkin-grafted watermelon, PGW), self-grafted watermelon (SGW), and non-grafted watermelon (NGW). The results showed variation in the lycopene content in the flesh of PGW fruits, first increasing and then decreasing in the four stages, which was different from the trend in the flesh of NGW and SGW fruits. The transcriptome profiling data provided new information on the grafting-induced gene regulation of lycopene biosynthesis during fruit growth and development. The expression levels of 33 genes from 8 gene families (GGPS, PSY, PDS, ZDS, CRTISO, LCYb, LCYe, and CHY) related to lycopene biosynthesis, which play critical roles in fruit coloration and contribute significantly to fruit phytonutrient values, were monitored during the four periods of fruit development in watermelon. Compared with those of NGW and SGW, 14 genes were differentially expressed in PGW during fruit development, suggesting that these genes possibly help to mediate lycopene biosynthesis in grafted watermelon fruit. Our work provides some novel insights into grafting-responsive carotenoid metabolism and its potential roles during PGW fruit development and ripening. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

PubMed

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

PubMed

Ponchel, Frederique; Toomes, Carmel; Bransfield, Kieran; Leong, Fong T; Douglas, Susan H; Field, Sarah L; Bell, Sandra M; Combaret, Valerie; Puisieux, Alain; Mighell, Alan J; Robinson, Philip A; Inglehearn, Chris F; Isaacs, John D; Markham, Alex F

2003-10-13

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Whole farm quantification of GHG emissions within smallholder farms in developing countries

NASA Astrophysics Data System (ADS)

Seebauer, Matthias

2014-03-01

The IPCC has compiled the best available scientific methods into published guidelines for estimating greenhouse gas emissions and emission removals from the land-use sector. In order to evaluate existing GHG quantification tools to comprehensively quantify GHG emissions and removals in smallholder conditions, farm scale quantification was tested with farm data from Western Kenya. After conducting a cluster analysis to identify different farm typologies GHG quantification was exercised using the VCS SALM methodology complemented with IPCC livestock emission factors and the cool farm tool. The emission profiles of four farm clusters representing the baseline conditions in the year 2009 are compared with 2011 where farmers adopted sustainable land management practices (SALM). The results demonstrate the variation in both the magnitude of the estimated GHG emissions per ha between different smallholder farm typologies and the emissions estimated by applying two different accounting tools. The farm scale quantification further shows that the adoption of SALM has a significant impact on emission reduction and removals and the mitigation benefits range between 4 and 6.5 tCO2 ha-1 yr-1 with significantly different mitigation benefits depending on typologies of the crop-livestock systems, their different agricultural practices, as well as adoption rates of improved practices. However, the inherent uncertainty related to the emission factors applied by accounting tools has substantial implications for reported agricultural emissions. With regard to uncertainty related to activity data, the assessment confirms the high variability within different farm types as well as between different parameters surveyed to comprehensively quantify GHG emissions within smallholder farms.

Transcription Tales or Let Not Love's Labour Be Lost

ERIC Educational Resources Information Center

Downs, Yvonne

2010-01-01

Drawing heavily on my MA dissertation but influenced by subsequent transcription experience, I relate how a technical problem in the recording of an interview necessitated deliberations on the nature and purpose of transcription that continue to have repercussions for my transcription practice and, furthermore, for my understanding of research as…

18 CFR 1b.12 - Transcripts.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any, of...

18 CFR 1b.12 - Transcripts.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any, of...

18 CFR 1b.12 - Transcripts.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any, of...

18 CFR 1b.12 - Transcripts.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any, of...

Stereochemical analysis of the functional significance of the conserved inverted CCAAT and TATA elements in the rat bone sialoprotein gene promoter.

PubMed

Su, Ming; Lee, Daniel; Ganss, Bernhard; Sodek, Jaro

2006-04-14

Basal transcription of the bone sialoprotein gene is mediated by highly conserved inverted CCAAT (ICE; ATTGG) and TATA elements (TTTATA) separated by precisely 21 nucleotides. Here we studied the importance of the relative position and orientation of the CCAAT and TATA elements in the proximal promoter by measuring the transcriptional activity of a series of mutated reporter constructs in transient transfection assays. Whereas inverting the TTTATA (wild type) to a TATAAA (consensus TATA) sequence increased transcription slightly, transcription was reduced when the flanking dinucleotides were also inverted. In contrast, reversing the ATTGG (wild type; ICE) to a CCAAT (RICE) sequence caused a marked reduction in transcription, whereas both transcription and NF-Y binding were progressively increased with the simultaneous inversion of flanking nucleotides (f-RICE-f). Reducing the distance between the ICE and TATA elements produced cyclical changes in transcriptional activity that correlated with progressive alterations in the relative positions of the CCAAT and TATA elements on the face of the DNA helix. Minimal transcription was observed after 5 nucleotides were deleted (equivalent to approximately one half turn of the helix), whereas transcription was fully restored after deleting 10 nucleotides (approximately one full turn of the DNA helix), transcriptional activity being progressively lost with deletions beyond 10 nucleotides. In comparison, when deletions were made with the ICE in the reversed (f-RICE-f) orientation transcriptional activity was progressively lost with no recovery. These results show that, although transcription can still occur when the CCAAT box is reversed and/or displaced relative to the TATA box, the activity is dependent upon the flexibility of the intervening DNA helix needed to align the NF-Y complex on the CCAAT box with preinitiation complex proteins that bind to the TATA box. Thus, the precise location and orientation of the CCAAT element is necessary for optimizing basal transcription of the bone sialoprotein gene.

Transcriptional analysis of sulfate reducing and chemolithoautotrophic sulfur oxidizing bacteria in the deep subseafloor.

PubMed

Orsi, William D; Barker Jørgensen, Bo; Biddle, Jennifer F

2016-08-01

Sulfate reducing bacteria (SRB) oxidize a significant proportion of subseafloor organic carbon, but their metabolic activities and subsistence mechanisms are poorly understood. Here, we report in depth phylogenetic and metabolic analyses of SRB transcripts in the Peru Margin subseafloor and interpret these results in the context of sulfate reduction activity in the sediment. Relative abundance of overall SRB gene transcripts declines strongly whereas relative abundance of ribosomal protein transcripts from sulfate reducing δ-Proteobacteria peak at 90 m below seafloor (mbsf) within a deep sulfate methane transition zone. This coincides with isotopically heavy δ(34) S values of pore water sulfate (70‰), indicating active subseafloor microbial sulfate reduction. Within the shallow sulfate reduction zone (0-5 mbsf), a transcript encoding the beta subunit of dissimilatory sulfite reductase (dsrB) was related to Desulfitobacterium dehalogenans and environmental sequences from Aarhus Bay (Denmark). At 159 mbsf we discovered a transcript encoding the reversely operating dissimilatory sulfite reductase α-subunit (rdsrA), with basal phylogenetic relation to the chemolithoautotrophic SUP05 Group II clade. A diversity of SRB transcripts involved in cellular maintenance point toward potential subsistence mechanisms under low-energy over long time periods, and provide a detailed new picture of SRB activities underlying sulfur cycling in the deep subseafloor. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Localized 2D COSY sequences: Method and experimental evaluation for a whole metabolite quantification approach

NASA Astrophysics Data System (ADS)

Martel, Dimitri; Tse Ve Koon, K.; Le Fur, Yann; Ratiney, Hélène

2015-11-01

Two-dimensional spectroscopy offers the possibility to unambiguously distinguish metabolites by spreading out the multiplet structure of J-coupled spin systems into a second dimension. Quantification methods that perform parametric fitting of the 2D MRS signal have recently been proposed for resolved PRESS (JPRESS) but not explicitly for Localized Correlation Spectroscopy (LCOSY). Here, through a whole metabolite quantification approach, correlation spectroscopy quantification performances are studied. The ability to quantify metabolite relaxation constant times is studied for three localized 2D MRS sequences (LCOSY, LCTCOSY and the JPRESS) in vitro on preclinical MR systems. The issues encountered during implementation and quantification strategies are discussed with the help of the Fisher matrix formalism. The described parameterized models enable the computation of the lower bound for error variance - generally known as the Cramér Rao bounds (CRBs), a standard of precision - on the parameters estimated from these 2D MRS signal fittings. LCOSY has a theoretical net signal loss of two per unit of acquisition time compared to JPRESS. A rapid analysis could point that the relative CRBs of LCOSY compared to JPRESS (expressed as a percentage of the concentration values) should be doubled but we show that this is not necessarily true. Finally, the LCOSY quantification procedure has been applied on data acquired in vivo on a mouse brain.

Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora.

PubMed

Ott, Stephan J; Musfeldt, Meike; Ullmann, Uwe; Hampe, Jochen; Schreiber, Stefan

2004-06-01

The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10(1) to 10(3) bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.

Food for thought: Selecting the right enzyme for the digestion of gluten.

PubMed

Colgrave, Michelle L; Byrne, Keren; Howitt, Crispin A

2017-11-01

Gluten describes a complex mixture of proteins found in wheat, rye, barley and oats that pose a health risk to people affected by conditions such as coeliac disease and non-coeliac gluten sensitivity. Complete digestion of gluten proteins is of critical importance during quantitative analysis. To this end, chymotrypsin was investigated for its ability to efficiently and reproducibly digest specific classes of gluten in barley. Using proteomics a chymotryptic peptide marker panel was elucidated and subjected to relative quantification using LC-MRM-MS. Thorough investigation of peptide markers revealed robust and reproducible quantification with CVs <15% was possible, however a greater proportion of non-specific cleavage variants were observed relative to trypsin. The selected peptide markers were assessed to ensure their efficient liberation from their parent proteins. While trypsin remains the preferred enzyme for quantification of the avenin-like A proteins, the B-, D- and γ-hordeins, chymotrypsin was the enzyme of choice for the C-hordeins. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

A Statistics-based Platform for Quantitative N-terminome Analysis and Identification of Protease Cleavage Products*

PubMed Central

auf dem Keller, Ulrich; Prudova, Anna; Gioia, Magda; Butler, Georgina S.; Overall, Christopher M.

2010-01-01

Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed. PMID:20305283

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

PubMed

Oberbach, Andreas; Schlichting, Nadine; Neuhaus, Jochen; Kullnick, Yvonne; Lehmann, Stefanie; Heinrich, Marco; Dietrich, Arne; Mohr, Friedrich Wilhelm; von Bergen, Martin; Baumann, Sven

2014-12-05

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Relative quantification of N(epsilon)-(Carboxymethyl)lysine, imidazolone A, and the Amadori product in glycated lysozyme by MALDI-TOF mass spectrometry.

PubMed

Kislinger, Thomas; Humeny, Andreas; Peich, Carlo C; Zhang, Xiaohong; Niwa, Toshimitsu; Pischetsrieder, Monika; Becker, Cord-Michael

2003-01-01

The nonenzymatic glycation of proteins by reducing sugars, also known as the Maillard reaction, has received increasing recognition from nutritional science and medical research. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to perform relative and simultaneous quantification of the Amadori product, which is an early glycation product, and of N(epsilon)-(carboxymethyl)lysine and imidazolone A, two important advanced glycation end products. Therefore, native lysozyme was incubated with d-glucose for increasing periods of time (1, 4, 8, and 16 weeks) in phosphate-buffered saline pH 7.8 at 50 degrees C. After enzymatic digestion with endoproteinase Glu-C, the N-terminal peptide fragment (m/z 838; amino acid sequence KVFGRCE) and the C-terminal peptide fragment (m/z 1202; amino acid sequence VQAWIRGCRL) were used for relative quantification of the three Maillard products. Amadori product, N(epsilon)-(carboxymethyl)lysine, and imidazolone A were the main glycation products formed under these conditions. Their formation was dependent on glucose concentration and reaction time. The kinetics were similar to those obtained by competitive ELISA, an established method for quantification of N(epsilon)-(carboxymethyl)lysine and imidazolone A. Inhibition experiments showed that coincubation with N(alpha)-acetylargine suppressed formation of imidazolone A but not of the Amadori product or N(epsilon)-(carboxymethyl)lysine. The presence of N(alpha)-acetyllysine resulted in the inhibition of lysine modifications but in higher concentrations of imidazolone A. o-Phenylenediamine decreased the yield of the Amadori product and completely inhibited the formation of N(epsilon)-(carboxymethyl)lysine and imidazolone A. MALDI-TOF-MS proved to be a new analytical tool for the simultaneous, relative quantification of specific products of the Maillard reaction. For the first time, kinetic data of defined products on specific sites of glycated protein could be measured. This characterizes MALDI-TOF-MS as a valuable method for monitoring the Maillard reaction in the course of food processing.

Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation.

PubMed

Krin, Evelyne; Pierlé, Sebastian Aguilar; Sismeiro, Odile; Jagla, Bernd; Dillies, Marie-Agnès; Varet, Hugo; Irazoki, Oihane; Campoy, Susana; Rouy, Zoé; Cruveiller, Stéphane; Médigue, Claudine; Coppée, Jean-Yves; Mazel, Didier

2018-05-21

The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment. Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection. We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria.

Molecular characterization and transcriptional regulation of the Na +/K+ ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis).

PubMed

Armesto, Paula; Campinho, Marco A; Rodríguez-Rúa, Ana; Cousin, Xavier; Power, Deborah M; Manchado, Manuel; Infante, Carlos

2014-09-01

In the present work, five genes encoding different Na(+),K(+) ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills. Copyright © 2014 Elsevier Inc. All rights reserved.

Synchronized arousal between performers and related spectators in a fire-walking ritual

PubMed Central

Konvalinka, Ivana; Xygalatas, Dimitris; Bulbulia, Joseph; Schjødt, Uffe; Jegindø, Else-Marie; Wallot, Sebastian; Van Orden, Guy; Roepstorff, Andreas

2011-01-01

Collective rituals are present in all known societies, but their function is a matter of long-standing debates. Field observations suggest that they may enhance social cohesion and that their effects are not limited to those actively performing but affect the audience as well. Here we show physiological effects of synchronized arousal in a Spanish fire-walking ritual, between active participants and related spectators, but not participants and other members of the audience. We assessed arousal by heart rate dynamics and applied nonlinear mathematical analysis to heart rate data obtained from 38 participants. We compared synchronized arousal between fire-walkers and spectators. For this comparison, we used recurrence quantification analysis on individual data and cross-recurrence quantification analysis on pairs of participants' data. These methods identified fine-grained commonalities of arousal during the 30-min ritual between fire-walkers and related spectators but not unrelated spectators. This indicates that the mediating mechanism may be informational, because participants and related observers had very different bodily behavior. This study demonstrates that a collective ritual may evoke synchronized arousal over time between active participants and bystanders. It links field observations to a physiological basis and offers a unique approach for the quantification of social effects on human physiology during real-world interactions. PMID:21536887

Cognitive-behavioral stress management reverses anxiety-related leukocyte transcriptional dynamics

PubMed Central

Antoni, Michael H.; Lutgendorf, Susan K.; Blomberg, Bonnie; Carver, Charles S.; Lechner, Suzanne; Diaz, Alain; Stagl, Jamie; Arevalo, Jesusa M.G.; Cole, Steven W.

2011-01-01

Background Chronic threat and anxiety are associated with pro-inflammatory transcriptional profiles in circulating leukocytes, but the causal direction of that relationship has not been established. This study tested whether a Cognitive-Behavioral Stress Management (CBSM) intervention targeting negative affect and cognition might counteract anxiety-related transcriptional alterations in people confronting a major medical threat. Methods 199 women undergoing primary treatment of Stage 0–III breast cancer were randomized to a 10-week CBSM protocol or an active control condition. 79 provided peripheral blood leukocyte samples for genome-wide transcriptional profiling and bioinformatic analyses at baseline, 6-, and 12-month follow-ups. Results Baseline negative affect was associated with > 50% differential expression of 201 leukocyte transcripts, including up-regulated expression of pro-inflammatory and metastasis-related genes. CBSM altered leukocyte expression of 91 genes by > 50% at follow-up (Group × Time interaction), including down-regulation of pro-inflammatory and metastasis-related genes and up-regulation of Type I interferon response genes. Promoter-based bioinformatic analyses implicated decreased activity of NF-κB/Rel and GATA family transcription factors and increased activity of Interferon Response Factors and the Glucocorticoid Receptor (GR) as potential mediators of CBSM-induced transcriptional alterations. Conclusions In early stage breast cancer patients, a 10-week CBSM intervention can reverse anxiety-related up-regulation of pro-inflammatory gene expression in circulating leukocytes. These findings clarify the molecular signaling pathways by which behavioral interventions can influence physical health and alter peripheral inflammatory processes that may reciprocally affect brain affective and cognitive processes. PMID:22088795

Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

NASA Astrophysics Data System (ADS)

House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

2015-06-01

Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

Acclimation of the crucifer Eutrema salsugineum to phosphate limitation is associated with constitutively high expression of phosphate-starvation genes.

PubMed

Velasco, Vera Marjorie Elauria; Mansbridge, John; Bremner, Samantha; Carruthers, Kimberley; Summers, Peter S; Sung, Wilson W L; Champigny, Marc J; Weretilnyk, Elizabeth A

2016-08-01

Eutrema salsugineum, a halophytic relative of Arabidopsis thaliana, was subjected to varying phosphate (Pi) treatments. Arabidopsis seedlings grown on 0.05 mm Pi displayed shortened primary roots, higher lateral root density and reduced shoot biomass allocation relative to those on 0.5 mm Pi, whereas Eutrema seedlings showed no difference in lateral root density and shoot biomass allocation. While a low Fe concentration mitigated the Pi deficiency response for Arabidopsis, Eutrema root architecture was unaltered, but adding NaCl increased Eutrema lateral root density almost 2-fold. Eutrema and Arabidopsis plants grown on soil without added Pi for 4 weeks had low shoot and root Pi content. Pi-deprived, soil-grown Arabidopsis plants were stunted with senescing older leaves, whereas Eutrema plants were visually indistinguishable from 2.5 mm Pi-supplemented plants. Genes associated with Pi starvation were analysed by RT-qPCR. EsIPS2, EsPHT1;4 and EsPAP17 showed up-regulated expression in Pi-deprived Eutrema plants, while EsPHR1, EsWRKY75 and EsRNS1 showed no induction. Absolute quantification of transcripts indicated that PHR1, WRKY75 and RNS1 were expressed at higher levels in Eutrema plants relative to those in Arabidopsis regardless of external Pi. The low phenotypic plasticity Eutrema displays to Pi supply is consistent with adaptation to chronic Pi deprivation in its extreme natural habitat. © 2016 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

Fatigue and gene expression in human leukocytes: Increased NF-κB and decreased glucocorticoid signaling in breast cancer survivors with persistent fatigue

PubMed Central

Bower, Julienne E.; Ganz, Patricia A.; Irwin, Michael R.; Arevalo, Jesusa M.G.; Cole, Steve W.

2013-01-01

Fatigue is highly prevalent in the general population and is one of the most common side effects of cancer treatment. There is growing evidence that pro-inflammatory cytokines play a role in cancer-related fatigue, although the molecular mechanisms for chronic inflammation and fatigue have not been determined. The current study utilized genome-wide expression microarrays to identify differences in gene expression and associated alterations in transcriptional activity in leukocytes from breast cancer survivors with persistent fatigue (n = 11) and non-fatigued controls (n = 10). We focused on transcription of inflammation-related genes, particularly those responsive to the pro-inflammatory NF-κB transcription control pathway. Further, given the role of glucocorticoids as key regulators of inflammatory processes, we examined transcription of glucocorticoid-responsive genes indicative of potential glucocorticoid receptor (GR) desensitization. Plasma levels of cortisol were also assessed. Consistent with hypotheses, results showed increased expression of transcripts with response elements for NF-κB, and reduced expression of transcripts with response elements for glucocorticoids (p < .05) in fatigued breast cancer survivors. No differences in plasma levels of cortisol were observed. These data indicate that increased activity of pro-inflammatory transcription factors may contribute to persistent cancer-related fatigue and provide insight into potential mechanisms for tonic increases in NF-κB activity, specifically decreased expression of GR anti-inflammatory transcription factors. PMID:20854893

High-throughput microfluidic single-cell digital polymerase chain reaction.

PubMed

White, A K; Heyries, K A; Doolin, C; Vaninsberghe, M; Hansen, C L

2013-08-06

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

NASA Astrophysics Data System (ADS)

Pu, Fei; Yang, Bingye; Ke, Caihuan

2015-07-01

Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 ( ACT-2), elongation factor 1 alpha ( EF-1α), elongation factor 1 beta ( EF-1β), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), ubiquitin ( UBQ), β-tubulin ( β-TUB), and 18S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene ( Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ and β-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.

