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Development of a DNA macroarray for the molecular biological identification of trade-relevant tropical CITES timber species and their look-alikes

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HOLZFORSCHUNG 2024年第9期78卷 471-486页

作者： Bogun, Anna C. Paredes-Villanueva, Kathelyn Mascarello, Maurizio Magel, Elisabeth A. Univ Hamburg Inst Wood Sci Leuschnerstr 91d D-21031 Hamburg Germany Columbia Univ Lamont Doherty Earth Observ Palisades NY USA Univ Autonoma Gabriel Rene Moreno Fac Ciencias Agr Carrera Ingn Forestal Km 9 Al Norte El Vallecito Santa Cruz Bolivia Meise Bot Garden Meise Belgium Katholieke Univ Leuven Dept Biol Leuven Belgium Fdn Edmund Mach Res & Innovat Ctr Unity Plant Epigenet San Michele All Adige Italy

The control authorities of timber trade lack quick and easy methods to verify the legality of the traded product. This includes the clear identification of the timber species. In this study, a simple genetic method, the macroarray technique, was modified and optimised for this purpose. The internal transcribed spacer (ITS) was used as barcode region. Specific probes were designed and tested using this barcode of trade-relevant, CITES protected wood species and their look-alikes. The results reveal that 61 of these probes are suitable for identification purposes. Hence, 32 of the 50 investigated species can be clearly identified using the developed macroarray. Additional 15 species are represented by probes in groups of two to three species, which enables a delimitation of the species present. For four species, no identification was possible applying the probes created. The findings indicate the potential of time and cost savings compared to currently established methods in species control in the timber trade.

关键词： DNA barcoding illegal logging internal transcribed spacer (ITS) macroarray wood identification

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Screening asthmatics for atopic status using the ALergy EXplorer (ALEX²) macroarray

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JOURNAL OF ASTHMA 2024年第9期61卷 1050-1057页

作者： Hamilton, Robert G. Holbreich, Mark Bronzert, Charles Anderson, Rebecca L. Schoettler, Nathan Ober, Carole Johns Hopkins Univ Sch Med Johns Hopkins Asthma & Allergy Ctr Baltimore MD USA Allergy & Asthma Consultants Indianapolis IN USA Univ Chicago Dept Human Genet Chicago IL USA Univ Chicago Chicago IL USA Johns Hopkins Univ Johns Hopkins Asthma & Allergy Ctr Sch Med Room 1A20 Baltimore MD 21218 USA

Background :Screening asthma patients for atopy facilitates management. Since 2010, the core biomarker for screening asthma subjects for atopic status has been the qualitative Phadiatop. multi-aeroallergen screen. A more quantitative macroarray, the Allergy Explorer (ALEX(2)), shows promise as an alternative. Objective: The study's goal was to examine the pros and cons of the use of ALEX(2) in the screening of asthma patients for atopic status. Methods: We evaluated the atopic (IgE-sensitization) status in asthmatic Amish and Hutterite farm children using the ImmunoCAP and ALEX2 assays in Phadiatop equivocal and positive subjects. Results: All 42 asthmatic children were analyzed by Phadiatop and total serum IgE. Of these, 22 had a negative Phadiatop (<0.1 kUa/L) and total IgE <100 kU/L which defined them as non-atopic and they were excluded from ALEX(2) testing. Of six children with equivocal Phadiatops (0.1-0.2 kUa/L-Group 1) and three children with a negative Phadiatop but total IgE >100 kUa/L (group 3), 44% (n = 4) had detectable IgE antibody by ALEX(2) to mite, tree pollen, and other allergens not detected by Phadiatop, but confirmed by allergen-specific ImmunoCAP testing. In 11 Phadiatop positive subjects (>0.2 kUa/L-group 2), all but one were positive by ALEX(2). IgE antibody specific for mold and rabbit aeroallergens matched their agricultural and pet exposure history. Three children were positive for IgE antibody to allergens in the profilin, nsLTP, or PR-10 cross-reactive protein families. Conclusion: Judicious use of ALEX(2)'s enhanced specificity data not provided by the Phadiatop can aid in the interpretation of sensitization patterns and planning management of atopic asthmatics, but sensitization relevance must be confirmed by the patient's clinical history.

