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Systematic comparison of RNA-Seq normalization methods using measurement error models

BIOINFORMATICS 2012年第20期28卷 2584-2591页

作者： Sun, Zhaonan Zhu, Yu Purdue Univ Dept Stat W Lafayette IN 47906 USA

Motivation: Further advancement of RNA-Seq technology and its application call for the development of effective normalization methods for RNA-Seq data. Currently, different normalization methods are compared and validated by their correlations with a certain gold standard. Gene expression measurements generated by a different technology or platform such as Real-time reverse transcription polymerase chain reaction (qRT-PCR) or microarray are usually used as the gold standard. Although the current approach is intuitive and easy to implement, it becomes statistically inadequate when the gold standard is also subject to measurement error (ME). Furthermore, the current approach is not informative, because the correlation of a normalization method with a certain gold standard does not provide much information about the exact quality of the normalized RNA-Seq measurements. Results: We propose to use the system of ME models based on qRT-PCR, microarray and RNA-Seq gene expression data to compare and validate RNA-Seq normalization methods. This approach does not assume the existence of a gold standard. The performance of a normalization method can be characterized by a group of parameters of the system, which are referred to as the performance parameters, and these performance parameters can be consistently estimated. Different normalization methods can thus be compared by comparing their corresponding estimated performance parameters. We applied the proposed approach to compare five existing RNA-Seq normalization methods using the gene expression data of two RNA samples from the microarray Quality Control and Sequencing Quality Control projects and gained much insight about the pros and cons of these methods.

关键词： SYSTEMATIC reviews (Medical research) COMPARATIVE studies NUCLEOTIDE sequence ERRORS-in-variables models GENE expression REVERSE transcriptase polymerase chain reaction microarray technology

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Gene profiling of chikungunya virus arthritis in a mouse model reveals significant overlap with rheumatoid arthritis

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ARTHRITIS AND RHEUMATISM 2012年第11期64卷 3553-3563页

作者： Nakaya, Helder I. Gardner, Joy Poo, Yee-Suan Major, Lee Pulendran, Bali Suhrbier, Andreas Univ Queensland Queensland Inst Med Res Brisbane Qld 4029 Australia Australian Ctr Vaccine Dev Brisbane Qld Australia Emory Univ Atlanta GA 30322 USA Yerkes Natl Primate Res Ctr Emory Vaccine Ctr Atlanta GA USA

Objective Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a chronic debilitating polyarthralgia/polyarthritis, for which current treatments are often inadequate. To assess whether new drugs being developed for rheumatoid arthritis (RA) might find utility in the treatment of alphaviral arthritides, we sought to determine whether the inflammatory gene expression signature of CHIKV arthritis shows any similarities with RA or collagen-induced arthritis (CIA), a mouse model of RA. Methods Using a recently developed animal model of CHIKV arthritis in adult wild-type mice, we generated a consensus CHIKV arthritis gene expression signature, which was used to interrogate publicly available microarray studies of RA and CIA. Pathway analyses were then performed using the overlapping gene signatures. Results Gene set enrichment analysis showed that there was a highly significant overlap in the differentially expressed genes in the CHIKV arthritis model and in RA. This concordance also increased with the severity of RA, as measured by the inflammation score. A highly significant overlap was also seen between CHIKV arthritis and CIA. Pathway analysis revealed that the overlap between these arthritides was spread over a range of different inflammatory processes. Involvement of T cells and interferon-gamma (IFN gamma) in CHIKV arthritis was confirmed in studies of MHCII-deficient mice and IFN gamma-deficient mice, respectively. Conclusion These results suggest that RA, a chronic autoimmune arthritis, and CHIKV disease, usually a self-limiting viral arthropathy, share multiple inflammatory processes. New drugs and biologic therapies being developed for RA may thus find application in the treatment of alphaviral arthritides.