Proteomic profiling of early degenerative retina of RCS rats

PubMed Central

Zhu, Zhi-Hong; Fu, Yan; Weng, Chuan-Huang; Zhao, Cong-Jian; Yin, Zheng-Qin

2017-01-01

AIM To identify the underlying cellular and molecular changes in retinitis pigmentosa (RP). METHODS Label-free quantification-based proteomics analysis, with its advantages of being more economic and consisting of simpler procedures, has been used with increasing frequency in modern biological research. Dystrophic RCS rats, the first laboratory animal model for the study of RP, possess a similar pathological course as human beings with the diseases. Thus, we employed a comparative proteomics analysis approach for in-depth proteome profiling of retinas from dystrophic RCS rats and non-dystrophic congenic controls through Linear Trap Quadrupole - orbitrap MS/MS, to identify the significant differentially expressed proteins (DEPs). Bioinformatics analyses, including Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and upstream regulatory analysis, were then performed on these retina proteins. Finally, a Western blotting experiment was carried out to verify the difference in the abundance of transcript factor E2F1. RESULTS In this study, we identified a total of 2375 protein groups from the retinal protein samples of RCS rats and non-dystrophic congenic controls. Four hundred thirty-four significantly DEPs were selected by Student's t-test. Based on the results of the bioinformatics analysis, we identified mitochondrial dysfunction and transcription factor E2F1 as the key initiation factors in early retinal degenerative process. CONCLUSION We showed that the mitochondrial dysfunction and the transcription factor E2F1 substantially contribute to the disease etiology of RP. The results provide a new potential therapeutic approach for this retinal degenerative disease. PMID:28730077

E2F mediates enhanced alternative polyadenylation in proliferation.

PubMed

Elkon, Ran; Drost, Jarno; van Haaften, Gijs; Jenal, Mathias; Schrier, Mariette; Oude Vrielink, Joachim A F; Agami, Reuven

2012-07-02

The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

Prediction of response factors for gas chromatography with flame ionization detection: Algorithm improvement, extension to silylated compounds, and application to the quantification of metabolites

PubMed Central

de Saint Laumer, Jean‐Yves; Leocata, Sabine; Tissot, Emeline; Baroux, Lucie; Kampf, David M.; Merle, Philippe; Boschung, Alain; Seyfried, Markus

2015-01-01

We previously showed that the relative response factors of volatile compounds were predictable from either combustion enthalpies or their molecular formulae only 1. We now extend this prediction to silylated derivatives by adding an increment in the ab initio calculation of combustion enthalpies. The accuracy of the experimental relative response factors database was also improved and its population increased to 490 values. In particular, more brominated compounds were measured, and their prediction accuracy was improved by adding a correction factor in the algorithm. The correlation coefficient between predicted and measured values increased from 0.936 to 0.972, leading to a mean prediction accuracy of ± 6%. Thus, 93% of the relative response factors values were predicted with an accuracy of better than ± 10%. The capabilities of the extended algorithm are exemplified by (i) the quick and accurate quantification of hydroxylated metabolites resulting from a biodegradation test after silylation and prediction of their relative response factors, without having the reference substances available; and (ii) the rapid purity determinations of volatile compounds. This study confirms that Gas chromatography with a flame ionization detector and using predicted relative response factors is one of the few techniques that enables quantification of volatile compounds without calibrating the instrument with the pure reference substance. PMID:26179324

Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

PubMed

Jacchia, Sara; Nardini, Elena; Savini, Christian; Petrillo, Mauro; Angers-Loustau, Alexandre; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

2015-02-18

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

Development of a qPCR Method for the Identification and Quantification of Two Closely Related Tuna Species, Bigeye Tuna (Thunnus obesus) and Yellowfin Tuna (Thunnus albacares), in Canned Tuna.

PubMed

Bojolly, Daline; Doyen, Périne; Le Fur, Bruno; Christaki, Urania; Verrez-Bagnis, Véronique; Grard, Thierry

2017-02-01

Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, as juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional substitutions may occur during the canning process. In this study, two mitochondrial markers from NADH dehydrogenase subunit 2 and cytochrome c oxidase subunit II genes were used to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate these two closely related tuna species when used in a binary mix in tuna cans.

Quantification of Global DNA Methylation Levels by Mass Spectrometry.

PubMed

Fernandez, Agustin F; Valledor, Luis; Vallejo, Fernando; Cañal, Maria Jesús; Fraga, Mario F

2018-01-01

Global DNA methylation was classically considered the relative percentage of 5-methylcysine (5mC) with respect to total cytosine (C). Early approaches were based on the use of high-performance separation technologies and UV detection. However, the recent development of protocols using mass spectrometry for the detection has increased sensibility and permitted the precise identification of peak compounds based on their molecular masses. This allows work to be conducted with much less genomic DNA starting material and also to quantify 5-hydroxymethyl-cytosine (5hmC), a recently identified form of methylated cytosine that could play an important role in active DNA demethylation. Here, we describe the protocol that we currently use in our laboratory to analyze 5mC and 5hmC by mass spectrometry. The protocol, which is based on the method originally developed by Le and colleagues using Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry (triple Quadrupole (QqQ)) detection, allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels starting from just 1 μg of genomic DNA, which allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels.

A simple and selective method for the measurement of azadirachtin and related azadirachtoid levels in fruits and vegetables using liquid chromatography electrospray ionization tandem mass spectrometry.

PubMed

Sarais, Giorgia; Caboni, Pierluigi; Sarritzu, Erika; Russo, Mariateresa; Cabras, Paolo

2008-05-14

Neem-based insecticides containing azadirachtin and related azadirachtoids are widely used in agriculture. Here, we report an analytical method for the rapid and accurate quantification of the insecticide azadirachtin A and B and other azadirachtoids such as salannin, nimbin, and their deacetylated analogues on tomatoes and peaches. Azadirachtoids were extracted from fruits and vegetables with acetonitrile. Using high-performance liquid chromatography/electrospray ionization tandem mass spectrometer, azadirachtoids were selectively detected monitoring the multiple reaction transitions of sodium adduct precursor ions. For azadirachtin A, calibration was linear over a working range of 1-1000 microg/L with r > 0.996. The limit of detection and limit of quantification for azadirachtin A were 0.4 and 0.8 microg/kg, respectively. The presence of interfering compounds in the peach and tomato extracts was evaluated and found to be minimal. Because of the linear behavior, it was concluded that the multiple reaction transitions of sodium adduct ions can be used for analytical purposes, that is, for the identification and quantification of azadirachtin A and B and related azadirachtoids in fruit and vegetable extracts at trace levels.

Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters.

PubMed

Verma, Neha; Hung, Ko-Hsuan; Kang, Jin Joo; Barakat, Nermeen H; Stumph, William E

2013-09-20

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

Transcriptomic analysis of genetically defined autism candidate genes reveals common mechanisms of action

PubMed Central

2013-01-01

Background Austism spectrum disorder (ASD) is a heterogeneous behavioral disorder or condition characterized by severe impairment of social engagement and the presence of repetitive activities. The molecular etiology of ASD is still largely unknown despite a strong genetic component. Part of the difficulty in turning genetics into disease mechanisms and potentially new therapeutics is the sheer number and diversity of the genes that have been associated with ASD and ASD symptoms. The goal of this work is to use shRNA-generated models of genetic defects proposed as causative for ASD to identify the common pathways that might explain how they produce a core clinical disability. Methods Transcript levels of Mecp2, Mef2a, Mef2d, Fmr1, Nlgn1, Nlgn3, Pten, and Shank3 were knocked-down in mouse primary neuron cultures using shRNA constructs. Whole genome expression analysis was conducted for each of the knockdown cultures as well as a mock-transduced culture and a culture exposed to a lentivirus expressing an anti-luciferase shRNA. Gene set enrichment and a causal reasoning engine was employed to identify pathway level perturbations generated by the transcript knockdown. Results Quantification of the shRNA targets confirmed the successful knockdown at the transcript and protein levels of at least 75% for each of the genes. After subtracting out potential artifacts caused by viral infection, gene set enrichment and causal reasoning engine analysis showed that a significant number of gene expression changes mapped to pathways associated with neurogenesis, long-term potentiation, and synaptic activity. Conclusions This work demonstrates that despite the complex genetic nature of ASD, there are common molecular mechanisms that connect many of the best established autism candidate genes. By identifying the key regulatory checkpoints in the interlinking transcriptional networks underlying autism, we are better able to discover the ideal points of intervention that provide the broadest efficacy across the diverse population of autism patients. PMID:24238429

Transcriptomic analysis of genetically defined autism candidate genes reveals common mechanisms of action.

PubMed

Lanz, Thomas A; Guilmette, Edward; Gosink, Mark M; Fischer, James E; Fitzgerald, Lawrence W; Stephenson, Diane T; Pletcher, Mathew T

2013-11-15

Austism spectrum disorder (ASD) is a heterogeneous behavioral disorder or condition characterized by severe impairment of social engagement and the presence of repetitive activities. The molecular etiology of ASD is still largely unknown despite a strong genetic component. Part of the difficulty in turning genetics into disease mechanisms and potentially new therapeutics is the sheer number and diversity of the genes that have been associated with ASD and ASD symptoms. The goal of this work is to use shRNA-generated models of genetic defects proposed as causative for ASD to identify the common pathways that might explain how they produce a core clinical disability. Transcript levels of Mecp2, Mef2a, Mef2d, Fmr1, Nlgn1, Nlgn3, Pten, and Shank3 were knocked-down in mouse primary neuron cultures using shRNA constructs. Whole genome expression analysis was conducted for each of the knockdown cultures as well as a mock-transduced culture and a culture exposed to a lentivirus expressing an anti-luciferase shRNA. Gene set enrichment and a causal reasoning engine was employed to identify pathway level perturbations generated by the transcript knockdown. Quantification of the shRNA targets confirmed the successful knockdown at the transcript and protein levels of at least 75% for each of the genes. After subtracting out potential artifacts caused by viral infection, gene set enrichment and causal reasoning engine analysis showed that a significant number of gene expression changes mapped to pathways associated with neurogenesis, long-term potentiation, and synaptic activity. This work demonstrates that despite the complex genetic nature of ASD, there are common molecular mechanisms that connect many of the best established autism candidate genes. By identifying the key regulatory checkpoints in the interlinking transcriptional networks underlying autism, we are better able to discover the ideal points of intervention that provide the broadest efficacy across the diverse population of autism patients.

Physiological and transcriptional analyses of developmental stages along sugarcane leaf.

PubMed

Mattiello, Lucia; Riaño-Pachón, Diego Mauricio; Martins, Marina Camara Mattos; da Cruz, Larissa Prado; Bassi, Denis; Marchiori, Paulo Eduardo Ribeiro; Ribeiro, Rafael Vasconcelos; Labate, Mônica T Veneziano; Labate, Carlos Alberto; Menossi, Marcelo

2015-12-29

Sugarcane is one of the major crops worldwide. It is cultivated in over 100 countries on 22 million ha. The complex genetic architecture and the lack of a complete genomic sequence in sugarcane hamper the adoption of molecular approaches to study its physiology and to develop new varieties. Investments on the development of new sugarcane varieties have been made to maximize sucrose yield, a trait dependent on photosynthetic capacity. However, detailed studies on sugarcane leaves are scarce. In this work, we report the first molecular and physiological characterization of events taking place along a leaf developmental gradient in sugarcane. Photosynthetic response to CO2 indicated divergence in photosynthetic capacity based on PEPcase activity, corroborated by activity quantification (both in vivo and in vitro) and distinct levels of carbon discrimination on different segments along leaf length. Additionally, leaf segments had contrasting amount of chlorophyll, nitrogen and sugars. RNA-Seq data indicated a plethora of biochemical pathways differentially expressed along the leaf. Some transcription factors families were enriched on each segment and their putative functions corroborate with the distinct developmental stages. Several genes with higher expression in the middle segment, the one with the highest photosynthetic rates, were identified and their role in sugarcane productivity is discussed. Interestingly, sugarcane leaf segments had a different transcriptional behavior compared to previously published data from maize. This is the first report of leaf developmental analysis in sugarcane. Our data on sugarcane is another source of information for further studies aiming to understand and/or improve C4 photosynthesis. The segments used in this work were distinct in their physiological status allowing deeper molecular analysis. Although limited in some aspects, the comparison to maize indicates that all data acquired on one C4 species cannot always be easily extrapolated to other species. However, our data indicates that some transcriptional factors were segment-specific and the sugarcane leaf undergoes through the process of suberizarion, photosynthesis establishment and senescence.

New Face for Chromatin-Related Mesenchymal Modulator: n-CHD9 Localizes to Nucleoli and Interacts With Ribosomal Genes.

PubMed

Salomon-Kent, Ronit; Marom, Ronit; John, Sam; Dundr, Miroslav; Schiltz, Louis R; Gutierrez, Jose; Workman, Jerry; Benayahu, Dafna; Hager, Gordon L

2015-09-01

Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co-regulators which bind to specific gene promoters. The Chromatin-Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP-dependent manner. Epigenetically, CHD9 binds with modified H3-(K9me2/3 and K27me3). Previously, we presented a role for CHD9 with RNA Polymerase II (Pol II)-dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n-CHD9) that is dynamically associated with Pol I, fibrillarin, and upstream binding factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n-CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP- chip), we find an association of n-CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes. © 2015 Wiley Periodicals, Inc.

The impact of carbon-13 and deuterium on relative quantification of proteins using stable isotope diethyl labeling.

PubMed

Koehler, Christian J; Arntzen, Magnus Ø; Thiede, Bernd

2015-05-15

Stable isotopic labeling techniques are useful for quantitative proteomics. A cost-effective and convenient method for diethylation by reductive amination was established. The impact using either carbon-13 or deuterium on quantification accuracy and precision was investigated using diethylation. We established an effective approach for stable isotope labeling by diethylation of amino groups of peptides. The approach was validated using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nanospray liquid chromatography/electrospray ionization (nanoLC/ESI)-ion trap/orbitrap for mass spectrometric analysis as well as MaxQuant for quantitative data analysis. Reaction conditions with low reagent costs, high yields and minor side reactions were established for diethylation. Furthermore, we showed that diethylation can be applied to up to sixplex labeling. For duplex experiments, we compared diethylation in the analysis of the proteome of HeLa cells using acetaldehyde-(13) C(2)/(12) C(2) and acetaldehyde-(2) H(4)/(1) H(4). Equal numbers of proteins could be identified and quantified; however, (13) C(4)/(12) C(4) -diethylation revealed a lower variance of quantitative peptide ratios within proteins resulting in a higher precision of quantified proteins and less falsely regulated proteins. The results were compared with dimethylation showing minor effects because of the lower number of deuteriums. The described approach for diethylation of primary amines is a cost-effective and accurate method for up to sixplex relative quantification of proteomes. (13) C(4)/(12) C(4) -diethylation enables duplex quantification based on chemical labeling without using deuterium which reduces identification of false-negatives and increases the quality of the quantification results. Copyright © 2015 John Wiley & Sons, Ltd.

Proteomic Comparison and MRM-Based Comparative Analysis of Metabolites Reveal Metabolic Shift in Human Prostate Cancer Cell Lines.

PubMed

Shu, Qingbo; Cai, Tanxi; Chen, Xiulan; Zhu, Helen He; Xue, Peng; Zhu, Nali; Xie, Zhensheng; Wei, Shasha; Zhang, Qing; Niu, Lili; Gao, Wei-Qiang; Yang, Fuquan

2015-08-07

One of the major challenges in prostate cancer therapy remains the development of effective treatments for castration-resistant prostate cancer (CRPC), as the underlying mechanisms for its progression remain elusive. Previous studies showed that androgen receptor (AR) is crucially involved in regulation of metabolism in prostate cancer (PCa) cells throughout the transition from early stage, androgen-sensitive PCa to androgen-independent CRPC. AR achieves such metabolic rewiring directively either via its transcriptional activity or via interactions with AMP-activated protein kinase (AMPK). However, due to the heterogeneous expression and activity status of AR in PCa cells, it remains a challenge to investigate the links between AR status and metabolic alterations. To this end, we compared the proteomes of three pairs of androgen-sensitive (AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR(+)/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling approach. Our results revealed that most of the differentially expressed proteins between each pair function in metabolism, indicating a metabolic shift between AS and AI cells, as further validated by multiple reaction monitoring (MRM)-based quantification of nucleotides and relative comparison of fatty acids between these cell lines. Furthermore, increased adenylate kinase isoenzyme 1 (AK1) in AS relative to AI cells may result in activation of AMPK, representing a major regulatory factor involved in the observed metabolic shift in PCa cells.

Transcriptional Pathways Altered in Response to Vibration in a Model of Hand-Arm Vibration Syndrome.

PubMed

Waugh, Stacey; Kashon, Michael L; Li, Shengqiao; Miller, Gerome R; Johnson, Claud; Krajnak, Kristine

2016-04-01

The aim of this study was to use an established model of vibration-induced injury to assess frequency-dependent changes in transcript expression in skin, artery, and nerve tissues. Transcript expression in tissues from control and vibration-exposed rats (4 h/day for 10 days at 62.5, 125, or 250 Hz; 49 m/s, rms) was measured. Transcripts affected by vibration were used in bioinformatics analyses to identify molecular- and disease-related pathways associated with exposure to vibration. Analyses revealed that cancer-related pathways showed frequency-dependent changes in activation or inhibition. Most notably, the breast-related cancer-1 pathway was affected. Other pathways associated with breast cancer type 1 susceptibility protein related signaling, or associated with cancer and cell cycle/cell survivability were also affected. Occupational exposure to vibration may result in DNA damage and alterations in cell signaling pathways that have significant effects on cellular division.

Measures of Residual Risk with Connections to Regression, Risk Tracking, Surrogate Models, and Ambiguity

DTIC Science & Technology

2015-04-15

manage , predict, and mitigate the risk in the original variable. Residual risk can be exemplified as a quantification of the improved... the random variable of interest is viewed in concert with a related random vector that helps to manage , predict, and mitigate the risk in the original... manage , predict and mitigate the risk in the original variable. Residual risk can be exemplified as a quantification of the improved situation faced

Methods and insights from the characterization of osteoprogenitor cells of bats (Mammalia: Chiroptera).

PubMed

Ball, H C; Moussa, F M; Mbimba, T; Orman, R; Safadi, F F; Cooper, L N

2016-07-01

Osteoprogenitor cells contribute to the development and maintenance of skeletal tissues. Bats are unique model taxa whose cellular processes are poorly understood, especially in regards to skeletal biology. Forelimb bones of bats, unlike those of terrestrial mammals, bend during flight and function in controlled deformation. As a first step towards understanding the molecular processes governing deposition of this flexible bone matrix, we provide the first method for isolation and differentiation of cell populations derived from the bone marrow and cortical bone of bats, and compare results with those harvested from C57BL/6J mice. Osteogenic capacity of these cells was assessed via absolute quantitative real-time PCR (qPCR) and through quantification of in vitro mineral deposition. Results indicate the differentiated bone cells of bats display significantly lower gene expression of known osteogenic markers (Runt-related transcription factor (RUNX2), osteocalcin (BGLAP) and osterix (SP7)), and deposit a less-mineralized matrix compared with murine controls. By characterizing the in vitro performance of osteoprogenitor cells throughout differentiation and matrix production, this study lays the ground work for in vitro manipulations of bat stem and osteoprogenitor cells and extends our understanding of the cellular diversity across mammals that occupy different habitats. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Molecular events of apical bud formation in white spruce, Picea glauca.

PubMed

El Kayal, Walid; Allen, Carmen C G; Ju, Chelsea J-T; Adams, Eri; King-Jones, Susanne; Zaharia, L Irina; Abrams, Suzanne R; Cooke, Janice E K

2011-03-01

Bud formation is an adaptive trait that temperate forest trees have acquired to facilitate seasonal synchronization. We have characterized transcriptome-level changes that occur during bud formation of white spruce [Picea glauca (Moench) Voss], a primarily determinate species in which preformed stem units contained within the apical bud constitute most of next season's growth. Microarray analysis identified 4460 differentially expressed sequences in shoot tips during short day-induced bud formation. Cluster analysis revealed distinct temporal patterns of expression, and functional classification of genes in these clusters implied molecular processes that coincide with anatomical changes occurring in the developing bud. Comparing expression profiles in developing buds under long day and short day conditions identified possible photoperiod-responsive genes that may not be essential for bud development. Several genes putatively associated with hormone signalling were identified, and hormone quantification revealed distinct profiles for abscisic acid (ABA), cytokinins, auxin and their metabolites that can be related to morphological changes to the bud. Comparison of gene expression profiles during bud formation in different tissues revealed 108 genes that are differentially expressed only in developing buds and show greater transcript abundance in developing buds than other tissues. These findings provide a temporal roadmap of bud formation in white spruce. © 2011 Blackwell Publishing Ltd.

SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis

PubMed Central

Jia, Fengjuan; Wang, Chunyan; Huang, Jinguang; Yang, Guodong; Wu, Changai; Zheng, Chengchao

2015-01-01

Skp1–Cullin–F-box (SCF) E3 ligases are essential to the post-translational regulation of many important factors involved in cellular signal transduction. In this study, we identified an F-box protein from Arabidopsis thaliana, AtPP2-B11, which was remarkably induced with increased duration of salt treatment in terms of both transcript and protein levels. Transgenic Arabidopsis plants overexpressing AtPP2-B11 exhibited obvious tolerance to high salinity, whereas the RNA interference line was more sensitive to salt stress than wild-type plants. Isobaric tag for relative and absolute quantification analysis revealed that 4311 differentially expressed proteins were regulated by AtPP2-B11 under salt stress. AtPP2-B11 could upregulate the expression of annexin1 (AnnAt1) and function as a molecular link between salt stress and reactive oxygen species accumulation in Arabidopsis. Moreover, AtPP2-B11 influenced the expression of Na+ homeostasis genes under salt stress, and the AtPP2-B11 overexpressing lines exhibited lower Na+ accumulation. These results suggest that AtPP2-B11 functions as a positive regulator in response to salt stress in Arabidopsis. PMID:26041321

Technologies in the Whole-Genome Age: MALDI-TOF-Based Genotyping.

PubMed

Vogel, Nicolas; Schiebel, Katrin; Humeny, Andreas

2009-01-01

With the decipherment of the human genome, new questions have moved into the focus of today's research. One key aspect represents the discovery of DNA variations capable to influence gene transcription, RNA splicing, or regulating processes, and their link to pathology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a powerful tool for the qualitative investigation and relative quantification of variations like single nucleotide polymorphisms, DNA methylation, microsatellite instability, or loss of heterozygosity. After its introduction into proteomics, efforts were made to adopt this technique to DNA analysis. Initially intended for peptide/protein analysis, it held several difficulties for application to nucleic acids. Today, MALDI-TOF-MS has reached worldwide acceptance and application in nucleic acid research, with a wide spectrum of methods being available. One of the most versatile approaches relies on primer extension to genotype single alleles, microsatellite repeat lengths or the methylation status of a given cytosine. Optimized methods comprising intelligent primer design and proper nucleotide selection for primer extension enabled multiplexing of reactions, rendering the analysis more economic due to parallel genotyping of several alleles in a single experiment. Laboratories equipped with MALDI-TOF-MS possess a universal technical platform for the analysis of a large variety of different molecules.

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

PubMed

Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

Quantitative Proteomic Profiling of Low-Dose Ionizing Radiation Effects in a Human Skin Model

PubMed Central

Hengel, Shawna M.; Aldrich, Joshua T.; Waters, Katrina M.; Pasa-Tolic, Ljiljana; Stenoien, David L.

2014-01-01

To assess responses to low-dose ionizing radiation (LD-IR) exposures potentially encountered during medical diagnostic procedures, nuclear accidents or terrorist acts, a quantitative proteomic approach was used to identify changes in protein abundance in a reconstituted human skin tissue model treated with 0.1 Gy of ionizing radiation. To improve the dynamic range of the assay, subcellular fractionation was employed to remove highly abundant structural proteins and to provide insight into radiation-induced alterations in protein localization. Relative peptide quantification across cellular fractions, control and irradiated samples was performing using 8-plex iTRAQ labeling followed by online two-dimensional nano-scale liquid chromatography and high resolution MS/MS analysis. A total of 107 proteins were detected with statistically significant radiation-induced change in abundance (>1.5 fold) and/or subcellular localization compared to controls. The top biological pathways identified using bioinformatics include organ development, anatomical structure formation and the regulation of actin cytoskeleton. From the proteomic data, a change in proteolytic processing and subcellular localization of the skin barrier protein, filaggrin, was identified, and the results were confirmed by western blotting. This data indicate post-transcriptional regulation of protein abundance, localization and proteolytic processing playing an important role in regulating radiation response in human tissues. PMID:28250387

What is in a beer? Proteomic characterization and relative quantification of hordein (gluten) in beer.

PubMed

Colgrave, Michelle L; Goswami, Hareshwar; Howitt, Crispin A; Tanner, Gregory J

2012-01-01

The suite of prolamin proteins present in barley flour was characterized in this study, in which we provide spectral evidence for 3 previously characterized prolamins, 8 prolamins with only transcript evidence, and 19 genome-derived predicted prolamins. An additional 9 prolamins were identified by searching the complete spectral set against an unannotated translated EST database. Analyses of wort, the liquid extracted from the mashing process during beer production, and beer were undertaken and a similar suite of prolamins were identified. We have demonstrated by using tandem mass spectrometry that hordeins are indeed present in beer despite speculation to the contrary. Multiple reaction monitoring (MRM) mass spectrometry was used for the rapid analyses of hordein in barley (Hordeum vulgare L.) beer. A selection of international beers were analyzed and compared to the results obtained with hordein deletion beers. The hordein deletion beers were brewed from grains carrying mutations that prevented the accumulation of either B-hordeins (Risø 56) or C-hordeins (Risø 1508). No intact C-hordeins were detected in beer, although fragments of C-hordeins were present in wort. Multiple reaction monitoring analysis of non-barley based gluten (hordein)-free beers targeting the major hordein protein families was performed and confirmed the absence of hordein in several gluten-free commercial beers.

MALDI Mass Spectrometry Imaging of Lipids and Gene Expression Reveals Differences in Fatty Acid Metabolism between Follicular Compartments in Porcine Ovaries

PubMed Central

Uzbekova, Svetlana; Elis, Sebastien; Teixeira-Gomes, Ana-Paula; Desmarchais, Alice; Maillard, Virginie; Labas, Valerie

2015-01-01

In mammals, oocytes develop inside the ovarian follicles; this process is strongly supported by the surrounding follicular environment consisting of cumulus, granulosa and theca cells, and follicular fluid. In the antral follicle, the final stages of oogenesis require large amounts of energy that is produced by follicular cells from substrates including glucose, amino acids and fatty acids (FAs). Since lipid metabolism plays an important role in acquiring oocyte developmental competence, the aim of this study was to investigate site-specificity of lipid metabolism in ovaries by comparing lipid profiles and expression of FA metabolism-related genes in different ovarian compartments. Using MALDI Mass Spectrometry Imaging, images of porcine ovary sections were reconstructed from lipid ion signals for the first time. Cluster analysis of ion spectra revealed differences in spatial distribution of lipid species among ovarian compartments, notably between the follicles and interstitial tissue. Inside the follicles analysis differentiated follicular fluid, granulosa, theca and the oocyte-cumulus complex. Moreover, by transcript quantification using real time PCR, we showed that expression of five key genes in FA metabolism significantly varied between somatic follicular cells (theca, granulosa and cumulus) and the oocyte. In conclusion, lipid metabolism differs between ovarian and follicular compartments. PMID:25756245

Validation and evaluation of an HPLC methodology for the quantification of the potent antimitotic compound (+)-discodermolide in the Caribbean marine sponge Discodermia dissoluta.

PubMed

Valderrama, Katherine; Castellanos, Leonardo; Zea, Sven

2010-08-01

The sponge Discodermia dissoluta is the source of the potent antimitotic compound (+)-discodermolide. The relatively abundant and shallow populations of this sponge in Santa Marta, Colombia, allow for studies to evaluate the natural and biotechnological supply options of (+)-discodermolide. In this work, an RP-HPLC-UV methodology for the quantification of (+)-discodermolide from sponge samples was tested and validated. Our protocol for extracting this compound from the sponge included lyophilization, exhaustive methanol extraction, partitioning using water and dichloromethane, purification of the organic fraction in RP-18 cartridges and then finally retrieving the (+)-discodermolide in the methanol-water (80:20 v/v) fraction. This fraction was injected into an HPLC system with an Xterra RP-18 column and a detection wavelength of 235 nm. The calibration curve was linear, making it possible to calculate the LODs and quantification in these experiments. The intra-day and inter-day precision showed relative standard deviations lower than 5%. The accuracy, determined as the percentage recovery, was 99.4%. Nine samples of the sponge from the Bahamas, Bonaire, Curaçao and Santa Marta had concentrations of (+)-discodermolide ranging from 5.3 to 29.3 microg/g(-1) of wet sponge. This methodology is quick and simple, allowing for the quantification in sponges from natural environments, in situ cultures or dissociated cells.

Comparative quantification of dietary supplemented neural creatine concentrations with (1)H-MRS peak fitting and basis spectrum methods.

PubMed

Turner, Clare E; Russell, Bruce R; Gant, Nicholas

2015-11-01

Magnetic resonance spectroscopy (MRS) is an analytical procedure that can be used to non-invasively measure the concentration of a range of neural metabolites. Creatine is an important neurometabolite with dietary supplementation offering therapeutic potential for neurological disorders with dysfunctional energetic processes. Neural creatine concentrations can be probed using proton MRS and quantified using a range of software packages based on different analytical methods. This experiment examines the differences in quantification performance of two commonly used analysis packages following a creatine supplementation strategy with potential therapeutic application. Human participants followed a seven day dietary supplementation regime in a placebo-controlled, cross-over design interspersed with a five week wash-out period. Spectroscopy data were acquired the day immediately following supplementation and analyzed with two commonly-used software packages which employ vastly different quantification methods. Results demonstrate that neural creatine concentration was augmented following creatine supplementation when analyzed using the peak fitting method of quantification (105.9%±10.1). In contrast, no change in neural creatine levels were detected with supplementation when analysis was conducted using the basis spectrum method of quantification (102.6%±8.6). Results suggest that software packages that employ the peak fitting procedure for spectral quantification are possibly more sensitive to subtle changes in neural creatine concentrations. The relative simplicity of the spectroscopy sequence and the data analysis procedure suggest that peak fitting procedures may be the most effective means of metabolite quantification when detection of subtle alterations in neural metabolites is necessary. The straightforward technique can be used on a clinical magnetic resonance imaging system. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantification of Lignin and Its Structural Features in Plant Biomass Using 13C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS.

PubMed

van Erven, Gijs; de Visser, Ries; Merkx, Donny W H; Strolenberg, Willem; de Gijsel, Peter; Gruppen, Harry; Kabel, Mirjam A

2017-10-17

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13 C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12 C and 13 C lignin were isolated from nonlabeled and uniformly 13 C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13 C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12 C lignin analogue and was shown to be extremely accurate (>99.9%, R 2 > 0.999) and precise (RSD < 1.5%). Third, 13 C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.

Quantification of Lignin and Its Structural Features in Plant Biomass Using 13C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS

PubMed Central

2017-01-01

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12C and 13C lignin were isolated from nonlabeled and uniformly 13C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12C lignin analogue and was shown to be extremely accurate (>99.9%, R2 > 0.999) and precise (RSD < 1.5%). Third, 13C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin. PMID:28926698

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

PubMed Central

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna MY; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease. PMID:26011746

Functional assessment of human enhancer activities using whole-genome STARR-sequencing.

PubMed

Liu, Yuwen; Yu, Shan; Dhiman, Vineet K; Brunetti, Tonya; Eckart, Heather; White, Kevin P

2017-11-20

Genome-wide quantification of enhancer activity in the human genome has proven to be a challenging problem. Recent efforts have led to the development of powerful tools for enhancer quantification. However, because of genome size and complexity, these tools have yet to be applied to the whole human genome.  In the current study, we use a human prostate cancer cell line, LNCaP as a model to perform whole human genome STARR-seq (WHG-STARR-seq) to reliably obtain an assessment of enhancer activity. This approach builds upon previously developed STARR-seq in the fly genome and CapSTARR-seq techniques in targeted human genomic regions. With an improved library preparation strategy, our approach greatly increases the library complexity per unit of starting material, which makes it feasible and cost-effective to explore the landscape of regulatory activity in the much larger human genome. In addition to our ability to identify active, accessible enhancers located in open chromatin regions, we can also detect sequences with the potential for enhancer activity that are located in inaccessible, closed chromatin regions. When treated with the histone deacetylase inhibitor, Trichostatin A, genes nearby this latter class of enhancers are up-regulated, demonstrating the potential for endogenous functionality of these regulatory elements. WHG-STARR-seq provides an improved approach to current pipelines for analysis of high complexity genomes to gain a better understanding of the intricacies of transcriptional regulation.

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

PubMed

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

PubMed

Li, Xin; Ben-Dov, Iddo Z; Mauro, Maurizio; Williams, Zev

2015-05-06

RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

Assessment of cardiac fibrosis: a morphometric method comparison for collagen quantification.

PubMed

Schipke, Julia; Brandenberger, Christina; Rajces, Alexandra; Manninger, Martin; Alogna, Alessio; Post, Heiner; Mühlfeld, Christian

2017-04-01

Fibrotic remodeling of the heart is a frequent condition linked to various diseases and cardiac dysfunction. Collagen quantification is an important objective in cardiac fibrosis research; however, a variety of different histological methods are currently used that may differ in accuracy. Here, frequently applied collagen quantification techniques were compared. A porcine model of early stage heart failure with preserved ejection fraction was used as an example. Semiautomated threshold analyses were imprecise, mainly due to inclusion of noncollagen structures or failure to detect certain collagen deposits. In contrast, collagen assessment by automated image analysis and light microscopy (LM)-stereology was more sensitive. Depending on the quantification method, the amount of estimated collagen varied and influenced intergroup comparisons. PicroSirius Red, Masson's trichrome, and Azan staining protocols yielded similar results, whereas the measured collagen area increased with increasing section thickness. Whereas none of the LM-based methods showed significant differences between the groups, electron microscopy (EM)-stereology revealed a significant collagen increase between cardiomyocytes in the experimental group, but not at other localizations. In conclusion, in contrast to the staining protocol, section thickness and the quantification method being used directly influence the estimated collagen content and thus, possibly, intergroup comparisons. EM in combination with stereology is a precise and sensitive method for collagen quantification if certain prerequisites are considered. For subtle fibrotic alterations, consideration of collagen localization may be necessary. Among LM methods, LM-stereology and automated image analysis are appropriate to quantify fibrotic changes, the latter depending on careful control of algorithm and comparable section staining. NEW & NOTEWORTHY Direct comparison of frequently applied histological fibrosis assessment techniques revealed a distinct relation of measured collagen and utilized quantification method as well as section thickness. Besides electron microscopy-stereology, which was precise and sensitive, light microscopy-stereology and automated image analysis proved to be appropriate for collagen quantification. Moreover, consideration of collagen localization might be important in revealing minor fibrotic changes. Copyright © 2017 the American Physiological Society.

Antisense transcription is pervasive but rarely conserved in enteric bacteria.

PubMed

Raghavan, Rahul; Sloan, Daniel B; Ochman, Howard

2012-01-01

Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional.

RNA-Seq Profiling Reveals Novel Hepatic Gene Expression Pattern in Aflatoxin B1 Treated Rats

PubMed Central

Merrick, B. Alex; Phadke, Dhiral P.; Auerbach, Scott S.; Mav, Deepak; Stiegelmeyer, Suzy M.; Shah, Ruchir R.; Tice, Raymond R.

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1’s carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT’s) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq’s capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma. PMID:23630614

RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

PubMed

Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq's capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma.

Elucidation of biocontrol mechanisms of Trichoderma harzianum against different plant fungal pathogens: Universal yet host specific response.

PubMed

Sharma, Vivek; Salwan, Richa; Sharma, Prem N; Kanwar, S S

2017-02-01

In the present study, different transcripts of Trichoderma harzianum ThHP-3 were evaluated for their response against four fungal pathogens Fusarium oxysporum, Colletotrichum capsici, Colletotrichum truncatum and Gloesercospora sorghi using RT-qPCR. The time course study of T. harzianum transcripts related to signal transduction, lytic enzymes, secondary metabolites and various transporters revealed variation in expression against four fungal pathogens. In a broader term, the transcripts were upregulated at various time intervals but the optimum expression of cyp3, abc, nrp, tga1, pmk, ech42 and glh20 varied with respect to host fungi. Additionally, the expression of transcripts related to transporters/cytochromes was also observed against Fusarium oxysporum after 96h whereas transcripts related to secondary metabolites and lytic enzymes showed significant difference in expression against Colletotrichum spp. from 72 to 96h. This is first study on transcriptomic response of T. harzianum against pathogenic fungi which shows their host specific response. Copyright © 2016 Elsevier B.V. All rights reserved.

Air-liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2.

PubMed

Klasvogt, Sonja; Zuschratter, Werner; Schmidt, Anke; Kröber, Andrea; Vorwerk, Sandra; Wolter, Romina; Isermann, Berend; Wimmers, Klaus; Rothkötter, Hermann-Josef; Nossol, Constanze

2017-01-01

The intestinal porcine epithelial cell line IPEC-J2, cultured under the air-liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo . Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium-cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1 α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1 α , demonstrating a major change in the transcriptional response.

A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

PubMed

Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

2017-10-30

Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods

DOE PAGES

He, Jintang; Schepmoes, Athena A.; Shi, Tujin; ...

2015-01-01

Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. Methods: Therefore, an antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERGmore » negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Results: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was around approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house enzyme-linked immunosorbent assay (ELISA) was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostics and prognostics assays for prostate cancer given their sensitivity, specificity, and reproducibility.« less

PARRoT- a homology-based strategy to quantify and compare RNA-sequencing from non-model organisms.

PubMed

Gan, Ruei-Chi; Chen, Ting-Wen; Wu, Timothy H; Huang, Po-Jung; Lee, Chi-Ching; Yeh, Yuan-Ming; Chiu, Cheng-Hsun; Huang, Hsien-Da; Tang, Petrus

2016-12-22

Next-generation sequencing promises the de novo genomic and transcriptomic analysis of samples of interests. However, there are only a few organisms having reference genomic sequences and even fewer having well-defined or curated annotations. For transcriptome studies focusing on organisms lacking proper reference genomes, the common strategy is de novo assembly followed by functional annotation. However, things become even more complicated when multiple transcriptomes are compared. Here, we propose a new analysis strategy and quantification methods for quantifying expression level which not only generate a virtual reference from sequencing data, but also provide comparisons between transcriptomes. First, all reads from the transcriptome datasets are pooled together for de novo assembly. The assembled contigs are searched against NCBI NR databases to find potential homolog sequences. Based on the searched result, a set of virtual transcripts are generated and served as a reference transcriptome. By using the same reference, normalized quantification values including RC (read counts), eRPKM (estimated RPKM) and eTPM (estimated TPM) can be obtained that are comparable across transcriptome datasets. In order to demonstrate the feasibility of our strategy, we implement it in the web service PARRoT. PARRoT stands for Pipeline for Analyzing RNA Reads of Transcriptomes. It analyzes gene expression profiles for two transcriptome sequencing datasets. For better understanding of the biological meaning from the comparison among transcriptomes, PARRoT further provides linkage between these virtual transcripts and their potential function through showing best hits in SwissProt, NR database, assigning GO terms. Our demo datasets showed that PARRoT can analyze two paired-end transcriptomic datasets of approximately 100 million reads within just three hours. In this study, we proposed and implemented a strategy to analyze transcriptomes from non-reference organisms which offers the opportunity to quantify and compare transcriptome profiles through a homolog based virtual transcriptome reference. By using the homolog based reference, our strategy effectively avoids the problems that may cause from inconsistencies among transcriptomes. This strategy will shed lights on the field of comparative genomics for non-model organism. We have implemented PARRoT as a web service which is freely available at http://parrot.cgu.edu.tw .

Air–liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2

PubMed Central

Klasvogt, Sonja; Zuschratter, Werner; Schmidt, Anke; Kröber, Andrea; Vorwerk, Sandra; Wolter, Romina; Isermann, Berend; Wimmers, Klaus; Rothkötter, Hermann-Josef; Nossol, Constanze

2017-01-01

The intestinal porcine epithelial cell line IPEC-J2, cultured under the air–liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium–cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response. PMID:28250970

Fatigue and gene expression in human leukocytes: increased NF-κB and decreased glucocorticoid signaling in breast cancer survivors with persistent fatigue.

