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Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

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NEW BIOtechnology 2016年第5期33卷 574-581页

作者： Kibat, Janek Schirrmann, Thomas Knape, Matthias J. Helmsing, Saskia Meier, Doris Hust, Michael Schroeder, Christoph Bertinetti, Daniela Winter, Gerhard Pardes, Khalid Funk, Mia Vala, Andrea Giese, Nathalia Herberg, Friedrich W. Duebel, Stefan Hoheisel, Joerg D. Deutsch Krebsforschungszentrum DKFZ Div Funct Genome Anal Neuenheimer Feld 580 D-69120 Heidelberg Germany Univ Munich Dept Pharm Pharmaceut Technol & Biopharmaceut Butenandtstr 5 D-81377 Munich Germany Tech Univ Carolo Wilhelmina Braunschweig Inst Biochem Biotechnol & Bioinformat Dept Biotechnol Spielmannstr 7 D-38106 Braunschweig Germany YUMAB GmbH Rebenring 33 D-38106 Braunschweig Germany Univ Kassel Dept Biochem Heinrich Plett Str 40 D-34132 Kassel Germany Sciom GmbH Neuenheimer Feld 583 D-69120 Heidelberg Germany Univ Copenhagen Fac Hlth & Med Sci Prot Struct & Funct Program Novo Nordisk Fdn Ctr Prot Res Blegdamsvej 3B DK-2200 Copenhagen Denmark Univ Heidelberg Hosp Dept Surg Neuenheimer Feld 110 D-69120 Heidelberg Germany

Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis.

关键词： microarray technology IMMUNOGLOBULINS GLUTAMATE receptors IMMUNOHISTOCHEMISTRY ADENOCARCINOMA

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Differential Site-Based Expression of Pentose Phosphate Pathway-Related Proteins among Breast Cancer Metastases

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DISEASE MARKERS 2017年第20170101期2017卷 7062517-7062517页

作者： Cha, Yoon Jin Jung, Woo Hee Koo, Ja Seung Yonsei Univ Coll Med Gangnam Severance Hosp Dept Pathol 211 Eonju Ro Seoul 06273 South Korea Yonsei Univ Coll Med Severance Hosp Dept Pathol 50-1 Yonsei Ro Seoul 03722 South Korea

Purpose. We aimed to investigate the expression of pentose phosphate pathway-(PPP-) related proteins in metastatic breast cancer and its relationship with clinicopathologic factors. Methods. Tissue samples from 126 metastatic breast cancers were included in a tissuemicroarray. Expression of PPP-related proteins [glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), 6-phosphogluconate dehydrogenase (6PGDH), and nuclear factor erythroid 2-related factor (NRF2)] was determined by immunohistochemistry. Results. G6PDH (p = 0.011) and cytoplasmic NRF2 (p = 0.001) showed the highest expression in brain metastases. Human epidermal growth factor receptor (HER-2) positivity was associated with G6PDH (p < 0.001) and cytoplasmic NRF2 (p = 0.015) positivity. A high Ki-67 labeling index (LI) was correlated with nuclear NRF2 positivity (p = 0.037), and HER-2-positive luminal B type was associated with G6PDH positivity (p = 0.001). On multivariate Cox analysis, independent risk factors of short overall survival were 6PGL positivity in bone metastasis (HR 4.180, 95% CI 1.160-15.06, p = 0.029) and low Ki-67 LI in lung metastasis (HR 11.853, 95% CI 1.841-76.30, p = 0.009). Conclusion. Differential expression of PPP-related proteins correlated with different prognoses and metastatic sites, with the highest expression in brain metastases, and could be a potential therapeutic target.