关键词： Phadiatop multiallergen screen singleplex ALEX(2) macroarray atopy

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A microarray-based IgE-molecular mimicry index (IgE-MMI): A biomarker for disease severity, clinical phenotypes, and therapeutic response in atopic dermatitis?

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ALLERGY 2024年第12期79卷 3415-3429页

作者： Scala, Enrico Madonna, Stefania Abeni, Damiano Cecchi, Lorenzo Cocuroccia, Barbara Dattolo, Anna Moretta, Gaia Provini, Alessia Russo, Filomena Sordi, Donatella Pallotta, Sabatino Galluzzo, Marco Talamonti, Marina Villella, Valeria Giani, Mauro Caprini, Elisabetta Albanesi, Cristina Villalta, Danilo Asero, Riccardo Matricardi, Paolo Maria Fdn Luigi Maria Monti IDI IRCCS Rome Italy USL Toscana Ctr SOSD Allergol & Clin Immunol Prato Italy Asl Roma2 Osped Sandro Pertini Rome Italy Fdn Policlin Tor Vergata Dermatol Unit Rome Italy Tor Vergata Univ Rome Dept Syst Med Rome Italy PO S Maria degli Angeli SC Immunol & Allergol Lab Pordenone Italy Clin San Carlo Ambulatorio Allergol Milan Italy Charite Univ Med Berlin Dept Pediat Resp Med Immunol & Crit Care Med Berlin Germany Free Univ Berlin Berlin Germany Humboldt Univ Berlin Germany Charite Univ Med Berlin Inst Allergol Berlin Germany Fraunhofer Inst Translat Med & Pharmacol ITMP Dept Allergol & Immunol Berlin Germany

BackgroundThe role of autoimmune IgE responses in atopic dermatitis (AD) is highly debated. While IgE targeting self-proteins has been extensively studied, IgE responses induced by human-homologous exogenous molecular allergens (HEMAs) remains less *** investigate whether IgE antibody responses to HEMAs are associated with AD, its severity, and response to *** enrolled 3325 participants with a history of allergic diseases, including 577 (17.3%) diagnosed with AD. Serum IgE antibodies against 183 exogenous allergenic molecules were measured using the IgE microarray (Allergy Explorer-ALEX-2 (R), MADX, Vienna). Based on international classification criteria, participants were stratified by AD severity and clinical phenotypes. For each patient, we developed an 'IgE molecular-mimicry index' (IgE-MMI), calculated from IgE reactivity to a panel of five HEMA protein families: arginine kinase, enolase (ENO), cyclophilin (CYP), lipocalin, and MnSOD. Logistic regression was employed to assess the association between IgE to HEMAs or IgE-MMI and AD, its severity, and response to *** sensitization to most HEMAs (32/48, 67%), but only to a small fraction of non-HEMAs (3/135, 2.2%), was significantly more common in patients with severe AD compared to other patient groups. The IgE-MMI was positive in 295/2748 (10.7%) of allergic patients without AD, and in 58/283 (20%), 52/134 (39%), and 86/160 (54%) of patients with remitting, moderate, or severe AD, respectively. It was strongly associated with specific phenotypes, such as flexural dermatitis (OR 8.4, 95% CI: 6.3-11.2), head and neck dermatitis (OR: 16.5, 95% CI: 7.4-37.2), and generalized eczema (OR: 8.6, 95% CI: 4.9-15.6). Poor response to dupilumab was associated with IgE antibodies to ENO (OR: 22.7, 95% CI: 1.7-302.9), but inversely associated with IgE antibodies to MnSOD (OR: 0.1, 95% CI: 0.02-0.8) and NPC-2 from dust mites (OR: 0.1, 95% CI: 0.01-0.9).ConclusionIgE microarrays are

关键词： atopic dermatitis autoreactivity IgE macroarray molecular mimicry

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macroarray assay for differential diagnosis of meningoencephalitis in southern Africa

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JOURNAL OF CLINICAL VIROLOGY 2014年第1期60卷 50-56页

作者： Venter, Marietjie Zaayman, Dewald van Niekerk, Stephanie Stivaktas, Voula Goolab, Shivani Weyer, Jacqueline Paweska, Janusz T. Swanepoel, Robert Univ Pretoria Zoonoses Res Unit ZA-0001 Pretoria South Africa Natl Hlth Lab Serv Ctr Emerging & Zoonot Dis Natl Inst Communicable Dis Special Viral Pathogens Reference Lab Johannesburg South Africa Natl Hlth Lab Serv Ctr Resp Dis & Meningitis Natl Inst Communicable Dis Johannesburg South Africa