PubMed

Bower, Julienne E; Ganz, Patricia A; Irwin, Michael R; Arevalo, Jesusa M G; Cole, Steve W

2011-01-01

Fatigue is highly prevalent in the general population and is one of the most common side effects of cancer treatment. There is growing evidence that pro-inflammatory cytokines play a role in cancer-related fatigue, although the molecular mechanisms for chronic inflammation and fatigue have not been determined. The current study utilized genome-wide expression microarrays to identify differences in gene expression and associated alterations in transcriptional activity in leukocytes from breast cancer survivors with persistent fatigue (n=11) and non-fatigued controls (n=10). We focused on transcription of inflammation-related genes, particularly those responsive to the pro-inflammatory NF-κB transcription control pathway. Further, given the role of glucocorticoids as key regulators of inflammatory processes, we examined transcription of glucocorticoid-responsive genes indicative of potential glucocorticoid receptor (GR) desensitization. Plasma levels of cortisol were also assessed. Consistent with hypotheses, results showed increased expression of transcripts with response elements for NF-κB, and reduced expression of transcripts with response elements for glucocorticoids (p<.05) in fatigued breast cancer survivors. No differences in plasma levels of cortisol were observed. These data indicate that increased activity of pro-inflammatory transcription factors may contribute to persistent cancer-related fatigue and provide insight into potential mechanisms for tonic increases in NF-κB activity, specifically decreased expression of GR anti-inflammatory transcription factors. Copyright © 2010 Elsevier Inc. All rights reserved.

smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.

PubMed

Tsanov, Nikolay; Samacoits, Aubin; Chouaib, Racha; Traboulsi, Abdel-Meneem; Gostan, Thierry; Weber, Christian; Zimmer, Christophe; Zibara, Kazem; Walter, Thomas; Peter, Marion; Bertrand, Edouard; Mueller, Florian

2016-12-15

Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

PubMed Central

Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

2006-01-01

Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

Estimating a structured covariance matrix from multi-lab measurements in high-throughput biology.

PubMed

Franks, Alexander M; Csárdi, Gábor; Drummond, D Allan; Airoldi, Edoardo M

2015-03-01

We consider the problem of quantifying the degree of coordination between transcription and translation, in yeast. Several studies have reported a surprising lack of coordination over the years, in organisms as different as yeast and human, using diverse technologies. However, a close look at this literature suggests that the lack of reported correlation may not reflect the biology of regulation. These reports do not control for between-study biases and structure in the measurement errors, ignore key aspects of how the data connect to the estimand, and systematically underestimate the correlation as a consequence. Here, we design a careful meta-analysis of 27 yeast data sets, supported by a multilevel model, full uncertainty quantification, a suite of sensitivity analyses and novel theory, to produce a more accurate estimate of the correlation between mRNA and protein levels-a proxy for coordination. From a statistical perspective, this problem motivates new theory on the impact of noise, model mis-specifications and non-ignorable missing data on estimates of the correlation between high dimensional responses. We find that the correlation between mRNA and protein levels is quite high under the studied conditions, in yeast, suggesting that post-transcriptional regulation plays a less prominent role than previously thought.

Estimating a structured covariance matrix from multi-lab measurements in high-throughput biology

PubMed Central

Franks, Alexander M.; Csárdi, Gábor; Drummond, D. Allan; Airoldi, Edoardo M.

2015-01-01

We consider the problem of quantifying the degree of coordination between transcription and translation, in yeast. Several studies have reported a surprising lack of coordination over the years, in organisms as different as yeast and human, using diverse technologies. However, a close look at this literature suggests that the lack of reported correlation may not reflect the biology of regulation. These reports do not control for between-study biases and structure in the measurement errors, ignore key aspects of how the data connect to the estimand, and systematically underestimate the correlation as a consequence. Here, we design a careful meta-analysis of 27 yeast data sets, supported by a multilevel model, full uncertainty quantification, a suite of sensitivity analyses and novel theory, to produce a more accurate estimate of the correlation between mRNA and protein levels—a proxy for coordination. From a statistical perspective, this problem motivates new theory on the impact of noise, model mis-specifications and non-ignorable missing data on estimates of the correlation between high dimensional responses. We find that the correlation between mRNA and protein levels is quite high under the studied conditions, in yeast, suggesting that post-transcriptional regulation plays a less prominent role than previously thought. PMID:25954056

Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.

PubMed

Cheng, Yangjian; Niu, Jianjun; Zhang, Yongyou; Huang, Jianwei; Li, Qingge

2006-10-01

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

Dynamic expression of 3′ UTRs revealed by Poisson hidden Markov modeling of RNA-Seq: Implications in gene expression profiling

PubMed Central

Lu, Jun; Bushel, Pierre R.

2013-01-01

RNA sequencing (RNA-Seq) allows for the identification of novel exon-exon junctions and quantification of gene expression levels. We show that from RNA-Seq data one may also detect utilization of alternative polyadenylation (APA) in 3′ untranslated regions (3′ UTRs) known to play a critical role in the regulation of mRNA stability, cellular localization and translation efficiency. Given the dynamic nature of APA, it is desirable to examine the APA on a sample by sample basis. We used a Poisson hidden Markov model (PHMM) of RNA-Seq data to identify potential APA in human liver and brain cortex tissues leading to shortened 3′ UTRs. Over three hundred transcripts with shortened 3′ UTRs were detected with sensitivity >75% and specificity >60%. tissue-specific 3′ UTR shortening was observed for 32 genes with a q-value ≤ 0.1. When compared to alternative isoforms detected by Cufflinks or MISO, our PHMM method agreed on over 100 transcripts with shortened 3′ UTRs. Given the increasing usage of RNA-Seq for gene expression profiling, using PHMM to investigate sample-specific 3′ UTR shortening could be an added benefit from this emerging technology. PMID:23845781

A cloud-based workflow to quantify transcript-expression levels in public cancer compendia

PubMed Central

Tatlow, PJ; Piccolo, Stephen R.

2016-01-01

Public compendia of sequencing data are now measured in petabytes. Accordingly, it is infeasible for researchers to transfer these data to local computers. Recently, the National Cancer Institute began exploring opportunities to work with molecular data in cloud-computing environments. With this approach, it becomes possible for scientists to take their tools to the data and thereby avoid large data transfers. It also becomes feasible to scale computing resources to the needs of a given analysis. We quantified transcript-expression levels for 12,307 RNA-Sequencing samples from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas. We used two cloud-based configurations and examined the performance and cost profiles of each configuration. Using preemptible virtual machines, we processed the samples for as little as $0.09 (USD) per sample. As the samples were processed, we collected performance metrics, which helped us track the duration of each processing step and quantified computational resources used at different stages of sample processing. Although the computational demands of reference alignment and expression quantification have decreased considerably, there remains a critical need for researchers to optimize preprocessing steps. We have stored the software, scripts, and processed data in a publicly accessible repository (https://osf.io/gqrz9). PMID:27982081

A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.

PubMed

Adamski, Mateusz G; Gumann, Patryk; Baird, Alison E

2014-01-01

Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR) have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR) and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells) and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA)) permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1) the achievement of absolute quantification and (2) a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

PubMed Central

Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

2017-01-01

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. DOI: http://dx.doi.org/10.7554/eLife.23326.001 PMID:28425915

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos.

PubMed

Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

2017-04-20

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

VITELLOGENIN GENE TRANSCRIPTION: A RELATIVE QUANTITATIVE EXPOSURE INDICATOR OF ENVIRONMENTAL ESTROGENS

EPA Science Inventory

We report the development of a quantifiable exposure indicator for measuring the presence of environmental estrogens in aquatic systems. Synthetic oligonucleotides, designed specifically for the vitellogenin gene (Vg) transcription product, were used in a Reverse Transcription Po...

Method to determine transcriptional regulation pathways in organisms

DOEpatents

Gardner, Timothy S.; Collins, James J.; Hayete, Boris; Faith, Jeremiah

2012-11-06

The invention relates to computer-implemented methods and systems for identifying regulatory relationships between expressed regulating polypeptides and targets of the regulatory activities of such regulating polypeptides. More specifically, the invention provides a new method for identifying regulatory dependencies between biochemical species in a cell. In particular embodiments, provided are computer-implemented methods for identifying a regulatory interaction between a transcription factor and a gene target of the transcription factor, or between a transcription factor and a set of gene targets of the transcription factor. Further provided are genome-scale methods for predicting regulatory interactions between a set of transcription factors and a corresponding set of transcriptional target substrates thereof.

c-Rel, an NF-[kappa]B Family Transcription Factor, Is Required for Hippocampal Long-Term Synaptic Plasticity and Memory Formation

ERIC Educational Resources Information Center

Ahn, Hyung Jin; Hernandez, Caterina M.; Levenson, Jonathan M.; Lubin, Farah D.; Liou, Hsiou-Chi; Sweatt, J. David

2008-01-01

Transcription is a critical component for consolidation of long-term memory. However, relatively few transcriptional mechanisms have been identified for the regulation of gene expression in memory formation. In the current study, we investigated the activity of one specific member of the NF-[kappa]B transcription factor family, c-Rel, during…

Staying alive in adversity: transcriptome dynamics in the stress-resistant dauer larva.

PubMed

Holt, Suzan J

2006-10-01

In response to food depletion and overcrowding, the soil nematode Caenorhabditis elegans can arrest development and form an alternate third larval stage called the dauer. Though nonfeeding, the dauer larva is long lived and stress resistant. Metabolic and transcription rates are lowered but the transcriptome of the dauer is complex. In this study, distribution analysis of transcript profiles generated by Serial Analysis of Gene Expression (SAGE) in dauer larvae and in mixed developmental stages is presented. An inverse relationship was observed between frequency and abundance/copy number of SAGE tag types (transcripts) in both profiles. In the dauer profile, a relatively greater proportion of highly abundant transcripts was counterbalanced by a smaller fraction of low to moderately abundant transcripts. Comparisons of abundant tag counts between the two profiles revealed relative enrichment in the dauer profile of transcripts with predicted or known involvement in ribosome biogenesis and protein synthesis, membrane transport, and immune responses. Translation-coupled mRNA decay is proposed as part of an immune-like stress response in the dauer larva. An influence of genomic region on transcript level may reflect the coordination of transcription and mRNA turnover.

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li

2013-02-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently ofmore » its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.« less

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

PubMed

Ribeiro, José M C; Genta, Fernando A; Sorgine, Marcos H F; Logullo, Raquel; Mesquita, Rafael D; Paiva-Silva, Gabriela O; Majerowicz, David; Medeiros, Marcelo; Koerich, Leonardo; Terra, Walter R; Ferreira, Clélia; Pimentel, André C; Bisch, Paulo M; Leite, Daniel C; Diniz, Michelle M P; da S G V Junior, João Lídio; Da Silva, Manuela L; Araujo, Ricardo N; Gandara, Ana Caroline P; Brosson, Sébastien; Salmon, Didier; Bousbata, Sabrina; González-Caballero, Natalia; Silber, Ariel Mariano; Alves-Bezerra, Michele; Gondim, Katia C; Silva-Neto, Mário Alberto C; Atella, Georgia C; Araujo, Helena; Dias, Felipe A; Polycarpo, Carla; Vionette-Amaral, Raquel J; Fampa, Patrícia; Melo, Ana Claudia A; Tanaka, Aparecida S; Balczun, Carsten; Oliveira, José Henrique M; Gonçalves, Renata L S; Lazoski, Cristiano; Rivera-Pomar, Rolando; Diambra, Luis; Schaub, Günter A; Garcia, Elói S; Azambuja, Patrícia; Braz, Glória R C; Oliveira, Pedro L

2014-01-01

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

An extensive requirement for transcription factor IID-specific TAF-1 in Caenorhabditis elegans embryonic transcription.

PubMed

Walker, Amy K; Shi, Yang; Blackwell, T Keith

2004-04-09

The general transcription factor TFIID sets the mRNA start site and consists of TATA-binding protein and associated factors (TAF(II)s), some of which are also present in SPT-ADA-GCN5 (SAGA)-related complexes. In yeast, results of multiple studies indicate that TFIID-specific TAF(II)s are not required for the transcription of most genes, implying that intact TFIID may have a surprisingly specialized role in transcription. Relatively little is known about how TAF(II)s contribute to metazoan transcription in vivo, especially at developmental and tissue-specific genes. Previously, we investigated functions of four shared TFIID/SAGA TAF(II)s in Caenorhabditis elegans. Whereas TAF-4 was required for essentially all embryonic transcription, TAF-5, TAF-9, and TAF-10 were dispensable at multiple developmental and other metazoan-specific promoters. Here we show evidence that in C. elegans embryos transcription of most genes requires TFIID-specific TAF-1. TAF-1 is not as universally required as TAF-4, but it is essential for a greater proportion of transcription than TAF-5, -9, or -10 and is important for transcription of many developmental and other metazoan-specific genes. TAF-2, which binds core promoters with TAF-1, appears to be required for a similarly substantial proportion of transcription. C. elegans TAF-1 overlaps functionally with the coactivator p300/CBP (CBP-1), and at some genes it is required along with the TBP-like protein TLF(TRF2). We conclude that during C. elegans embryogenesis TAF-1 and TFIID have broad roles in transcription and development and that TFIID and TLF may act together at certain promoters. Our findings imply that in metazoans TFIID may be of widespread importance for transcription and for expression of tissue-specific genes.

Cross-species approaches to seed dormancy and germination: conservation and biodiversity of ABA-regulated mechanisms and the Brassicaceae DOG1 genes.

PubMed

Graeber, Kai; Linkies, Ada; Müller, Kerstin; Wunchova, Andrea; Rott, Anita; Leubner-Metzger, Gerhard

2010-05-01

Seed dormancy is genetically determined with substantial environmental influence mediated, at least in part, by the plant hormone abscisic acid (ABA). The ABA-related transcription factor ABI3/VP1 (ABA INSENSITIVE3/VIVIPAROUS1) is widespread among green plants. Alternative splicing of its transcripts appears to be involved in regulating seed dormancy, but the role of ABI3/VP1 goes beyond seeds and dormancy. In contrast, DOG1 (DELAY OF GERMINATION 1), a major quantitative trait gene more specifically involved in seed dormancy, was so far only known from Arabidopsis thaliana (AtDOG1) and whether it also has roles during the germination of non-dormant seeds was not known. Seed germination of Lepidium sativum ('garden cress') is controlled by ABA and its antagonists gibberellins and ethylene and involves the production of apoplastic hydroxyl radicals. We found orthologs of AtDOG1 in the Brassicaceae relatives L. sativum (LesaDOG1) and Brassica rapa (BrDOG1) and compared their gene structure and the sequences of their transcripts expressed in seeds. Tissue-specific analysis of LesaDOG1 transcript levels in L. sativum seeds showed that they are degraded upon imbibition in the radicle and the micropylar endosperm. ABA inhibits germination in that it delays radicle protrusion and endosperm weakening and it increased LesaDOG1 transcript levels during early germination due to enhanced transcription and/or inhibited degradation. A reduced decrease in LesaDOG1 transcript levels upon ABA treatment is evident in the late germination phase in both tissues. This temporal and ABA-related transcript expression pattern suggests a role for LesaDOG1 in the control of germination timing of non-dormant L. sativum seeds. The possible involvement of the ABA-related transcription factors ABI3 and ABI5 in the regulation of DOG1 transcript expression is discussed. Other species of the monophyletic genus Lepidium showed coat or embryo dormancy and are therefore highly suited for comparative seed biology.

Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis

PubMed Central

Santangeloyz, K.S.; Bertoneyz, A.L.

2011-01-01

summary Objective To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1β (IL-1β) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. Methods Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1β transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RTPCR) was used to quantify in vitro transcript changes of IL-1β and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2−ΔΔCT) method. Results Statistically significant decreases in IL-1β expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-β (TGF-β) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P= 0.0045) or >90% (P= 0.0001) of the IL-1β transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. Conclusions Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1β transcript in vivo are warranted. PMID:21945742

24 CFR 1720.510 - Reporting and transcription.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 5 2011-04-01 2011-04-01 false Reporting and transcription. 1720.510 Section 1720.510 Housing and Urban Development Regulations Relating to Housing and Urban... PRACTICE Adjudicatory Proceedings Hearings § 1720.510 Reporting and transcription. Hearings shall be...

24 CFR 1720.510 - Reporting and transcription.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Reporting and transcription. 1720.510 Section 1720.510 Housing and Urban Development Regulations Relating to Housing and Urban... PRACTICE Adjudicatory Proceedings Hearings § 1720.510 Reporting and transcription. Hearings shall be...

Automated Quantification of Pneumothorax in CT

PubMed Central

Do, Synho; Salvaggio, Kristen; Gupta, Supriya; Kalra, Mannudeep; Ali, Nabeel U.; Pien, Homer

2012-01-01

An automated, computer-aided diagnosis (CAD) algorithm for the quantification of pneumothoraces from Multidetector Computed Tomography (MDCT) images has been developed. Algorithm performance was evaluated through comparison to manual segmentation by expert radiologists. A combination of two-dimensional and three-dimensional processing techniques was incorporated to reduce required processing time by two-thirds (as compared to similar techniques). Volumetric measurements on relative pneumothorax size were obtained and the overall performance of the automated method shows an average error of just below 1%. PMID:23082091

Assessing senescence in Drosophila using video tracking.

PubMed

Ardekani, Reza; Tavaré, Simon; Tower, John

2013-01-01

Senescence is associated with changes in gene expression, including the upregulation of stress response- and innate immune response-related genes. In addition, aging animals exhibit characteristic changes in movement behaviors including decreased gait speed and a deterioration in sleep/wake rhythms. Here, we describe methods for tracking Drosophila melanogaster movements in 3D with simultaneous quantification of fluorescent transgenic reporters. This approach allows for the assessment of correlations between behavior, aging, and gene expression as well as for the quantification of biomarkers of aging.

Comparison of hemagglutination inhibition test and ELISA in quantification of antibodies to egg drop syndrome virus.

PubMed

Raj, G Dhinakar; Ratnapraba, S; Matheswaran, K; Nachimuthu, K

2004-01-01

A single-serum dilution ELISA for egg drop syndrome (EDS) virus-specific antibodies was developed. In testing 425 chicken sera it was found to have a 93.6% sensitivity and 98.7% specificity relative to a hemagglutination inhibition (HI) test. The correlation coefficient for ELISA and HI titers was 0.793. The ELISA was efficacious in quantification of both vaccinal and infection antibodies and could routinely be used for screening large numbers of field sera.

Consequences of changes to the NRCS rainfall-runoff relations on hydrologic design

USDA-ARS?s Scientific Manuscript database

A proposed quantification of the fundamental concepts in the Natural Resources Conservation Service (NRCS) rainfall-runoff relation is examined to determine changes relevant to peak discharge estimation and drainage design. Changes to the NRCS curve number, storage, and initial abstraction relations...

A hybrid segmentation approach for geographic atrophy in fundus auto-fluorescence images for diagnosis of age-related macular degeneration.

PubMed

Lee, Noah; Laine, Andrew F; Smith, R Theodore

2007-01-01

Fundus auto-fluorescence (FAF) images with hypo-fluorescence indicate geographic atrophy (GA) of the retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Manual quantification of GA is time consuming and prone to inter- and intra-observer variability. Automatic quantification is important for determining disease progression and facilitating clinical diagnosis of AMD. In this paper we describe a hybrid segmentation method for GA quantification by identifying hypo-fluorescent GA regions from other interfering retinal vessel structures. First, we employ background illumination correction exploiting a non-linear adaptive smoothing operator. Then, we use the level set framework to perform segmentation of hypo-fluorescent areas. Finally, we present an energy function combining morphological scale-space analysis with a geometric model-based approach to perform segmentation refinement of false positive hypo- fluorescent areas due to interfering retinal structures. The clinically apparent areas of hypo-fluorescence were drawn by an expert grader and compared on a pixel by pixel basis to our segmentation results. The mean sensitivity and specificity of the ROC analysis were 0.89 and 0.98%.

Genomic and transcriptomic characterization of the transcription factor family R2R3-MYB in soybean and its involvement in the resistance responses to Phakopsora pachyrhizi.