关键词： BREAST cancer PENTOSE phosphate pathway PROTEIN expression microarray technology IMMUNOHISTOCHEMISTRY

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Gastric Cancer Associated Genes Identified by an Integrative Analysis of Gene Expression Data

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BIOMED RESEARCH INTERNATIONAL 2017年第20170101期2017卷 7259097-7259097页

作者： Jiang, Bing Li, Shuwen Jiang, Zhi Shao, Ping Hosp Tradit Chinese Med Dept Spleen & Stomach Dis Yixing 214200 Jiangsu Peoples R China Soochow Univ Affiliated Hosp 1 Dept Gastroenterol Suzhou 215006 Peoples R China Soochow Univ Sch Med Dept Biochem & Mol Biol Suzhou 215123 Jiangsu Peoples R China

Gastric cancer is one of the most severe complex diseases with high morbidity and mortality in the world. The molecular mechanisms and risk factors for this disease are still not clear since the cancer heterogeneity caused by different genetic and environmental factors. With more and more expression data accumulated nowadays, we can perform integrative analysis for these data to understand the complexity of gastric cancer and to identify consensus players for the heterogeneous cancer. In the present work, we screened the published gene expression data and analyzed them with integrative tool, combined with pathway and gene ontology enrichment investigation. We identified several consensus differentially expressed genes and these genes were further confirmed with literature mining;at last, two genes, that is, immunoglobulin J chain and C-X-C motif chemokine ligand 17, were screened as novel gastric cancer associated genes. Experimental validation is proposed to further confirm this finding.

关键词： STOMACH tumors GENE expression META-analysis PATH analysis (Statistics) FINANCING of research DATA analysis software microarray technology DESCRIPTIVE statistics GENETIC aspects

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Elevated IgA Plasmablast Levels in Subjects at Risk of Developing Rheumatoid Arthritis

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ARTHRITIS & RHEUMATOLOGY 2016年第10期68卷 2372-2383页

作者： Kinslow, Jennifer D. Blum, Lisa K. Deane, Kevin D. Demoruelle, M. Kristen Okamoto, Yuko Parish, Mark C. Kongpachith, Sarah Lahey, Lauren J. Norris, Jill M. Robinson, William H. Holers, V. Michael Univ Colorado Sch Med Aurora CO USA Stanford Univ Stanford CA 94305 USA VA Palo Alto Hlth Care Syst Palo Alto CA USA Colorado Sch Publ Hlth Aurora CO USA Univ Colorado Aurora CO USA

ObjectiveThe disease process in rheumatoid arthritis (RA) starts years before the clinical diagnosis is made, and elevated levels of disease-specific autoantibodies can be detected during this period. Early responses to known or novel autoantigens likely drive the eventual production of pathogenic autoimmunity. Importantly, the presence of disease-specific autoantibodies can identify individuals who are at high risk of developing RA but who do not currently have arthritis. The goal of the current study was to characterize plasmablasts from individuals at risk of developing RA. MethodsWe investigated antibody-secreting plasmablasts derived from a well-characterized cohort of individuals who were at risk of developing RA, based on RA-related serum autoantibody positivity, as compared to patients with early (<1 year) seropositive RA as well as healthy control subjects. The plasmablast antibody repertoires of at-risk subjects were analyzed using DNA barcode-based methods with paired heavy- and light-chain gene sequencing. Cells were single-cell sorted, the cell- and plate-specific DNA barcodes were sequentially added, and next-generation sequencing was performed. ResultsTotal plasmablast levels were similar in the antibody-positive (1%) and control (0.4-1.6%) groups. However, increased frequencies of IgA+ versus IgG+ plasmablasts were observed in the antibody-positive group (39% IgA+ and 37% IgG+) as compared to other groups (1-9% IgA+ and 71-87% IgG+). Paired antibody sequences from antibody-positive subjects revealed cross-isotype clonal families and similar sequence characteristics in the IgA and IgG plasmablast repertoires. Antibody-positive individuals also demonstrated elevated serum levels of IgA isotype anti-cyclic citrullinated peptide 3 antibodies. ConclusionThe IgA plasmablast dominance in these antibody-positive individuals suggests that a subset of RA-related autoantibodies may arise from mucosal immune responses and may be involved in early disease pathoge

关键词： AUTOANTIBODY analysis RHEUMATOID arthritis risk factors ANALYSIS of DNA AUTOANTIBODIES B cells CHI-squared test COMPARATIVE studies FISHER exact test PROBABILITY theory FINANCING of research RHEUMATOID arthritis STATISTICAL hypothesis testing T-test (Statistics) EARLY diagnosis DATA analysis software microarray technology GENE expression profiling DESCRIPTIVE statistics MANN Whitney U Test KRUSKAL-Wallis Test ONE-way analysis of variance