关键词： GENETICS of rheumatoid arthritis GENETICS of virus diseases ACADEMIC medical centers ANIMAL experimentation MICE POLYMERASE chain reaction FINANCING of research T-test (Statistics) GENOMICS EQUIPMENT & supplies REVERSE transcriptase polymerase chain reaction microarray technology

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Safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus Results of a phase I, placebo-controlled, double-blind, dose-escalation study

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ARTHRITIS AND RHEUMATISM 2012年第11期64卷 3666-3676页

作者： McBride, Jacqueline M. Jiang, Jenny Abbas, Alexander R. Morimoto, Alyssa Li, Jing Maciuca, Romeo Townsend, Michael Wallace, Daniel J. Kennedy, William P. Drappa, Jorn Genentech Inc San Francisco CA 94080 USA Univ Calif Los Angeles David Geffen Sch Med Los Angeles CA 90095 USA Cedars Sinai Med Ctr Los Angeles CA 90048 USA

Objective. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies and inflammation in multiple organ systems. Elevation of messenger RNA levels of interferon (IFN)-regulated genes (IRGs) has been described in the peripheral blood of SLE patients and has been associated with disease activity. The safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of rontalizumab, a humanized IgG1 monoclonal antibody that neutralizes IFN alpha, were assessed in a phase I dose-escalation study of single and repeat doses of rontalizumab in adults with mildly active SLE. The present report describes the safety results and the impact of rontalizumab on expression of IRGs, IFN-inducible proteins, and autoantibodies. Methods. Patients were enrolled into dose groups ranging from 0.3 to 10 mg/kg, administered via intravenous (IV) or subcutaneous routes. Expression levels of 7 IRGs and IFN-inducible serum proteins were monitored as potential biomarkers for the PD activity of rontalizumab. Results. An acceptable safety profile was demonstrated for rontalizumab in patients with SLE. Prespecified criteria for dose-limiting toxicity were not met. The incidence of serious adverse events was comparable across cohorts. The PK properties were as expected for an IgG1 monoclonal antibody and were proportional to dose. Following administration of rontalizumab, a rapid decline in the expression of IRGs was observed in the 3 mg/kg and 10 mg/kg IV cohorts, and this effect could be sustained with repeat dosing. There was no apparent decline in the levels of IFN-inducible proteins or levels of anti-double-stranded DNA and anti-extractable nuclear antigen autoantibodies following treatment with rontalizumab. Conclusion. The preliminary safety, PK profile, and observed PD effects of rontalizumab support further evaluation of its safety and efficacy in SLE.

关键词： BLOOD analysis CONFIDENCE intervals DOSE-response relationship (Biochemistry) GENETIC polymorphisms MONOCLONAL antibodies PHARMACOKINETICS PLACEBOS (Medicine) POLYMERASE chain reaction FINANCING of research SAFETY SYSTEMIC lupus erythematosus EQUIPMENT & supplies RANDOMIZED controlled trials BLIND experiment microarray technology

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Expression of COX-2 on Reed-Sternberg cells is an independent unfavorable prognostic factor in Hodgkin lymphoma treated with ABVD

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BLOOD 2012年第25期119卷 6072-6079页

作者： Mestre, Francisco Gutierrez, Antonio Ramos, Rafael Martinez-Serra, Jordi Sanchez, Lydia Matheu, Gabriel Ros, Teresa Garcia, Juan Fernando Rodriguez, Jose Univ Hosp Severo Ochoa Dept Internal Med Madrid 28911 Spain Univ Hosp Son Espases Dept Radiotherapy Palma De Mallorca Balearic Island Spain Univ Hosp Son Espases Dept Hematol Palma De Mallorca Balearic Island Spain Univ Hosp Son Espases Dept Pathol Palma De Mallorca Balearic Island Spain Spanish Natl Canc Res Ctr Madrid Spain