PubMed

Aoyagi, Luciano N; Lopes-Caitar, Valéria S; de Carvalho, Mayra C C G; Darben, Luana M; Polizel-Podanosqui, Adriana; Kuwahara, Marcia K; Nepomuceno, Alexandre L; Abdelnoor, Ricardo V; Marcelino-Guimarães, Francismar C

2014-12-01

Myb genes constitute one of the largest transcription factor families in the plant kingdom. Soybean MYB transcription factors have been related to the plant response to biotic stresses. Their involvement in response to Phakopsora pachyrhizi infection has been reported by several transcriptional studies. Due to their apparently highly diverse functions, these genes are promising targets for developing crop varieties resistant to diseases. In the present study, the identification and phylogenetic analysis of the soybean R2R3-MYB (GmMYB) transcription factor family was performed and the expression profiles of these genes under biotic stress were determined. GmMYBs were identified from the soybean genome using bioinformatic tools, and their putative functions were determined based on the phylogenetic tree and classified into subfamilies using guides AtMYBs describing known functions. The transcriptional profiles of GmMYBs upon infection with different pathogen were revealed by in vivo and in silico analyses. Selected target genes potentially involved in disease responses were assessed by RT-qPCR after different times of inoculation with P. pachyrhizi using different genetic backgrounds related to resistance genes (Rpp2 and Rpp5). R2R3-MYB transcription factors related to lignin synthesis and genes responsive to chitin were significantly induced in the resistant genotypes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Identification and molecular cloning of novel transcripts of the human kallikrein-related peptidase 10 (KLK10) gene using next-generation sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adamopoulos, Panagiotis G.; Kontos, Christos K.; Scorilas, Andreas

Tissue kallikrein and kallikrein-related peptidases (KLKs) form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. Multiple alternative transcripts have been reported for the most human KLK genes, while many of them are aberrantly expressed in various malignancies, thus possessing significant prognostic and/or diagnostic value. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity, as it affects cell cycle control, proliferation, apoptosis, invasion, and metastasis. In this study, we describe the identification and molecular cloning of eight novel transcripts of the human KLK10 gene using 3′more » rapid amplification of cDNA ends (3′ RACE) and next-generation sequencing (NGS), as well as their expression analysis in a wide panel of cell lines, originating from several distinct cancerous and normal tissues. Bioinformatic analysis revealed that the novel KLK10 transcripts contain new alternative splicing events between already annotated exons as well as novel exons. In addition, investigation of their expression profile in a wide panel of cell lines was performed with nested RT-PCR using variant-specific pairs of primers. Since many KLK mRNA transcripts possess clinical value, these newly discovered alternatively spliced KLK10 transcripts appear as new potential biomarkers for diagnostic and/or prognostic purposes or as targets for therapeutic strategies. - Highlights: • NGS was used to identify novel transcripts of the human KLK10 gene. • 8 novel KLK10 transcripts were identified. • A novel 3′UTR was detected and characterized. • The expression profiles of all 8 novel KLK10 transcripts were identified.« less

In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Boo-Ja; Park, Chang-Jin; Kim, Sung-Kyu

2006-05-26

We find that salicylic acid and ethephon treatment in hot pepper increases the expression of a putative basic/leucine zipper (bZIP) transcription factor gene, CabZIP1. CabZIP1 mRNA is expressed ubiquitously in various organs. The green fluorescent protein-fused transcription factor, CabZIP1::GFP, can be specifically localized to the nucleus, an action that is consistent with the presence of a nuclear localization signal in its protein sequence. Transient overexpression of the CabZIP1 transcription factor results in an increase in PR-1 transcripts level in Nicotiana benthamiana leaves. Using chromatin immunoprecipitation, we demonstrate that CabZIP1 binds to the G-box elements in native promoter of the hotmore » pepper pathogenesis-related protein 1 (CaPR-1) gene in vivo. Taken together, our results suggest that CabZIP1 plays a role as a transcriptional regulator of the CaPR-1 gene.« less

Physiological plasticity related to zonation affects hsp70 expression in the reef-building coral Pocillopora verrucosa

PubMed Central

Poli, Davide; Fabbri, Elena; Goffredo, Stefano; Airi, Valentina

2017-01-01

This study investigates for the first time the transcriptional regulation of a stress-inducible 70-kDa heat shock protein (hsp70) in the scleractinian coral Pocillopora verrucosa sampled at three locations and two depths (3 m and 12 m) in Bangka Island waters (North Sulawesi, Indonesia). Percentage of coral cover indicated reduced habitat suitability with depth and at the Tanjung Husi (TA) site, which also displayed relatively higher seawater temperatures. Expression of the P. verrucosa hsp70 transcript evaluated under field conditions followed a depth-related profile, with relatively higher expression levels in 3-m collected nubbins compared to the 12-m ones. Expression levels of metabolism-related transcripts ATP synthase and NADH dehydrogenase indicated metabolic activation of nubbins to cope with habitat conditions of the TA site at 3 m. After a 14-day acclimatization to common and fixed temperature conditions in the laboratory, corals were subjected for 7 days to an altered thermal regime, where temperature was elevated at 31°C during the light phase and returned to 28°C during the dark phase. Nubbins collected at 12 m were relatively more sensitive to thermal stress, as they significantly over-expressed the selected transcripts. Corals collected at 3 m appeared more resilient, as they showed unaffected mRNA expressions. The results indicated that local habitat conditions may influence transcription of stress-related genes in P. verrucosa. Corals exhibiting higher basal hsp70 levels may display enhanced tolerance towards environmental stressors. PMID:28199351

Measuring mumbo jumbo: A preliminary quantification of the use of jargon in science communication.

PubMed

Sharon, Aviv J; Baram-Tsabari, Ayelet

2014-07-01

Leaders of the scientific community encourage scientists to learn effective science communication, including honing the skill to discuss science with little professional jargon. However, avoiding jargon is not trivial for scientists for several reasons, and this demands special attention in teaching and evaluation. Despite this, no standard measurement for the use of scientific jargon in speech has been developed to date. Here a standard yardstick for the use of scientific jargon in spoken texts, using a computational linguistics approach, is proposed. Analyzed transcripts included academic speech, scientific TEDTalks, and communication about the discovery of a Higgs-like boson at CERN. Findings suggest that scientists use less jargon in communication with a general audience than in communication with peers, but not always less obscure jargon. These findings may lay the groundwork for evaluating the use of jargon.

iSeq: Web-Based RNA-seq Data Analysis and Visualization.

PubMed

Zhang, Chao; Fan, Caoqi; Gan, Jingbo; Zhu, Ping; Kong, Lei; Li, Cheng

2018-01-01

Transcriptome sequencing (RNA-seq) is becoming a standard experimental methodology for genome-wide characterization and quantification of transcripts at single base-pair resolution. However, downstream analysis of massive amount of sequencing data can be prohibitively technical for wet-lab researchers. A functionally integrated and user-friendly platform is required to meet this demand. Here, we present iSeq, an R-based Web server, for RNA-seq data analysis and visualization. iSeq is a streamlined Web-based R application under the Shiny framework, featuring a simple user interface and multiple data analysis modules. Users without programming and statistical skills can analyze their RNA-seq data and construct publication-level graphs through a standardized yet customizable analytical pipeline. iSeq is accessible via Web browsers on any operating system at http://iseq.cbi.pku.edu.cn .

Real time non invasive imaging of fatty acid uptake in vivo

PubMed Central

Henkin, Amy H.; Cohen, Allison S.; Dubikovskaya, Elena A.; Park, Hyo Min; Nikitin, Gennady F.; Auzias, Mathieu G.; Kazantzis, Melissa; Bertozzi, Carolyn R.; Stahl, Andreas

2012-01-01

Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin-sensitivity. To enable non-invasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real-time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic metabolic processes and pathological alterations in multiple disease models. PMID:22928772

Expression of growth hormone gene during early development of Siberian sturgeon (Acipenser baerii)

PubMed Central

Abdolahnejad, Zeinab; Pourkazemi, Mohammad; Khoshkholgh, Majid Reza; Yarmohammadi, Mahtab

2015-01-01

The mRNA expression of growth hormone (GH) gene in early development stages of Siberian sturgeon was investigated using RT-PCR method. Samples were collected from unfertilized eggs up to 50 days post hatched (dph) larvae in 11 different times. Ribosomal protein L6 (RPL6) transcripts were used as the internal standard during quantification of GH mRNA expression. The results showed that the GH mRNA could be observed in the eyed eggs and even at unfertilized eggs of Siberian sturgeon. The highest amounts of GH mRNA were found at 25 and 50 dph larvae, while the lowest levels were detected at 1 and 3 dph larvae stage. These findings suggest that, the GH mRNA play a key role during developmental stages of Siberian sturgeon. PMID:27844010

Whole-genome transcriptional analysis of Escherichia coli during heat inactivation processes related to industrial cooking.

PubMed

Guernec, A; Robichaud-Rincon, P; Saucier, L

2013-08-01

Escherichia coli K-12 was grown to the stationary phase, for maximum physiological resistance, in brain heart infusion (BHI) broth at 37°C. Cells were then heated at 58°C or 60°C to reach a process lethality value \\[\\mathbf{\\left(}{{\\mathit{F}}^{\\mathit{o}}}_{\\mathbf{70}}^{\\mathbf{10}}\\mathbf{\\right)} \\] of 2 or 3 or to a core temperature of 71°C (control industrial cooking temperature). Growth recovery and cell membrane integrity were evaluated immediately after heating, and a global transcription analysis was performed using gene expression microarrays. Only cells heated at 58°C with F(o) = 2 were still able to grow on liquid or solid BHI broth after heat treatment. However, their transcriptome did not differ from that of bacteria heated at 58°C with F(o) = 3 (P value for the false discovery rate [P-FDR] > 0.01), where no growth recovery was observed posttreatment. Genome-wide transcriptomic data obtained at 71°C were distinct from those of the other treatments without growth recovery. Quantification of heat shock gene expression by real-time PCR revealed that dnaK and groEL mRNA levels decreased significantly above 60°C to reach levels similar to those of control cells at 37°C (P < 0.0001). Furthermore, despite similar levels of cell inactivation measured by growth on BHI media after heating, 132 and 8 genes were differentially expressed at 71°C compared to 58°C and 60°C at F(o) = 3, respectively (P-FDR < 0.01). Among them, genes such as aroA, citE, glyS, oppB, and asd, whose expression was upregulated at 71°C, may be worth investigating as good biomarkers for accurately determining the efficiency of heat treatments, especially when cells are too injured to be enumerated using growth media.

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

PubMed Central

Okello, John B. A.; Rodriguez, Linda; Poinar, Debi; Bos, Kirsten; Okwi, Andrew L.; Bimenya, Gabriel S.; Sewankambo, Nelson K.; Henry, Kenneth R.; Kuch, Melanie; Poinar, Hendrik N.

2010-01-01

Background The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues. Methodology/Principal Findings We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays. Conclusions/Significance We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation. PMID:21085668

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

PubMed

Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

Solid-phase microextraction method development for headspace analysis of volatile flavor compounds.

PubMed

Roberts, D D; Pollien, P; Milo, C

2000-06-01

Solid-phase microextraction (SPME) fibers were evaluated for their ability to adsorb volatile flavor compounds under various conditions with coffee and aqueous flavored solutions. Experiments comparing different fibers showed that poly(dimethylsiloxane)/divinylbenzene had the highest overall sensitivity. Carboxen/poly(dimethylsiloxane) was the most sensitive to small molecules and acids. As the concentrations of compounds increased, the quantitative linear range was exceeded as shown by competition effects with 2-isobutyl-3-methoxypyrazine at concentrations above 1 ppm. A method based on a short-time sampling of the headspace (1 min) was shown to better represent the equilibrium headspace concentration. Analysis of coffee brew with a 1-min headspace adsorption time was verified to be within the linear range for most compounds and thus appropriate for relative headspace quantification. Absolute quantification of volatiles, using isotope dilution assays (IDA), is not subject to biases caused by excess compound concentrations or complex matrices. The degradation of coffee aroma volatiles during storage was followed by relative headspace measurements and absolute quantifications. Both methods gave similar values for 3-methylbutanal, 4-ethylguaiacol, and 2,3-pentanedione. Acetic acid, however, gave higher values during storage upon relative headspace measurements due to concurrent pH decreases that were not seen with IDA.

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

PubMed Central

Schouten, Jan P.; McElgunn, Cathal J.; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-01-01

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down’s syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50–70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences. PMID:12060695

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.

PubMed

Schouten, Jan P; McElgunn, Cathal J; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-06-15

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.

Trace quantification of selected sulfonamides in aqueous media by implementation of a new dispersive solid-phase extraction method using a nanomagnetic titanium dioxide graphene-based sorbent and HPLC-UV.

PubMed

Izanloo, Maryam; Esrafili, Ali; Behbahani, Mohammad; Ghambarian, Mahnaz; Reza Sobhi, Hamid

2018-02-01

Herein, a new dispersive solid-phase extraction method using a nano magnetic titanium dioxide graphene-based sorbent in conjunction with high-performance liquid chromatography and ultraviolet detection was successfully developed. The method was proved to be simple, sensitive, and highly efficient for the trace quantification of sulfacetamide, sulfathiazole, sulfamethoxazole, and sulfadiazine in relatively large volume of aqueous media. Initially, the nano magnetic titanium dioxide graphene-based sorbent was successfully synthesized and subsequently characterized by scanning electron microscopy and X-ray diffraction. Then, the sorbent was used for the sorption and extraction of the selected sulfonamides mainly through π-π stacking hydrophobic interactions. Under the established conditions, the calibration curves were linear over the concentration range of 1-200 μg/L. The limit of quantification (precision of 20%, and accuracy of 80-120%) for the detection of each sulfonamide by the proposed method was 1.0 μg/L. To test the extraction efficiency, the method was applied to various fortified real water samples. The average relative recoveries obtained from the fortified samples varied between 90 and 108% with the relative standard deviations of 5.3-10.7%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification of allantoin in various Zea mays L. hybrids by RP-HPLC with UV detection.

PubMed

Maksimović, Z; Malenović, A; Jancić, B; Kovacević, N

2004-07-01

A RP-HPLC method for quantification of allantoin in silk of fifteen maize hybrids (Zea mays L., Poaceae) was described. Following extraction of the plant material with an acetone-water (7:3, VN) mixture, filtration and dilution, the extracts were analyzed without previous chemical derivatization. Separation and quantification were achieved using an Alltech Econosil C18 column under isocratic conditions at 40 degrees C. The mobile phase flow (20% methanol--80% water with 5 mM sodium laurylsulfate added at pH 2.5, adjusted with 85% orthophosphoric acid; pH of water phase was finally adjusted at 6.0 by addition of triethylamine) was maintained at 1.0 mL/min. Column effluent was monitored at 235 nm. This simple procedure afforded efficient separation and quantification of allantoin in plant material, without interference of polyphenols or other plant constituents of medium to high polarity, or similar UV absorption. Our study revealed that the silk of all investigated maize hybrids could be considered relatively rich in allantoin, covering the concentration range between 215 and 289 mg per 100 g of dry plant material.

Standardless quantification by parameter optimization in electron probe microanalysis

NASA Astrophysics Data System (ADS)

Limandri, Silvina P.; Bonetto, Rita D.; Josa, Víctor Galván; Carreras, Alejo C.; Trincavelli, Jorge C.

2012-11-01

A method for standardless quantification by parameter optimization in electron probe microanalysis is presented. The method consists in minimizing the quadratic differences between an experimental spectrum and an analytical function proposed to describe it, by optimizing the parameters involved in the analytical prediction. This algorithm, implemented in the software POEMA (Parameter Optimization in Electron Probe Microanalysis), allows the determination of the elemental concentrations, along with their uncertainties. The method was tested in a set of 159 elemental constituents corresponding to 36 spectra of standards (mostly minerals) that include trace elements. The results were compared with those obtained with the commercial software GENESIS Spectrum® for standardless quantification. The quantifications performed with the method proposed here are better in the 74% of the cases studied. In addition, the performance of the method proposed is compared with the first principles standardless analysis procedure DTSA for a different data set, which excludes trace elements. The relative deviations with respect to the nominal concentrations are lower than 0.04, 0.08 and 0.35 for the 66% of the cases for POEMA, GENESIS and DTSA, respectively.

Consistency of flow quantifications in tridirectional phase-contrast MRI

NASA Astrophysics Data System (ADS)

Unterhinninghofen, R.; Ley, S.; Dillmann, R.

2009-02-01

Tridirectionally encoded phase-contrast MRI is a technique to non-invasively acquire time-resolved velocity vector fields of blood flow. These may not only be used to analyze pathological flow patterns, but also to quantify flow at arbitrary positions within the acquired volume. In this paper we examine the validity of this approach by analyzing the consistency of related quantifications instead of comparing it with an external reference measurement. Datasets of the thoracic aorta were acquired from 6 pigs, 1 healthy volunteer and 3 patients with artificial aortic valves. Using in-house software an elliptical flow quantification plane was placed manually at 6 positions along the descending aorta where it was rotated to 5 different angles. For each configuration flow was computed based on the original data and data that had been corrected for phase offsets. Results reveal that quantifications are more dependent on changes in position than on changes in angle. Phase offset correction considerably reduces this dependency. Overall consistency is good with a maximum variation coefficient of 9.9% and a mean variation coefficient of 7.2%.

Elemental labelling combined with liquid chromatography inductively coupled plasma mass spectrometry for quantification of biomolecules: A review

PubMed Central

Kretschy, Daniela; Koellensperger, Gunda; Hann, Stephan

2012-01-01

This article reviews novel quantification concepts where elemental labelling is combined with flow injection inductively coupled plasma mass spectrometry (FI-ICP-MS) or liquid chromatography inductively coupled plasma mass spectrometry (LC–ICP-MS), and employed for quantification of biomolecules such as proteins, peptides and related molecules in challenging sample matrices. In the first sections an overview on general aspects of biomolecule quantification, as well as of labelling will be presented emphasizing the potential, which lies in such methodological approaches. In this context, ICP-MS as detector provides high sensitivity, selectivity and robustness in biological samples and offers the capability for multiplexing and isotope dilution mass spectrometry (IDMS). Fundamental methodology of elemental labelling will be highlighted and analytical, as well as biomedical applications will be presented. A special focus will lie on established applications underlining benefits and bottlenecks of such approaches for the implementation in real life analysis. Key research made in this field will be summarized and a perspective for future developments including sophisticated and innovative applications will given. PMID:23062431

Simple and rapid quantification of brominated vegetable oil in commercial soft drinks by LC–MS

PubMed Central

Chitranshi, Priyanka; da Costa, Gonçalo Gamboa

2016-01-01

We report here a simple and rapid method for the quantification of brominated vegetable oil (BVO) in soft drinks based upon liquid chromatography–electrospray ionization mass spectrometry. Unlike previously reported methods, this novel method does not require hydrolysis, extraction or derivatization steps, but rather a simple “dilute and shoot” sample preparation. The quantification is conducted by mass spectrometry in selected ion recording mode and a single point standard addition procedure. The method was validated in the range of 5–25 μg/mL BVO, encompassing the legal limit of 15 μg/mL established by the US FDA for fruit-flavored beverages in the US market. The method was characterized by excellent intra- and inter-assay accuracy (97.3–103.4%) and very low imprecision [0.5–3.6% (RSD)]. The direct nature of the quantification, simplicity, and excellent statistical performance of this methodology constitute clear advantages in relation to previously published methods for the analysis of BVO in soft drinks. PMID:27451219

Involvement of Human Estrogen Related Receptor Alpha 1 (hERR 1) in Breast Cancer and Hormonally Insensitive Disease

DTIC Science & Technology

2000-08-01

SV40 early-to-late switch involves titration of cellular transcriptional repressors, Genes Dev. 7: 2206-19, 1993. 6. Bonnelye, E., Vanacker , J. M ...transcriptional regulator of the human medium-chain acyl coenzyme A dehydrogenase gene, Mol Cell Biol. 17: 5400-9, 1997. 8. Vanacker , J. M ., Bonnelye, E...related receptor-alpha), Mol Endocrinol. 13: 764-73, 1999. 9. Vanacker , J. M ., Pettersson, K., Gustafsson, J. A., and Laudet, V. Transcriptional

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

Driving under the influence of cannabis: pitfalls, validation, and quality control of a UPLC-MS/MS method for the quantification of tetrahydrocannabinol in oral fluid collected with StatSure, Quantisal, or Certus collector.