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Regulation of Cholesterol Homeostasis by Hedgehog Signaling in Osteoarthritic Cartilage

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ARTHRITIS & RHEUMATOLOGY 2016年第1期68卷 127-137页

作者： Ali, Shabana Amanda Al-Jazrawe, Mushriq Ma, Henry Whetstone, Heather Poon, Raymond Farr, Sarah Naples, Mark Adeli, Khosrow Alman, Benjamin A. Univ Toronto Toronto ON Canada Hosp Sick Children Toronto ON M5G 1X8 Canada Duke Univ Durham NC USA

ObjectiveWith no effective therapies to attenuate cartilage degeneration in osteoarthritis (OA), the result is pain and disability. Activation of hedgehog (HH) signaling causes changes related to the progression of OA, with higher levels of Gli-mediated transcriptional activation associated with increased disease severity. To elucidate the mechanism through which this occurs, this study sought to identify genes regulated by HH signaling in human OA chondrocytes. MethodsUsing human OA cartilage samples, microarray analyses were performed to detect changes in gene expression when the HH pathway was modulated. Results were analyzed for differentially expressed genes, grouped into functional networks, and validated in independent samples. To investigate the effects of chondrocyte-specific sterol accumulation, we generated mice lacking Insig1 and Insig2, which are major negative regulators of cholesterol homeostasis, under Col2a1 regulatory elements. ResultsHH signaling was found to regulate genes that govern cholesterol homeostasis, and this led to alterations in cholesterol accumulation in chondrocytes. A higher level of Gli-mediated transcription resulted in accumulation of intracellular cholesterol. In genetically modified mice, chondrocyte-specific cholesterol accumulation was associated with an OA phenotype. Reducing cholesterol accumulation attenuated the severity of OA in mice in vivo and decreased the expression of proteases in human OA cartilage in vitro. ConclusionHH signaling regulates cholesterol homeostasis in chondrocytes, and intracellular cholesterol accumulation contributes to the severity of OA. Our findings have therapeutic implications, since reduction of HH signaling reversed cholesterol accumulation and statin treatment attenuated cartilage degeneration.

关键词： ANIMAL experimentation CARTILAGE diseases CARTILAGE cells CHOLESTEROL CONFIDENCE intervals GENE expression GROWTH factors HISTOLOGICAL technique HOMEOSTASIS MICE OSTEOARTHRITIS POLYMERASE chain reaction FINANCING of research T-test (Statistics) WESTERN immunoblotting microarray technology GENE expression profiling DESCRIPTIVE statistics MANN Whitney U Test DISEASE complications

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RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site

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BIOMED RESEARCH INTERNATIONAL 2017年第20170101期2017卷 9596152-9596152页

作者： Cheng, Feng Pan, Ying Lu, Yi-Min Zhu, Lei Chen, Shuzheng Wenzhou Med Univ Affiliated Hosp 5 Zhejiang UnivDept Thyroid Breast Surg Lishui HospLishui Municipal Cent Hosp Wenzhou 323000 Zhejiang Peoples R China Zhejiang Univ Coll Med Affiliated Hosp 1 Dept Oncol Surg Hangzhou 310003 Zhejiang Peoples R China

RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated (p = 0.04). Patients with higher Dnd1 expression level had longer overall survival (p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim-mRNA 3' UTR, the stability of Bim-5' UTR was not affected. In addition, mutation of miR-221-binding site in Bim3' UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221 inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3' UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

关键词： RNA physiology APOPTOSIS BINDING sites (Biochemistry) BIOCHEMISTRY BREAST tumors CARRIER proteins CELL culture ETHICS GENOMES GERM cells POLYMERASE chain reaction FINANCING of research T-test (Statistics) WESTERN immunoblotting microarray technology

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Joint Covariate Detection on Expression Profiles for Selecting Prognostic miRNAs in Glioblastoma

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BIOMED RESEARCH INTERNATIONAL 2017年第20170101期2017卷 3017948-3017948页

作者： Sun, Chengqi Zhao, Xudong Northeast Forestry Univ Coll Informat & Comp Engn Harbin 150001 Peoples R China