Cyclooxygenase 2 (COX-2) is an inflammatory enzyme involved in the pathogenesis and prognosis of several malignancies. In the present study, we investigated the prognostic value of COX-2 expression in a large (N = 242), uniformly treated Hodgkin lymphoma (HL) population from the Spanish Network of HL using tissue microarrays. Univariate and multivariate analysis was done, including comparing the most recognized clinical variables: the early- and advanced-stage subgroups. COX-2 was expressed on Reed-Sternberg cells in 37% of patients. There were no differences in the distribution of clinical variables according to COX-2 expression. With a median follow-up time of 58 months, PFS at 5 years was 60% and 79% for COX-2(+) and COX-2(-) patients, respectively (P = .003). The overall survival was 73% and 91%, respectively (P < .001). The major impact on prognosis was observed in the early AA stage (I-II) group. In fact, in these low-risk groups the expression of COX-2 defined a group with significantly worse progression-free and overall survival. In conclusion, COX-2 was expressed on Reed-Sternberg cells in one-third of HL patients and was a major independent, unfavorable prognostic factor in early-stage HL. We conclude that COX-2 may be a major prognostic variable in HL and a potential therapeutic target. (Blood. 2012;119(25):6072-6079)

关键词： CYCLOOXYGENASE 2 HODGKIN'S disease -- Treatment microarray technology FOLLOW-up studies (Medicine) DRUG therapy MULTIVARIATE analysis LYMPHOID tissue

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Genome-wide analysis of longevity in nutrient-deprived Saccharomyces cerevisiae reveals importance of recycling in maintaining cell viability

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ENVIRONMENTAL MICROBIOLOGY 2012年第5期14卷 1249-1260页

作者： Davey, Hazel M. Cross, Emma J. M. Davey, Christopher L. Gkargkas, Konstantinos Delneri, Daniela Hoyle, David C. Oliver, Stephen G. Kell, Douglas B. Griffith, Gareth W. Aberystwyth Univ Inst Biol Environm & Rural Sci Aberystwyth SY23 3DA Dyfed Wales Univ Cambridge Cambridge Syst Biol Ctr Cambridge CB2 1GA England Univ Cambridge Dept Biochem Cambridge CB2 1GA England Univ Manchester Fac Life Sci Manchester M13 9PT Lancs England Univ Manchester Manchester Interdisciplinary Bioctr Manchester M1 7DN Lancs England Univ Manchester Sch Chem Manchester M1 7DN Lancs England

Although typically cosseted in the laboratory with constant temperatures and plentiful nutrients, microbes are frequently exposed to much more stressful conditions in their natural environments where survival and competitive fitness depend upon both growth rate when conditions are favourable and on persistence in a viable and recoverable state when they are not. In order to determine the role of genetic heterogeneity in environmental fitness we present a novel approach that combines the power of fluorescence-activated cell sorting with barcode microarray analysis and apply this to determining the importance of every gene in the Saccharomyces cerevisiae genome in a high-throughput, genome-wide fitness screen. We have grown > 6000 heterozygous mutants together and exposed them to a starvation stress before using fluorescence-activated cell sorting to identify and isolate those individual cells that have not survived the stress applied. Barcode array analysis of the sorted and total populations reveals the importance of cellular recycling mechanisms (autophagy, pexophagy and ribosome breakdown) in maintaining cell viability during starvation and provides compelling evidence for an important role for fatty acid degradation in maintaining viability. In addition, we have developed a semi-batch fermentor system that is a more realistic model of environmental fitness than either batch or chemostat culture. Barcode array analysis revealed that arginine biosynthesis was important for fitness in semi-batch culture and modelling of this regime showed that rapid emergence from lag phase led to greatly increased fitness. One hundred and twenty-five strains with deletions in unclassified proteins were identified as being over-represented in the sorted fraction, while 27 unclassified proteins caused a haploinsufficient phenotype in semi-batch culture. These methods thus provide a screen to identifying other genes and pathways that have a role in maintaining cell viability.

关键词： SACCHAROMYCES cerevisiae FUNGAL genomes MICROBIAL viability counts MICROBIAL ecology BAR codes microarray technology FATTY acids CHEMOSTAT

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Fabrication of Microfluidic Devices Containing Patterned Microwell Arrays

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ANALYTICAL CHEMISTRY 2012年第3期84卷 1776-1780页

作者： Henley, W. Hampton Dennis, Patty J. Ramsey, J. Michael Univ N Carolina Dept Chem Chapel Hill NC 27599 USA