Background: Many cases of acute febrile illness with nervous manifestations go undiagnosed, partly because the potential pathogens are not investigated routinely. Objective: To develop a multiplex PCR-based macroarray for detection of 29 pathogens associated with febrile disease, aseptic meningitis and meningoencephalitis in southern Africa, including common viruses, bacteria, parasites and selected arboviruses. Study design: Pathogens were identified by hybridization of PCR amplicons to probes on a macroarray chip, followed by colorimetric detection. Results: Positive control specimens for all 29 targets were detected with high sensitivity. Twenty-seven clinical samples previously found positive for various etiologies of febrile disease and meningoencephalitis, including less common infections such as Crimean Congo haemorrhagic fever, Rift Valley fever, West Nile and rabies were all identified. Testing of a blinded panel of 16 specimens in triplicate demonstrated high repeatability. Screening of 138 specimens from patients with febrile and/or neurological signs that could not be solved in routine investigations yielded 5 additional diagnoses. Conclusion: A highly sensitive, specific and easily interpreted test was developed to detect 29 pathogens associated with febrile and nervous disease. (c) 2014 Published by Elsevier B.V.

关键词： Differential diagnosis Meningoencephalitis multiplex PCR macroarray

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Detection of Plant Viruses in Mixed Infection by a macroarray-Assisted Method

Detection of Plant Viruses in Mixed Infection by a Macroarra...

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作者： Hanako Shimura Kazuyoshi Furuta Chikara Masuta

The protocol for a simple, sensitive, and specific method using a cDNA macroarray to detect multiple viruses is provided. The method can be used even at the production sites for crops, which need a reliable routine diagnosis for mixed infection of plant viruses. The method consists of three steps: RNA extraction, duplex RT-PCR, and “microtube hybridization” (MTH). Biotinylated cDNA probes are prepared using RT-PCR and used to hybridize a nylon membrane containing target viral cDNAs by MTH. Positive signals can be visualized by colorimetric reaction and judged by eyes. We here demonstrate this method to detect asparagus viruses (Asparagus virus 1 and Asparagus virus 2) from latently infected asparagus plants. less

关键词： RNA Extraction Reverse Transcription PCR Array Printing Liquid Handling Electrophoresis Asparagus virus 1 Asparagus virus 2 Viruses Escherichia coli macroarray Microtube hybridization

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Tomato chlorosis virus infection represses chloroplast related genes in tomato.

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PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY 2021年 116卷 101722-101722页

作者： Cevik, Bayram Sonmez, Ezgi Sahin-Cevik, Mehtap Isparta Univ Appl Sci Dept Plant Protect Fac Agr TR-32260 Isparta Turkey Isparta Univ Appl Sci Dept Agr Biotechnol Fac Agr TR-32260 Isparta Turkey

Tomato chlorosis virus (ToCV) causes mild to severe chlorosis on tomato leaves. Although it has been postulated that chlorosis is caused by changes in biosynthesis, degradation, function and/or structure of chlorophyll, mechanisms of virus-induced chlorosis has not been explored to date. To understand the mechanisms of virus induced chlorosis, ToCV-tomato interaction was studied at transcriptome level using suppression subtractive hybridization (SSH) followed by macroarray hybridization and RT-qPCR. First, a susceptible tomato cultivar, MoneyMaker was graft-inoculated with ToCV. Next, cDNAs were synthesized from mRNAs of leaf samples from ToCV-inoculated and mock-inoculated control plants at 30-day of post inoculation (dpi). Then, a cDNA library enriched with ToCV-repressed genes was constructed using SSH. 250 randomly selected clones from library were sequenced and the expressions of 140 genes were determined by nylon macroarray hybridizations. The expression of 119 genes were significantly decreased by 1.5 or greater fold in response to ToCV infection. The expressions of many genes involved in transcription, defense response, metabolism, and photosynthesis, signaling and protein fate were repressed upon infection with ToCV. The genes repressed by ToCV in tomato were mostly associated with organelles, especially with the chloroplast. After expression analysis of 40 selected ToCV-repressed genes by RT-qPCR assay, the repression of 37 genes in response to ToCV infection was confirmed. Furthermore, a detail expression analysis of ferredoxin 2 (Fd2) gene between 0 dpi and 45 dpi in ToCV-inoculated and mock-inoculated control plants showed that the expression of Fd-2 gene was significantly repressed at 8 to 30 dpi in ToCV-infected plants.