An important application of expression profiles is to stratify patients into high-risk and low-risk groups using limited but key covariates associated with survival outcomes. Prior to that, variables considered to be associated with survival outcomes are selected. A combination of single variables, each of which is significantly related to survival outcomes, is always regarded to be candidates for posterior patient stratification. Instead of individually significant variables, a combination that contains not only significant but also insignificant variables is supposed to be concentrated on. By means of bottom-up enumeration on each pair of variables, we propose a joint covariate detection strategy to select candidates that not only correspond to close association with survival outcomes but also help to make a clear stratification of patients. Experimental results on a publicly available dataset of glioblastoma multiforme indicate that the selected pair composed of an individually significant and an insignificant miRNA keeps a better performance than the combination of significant single variables. The selected miRNA pair is ultimately regarded to be associated with the prognosis of glioblastoma multiforme by further pathway analysis.

关键词： GLIOMAS PROGNOSIS RNA analysis GENE expression SURVIVAL analysis (Biometry) SAMPLE size (Statistics) PROPORTIONAL hazards models microarray technology KAPLAN-Meier estimator LOG-rank test GENETIC aspects

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In Situ Ion-Transmission Mass Spectrometry for Paper-Based Analytical Devices

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ANALYTICAL CHEMISTRY 2016年第22期88卷 10805-10810页

作者： Zhao, Yaoyao Wei, Zhenwei Zhao, Hansen Jia, Jia Chen, Zhenzhen Zhang, Sichun Ouyang, Zheng Ma, Xiaoxiao Zhang, Xinrong Tsinghua Univ Dept Chem Beijing 100084 Peoples R China Shandong Normal Univ Collaborat Innovat Ctr Functionalized Probes Chem Jinan 250014 Peoples R China Tsinghua Univ Dept Precis Instrument Beijing 100084 Peoples R China

Current detection methods for paper-based analytical devices (PADs) rely on spectroscopic and electrochemical properties, which place special requirements on the analyte or need analyte labeling. Here, ion-transmission mass spectrometry (MS) was proposed for coupling with PADs to enable rapid in situ MS analysis of the sample on paper. The sample was analyzed directly on paper via analyte ionization by ions transmitted through the paper, generated by a low temperature plasma probe. Prior to MS analysis, the sample can be separated by paper electrophoresis or by paper chromatography, among a variety of other features offered by PADs. The versatility of this technique was demonstrated by MS analysis of a paper microarray, a mixture of amino acids, and whole blood doped with drugs on PADs.

关键词： MASS spectrometry NUCLEAR spectroscopy microarray technology ELECTROPHORESIS DESORPTION electrospray ionization

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Sexual Dimorphism in the Th17 Signature of Ankylosing Spondylitis

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ARTHRITIS & RHEUMATOLOGY 2016年第3期68卷 679-689页

作者： Gracey, Eric Yao, YuChen Green, Blerta Qaiyum, Zoya Baglaenko, Yuriy Lin, Aifeng Anton, Ammepa Ayearst, Renise Yip, Paul Inman, Robert D. Univ Toronto Toronto Western Hosp Toronto ON M5T 2S8 Canada Univ Hlth Network Toronto ON Canada Toronto Western Hosp Toronto ON M5T 2S8 Canada

Objective. To identify an immunologic basis for the male sex bias in ankylosing spondylitis (AS). Methods. Cohorts of male and female patients with AS and age- and sex-matched healthy control subjects were selected, and the levels of serum cytokines (interferon-gamma [IFN gamma], tumor necrosis factor alpha , interleukin-17A [IL-17A], and IL-6) were examined by enzyme-linked immunosorbent assay, the frequencies of Th1 and Th17 cells were assessed by flow cytometry, and whole blood gene expression was analyzed using both microarray and NanoString approaches. Results. The frequency of IL-17A and Th17 cells, both of which are key factors in the inflammatory Th17 axis, was elevated in male patients with AS but not in female patients with AS. In contrast, AS-associated alterations in the Th1 axis, such as the frequency of IFN gamma and Th1 cells in serum, were independent of a patient's sex. Results of microarray analysis supported an altered Th17 axis in male patients, with a specific increase in IL17RA. In addition, male and female patients with AS displayed shared gene expression patterns, while male patients with AS had additional alterations in gene expression that were not seen in female patients with AS. The differential sex-related immune profiles were independent of HLA-B27 status, clinical disease activity (as measured by the Bath Ankylosing Spondylitis Disease Activity Index), or treatment (with nonsteroidal antiinflammatory drugs or biologic agents), implicating intrinsic sexual dimorphism in AS. Conclusion. The results of this study demonstrate distinct sexual dimorphism in the activation status of the immune system in patients with AS, particularly in the Th17 axis. This dimorphism could underlie sex-related differences in the clinical features of AS and could provide a rationale for sex-specific treatment of AS.