A rapid fabrication and prototyping technique to incorporate microwell arrays with sub-10 mu m features within a single layer of microfluidic circuitry is presented. Typically, the construction of devices that incorporate very small architecture within larger components has required the assembly of multiple elements to form a working device. Rapid, facile production of a working device using only a single layer of molded polydimethylsiloxane (PDMS) and a glass support substrate is achieved with the reported fabrication technique. A combination of conventional wet-chemical etching for larger (>= 20 mu m) microchannel features and focused ion beam (FIB) milling for smaller (<= 10 mu m) microwell features was used to fabricate a monolithic glass master mold. PDMS/glass hybrid chips were then produced using simple molding and oxygen plasma bonding methods. Microwell structures were loaded with 3 pm antibody-functionalized dye-encoded polystyrene spheres, and a sandwich immunoassay for common cytokines was performed to demonstrate proof-of-principle. Potential applications for this device include highly parallel multiplexed sandwich immunoassays, DNA/RNA hybridization analyses, and enzyme linked immunosorbent assay (ELISA). The fabrication technique described can be used for rapid prototyping of devices wherever submicrometer- to micrometer-sized features are incorporated into a microfluidic device.

关键词： MICROFLUIDIC devices MICROFABRICATION RAPID prototyping microarray technology MICROFLUIDIC analytical techniques IMMUNOASSAY NUCLEIC acid hybridization ENZYME-linked immunosorbent assay DESIGN & construction

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Direct Lipid Profiling of Single Cells from Inkjet Printed microarrays

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ANALYTICAL CHEMISTRY 2012年第22期84卷 9679-9683页

作者： Ellis, Shane R. Ferris, Cameron J. Gilmore, Kerry J. Mitchell, Todd W. Blanksby, Stephen J. Panhuis, Marc In Het Univ Wollongong Sch Chem ARC Ctr Excellence Free Rad Chem & Biotechnol Wollongong NSW 2522 Australia Univ Wollongong Sch Chem Soft Mat Grp Wollongong NSW 2522 Australia Univ Wollongong AIIM Facil ARC Ctr Excellence Electromat Sci Intelligent Polymer Res Inst Wollongong NSW 2522 Australia Univ Wollongong Sch Hlth Sci Wollongong NSW 2522 Australia

The on-demand printing of living cells using inkjet technologies has recently been demonstrated and allows for the controlled deposition of cells in microarrays. Here, we show that such arrays can be interrogated directly by robot-controlled liquid microextraction coupled with chip-based nanoelectospray mass spectrometry. Such automated analyses generate a profile of abundant membrane lipids that are characteristic of cell type. Significantly, the spatial control in both deposition and extraction steps combined with the sensitivity of the mass spectrometric detection allows for robust molecular profiling of individual cells.

关键词： LIPIDS microarray technology INK-jet printing EXTRACTION (Chemistry) MASS spectrometry MEMBRANE lipids CELL membranes IONIZATION (Atomic physics)

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Comparison of transcript profiles in different life stages of the nematode Globodera pallida under different host potato genotypes

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MOLECULAR PLANT PATHOLOGY 2012年第9期13卷 1120-1134页

作者： Palomares-Rius, Juan E. Hedley, Pete E. Cock, Peter J. A. Morris, Jenny A. Jones, John T. Vovlas, Nikos Blok, Vivian James Hutton Inst Dundee DD2 5DA Scotland UOS Bari CNR IPP I-70126 Bari Italy

The potato cyst nematodes (PCNs) Globodera pallida and Globodera rostochiensis are important parasites of potato. PCNs undergo complex biotrophic interactions with their hosts that involve gene expression changes in both the nematode and the host plant. The aim of this study was to determine key genes that are differentially expressed in Globodera pallida life cycle stages and during the initiation of the feeding site in susceptible and partially resistant potato genotypes. For this purpose, two microarray experiments were designed: (i) a comparison of eggs, infective second-stage juveniles (J2s) and sedentary parasitic-stage J2s (SJ2);(ii) a comparison of SJ2s at 8?days after inoculation (DAI) in the susceptible cultivar (Desiree) and two partially resistant lines. The results showed differential expression of G.?pallida genes during the stages studied, including previously characterized effectors. In addition, a large number of genes changed their expression between SJ2s in the susceptible cultivar and those infecting partially resistant lines;the number of genes with modified expression was lower when the two partially resistant lines were compared. Moreover, a histopathological study was performed at several time points (7, 14 and 30?DAI) and showed the similarities between both partially resistant lines with a delay and degeneration in the formation of the syncytia in comparison with the susceptible cultivar. Females at 30?DAI in partially resistant lines showed a delay in their development in comparison with those in the susceptible cultivar.