PubMed

Wille, Sarah M R; Di Fazio, Vincent; Ramírez-Fernandez, Maria del Mar; Kummer, Natalie; Samyn, Nele

2013-02-01

"Driving under the influence of drugs" (DUID) has a large impact on the worldwide mortality risk. Therefore, DUID legislations based on impairment or analytical limits are adopted. Drug detection in oral fluid is of interest due to the ease of sampling during roadside controls. The prevalence of Δ9-tetrahydrocannabinol (THC) in seriously injured drivers ranges from 0.5% to 7.6% in Europe. For these reasons, the quantification of THC in oral fluid collected with 3 alternative on-site collectors is presented and discussed in this publication. An ultra-performance liquid chromatography-mass spectrometric quantification method for THC in oral fluid samples collected with the StatSure (Diagnostic Systems), Quantisal (Immunalysis), and Certus (Concateno) devices was validated according to the international guidelines. Small sample volumes of 100-200 μL were extracted using hexane. Special attention was paid to factors such as matrix effects, THC adsorption onto the collector, and stability in the collection fluid. A relatively high-throughput analysis was developed and validated according to ISO 17025 requirements. Although the effects of the matrix on the quantification could be minimized using a deuterated internal standard, and stability was acceptable according the validation data, adsorption of THC onto the collectors was a problem. For the StatSure device, THC was totally recovered from the collector pad after storage for 24 hours at room temperature or 7 days at 4°C. A loss of 15%-25% was observed for the Quantisal collector, whereas the recovery from the Certus device was irreproducible (relative standard deviation, 44%-85%) and low (29%-80%). During the roadside setting, a practical problem arose: small volumes of oral fluid (eg, 300 μL) were collected. However, THC was easily detected and concentrations ranged from 8 to 922 ng/mL in neat oral fluid. A relatively high-throughput analysis (40 samples in 4 hours) adapted for routine DUID analysis was developed and validated for THC quantification in oral fluid samples collected from drivers under the influence of cannabis.

Recent advances in hopanoids analysis: Quantification protocols overview, main research targets and selected problems of complex data exploration.

PubMed

Zarzycki, Paweł K; Portka, Joanna K

2015-09-01

Pentacyclic triterpenoids, particularly hopanoids, are organism-specific compounds and are generally considered as useful biomarkers that allow fingerprinting and classification of biological, environmental and geological samples. Simultaneous quantification of various hopanoids together with battery of related non-polar and low-molecular mass compounds may provide principal information for geochemical and environmental research focusing on both modern and ancient investigations. Target compounds can be derived from microbial biomass, water columns, sediments, coals, crude fossils or rocks. This create number of analytical problems due to different composition of the analytical matrix and interfering compounds and therefore, proper optimization of quantification protocols for such biomarkers is still the challenge. In this work we summarizing typical analytical protocols that were recently applied for quantification of hopanoids like compounds from different samples. Main steps including components of interest extraction, pre-purification, fractionation, derivatization and quantification involving gas (1D and 2D) as well as liquid separation techniques (liquid-liquid extraction, solid-phase extraction, planar and low resolution column chromatography, high-performance liquid chromatography) are described and discussed from practical point of view, mainly based on the experimental papers that were published within last two years, where significant increase in hopanoids research was noticed. The second aim of this review is to describe the latest research trends concerning determination of hopanoids and related low-molecular mass lipids analyzed in various samples including sediments, rocks, coals, crude oils and plant fossils as well as stromatolites and microbial biomass cultivated under different conditions. It has been found that majority of the most recent papers are based on uni- or bivariate approach for complex data analysis. Data interpretation involves number of physicochemical parameters and hopanoids quantities or given biomarkers mass ratios derived from high-throughput separation and detection systems, typically GC-MS and HPLC-MS. Based on quantitative data reported in recently published experimental works it has been demonstrated that multivariate data analysis using e.g. principal components computations may significantly extend our knowledge concerning proper biomarkers selection and samples classification by means of hopanoids and related non-polar compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

PubMed

Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

2017-02-14

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

29 CFR 417.21 - Transcript.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 2 2010-07-01 2010-07-01 false Transcript. 417.21 Section 417.21 Labor Regulations Relating to Labor OFFICE OF LABOR-MANAGEMENT STANDARDS, DEPARTMENT OF LABOR LABOR-MANAGEMENT STANDARDS... hearings. In the event he does so require, copies of the official transcript shall be made available upon...

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

PubMed

Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

2016-01-01

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure.

PubMed

Rodríguez-Fuentes, Gabriela; Rubio-Escalante, Fernando J; Noreña-Barroso, Elsa; Escalante-Herrera, Karla S; Schlenk, Daniel

2015-01-01

Organophosphate pesticides cause irreversible inhibition of AChE which leads to neuronal overstimulation and death. Thus, dogma indicates that the target of OP pesticides is AChE, but many authors postulate that these compounds also disturb cellular redox processes, and change the activities of antioxidant enzymes. Interestingly, it has also been reported that oxidative stress plays also a role in the regulation and activity of AChE. The aims of this study were to determine the effects of the antioxidant, vitamin C (VC), the oxidant, t-butyl hydroperoxide (tBOOH) and the organophosphate Chlorpyrifos (CPF), on AChE gene transcription and activity in zebrafish embryos after 72h exposure. In addition, oxidative stress was evaluated by measuring antioxidant enzymes activities and transcription, and quantification of total glutathione. Apical effects on the development of zebrafish embryos were also measured. With the exception of AChE inhibition and enhanced gene expression, limited effects of CPF on oxidative stress and apical endpoints were found at this developmental stage. Addition of VC had little effect on oxidative stress or AChE, but increased pericardial area and heartbeat rate through an unknown mechanism. TBOOH diminished AChE gene expression and activity, and caused oxidative stress when administered alone. However, in combination with CPF, only reductions in AChE activity were observed with no significant changes in oxidative stress suggesting the adverse apical endpoints in the embryos may have been due to AChE inhibition by CPF rather than oxidative stress. These results give additional evidence to support the role of prooxidants in AChE activity and expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Mice maintain predominantly maternal Gαs expression throughout life in brown fat tissue (BAT), but not other tissues.

PubMed

Tafaj, Olta; Hann, Steven; Ayturk, Ugur; Warman, Matthew L; Jüppner, Harald

2017-10-01

The murine Gnas (human GNAS) locus gives rise to Gαs and different splice variants thereof. The Gαs promoter is not methylated thus allowing biallelic expression in most tissues. In contrast, the alternative first Gnas/GNAS exons and their promoters undergo parent specific methylation, which limits transcription to the non-methylated allele. Pseudohypoparathyroidism type Ia (PHP1A) or type Ib (PHP1B) are caused by heterozygous maternal GNAS mutations suggesting that little or no Gαs is derived in some tissues from the non-mutated paternal GNAS thereby causing hormonal resistance. Previous data had indicated that Gαs is mainly derived from the maternal Gnas allele in brown adipose tissue (BAT) of newborn mice, yet it is biallelically expressed in adult BAT. This suggested that paternal Gαs expression is regulated by an unknown factor(s) that varies considerably with age. To extend these findings, we now used a strain-specific SNP in Gnas exon 11 (rs13460569) for evaluation of parent-specific Gαs expression through the densitometric quantification of BanII-digested RT-PCR products and digital droplet PCR (ddPCR). At all investigated ages, Gαs transcripts were derived in BAT predominantly from the maternal Gnas allele, while kidney and liver showed largely biallelic Gαs expression. Only low or undetectable levels of other paternally Gnas-derived transcripts were observed, making it unlikely that these are involved in regulating paternal Gαs expression. Our findings suggest that a cis-acting factor could be implicated in reducing paternal Gαs expression in BAT and presumably in proximal renal tubules, thereby causing PTH-resistance if the maternal GNAS/Gnas allele is mutated. Copyright © 2017 Elsevier Inc. All rights reserved.

Grape RNA-Seq analysis pipeline environment

PubMed Central

Knowles, David G.; Röder, Maik; Merkel, Angelika; Guigó, Roderic

2013-01-01

Motivation: The avalanche of data arriving since the development of NGS technologies have prompted the need for developing fast, accurate and easily automated bioinformatic tools capable of dealing with massive datasets. Among the most productive applications of NGS technologies is the sequencing of cellular RNA, known as RNA-Seq. Although RNA-Seq provides similar or superior dynamic range than microarrays at similar or lower cost, the lack of standard and user-friendly pipelines is a bottleneck preventing RNA-Seq from becoming the standard for transcriptome analysis. Results: In this work we present a pipeline for processing and analyzing RNA-Seq data, that we have named Grape (Grape RNA-Seq Analysis Pipeline Environment). Grape supports raw sequencing reads produced by a variety of technologies, either in FASTA or FASTQ format, or as prealigned reads in SAM/BAM format. A minimal Grape configuration consists of the file location of the raw sequencing reads, the genome of the species and the corresponding gene and transcript annotation. Grape first runs a set of quality control steps, and then aligns the reads to the genome, a step that is omitted for prealigned read formats. Grape next estimates gene and transcript expression levels, calculates exon inclusion levels and identifies novel transcripts. Grape can be run on a single computer or in parallel on a computer cluster. It is distributed with specific mapping and quantification tools, but given its modular design, any tool supporting popular data interchange formats can be integrated. Availability: Grape can be obtained from the Bioinformatics and Genomics website at: http://big.crg.cat/services/grape. Contact: david.gonzalez@crg.eu or roderic.guigo@crg.eu PMID:23329413

Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

PubMed

Zilberman-Peled, B; Appelbaum, L; Vallone, D; Foulkes, N S; Anava, S; Anzulovich, A; Coon, S L; Klein, D C; Falcón, J; Ron, B; Gothilf, Y

2007-01-01

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

High-throughput interpretation of gene structure changes in human and nonhuman resequencing data, using ACE

PubMed Central

Majoros, William H.; Campbell, Michael S.; Holt, Carson; DeNardo, Erin K.; Ware, Doreen; Allen, Andrew S.; Yandell, Mark; Reddy, Timothy E.

2017-01-01

Abstract Motivation: The accurate interpretation of genetic variants is critical for characterizing genotype–phenotype associations. Because the effects of genetic variants can depend strongly on their local genomic context, accurate genome annotations are essential. Furthermore, as some variants have the potential to disrupt or alter gene structure, variant interpretation efforts stand to gain from the use of individualized annotations that account for differences in gene structure between individuals or strains. Results: We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE (‘Assessing Changes to Exons’) converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detects gene-structure changes and their possible repercussions, and identifies several classes of possible loss of function. Novel transcripts predicted by ACE are commonly supported by spliced RNA-seq reads, and can be used to improve read alignment and transcript quantification when an individual-specific genome sequence is available. Using publicly available RNA-seq data, we show that ACE predictions confirm earlier results regarding the quantitative effects of nonsense-mediated decay, and we show that predicted loss-of-function events are highly concordant with patterns of intolerance to mutations across the human population. ACE can be readily applied to diverse species including animals and plants, making it a broadly useful tool for use in eukaryotic population-based resequencing projects, particularly for assessing the joint impact of all variants at a locus. Availability and Implementation: ACE is written in open-source C ++ and Perl and is available from geneprediction.org/ACE Contact: myandell@genetics.utah.edu or tim.reddy@duke.edu Supplementary information: Supplementary information is available at Bioinformatics online. PMID:28011790

High-throughput interpretation of gene structure changes in human and nonhuman resequencing data, using ACE.

PubMed

Majoros, William H; Campbell, Michael S; Holt, Carson; DeNardo, Erin K; Ware, Doreen; Allen, Andrew S; Yandell, Mark; Reddy, Timothy E

2017-05-15

The accurate interpretation of genetic variants is critical for characterizing genotype-phenotype associations. Because the effects of genetic variants can depend strongly on their local genomic context, accurate genome annotations are essential. Furthermore, as some variants have the potential to disrupt or alter gene structure, variant interpretation efforts stand to gain from the use of individualized annotations that account for differences in gene structure between individuals or strains. We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE ('Assessing Changes to Exons') converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detects gene-structure changes and their possible repercussions, and identifies several classes of possible loss of function. Novel transcripts predicted by ACE are commonly supported by spliced RNA-seq reads, and can be used to improve read alignment and transcript quantification when an individual-specific genome sequence is available. Using publicly available RNA-seq data, we show that ACE predictions confirm earlier results regarding the quantitative effects of nonsense-mediated decay, and we show that predicted loss-of-function events are highly concordant with patterns of intolerance to mutations across the human population. ACE can be readily applied to diverse species including animals and plants, making it a broadly useful tool for use in eukaryotic population-based resequencing projects, particularly for assessing the joint impact of all variants at a locus. ACE is written in open-source C ++ and Perl and is available from geneprediction.org/ACE. myandell@genetics.utah.edu or tim.reddy@duke.edu. Supplementary information is available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Global exosome transcriptome profiling reveals biomarkers for multiple sclerosis.

PubMed

Selmaj, Igor; Cichalewska, Maria; Namiecinska, Magdalena; Galazka, Grazyna; Horzelski, Wojciech; Selmaj, Krzysztof W; Mycko, Marcin P

2017-05-01

Accumulating evidence supports a role for exosomes in immune regulation. In this study, we investigated the total circulating exosome transcriptome in relapsing-remitting multiple sclerosis (RRMS) patients and healthy controls (HC). Next generation sequencing (NGS) was used to define the global RNA profile of serum exosomes in 19 RRMS patients (9 in relapse, 10 in remission) and 10 HC. We analyzed 5 million reads and >50,000 transcripts per sample, including a detailed analysis of microRNAs (miRNAs) differentially expressed in RRMS. The discovery set data were validated by quantification using digital quantitative polymerase chain reaction with an independent cohort of 63 RRMS patients (33 in relapse, 30 in remission) and 32 HC. Exosomal RNA NGS revealed that of 15 different classes of transcripts detected, 4 circulating exosomal sequences within the miRNA category were differentially expressed in RRMS patients versus HC: hsa-miR-122-5p, hsa-miR-196b-5p, hsa-miR-301a-3p, and hsa-miR-532-5p. Serum exosomal expression of these miRNAs was significantly decreased during relapse in RRMS. These miRNAs were also decreased in patients with a gadolinium enhancement on brain magnetic resonance imaging. In vitro secretion of these miRNAs by peripheral blood mononuclear cells was also significantly impaired in RRMS. These data show that circulating exosomes have a distinct RNA profile in RRMS. Because putative targets for these miRNAs include the signal transducer and activator of transcription 3 and the cell cycle regulator aryl hydrocarbon receptor, the data suggest a disturbed cell-to-cell communication in this disease. Thus, exosomal miRNAs might represent a useful biomarker to distinguish multiple sclerosis relapse. Ann Neurol 2017;81:703-717. © 2017 American Neurological Association.

Transcript Assembly and Quantification by RNA-Seq Reveals Differentially Expressed Genes between Soft-Endocarp and Hard-Endocarp Hawthorns

PubMed Central

Zhang, Feng; Liu, Zhongchi; Li, Xiaoming; Li, Wenran; Ma, Yue; Li, He; Liu, Yuexue; Zhang, Zhihong

2013-01-01

Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn. PMID:24039819

Human nutrigenomics of gene regulation by dietary fatty acids.

PubMed

Afman, Lydia A; Müller, Michael

2012-01-01

Nutrigenomics employs high-throughput genomics technologies to unravel how nutrients modulate gene and protein expression and ultimately influence cellular and organism metabolism. The most often-applied genomics technique so far is transcriptomics, which allows quantifying genome-wide changes in gene expression of thousands of genes at the same time in one sample. The performance of gene expression quantification requires sufficient high-quality homogenous cellular material, therefore research in healthy volunteers is restricted to biopsies from easy accessible tissues such as subcutaneous adipose tissue, skeletal muscle and intestinal biopsies or even more easily accessible cells such as peripheral blood mononuclear cells from blood. There is now significant evidence that fatty acids, in particular unsaturated fatty acids, exert many of their effects through modulation of gene transcription by regulating the activity of numerous transcription factors, including nuclear receptors such as peroxisome proliferator activated receptors, liver X receptor and sterol regulatory binding proteins. This review evaluates the human nutrigenomics studies performed on dietary fat since the initiation of nutrigenomics research around 10 years ago. Although the number of studies is still limited, all studies clearly suggest that changes in dietary fatty acids intake and composition can have a significant impact on cellular adaptive response capacity by gene transcription changes in humans. This adds important knowledge to our understanding of the strong effects that various fatty acids can have on numerous metabolic and inflammatory pathways, signaling routes and homeostatic control in the cell and ultimately on whole body health. It is important to use and integrate nutrigenomics in all future nutrition studies to build up the necessary framework for evidence-based nutrition in near future. Copyright © 2011 Elsevier Ltd. All rights reserved.

Assessing gene expression during pathogenesis: Use of qRT-PCR to follow toxin production in the entomopathogenic fungus Beauveria bassiana during infection and immune response of the insect host Triatoma infestans.

PubMed

Lobo, Luciana S; Luz, Christian; Fernandes, Éverton K K; Juárez, M Patricia; Pedrini, Nicolás

2015-06-01

Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions

PubMed Central

Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen

2012-01-01

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions. PMID:22952972

Making lineage decisions with biological noise: Lessons from the early mouse embryo.

PubMed

Simon, Claire S; Hadjantonakis, Anna-Katerina; Schröter, Christian

2018-04-30

Understanding how individual cells make fate decisions that lead to the faithful formation and homeostatic maintenance of tissues is a fundamental goal of contemporary developmental and stem cell biology. Seemingly uniform populations of stem cells and multipotent progenitors display a surprising degree of heterogeneity, primarily originating from the inherent stochastic nature of molecular processes underlying gene expression. Despite this heterogeneity, lineage decisions result in tissues of a defined size and with consistent proportions of differentiated cell types. Using the early mouse embryo as a model we review recent developments that have allowed the quantification of molecular intercellular heterogeneity during cell differentiation. We first discuss the relationship between these heterogeneities and developmental cellular potential. We then review recent theoretical approaches that formalize the mechanisms underlying fate decisions in the inner cell mass of the blastocyst stage embryo. These models build on our extensive knowledge of the genetic control of fate decisions in this system and will become essential tools for a rigorous understanding of the connection between noisy molecular processes and reproducible outcomes at the multicellular level. We conclude by suggesting that cell-to-cell communication provides a mechanism to exploit and buffer intercellular variability in a self-organized process that culminates in the reproducible formation of the mature mammalian blastocyst stage embryo that is ready for implantation into the maternal uterus. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Quantitative Methods and Models. © 2018 Wiley Periodicals, Inc.

Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine.

PubMed

Lemaître, Chloé; Bidet, Philippe; Bingen, Edouard; Bonacorsi, Stéphane

2012-06-21

The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose:fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light.

PubMed

Lu, Chungui; Koroleva, Olga A; Farrar, John F; Gallagher, Joe; Pollock, Chris J; Tomos, A Deri

2002-11-01

We describe a highly efficient two-step single-cell reverse transcriptase-polymerase chain reaction technique for analyzing gene expression at the single-cell level. Good reproducibility and a linear dose response indicated that the technique has high specificity and sensitivity for detection and quantification of rare RNA. Actin could be used as an internal standard. The expression of message for Rubisco small subunit (RbcS), chlorophyll a/b-binding protein (Cab), sucrose (Suc):fructan-6-fructosyl transferase (6-SFT), and Actin were measured in individual photosynthetic cells of the barley (Hordeum vulgare) leaf. Only Actin was found in the non-photosynthetic epidermal cells. Cab, RbcS, and 6-SFT genes were expressed at a low level in mesophyll and parenchymatous bundle sheath (BS) cells when sampled from plants held in dark for 40 h. Expression increased considerably after illumination. The amount of 6-SFT, Cab, and RbcS transcript increased more in mesophyll cells than in the parenchymatous BS cells. The difference may be caused by different chloroplast structure and posttranscriptional control in mesophyll and BS cells. When similar single-cell samples were assayed for Suc, glucose, and fructan, there was high correlation between 6-SFT gene expression and Suc and glucose concentrations. This is consistent with Suc concentration being the trigger for transcription. Together with earlier demonstrations that the mesophyll cells have a higher sugar threshold for fructan polymerization, our data may indicate separate control of transcription and enzyme activity. Values for the sugar concentrations of the individual cell types are reported.

E2F mediates enhanced alternative polyadenylation in proliferation

PubMed Central

2012-01-01

Background The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Results Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Conclusions Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation. PMID:22747694

Optically transmitted and inductively coupled electric reference to access in vivo concentrations for quantitative proton-decoupled ¹³C magnetic resonance spectroscopy.