关键词： ToCV Host-pathogen interaction Suppression subtractive hybridization macroarray Gene expression RT-qPCR

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The Use of macroarray as a Simple Tool to Follow the Metabolic Profile of Lactobacillus plantarum during Fermentation

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Advances in Microbiology 2014年第14期4卷 996-1016页

作者： Minna Kahala Virpi Ahola Elina Makimattila Lars Paulin Vesa Joutsjoki Biotechnology and Food Research MTT Agrifood Research FinlandJokioinenFinland Department of Biosciences University of HelsinkiHelsinkiFinland Institute of Biotechnology University of HelsinkiHelsinkiFinland

This study focused on defining the differences in L. plantarum gene expression levels in different media and in different growth phases using an easy and cost-efficient monitoring of gene expression. A macroarray based on a group of selected L. plantarum genes, 178 genes belonging to 18 main groups, printed onto a nitrocellulose filter was designed in this work. Using the macrofilters designed, the expression of a selected set of L. plantarum genes was assayed in synthetic MRS medium and in extracted carrot juice. To compare the potential differences of starter gene expression in hygienic and contaminated cultivation media, the L. plantarum strain was cultivated in both sterile and contaminated (yeast and Escherichia coli) MRS and carrot juice. The number of genes found to be regulated as a function of growth was clearly higher in MRS-based growth medium than in carrot juice, In carrot juice, expression of the gene encoding malolactic enzyme (MLE), which makes L. plantarum an advantageous microbe in e.g. wine making, was found to be upregulated in logarithmic phase of growth. The current study demonstrated that macroarrays printed on nitrocellulose filters with simple robotic systems can be analyzed by standard laboratory equipment and methods usually available in molecular laboratories. Using this technology, rapid and cost-efficient analysis of genome function of L. plantarum can be carried out e.g. in developing regions, where lactic acid fermentation of food and feed matrices is a common practice.

关键词： macroarray Gene Expression L. plantarum

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Spot quality control for protein macroarray on multiple detection of food-borne pathogens

Spot quality control for protein macroarray on multiple dete...

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2014年上海市研究生学术论坛:生物医疗仪器

作者： Shi Qiu Chunmei Song Jianwu Li Haijuan Zeng Qing Liu School of Medical Instrument and Food Engnieering University of Shanghai for Science and TechnologyShanghai 200093China

Protein macroarray is a new,simple and multiple detection system,whose spots are more than 1mm diameter and results are visible to naked eyes without any ***,especially applied in the detection of food-borne pathogens,only needs to achieve qualitative or semiquantitative detection of *** useful technology,however,possesses recognized spot quality and uniformity issues that can limit its *** methods were developed for spot quality control,however,they were complicated or required professional *** therefore generated,for the first time,spotting buffer for protein macroarray directly incorporated with Ponceau S as a simple means of visualizing spot morphology on nitrocellulose membrane prior to *** this report,1%(w/v)Ponceau S and 10%(v/v)glycerol in spotting buffer were found to be optimal for spot quality control and targets ***,the results of sensitivity and specificity for protein macroarray indicated that there was no difference between the protein macroarrays created by spotting buffer with and without Ponceau *** optimal conditions,we make the important observation that macroarray imaging prior to hybridization is predictive of hybridization performance.

关键词： Protein macroarray Spot quality control Ponceau S Nitrocellulose membrane

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Characterization of ripening-associated genes using a tomato DNA macroarray, 1-methylcyclopropene, and ripening-impaired mutants

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POSTHARVEST BIOLOGY AND TECHNOLOGY 2013年 86卷 159-170页

作者： Yan, Rui Yokotani, Naoki Yamaoka, Tatsuya Ushijima, Kohichiro Nakano, Ryohei Yano, Kentaro Aoki, Koh Kubo, Yasutaka Okayama Univ Grad Sch Nat Sci & Technol Okayama 7008530 Japan Okayama Univ Grad Sch Environm & Life Sci Okayama 7008530 Japan Meiji Univ Sch Agr Kawasaki Kanagawa 2148571 Japan Osaka Prefectural Univ Grad Sch Life & Environm Sci Sakai Osaka 5998531 Japan