关键词： ANKYLOSING spondylitis CYTOKINES ENZYME-linked immunosorbent assay FLOW cytometry GENE expression GENETIC polymorphisms INTERLEUKINS POLYMERASE chain reaction FINANCING of research SEX distribution (Demography) T-test (Statistics) TUMOR necrosis factor SEVERITY of illness index CASE-control method DATA analysis software microarray technology GENE expression profiling DESCRIPTIVE statistics GENETIC aspects

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Endochondral Growth Defect and Deployment of Transient Chondrocyte Behaviors Underlie Osteoarthritis Onset in a Natural Murine Model

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ARTHRITIS & RHEUMATOLOGY 2016年第4期68卷 880-891页

作者： Staines, K. A. Madi, K. Mirczuk, S. M. Parker, S. Burleigh, A. Poulet, B. Hopkinson, M. Bodey, A. J. Fowkes, R. C. Farquharson, C. Lee, P. D. Pitsillides, A. A. Univ London Royal Vet Coll London England Univ Edinburgh Roslin Inst Easter Bush England Univ Edinburgh Royal Dick Sch Vet Studies Easter Bush England Univ Manchester Xray Imaging Facil Manchester Lancs England UCL Sch Med London NW1 0TU England Harwell Sci & Innovat Campus Diamond Light Source Didcot Oxon England

ObjectiveTo explore whether aberrant transient chondrocyte behaviors occur in the joints of STR/Ort mice (which spontaneously develop osteoarthritis [OA]) and whether they are attributable to an endochondral growth defect. MethodsKnee joints from STR/Ort mice with advanced OA and age-matched CBA (control) mice were examined by Affymetrix microarray profiling, multiplex polymerase chain reaction (PCR) analysis, and immunohistochemical labeling of endochondral markers, including sclerostin and MEPE. The endochondral phenotype of STR/Ort mice was analyzed by histologic examination, micro-computed tomography, and ex vivo organ culture. A novel protocol for quantifying bony bridges across the murine epiphysis (growth plate fusion) using synchrotron x-ray computed microtomography was developed and applied. ResultsMeta-analysis of transcription profiles showed significant elevation in functions linked with endochondral ossification in STR/Ort mice (compared to CBA mice;P < 0.05). Consistent with this, immunolabeling revealed increased matrix metalloproteinase 13 (MMP-13) and type X collagen expression in STR/Ort mouse joints, and multiplex quantitative reverse transcriptase-PCR showed differential expression of known mineralization regulators, suggesting an inherent chondrocyte defect. Support for the notion of an endochondral defect included accelerated growth, increased zone of growth plate proliferative chondrocytes (P < 0.05), and widespread type X collagen/MMP-13 labeling beyond the expected hypertrophic zone distribution. OA development involved concomitant focal suppression of sclerostin/MEPE in STR/Ort mice. Our novel synchrotron radiation microtomography method showed increased numbers (P < 0.001) and mean areal growth plate bridge densities (P < 0.01) in young and aged STR/Ort mice compared to age-matched CBA mice. ConclusionTaken together, our data support the notion of an inherent endochondral defect that is linked to growth dynamics and subject to regulation b

关键词： ANIMAL experimentation CARTILAGE cells COMPUTED tomography ENZYME-linked immunosorbent assay HISTOLOGICAL technique HUMAN growth IMMUNOHISTOCHEMISTRY KNEE MICE OSTEOARTHRITIS POLYMERASE chain reaction FINANCING of research T-test (Statistics) PHENOTYPE THREE-dimensional imaging DATA analysis software microarray technology DESCRIPTIVE statistics MATRIX metalloproteinases

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