PubMed

Chen, Xing; Pavan, Matteo; Heinzer-Schweizer, Susanne; Boesiger, Peter; Henning, Anke

2012-01-01

This report describes our efforts on quantification of tissue metabolite concentrations in mM by nuclear Overhauser enhanced and proton decoupled (13) C magnetic resonance spectroscopy and the Electric Reference To access In vivo Concentrations (ERETIC) method. Previous work showed that a calibrated synthetic magnetic resonance spectroscopy-like signal transmitted through an optical fiber and inductively coupled into a transmit/receive coil represents a reliable reference standard for in vivo (1) H magnetic resonance spectroscopy quantification on a clinical platform. In this work, we introduce a related implementation that enables simultaneous proton decoupling and ERETIC-based metabolite quantification and hence extends the applicability of the ERETIC method to nuclear Overhauser enhanced and proton decoupled in vivo (13) C magnetic resonance spectroscopy. In addition, ERETIC signal stability under the influence of simultaneous proton decoupling is investigated. The proposed quantification method was cross-validated against internal and external reference standards on human skeletal muscle. The ERETIC signal intensity stability was 100.65 ± 4.18% over 3 months including measurements with and without proton decoupling. Glycogen and unsaturated fatty acid concentrations measured with the ERETIC method were in excellent agreement with internal creatine and external phantom reference methods, showing a difference of 1.85 ± 1.21% for glycogen and 1.84 ± 1.00% for unsaturated fatty acid between ERETIC and creatine-based quantification, whereas the deviations between external reference and creatine-based quantification are 6.95 ± 9.52% and 3.19 ± 2.60%, respectively. Copyright © 2011 Wiley Periodicals, Inc.

Direct quantification of lipopeptide biosurfactants in biological samples via HPLC and UPLC-MS requires sample modification with an organic solvent.

PubMed

Biniarz, Piotr; Łukaszewicz, Marcin

2017-06-01

The rapid and accurate quantification of biosurfactants in biological samples is challenging. In contrast to the orcinol method for rhamnolipids, no simple biochemical method is available for the rapid quantification of lipopeptides. Various liquid chromatography (LC) methods are promising tools for relatively fast and exact quantification of lipopeptides. Here, we report strategies for the quantification of the lipopeptides pseudofactin and surfactin in bacterial cultures using different high- (HPLC) and ultra-performance liquid chromatography (UPLC) systems. We tested three strategies for sample pretreatment prior to LC analysis. In direct analysis (DA), bacterial cultures were injected directly and analyzed via LC. As a modification, we diluted the samples with methanol and detected an increase in lipopeptide recovery in the presence of methanol. Therefore, we suggest this simple modification as a tool for increasing the accuracy of LC methods. We also tested freeze-drying followed by solvent extraction (FDSE) as an alternative for the analysis of "heavy" samples. In FDSE, the bacterial cultures were freeze-dried, and the resulting powder was extracted with different solvents. Then, the organic extracts were analyzed via LC. Here, we determined the influence of the extracting solvent on lipopeptide recovery. HPLC methods allowed us to quantify pseudofactin and surfactin with run times of 15 and 20 min per sample, respectively, whereas UPLC quantification was as fast as 4 and 5.5 min per sample, respectively. Our methods provide highly accurate measurements and high recovery levels for lipopeptides. At the same time, UPLC-MS provides the possibility to identify lipopeptides and their structural isoforms.

Quantification of pericardial effusions by echocardiography and computed tomography.

PubMed

Leibowitz, David; Perlman, Gidon; Planer, David; Gilon, Dan; Berman, Philip; Bogot, Naama

2011-01-15

Echocardiography is a well-accepted tool for the diagnosis and quantification of pericardial effusion (PEff). Given the increasing use of computed tomographic (CT) scanning, more PEffs are being initially diagnosed by computed tomography. No study has compared quantification of PEff by computed tomography and echocardiography. The objective of this study was to assess the accuracy of quantification of PEff by 2-dimensional echocardiography and computed tomography compared to the amount of pericardial fluid drained at pericardiocentesis. We retrospectively reviewed an institutional database to identify patients who underwent chest computed tomography and echocardiography before percutaneous pericardiocentesis with documentation of the amount of fluid withdrawn. Digital 2-dimensional echocardiographic and CT images were retrieved and quantification of PEff volume was performed by applying the formula for the volume of a prolate ellipse, π × 4/3 × maximal long-axis dimension/2 × maximal transverse dimension/2 × maximal anteroposterior dimension/2, to the pericardial sac and to the heart. Nineteen patients meeting study qualifications were entered into the study. The amount of PEff drained was 200 to 1,700 ml (mean 674 ± 340). Echocardiographically calculated pericardial effusion volume correlated relatively well with PEff volume (r = 0.73, p <0.001, mean difference -41 ± 225 ml). There was only moderate correlation between CT volume quantification and actual volume drained (r = 0.4, p = 0.004, mean difference 158 ± 379 ml). In conclusion, echocardiography appears a more accurate imaging technique than computed tomography in quantitative assessment of nonloculated PEffs and should continue to be the primary imaging in these patients. Copyright © 2011 Elsevier Inc. All rights reserved.

Customized Consensus Spectral Library Building for Untargeted Quantitative Metabolomics Analysis with Data Independent Acquisition Mass Spectrometry and MetaboDIA Workflow.

PubMed

Chen, Gengbo; Walmsley, Scott; Cheung, Gemmy C M; Chen, Liyan; Cheng, Ching-Yu; Beuerman, Roger W; Wong, Tien Yin; Zhou, Lei; Choi, Hyungwon

2017-05-02

Data independent acquisition-mass spectrometry (DIA-MS) coupled with liquid chromatography is a promising approach for rapid, automatic sampling of MS/MS data in untargeted metabolomics. However, wide isolation windows in DIA-MS generate MS/MS spectra containing a mixed population of fragment ions together with their precursor ions. This precursor-fragment ion map in a comprehensive MS/MS spectral library is crucial for relative quantification of fragment ions uniquely representative of each precursor ion. However, existing reference libraries are not sufficient for this purpose since the fragmentation patterns of small molecules can vary in different instrument setups. Here we developed a bioinformatics workflow called MetaboDIA to build customized MS/MS spectral libraries using a user's own data dependent acquisition (DDA) data and to perform MS/MS-based quantification with DIA data, thus complementing conventional MS1-based quantification. MetaboDIA also allows users to build a spectral library directly from DIA data in studies of a large sample size. Using a marine algae data set, we show that quantification of fragment ions extracted with a customized MS/MS library can provide as reliable quantitative data as the direct quantification of precursor ions based on MS1 data. To test its applicability in complex samples, we applied MetaboDIA to a clinical serum metabolomics data set, where we built a DDA-based spectral library containing consensus spectra for 1829 compounds. We performed fragment ion quantification using DIA data using this library, yielding sensitive differential expression analysis.

Quantification of maltol in Korean ginseng (Panax ginseng) products by high-performance liquid chromatography-diode array detector

PubMed Central

Jeong, Hyun Cheol; Hong, Hee-Do; Kim, Young-Chan; Rhee, Young Kyoung; Choi, Sang Yoon; Kim, Kyung-Tack; Kim, Sung Soo; Lee, Young-Chul; Cho, Chang-Won

2015-01-01

Background: Maltol, as a type of phenolic compounds, is produced by the browning reaction during the high-temperature treatment of ginseng. Thus, maltol can be used as a marker for the quality control of various ginseng products manufactured by high-temperature treatment including red ginseng. For the quantification of maltol in Korean ginseng products, an effective high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed. Materials and Methods: The HPLC-DAD method for maltol quantification coupled with a liquid-liquid extraction (LLE) method was developed and validated in terms of linearity, precision, and accuracy. An HPLC separation was performed on a C18 column. Results: The LLE methods and HPLC running conditions for maltol quantification were optimized. The calibration curve of the maltol exhibited good linearity (R2 = 1.00). The limit of detection value of maltol was 0.26 μg/mL, and the limit of quantification value was 0.79 μg/mL. The relative standard deviations (RSDs) of the data of the intra- and inter-day experiments were <1.27% and 0.61%, respectively. The results of the recovery test were 101.35–101.75% with an RSD value of 0.21–1.65%. The developed method was applied successfully to quantify the maltol in three ginseng products manufactured by different methods. Conclusion: The results of validation demonstrated that the proposed HPLC-DAD method was useful for the quantification of maltol in various ginseng products. PMID:26246746

Transcriptional enhancer from milk protein genes

DOEpatents

Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

1999-01-01

The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

Transcriptional Pathways Altered in Response to Vibration in a Model of Hand-Arm Vibration Syndrome

PubMed Central

Waugh, Stacey; Kashon, Michael L.; Li, Shengqiao; Miller, Gerome R.; Johnson, Claud; Krajnak, Kristine

2016-01-01

Objective The aim of this study was to use an established model of vibration-induced injury to assess frequency-dependent changes in transcript expression in skin, artery, and nerve tissues. Methods Transcript expression in tissues from control and vibration-exposed rats (4 h/day for 10 days at 62.5, 125, or 250 Hz; 49 m/s2, rms) was measured. Transcripts affected by vibration were used in bioinformatics analyses to identify molecular- and disease-related pathways associated with exposure to vibration. Results Analyses revealed that cancer-related pathways showed frequency-dependent changes in activation or inhibition. Most notably, the breast-related cancer-1 pathway was affected. Other pathways associated with breast cancer type 1 susceptibility protein related signaling, or associated with cancer and cell cycle/cell survivability were also affected. Conclusion Occupational exposure to vibration may result in DNA damage and alterations in cell signaling pathways that have significant effects on cellular division. PMID:27058473

Sirtuins: molecular traffic lights in the crossroad of oxidative stress, chromatin remodeling, and transcription.

PubMed

Rajendran, Ramkumar; Garva, Richa; Krstic-Demonacos, Marija; Demonacos, Constantinos

2011-01-01

Transcription is regulated by acetylation/deacetylation reactions of histone and nonhistone proteins mediated by enzymes called KATs and HDACs, respectively. As a major mechanism of transcriptional regulation, protein acetylation is a key controller of physiological processes such as cell cycle, DNA damage response, metabolism, apoptosis, and autophagy. The deacetylase activity of class III histone deacetylases or sirtuins depends on the presence of NAD(+) (nicotinamide adenine dinucleotide), and therefore, their function is closely linked to cellular energy consumption. This activity of sirtuins connects the modulation of chromatin dynamics and transcriptional regulation under oxidative stress to cellular lifespan, glucose homeostasis, inflammation, and multiple aging-related diseases including cancer. Here we provide an overview of the recent developments in relation to the diverse biological activities associated with sirtuin enzymes and stress responsive transcription factors, DNA damage, and oxidative stress and relate the involvement of sirtuins in the regulation of these processes to oncogenesis. Since the majority of the molecular mechanisms implicated in these pathways have been described for Sirt1, this sirtuin family member is more extensively presented in this paper.

Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

PubMed Central

Morisset, Dany; Štebih, Dejan; Milavec, Mojca; Gruden, Kristina; Žel, Jana

2013-01-01

In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed. PMID:23658750

Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

PubMed

Ballari, Rajashekhar V; Martin, Asha

2013-12-01

DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal. Copyright © 2013 Elsevier Ltd. All rights reserved.

39 CFR 963.16 - Transcript.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 39 Postal Service 1 2010-07-01 2010-07-01 false Transcript. 963.16 Section 963.16 Postal Service UNITED STATES POSTAL SERVICE PROCEDURES RULES OF PRACTICE IN PROCEEDINGS RELATIVE TO VIOLATIONS OF THE PANDERING ADVERTISEMENTS STATUTE, 39 U.S.C. 3008 § 963.16 Transcript. Testimony and argument at hearings...

17A-ETHYNYLESTRADIOL-INDUCED VITELLOGENIN GENE TRANSCRIPTION QUANTIFIED IN LIVERS OF ADULT MALES, LARVAE, AND GILLS OF FATHEAD MINNOW (PIMEPHALES PROMELAS)

EPA Science Inventory

We have applied a method for quantifying relative levels of messenger RNA (mRNA) transcription to assess chemically-induced gene expression in fathead minnows (Pimephales promelas). Synthetic oligonucleotides designed for the fathead minnow vitellogenin gene transcription (Vg) p...

29 CFR 1912a.11 - Minutes; transcript.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 29 Labor 7 2014-07-01 2014-07-01 false Minutes; transcript. 1912a.11 Section 1912a.11 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.11 Minutes; transcript. (a...

29 CFR 1912a.11 - Minutes; transcript.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 29 Labor 7 2013-07-01 2013-07-01 false Minutes; transcript. 1912a.11 Section 1912a.11 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.11 Minutes; transcript. (a...

45 CFR 150.449 - Cost of transcripts.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Cost of transcripts. 150.449 Section 150.449 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES REQUIREMENTS RELATING TO HEALTH CARE ACCESS CMS ENFORCEMENT IN GROUP AND INDIVIDUAL INSURANCE MARKETS Administrative Hearings § 150.449 Cost of transcripts...

29 CFR 1912a.11 - Minutes; transcript.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 7 2010-07-01 2010-07-01 false Minutes; transcript. 1912a.11 Section 1912a.11 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.11 Minutes; transcript. (a...

The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

PubMed

Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

2016-07-01

The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Mathematical simulations for bioanalytical assay development: the (un-)necessity and (im-)possibility of free drug quantification.

PubMed

Staack, Roland F; Jordan, Gregor; Heinrich, Julia

2012-02-01

For every drug development program it needs to be discussed whether discrimination between free and total drug concentrations is required to accurately describe its pharmacokinetic behavior. This perspective describes the application of mathematical simulation approaches to guide this initial decision based on available knowledge about target biology, binding kinetics and expected drug concentrations. We provide generic calculations that can be used to estimate the necessity of free drug quantification for different drug molecules. In addition, mathematical approaches are used to simulate various assay conditions in bioanalytical ligand-binding assays: it is demonstrated that due to the noncovalent interaction between the binding partners and typical assay-related interferences in the equilibrium, a correct quantification of the free drug concentration is highly challenging and requires careful design of different assay procedure steps.

Effect of Microbial Interaction on Urea Metabolism in Chinese Liquor Fermentation.

PubMed

Wu, Qun; Lin, Jianchun; Cui, Kaixiang; Du, Rubin; Zhu, Yang; Xu, Yan

2017-12-20

Urea is the primary precursor of the carcinogen ethyl carbamate in fermented foods. Understanding urea metabolism is important for controlling ethyl carbamate production. Using Chinese liquor as a model system, we used metatranscriptome analysis to investigate urea metabolism in spontaneous food fermentation processes. Saccharomyces cerevisiae was dominant in gene transcription for urea biosynthesis and degradation. Lysinibacillus sphaericus was dominant for urea degradation. S. cerevisiae degraded 18% and L. sphaericus degraded 13% of urea in their corresponding single cultures, whereas they degraded 56% of urea in coculture after 12 h. Compared to single cultures, transcription of CAR1, DAL2, and argA, which are related to urea biosynthesis, decreased by 51, 36, and 69% in coculture, respectively. Transcription of DUR1 and ureA, which are related to urea degradation, increased by 227 and 70%, respectively. Thus, coexistence of the two strains promoted degradation of urea via transcriptional regulation of genes related to urea metabolism.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Yanli; Li, Hui; Zhang, Xiaoju

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, A3G) exert antiviral defense as an important factor of innate immunity. A variety of cytokines such as IFN-γ,IL2,IL15,IL7 could induce the transcription of A3G. However, the regulation of other nuclear factor on the transcription of A3G have not been reported at the present. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G and investigate the modulation of USF1 gene on the transcription of A3G. We identified a 232 bp region that was sufficient to regulate the activity of full promoter. Transcriptionalmore » start sites (TSS) were identified by the luciferase reporter assays of plasmids containing full or shorter fragments of the A3G promoter. The results demonstrated that the core promoter of A3G is located within the region -159/-84 relative to the TSS. Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position -91/-86 relative to the major TSS) and was abolished after mutation of this DNA element. USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte, and the identified E-box represented a binding site for the USF1. - Highlights: • The core promoter of A3G is located within the region −159/−84 relative to the TSS. • Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position −91/−86 relative to the major TSS). • USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte.« less

Effect of postharvest ethylene treatment on sugar content, glycosidase activity and its gene expression in mango fruit.

PubMed

Chidley, Hemangi G; Deshpande, Ashish B; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

2017-03-01

Ripening-associated softening is one of the important attributes that largely determines the shelf-life of mango (Mangifera indica Linn.) fruits. To reveal the effect of pre-climacteric ethylene treatment on ripening-related softening of Alphonso mango, ethylene treatment was given to mature, raw Alphonso fruits. Changes in the pool of reducing and non-reducing sugars, enzymatic activity of three glycosidases: β-d-galactosidase, α-d-mannosidase and β-d-glucosidase and their relative transcript abundance were analysed for control and ethylene treated fruits during ripening. Early activity of all the three glycosidases and accelerated accumulation of reducing and non-reducing sugars on ethylene treatment was evident. β-d-Galactosidase showed the highest activity among three glycosidases in control fruits and marked increase in activity upon ethylene treatment. This was confirmed by the histochemical assay of its activity in control and ethylene treated ripe fruits. Relative transcript abundance revealed high transcript levels of β-d-galactosidase in control fruits. Ethylene-treated fruits showed early and remarkable increase in the β-d-galactosidase transcripts while α-d-mannosidase transcript variants displayed early accumulation. The findings suggest reduction in the shelf-life of Alphonso mango upon pre-climacteric ethylene treatment, a significant role of β-d-galactosidase and α-d-mannosidase in the ripening related softening of Alphonso fruits and transcriptional regulation of their expression by ethylene. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors.

PubMed

Heller, Elizabeth A; Cates, Hannah M; Peña, Catherine J; Sun, Haosheng; Shao, Ningyi; Feng, Jian; Golden, Sam A; Herman, James P; Walsh, Jessica J; Mazei-Robison, Michelle; Ferguson, Deveroux; Knight, Scott; Gerber, Mark A; Nievera, Christian; Han, Ming-Hu; Russo, Scott J; Tamminga, Carol S; Neve, Rachael L; Shen, Li; Zhang, H Steve; Zhang, Feng; Nestler, Eric J

2014-12-01

Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.

Aging of the skeletal muscle extracellular matrix drives a stem cell fibrogenic conversion.

PubMed

Stearns-Reider, Kristen M; D'Amore, Antonio; Beezhold, Kevin; Rothrauff, Benjamin; Cavalli, Loredana; Wagner, William R; Vorp, David A; Tsamis, Alkiviadis; Shinde, Sunita; Zhang, Changqing; Barchowsky, Aaron; Rando, Thomas A; Tuan, Rocky S; Ambrosio, Fabrisia

2017-06-01

Age-related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age-related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age-related increase in muscle stiffness drives YAP/TAZ-mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Modulation of adrenocorticotrophin hormone (ACTH)-induced expression of stress-related genes by PUFA in inter-renal cells from European sea bass (Dicentrarchus labrax).

PubMed

Montero, Daniel; Terova, Genciana; Rimoldi, Simona; Tort, Lluis; Negrin, Davinia; Zamorano, María Jesús; Izquierdo, Marisol

2015-01-01

Dietary fatty acids have been shown to exert a clear effect on the stress response, modulating the release of cortisol. The role of fatty acids on the expression of steroidogenic genes has been described in mammals, but little is known in fish. The effect of different fatty acids on the release of cortisol and expression of stress-related genes of European sea bass (Dicentrarchus labrax) head kidney, induced by a pulse of adenocorticotrophin hormone (ACTH), was studied. Tissue was maintained in superfusion with 60 min of incubation with EPA, DHA, arachidonic acid (ARA), linoleic acid or α-linolenic acid (ALA) during 490 min. Cortisol was measured by RIA. The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real-time RT-PCR. There was an effect of the type of fatty acid on the ACTH-induced release of cortisol, values from ALA treatment being elevated within all of the experimental period. The expression of some steroidogenic genes, such as the steroidogenic acute regulatory protein (StAR) and c-fos, were affected by fatty acids, ALA increasing the expression of StAR after 1 h of ACTH stimulation whereas DHA, ARA and ALA increased the expression of c-fos after 20 min. ARA increased expression of the 11β-hydroxylase gene. Expression of heat shock protein 70 (HSP70) was increased in all the experimental treatments except for ARA. Results corroborate previous studies of the effect of different fatty acids on the release of cortisol in marine fish and demonstrate that those effects are mediated by alteration of the expression of steroidogenic genes.

N-acetylcysteine treatment blocks the development of ethanol-induced behavioural sensitization and related ΔFosB alterations.