关键词： POTATOES -- Diseases & pests GLOBODERA pallida GOLDEN nematode GENE expression in plants microarray technology FERTILIZERS COMPARATIVE studies

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Chondrogenic progenitor cells respond to cartilage injury

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ARTHRITIS AND RHEUMATISM 2012年第11期64卷 3626-3637页

作者： Seol, Dongrim McCabe, Daniel J. Choe, Hyeonghun Zheng, Hongjun Yu, Yin Jang, Keewoong Walter, Morgan W. Lehman, Abigail D. Ding, Lei Buckwalter, Joseph A. Martin, James A. Univ Iowa Med Labs 1182 Iowa City IA 52242 USA VA Med Ctr Iowa City IA USA

Objective Hypocellularity resulting from chondrocyte death in the aftermath of mechanical injury is thought to contribute to posttraumatic osteoarthritis. However, we observed that nonviable areas in cartilage injured by blunt impact were repopulated within 714 days by cells that appeared to migrate from the surrounding matrix. The aim of this study was to assess our hypothesis that the migrating cell population included chondrogenic progenitor cells that were drawn to injured cartilage by alarmins. Methods Osteochondral explants obtained from mature cattle were injured by blunt impact or scratching, resulting in localized chondrocyte death. Injured sites were serially imaged by confocal microscopy, and migrating cells were evaluated for chondrogenic progenitor characteristics. Chemotaxis assays were used to measure the responses to chemokines, injury-conditioned medium, dead cell debris, and high mobility group box chromosomal protein 1 (HMGB-1). Results Migrating cells were highly clonogenic and multipotent and expressed markers associated with chondrogenic progenitor cells. Compared with chondrocytes, these cells overexpressed genes involved in proliferation and migration and underexpressed cartilage matrix genes. They were more active than chondrocytes in chemotaxis assays and responded to cell lysates, conditioned medium, and HMGB-1. Glycyrrhizin, a chelator of HMGB-1 and a blocking antibody to receptor for advanced glycation end products (RAGE), inhibited responses to cell debris and conditioned medium and reduced the numbers of migrating cells on injured explants. Conclusion Injuries that caused chondrocyte death stimulated the emergence and homing of chondrogenic progenitor cells, in part via HMGB-1 release and RAGE-mediated chemotaxis. Their repopulation of the matrix could promote the repair of chondral damage that might otherwise contribute to progressive cartilage loss.

关键词： CARTILAGE injuries ANALYSIS of variance ANIMAL experimentation CATTLE FLOW cytometry IMMUNOHISTOCHEMISTRY MICROSCOPY OSTEOARTHRITIS FINANCING of research STATISTICS T-test (Statistics) GENOMICS DATA analysis EQUIPMENT & supplies DATA analysis software microarray technology IN vitro studies

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Molecular diagnostic strategies: a role in the practice of dermatology

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INTERNATIONAL JOURNAL OF DERMATOLOGY 2012年第11期51卷 1292-1302页

作者： Elaba, Zendee Phelps, Amanda Murphy, Michael J. Univ Connecticut Ctr Hlth Dept Dermatol Farmington CT 06030 USA Hartford Hosp Dept Pathol Hartford CT 06115 USA

Molecular diagnostic strategies are gaining wider acceptance and use in dermatology and dermatopathology as more practitioners in this field develop an understanding of the principles and applications of genomic technologies. Molecular testing is facilitating more accurate diagnosis, staging, and prognostication, in addition to guiding the selection of appropriate treatment, monitoring of therapy, and identification of novel therapeutic targets, for a wide variety of skin diseases.

关键词： DERMATOLOGY MOLECULAR diagnosis PROGNOSIS SKIN diseases -- Diagnosis microarray technology

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