Using a tomato (Solanum lycopersicum) DNA macroarray consisting of 11,520 genes, we identified 419 ripening-associated genes (224 upregulated, 195 downregulated). Treatment with 1-methylcyclopropene (1-MCP) at the turning stage restored expression to levels of the mature green (MG) stage in 159 upregulated and 45 downregulated genes, suggesting a strong dependence on ethylene signaling during ripening. Among 194 ethylene-upregulated genes, 143 genes in ripening inhibitor (rin) fruit and 140 genes in non-ripening (nor) fruit responded poorly to exogenous ethylene, suggesting the necessity of RIN and NOR signals for their expression. In contrast, 36 and 70 genes responded to ethylene in rin and nor fruit, respectively, to a similar extent as in wild-type fruit, suggesting a clear independence of RIN and NOR signals for their response to ethylene. Ripening-associated expression of number of genes involved in respiratory, lipid and energy metabolism, and ethylene were upregulated during ripening under strong control of an ethylene signal. Large number of photosynthesis-related genes were downregulated during ripening in an ethylene-independent manner, whereas decreased expression of a few genes such as chloroplast thiazole biosynthetic protein and phosphoenolpyruvate carboxylase, was overturned by 1-MCP treatment, suggesting ethylene dependent regulation. Ripening-associated expression of several transcription and signal transduction factors, such as TDR4, GRAS, S-adenosyl-L-homocysteine hydrolase, BNK1, bZip, and BTB showed clear ethylene dependency, suggesting their involvement in regulation of fruit ripening downstream of ethylene signaling. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved.

关键词： Fruit ripening Ethylene 1-MCP rin nor macroarray

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MicroRNA expression profile in plasma from type 1 diabetic patients: Case-control study and bioinformatic analysis

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DIABETES RESEARCH AND CLINICAL PRACTICE 2018年 141卷 35-46页

作者： Assmann, Tais S. Recamonde-Mendoza, Mariana Punales, Marcia Tschiedel, Balduino Canani, Luis H. Crispim, Daisy Hosp Clin Porto Alegre Div Endocrine Porto Alegre RS Brazil Univ Fed Rio Grande do Sul Fac Med Postgrad Program Med Sci Endocrinol Porto Alegre RS Brazil Univ Fed Rio Grande do Sul Inst Informat Porto Alegre RS Brazil Hosp Clin Porto Alegre Expt Res Ctr Bioinformat Core Porto Alegre RS Brazil Hosp Nossa Senhora da Conceicao Inst Crianca Diabet Porto Alegre RS Brazil

Aims: To investigate a miRNA expression profile in plasma of type 1 diabetes (T1DM) patients and control subjects and analyze the putative pathways involved. Methods: Expressions of 48 miRNAs were analyzed in plasma of 33 T1DM patients and 26 age-and-gender-matched controls using Stem-loop RT-PreAmp PCR and TaqMan Low Density Arrays (Thermo Fisher Scientific). Five dysregulated miRNAs were then chosen for validation in an independent sample of 27 T1DM patients and 14 controls, using RT-qPCR. Bioinformatic analyses were performed to determine in which pathways these miRNAs are involved. Results: Nine miRNAs were differentially expressed between recently-diagnosed T1DM patients (<5 years of diagnosis) and controls. No differences were observed between patients with >= 5 years of diagnosis and controls. After validation in an independent sample of T1DM patients, miR-103a-3p, miR-155-5p, miR-200a-3p, and miR-210-3p were confirmed as being upregulated in recently-diagnosed T1DM patients compared with controls or patients with >= 5 years of diagnosis. Moreover, miR-146a-5p was downregulated in recently-diagnosed T1DM patients compared with the other groups. These five miRNAs regulate several genes from innate immune system-, MAPK-, apoptosis-, insulin- and cancer-related pathways. Conclusion: Five miRNAs are dysregulated in recently-diagnosed T1DM patients and target several genes involved in pathways related to T1DM pathogenesis, thus representing potential T1DM biomarkers. (C) 2018 Elsevier B.V. All rights reserved.

关键词： miRNAs Type 1 diabetes Epigenetics macroarray In silico analysis

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