PubMed

Morais-Silva, Gessynger; Alves, Gabrielle Cunha; Marin, Marcelo T

2016-11-01

Ethanol addiction is a serious public health problem that still needs more effective pharmacological treatment. A key factor in the development and maintenance of this disease is the advent of neuroadaptations in the mesocorticolimbic brain pathway upon chronic ethanol abuse. In general, these neuroadaptations are maladaptive and affect numerous neurotransmitter systems and intracellular molecules. One of these molecules is ΔFosB, a transcription factor that is altered after chronic drug use. Behavioural sensitization is a useful model for the study of the neuroadaptations related to addiction. Recent works have shown a role for the imbalance of glutamatergic neurotransmission in the symptoms found in addicted people. In this sense, the treatment with N-acetylcysteine, a l-cysteine prodrug that acts by restoring extrasynaptic concentrations of glutamate through the activation of cystine-glutamate antiporter, has shown promising results in the treatment of addiction. Thus, an animal model of behavioural sensitization was used to evaluate the effects of N-acetylcysteine treatment in the behavioural and molecular alterations induced by chronic ethanol administration. Swiss mice were subject to 13 days of daily ethanol administration to induce behavioural sensitization. Two hours before each ethanol administration and locomotor activity evaluation, the animals received intraperitoneally N-acetylcysteine injections. Immediately after the last test session, their brains were removed for ΔFosB and cystine-glutamate antiporter quantification. It was found that N-acetylcysteine treatment blocked ethanol-induced behavioural sensitization, the increase of ΔFosB content in the prefrontal cortex, and its reduction in the nucleus accumbens. The results suggest a possible use of N-acetylcysteine in ethanol-related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

The upregulation of immune responses in tyrosine hydroxylase (TH) silenced Litopenaeus vannamei.

PubMed

Mapanao, Ratchaneegorn; Chang, Chin-Chyuan; Cheng, Winton

2017-02-01

Catecholamines (CAs) play a crucial role in maintaining physiological and immune homeostasis in invertebrates and vertebrates under stressful conditions. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in CA synthesis. To develop an effective CA-related immunological defense system against stress and pathogen infection, various criteria, were evaluated in TH double-stranded (ds) RNA-injected white shrimp, Litopenaeus vannamei. Specifically, the relative transcript quantification of TH, dopamine β-hydroxylase (DBH), crustacean hyperglycemic hormone (CHH), and other immune-related genes; TH activity in the haemolymph; and the estimation of l-dihydroxyphenylalanine (l-DOPA), glucose, and lactate levels in the haemolymph were examined. TH depletion revealed a significant increase in the total haemocyte count; granular cells; semigranular cells; respiratory bursts (RBs, release of superoxide anion); superoxide dismutase (SOD) activity; phagocytic activity and clearance efficiency; and the expression of lipopolysaccharide and β-1,3-glucan-binding protein and peroxinectin, SOD, crustin, and lysozyme genes. In addition, the reduction of TH gene expression and activity was accompanied by a decline of phenoloxidase (PO) activity per granulocyte, lower glucose and lactate levels, and significantly low expression of DBH and CHH genes. However, the number of hyaline cells, activity of PO, RBs per haemocyte, and expression of POI and POII genes were not significantly different in the LvTH-silenced shrimp. Notably, the survival ratio of LvTH-silenced shrimp was significantly higher than that of shrimp injected with diethyl pyrocarbonate-water and nontargeting dsRNA when challenged with Vibrio alginolyticus. Therefore, the depletion of TH can enhance disease resistance in shrimp by upregulating specific immune parameters but downregulating the levels of carbohydrate metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ariadne's Thread: A Robust Software Solution Leading to Automated Absolute and Relative Quantification of SRM Data.

PubMed

Nasso, Sara; Goetze, Sandra; Martens, Lennart

2015-09-04

Selected reaction monitoring (SRM) MS is a highly selective and sensitive technique to quantify protein abundances in complex biological samples. To enhance the pace of SRM large studies, a validated, robust method to fully automate absolute quantification and to substitute for interactive evaluation would be valuable. To address this demand, we present Ariadne, a Matlab software. To quantify monitored targets, Ariadne exploits metadata imported from the transition lists, and targets can be filtered according to mProphet output. Signal processing and statistical learning approaches are combined to compute peptide quantifications. To robustly estimate absolute abundances, the external calibration curve method is applied, ensuring linearity over the measured dynamic range. Ariadne was benchmarked against mProphet and Skyline by comparing its quantification performance on three different dilution series, featuring either noisy/smooth traces without background or smooth traces with complex background. Results, evaluated as efficiency, linearity, accuracy, and precision of quantification, showed that Ariadne's performance is independent of data smoothness and complex background presence and that Ariadne outperforms mProphet on the noisier data set and improved 2-fold Skyline's accuracy and precision for the lowest abundant dilution with complex background. Remarkably, Ariadne could statistically distinguish from each other all different abundances, discriminating dilutions as low as 0.1 and 0.2 fmol. These results suggest that Ariadne offers reliable and automated analysis of large-scale SRM differential expression studies.

Quantification of taurine in energy drinks using ¹H NMR.

PubMed

Hohmann, Monika; Felbinger, Christine; Christoph, Norbert; Wachter, Helmut; Wiest, Johannes; Holzgrabe, Ulrike

2014-05-01

The consumption of so called energy drinks is increasing, especially among adolescents. These beverages commonly contain considerable amounts of the amino sulfonic acid taurine, which is related to a magnitude of various physiological effects. The customary method to control the legal limit of taurine in energy drinks is LC-UV/vis with postcolumn derivatization using ninhydrin. In this paper we describe the quantification of taurine in energy drinks by (1)H NMR as an alternative to existing methods of quantification. Variation of pH values revealed the separation of a distinct taurine signal in (1)H NMR spectra, which was applied for integration and quantification. Quantification was performed using external calibration (R(2)>0.9999; linearity verified by Mandel's fitting test with a 95% confidence level) and PULCON. Taurine concentrations in 20 different energy drinks were analyzed by both using (1)H NMR and LC-UV/vis. The deviation between (1)H NMR and LC-UV/vis results was always below the expanded measurement uncertainty of 12.2% for the LC-UV/vis method (95% confidence level) and at worst 10.4%. Due to the high accordance to LC-UV/vis data and adequate recovery rates (ranging between 97.1% and 108.2%), (1)H NMR measurement presents a suitable method to quantify taurine in energy drinks. Copyright © 2013 Elsevier B.V. All rights reserved.

Simultaneous quantification of Δ9-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol, and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in human plasma using two-dimensional gas chromatography, cryofocusing, and electron impact-mass spectrometry

PubMed Central

Lowe, Ross H.; Karschner, Erin L.; Schwilke, Eugene W.; Barnes, Allan J.; Huestis, Marilyn A.

2009-01-01

A two-dimensional (2D) gas chromatography/electron impact-mass spectrometry (GC/EI-MS) method for simultaneous quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in human plasma was developed and validated. The method employs 2D capillary GC and cryofocusing for enhanced resolution and sensitivity. THC, 11-OH-THC, and THCCOOH were extracted by precipitation with acetonitrile followed by solid-phase extraction. GC separation of trimethylsilyl derivatives of analytes was accomplished with two capillary columns in series coupled via a pneumatic Deans switch system. Detection and quantification were accomplished with a bench-top single quadrupole mass spectrometer operated in electron impact-selected ion monitoring mode. Limits of quantification (LOQ) were 0.125, 0.25 and 0.125 ng/mL for THC, 11-OH-THC, and THCCOOH, respectively. Accuracy ranged from 86.0 to 113.0% for all analytes. Intra- and inter-assay precision, as percent relative standard deviation, was less than 14.1% for THC, 11-OH-THC, and THCCOOH. The method was successfully applied to quantification of THC and its 11-OH-THC and THCCOOH metabolites in plasma specimens following controlled administration of THC. PMID:17640656

Quantification of free fatty acids in human stratum corneum using tandem mass spectrometry and surrogate analyte approach.

PubMed

Dapic, Irena; Kobetic, Renata; Brkljacic, Lidija; Kezic, Sanja; Jakasa, Ivone

2018-02-01

The free fatty acids (FFAs) are one of the major components of the lipids in the stratum corneum (SC), the uppermost layer of the skin. Relative composition of FFAs has been proposed as a biomarker of the skin barrier status in patients with atopic dermatitis (AD). Here, we developed an LC-ESI-MS/MS method for simultaneous quantification of a range of FFAs with long and very long chain length in the SC collected by adhesive tape (D-Squame). The method, based on derivatization with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide, allowed highly sensitive detection and quantification of FFAs using multiple reaction monitoring. For the quantification, we applied a surrogate analyte approach and internal standardization using isotope labeled derivatives of FFAs. Adhesive tapes showed the presence of several FFAs, which are also present in the SC, a problem encountered in previous studies. Therefore, the levels of FFAs in the SC were corrected using C12:0, which was present on the adhesive tape, but not detected in the SC. The method was applied to SC samples from patients with atopic dermatitis and healthy subjects. Quantification using multiple reaction monitoring allowed sufficient sensitivity to analyze FFAs of chain lengths C16-C28 in the SC collected on only one tape strip. Copyright © 2017 John Wiley & Sons, Ltd.

Ranking Fragment Ions Based on Outlier Detection for Improved Label-Free Quantification in Data-Independent Acquisition LC-MS/MS

PubMed Central

Bilbao, Aivett; Zhang, Ying; Varesio, Emmanuel; Luban, Jeremy; Strambio-De-Castillia, Caterina; Lisacek, Frédérique; Hopfgartner, Gérard

2016-01-01

Data-independent acquisition LC-MS/MS techniques complement supervised methods for peptide quantification. However, due to the wide precursor isolation windows, these techniques are prone to interference at the fragment ion level, which in turn is detrimental for accurate quantification. The “non-outlier fragment ion” (NOFI) ranking algorithm has been developed to assign low priority to fragment ions affected by interference. By using the optimal subset of high priority fragment ions these interfered fragment ions are effectively excluded from quantification. NOFI represents each fragment ion as a vector of four dimensions related to chromatographic and MS fragmentation attributes and applies multivariate outlier detection techniques. Benchmarking conducted on a well-defined quantitative dataset (i.e. the SWATH Gold Standard), indicates that NOFI on average is able to accurately quantify 11-25% more peptides than the commonly used Top-N library intensity ranking method. The sum of the area of the Top3-5 NOFIs produces similar coefficients of variation as compared to the library intensity method but with more accurate quantification results. On a biologically relevant human dendritic cell digest dataset, NOFI properly assigns low priority ranks to 85% of annotated interferences, resulting in sensitivity values between 0.92 and 0.80 against 0.76 for the Spectronaut interference detection algorithm. PMID:26412574

Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus

PubMed Central

Hao, Xiao-Dan; Chen, Zhao-Li; Qu, Ming-Li; Zhao, Xiao-Wen; Li, Su-Xia; Chen, Peng

2016-01-01

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC. PMID:27783701

Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis.

PubMed

Santangelo, K S; Bertone, A L

2011-12-01

To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1β (IL-1β) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1β transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify in vitro transcript changes of IL-1β and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2(-ΔΔCT)) method. Statistically significant decreases in IL-1β expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-β (TGF-β) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P=0.0045) or >90% (P=0.0001) of the IL-1β transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1β transcript in vivo are warranted. Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Telomere length analysis.

PubMed

Canela, Andrés; Klatt, Peter; Blasco, María A

2007-01-01

Most somatic cells of long-lived species undergo telomere shortening throughout life. Critically short telomeres trigger loss of cell viability in tissues, which has been related to alteration of tissue function and loss of regenerative capabilities in aging and aging-related diseases. Hence, telomere length is an important biomarker for aging and can be used in the prognosis of aging diseases. These facts highlight the importance of developing methods for telomere length determination that can be employed to evaluate telomere length during the human aging process. Telomere length quantification methods have improved greatly in accuracy and sensitivity since the development of the conventional telomeric Southern blot. Here, we describe the different methodologies recently developed for telomere length quantification, as well as their potential applications for human aging studies.

Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.

2014-12-17

Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reactionmore » monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed, model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.« less

Biochemical metabolic changes assessed by 31P magnetic resonance spectroscopy after radiation-induced hepatic injury in rabbits

PubMed Central

Yu, Ri-Sheng; Hao, Liang; Dong, Fei; Mao, Jian-Shan; Sun, Jian-Zhong; Chen, Ying; Lin, Min; Wang, Zhi-Kang; Ding, Wen-Hong

2009-01-01

AIM: To compare the features of biochemical metabolic changes detected by hepatic phosphorus-31 magnetic resonance spectroscopy (31P MRS) with the liver damage score (LDS) and pathologic changes in rabbits and to investigate the diagnostic value of 31P MRS in acute hepatic radiation injury. METHODS: A total of 30 rabbits received different radiation doses (ranging 5-20 Gy) to establish acute hepatic injury models. Blood biochemical tests, 31P MRS and pathological examinations were carried out 24 h after irradiation. The degree of injury was evaluated according to LDS and pathology. Ten healthy rabbits served as controls. The MR examination was performed on a 1.5 T imager using a 1H/31P surface coil by the 2D chemical shift imaging technique. The relative quantities of phosphomonoesters (PME), phosphodiesters (PDE), inorganic phosphate (Pi) and adenosine triphosphate (ATP) were measured. The data were statistically analyzed. RESULTS: (1) Relative quantification of phosphorus metabolites: (a) ATP: there were significant differences (P < 0.05) (LDS-groups: control group vs mild group vs moderate group vs severe group, 1.83 ± 0.33 vs 1.55 ± 0.24 vs 1.27 ± 0.09 vs 0.98 ± 0.18; pathological groups: control group vs mild group vs moderate group vs severe group, 1.83 ± 0.33 vs 1.58 ± 0.25 vs 1.32 ± 0.07 vs 1.02 ± 0.18) of ATP relative quantification among control group, mild injured group, moderate injured group, and severe injured group according to both LDS grading and pathological grading, respectively, and it decreased progressively with the increased degree of injury (r = -0.723, P = 0.000). (b) PME and Pi; the relative quantification of PME and Pi decreased significantly in the severe injured group, and the difference between the control group and severe injured group was significant (P < 0.05) (PME: LDS-control group vs LDS-severe group, 0.86 ± 0.23 vs 0.58 ± 0.22, P = 0.031; pathological control group vs pathological severe group, 0.86 ± 0.23 vs 0.60 ± 0.21, P = 0.037; Pi: LDS-control group vs LDS-severe group, 0.74 ± 0.18 vs 0.43 ± 0.14, P = 0.013; pathological control group vs pathological severe group, 0.74 ± 0.18 vs 0.43 ± 0.14, P = 0.005) according to LDS grading and pathological grading, respectively. (c) PDE; there were no significant differences among groups according to LDS grading, and no significant differences between the control group and experimental groups according to pathological grading. (2) The ratio of relative quantification of phosphorus metabolites: significant differences (P < 0.05) (LDS-moderate group and LDS-severe group vs LDS-control group and LDS-mild group, 1.94 ± 0.50 and 1.96 ± 0.72 vs 1.43 ± 0.31 and 1.40 ± 0.38) were only found in PDE/ATP between the moderate injured group, the severe injured group and the control group, the mild injured group. No significant difference was found in other ratios of relative quantification of phosphorus metabolites. CONCLUSION: 31P MRS is a useful method to evaluate early acute hepatic radiation injury. The relative quantification of hepatic ATP levels, which can reflect the pathological severity of acute hepatic radiation injury, is correlated with LDS. PMID:19522022

22 CFR 1500.9 - Transcripts, recording of closed meetings.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., closed to the public. (b) The Foundation, after review by the General Counsel, shall make promptly... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Transcripts, recording of closed meetings. 1500.9 Section 1500.9 Foreign Relations AFRICAN DEVELOPMENT FOUNDATION SUNSHINE REGULATIONS § 1500.9...

Transcriptional Profiling of Human Endogenous Retrovirus Group HERV-K(HML-2) Loci in Melanoma

PubMed Central

Schmitt, Katja; Reichrath, Jörg; Roesch, Alexander; Meese, Eckart; Mayer, Jens

2013-01-01

Recent studies suggested a role for the human endogenous retrovirus (HERV) group HERV-K(HML-2) in melanoma because of upregulated transcription and expression of HERV-K(HML-2)-encoded proteins. Very little is known about which HML-2 loci are transcribed in melanoma. We assigned >1,400 HML-2 cDNA sequences generated from various melanoma and related samples to genomic HML-2 loci, identifying a total of 23 loci as transcribed. Transcription profiles of loci differed significantly between samples. One locus was found transcribed only in melanoma-derived samples but not in melanocytes and might represent a marker for melanoma. Several of the transcribed loci harbor ORFs for retroviral Gag and/or Env proteins. Env-encoding loci were transcribed only in melanoma. Specific investigation of rec and np9 transcripts indicated transcription of protein encoding loci in melanoma and melanocytes hinting at the relevance of Rec and Np9 in melanoma. UVB irradiation changed transcription profiles of loci and overall transcript levels decreased in melanoma and melanocytes. We further identified transcribed HML-2 loci formed by reverse transcription of spliced HML-2 transcripts by L1 machinery or in a retroviral fashion, with loci potentially encoding HML-2-like proteins. We reveal complex, sample-specific transcription of HML-2 loci in melanoma and related samples. Identified HML-2 loci and proteins encoded by those loci are particularly relevant for further studying the role of HML-2 in melanoma. Transcription of HERVs appears as a complex mechanism requiring specific studies to elucidate which HERV loci are transcribed and how transcribed HERVs may be involved in disease. PMID:23338945

Transcriptome Analysis of Scrippsiella trochoidea CCMP 3099 Reveals Physiological Changes Related to Nitrate Depletion

PubMed Central

Cooper, Joshua T.; Sinclair, Geoffrey A.; Wawrik, Boris

2016-01-01

Dinoflagellates are a major component of marine phytoplankton and many species are recognized for their ability to produce harmful algal blooms (HABs). Scrippsiella trochoidea is a non-toxic, marine dinoflagellate that can be found in both cold and tropic waters where it is known to produce “red tide” events. Little is known about the genomic makeup of S. trochoidea and a transcriptome study was conducted to shed light on the biochemical and physiological adaptations related to nutrient depletion. Cultures were grown under N and P limiting conditions and transcriptomes were generated via RNAseq technology. De novo assembly reconstructed 107,415 putative transcripts of which only 41% could be annotated. No significant transcriptomic response was observed in response to initial P depletion, however, a strong transcriptional response to N depletion was detected. Among the down-regulated pathways were those for glutamine/glutamate metabolism as well as urea and nitrate/nitrite transporters. Transcripts for ammonia transporters displayed both up- and down-regulation, perhaps related to a shift to higher affinity transporters. Genes for the utilization of DON compounds were up-regulated. These included transcripts for amino acids transporters, polyamine oxidase, and extracellular proteinase and peptidases. N depletion also triggered down regulation of transcripts related to the production of Photosystems I & II and related proteins. These data are consistent with a metabolic strategy that conserves N while maximizing sustained metabolism by emphasizing the relative contribution of organic N sources. Surprisingly, the transcriptome also contained transcripts potentially related to secondary metabolite production, including a homolog to the Short Isoform Saxitoxin gene (sxtA) from Alexandrium fundyense, which was significantly up-regulated under N-depletion. A total of 113 unique hits to Sxt genes, covering 17 of the 34 genes found in C. raciborskii were detected, indicating that S. trochoidea has previously unrecognized potential for the production of secondary metabolites with potential toxicity. PMID:27242681

An Insight into the Transcriptome of the Digestive Tract of the Bloodsucking Bug, Rhodnius prolixus

PubMed Central

Ribeiro, José M. C.; Genta, Fernando A.; Sorgine, Marcos H. F.; Logullo, Raquel; Mesquita, Rafael D.; Paiva-Silva, Gabriela O.; Majerowicz, David; Medeiros, Marcelo; Koerich, Leonardo; Terra, Walter R.; Ferreira, Clélia; Pimentel, André C.; Bisch, Paulo M.; Leite, Daniel C.; Diniz, Michelle M. P.; Junior, João Lídio da S. G. V.; Da Silva, Manuela L.; Araujo, Ricardo N.; Gandara, Ana Caroline P.; Brosson, Sébastien; Salmon, Didier; Bousbata, Sabrina; González-Caballero, Natalia; Silber, Ariel Mariano; Alves-Bezerra, Michele; Gondim, Katia C.; Silva-Neto, Mário Alberto C.; Atella, Georgia C.; Araujo, Helena; Dias, Felipe A.; Polycarpo, Carla; Vionette-Amaral, Raquel J.; Fampa, Patrícia; Melo, Ana Claudia A.; Tanaka, Aparecida S.; Balczun, Carsten; Oliveira, José Henrique M.; Gonçalves, Renata L. S.; Lazoski, Cristiano; Rivera-Pomar, Rolando; Diambra, Luis; Schaub, Günter A.; Garcia, Elói S.; Azambuja, Patrícia; Braz, Glória R. C.; Oliveira, Pedro L.

2014-01-01

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7–8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet. PMID:24416461