The traditional approach with microarray data has been to apply transformations that approximately normalize them, with the drawback of losing the original scale. The alternative stand point taken here is to search for models that fit the data, characterized by the presence of negative values, preserving their scale; one advantage of this strategy is that it facilitates a direct interpretation of the results. A new family of distributions named gpower-normal indexed by p∈R is introduced and it is proven that these variables become normal or truncated normal when a suitable gpower transformation is applied. Expressions are given for moments and quantiles, in terms of the truncated normal density. This new family can be used to model asymmetric data that include non-positive values, as required for microarray analysis. Moreover, it has been proven that the gpower-normal family is a special case of pseudo-dispersion models, inheriting all the good properties of these models, such as asymptotic normality for small variances. A combined maximum likelihood method is proposed to estimate the model parameters, and it is applied to microarray and contamination data. Rcodes are available from the authors upon request.
The GLUT members belong to a family of glucose transporter proteins that facilitate glucose transport across the cell membrane. The mammalian GLUT family consists of thirteen members (GLUTs 1–12 and H+-myo-inositol transporter (HMIT)). Humans have a recently duplicated GLUT member, GLUT14. Avians express the majority of GLUT members. The arrangement of multiple GLUTs across all somatic tissues signifies the important role of glucose across all organisms. Defects in glucose transport have been linked to metabolic disorders, insulin resistance and diabetes. Despite the essential importance of these transporters, our knowledge regarding GLUT members in avians is fragmented. It is clear that there are no chicken orthologs of mammalian GLUT4 and GLUT7. Our examination of GLUT members in the chicken revealed that some chicken GLUT members do not have corresponding orthologs in mammals. We review the information regarding GLUT orthologs and their function and expression in mammals and birds, with emphasis on chickens and humans.
Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131).
Recently, peptide microarrays have been used to distinguish proteins, antibodies, viruses, and bacteria based on their binding to random sequence peptides. We reported on the use of peptide arrays to identify enzyme modulators that involve screening an array of 10,000 defined and addressable peptides on a microarray. Primary peptides were first selected to inhibit the enzyme at low μM concentrations. Then, new peptides were found to only bind strongly with the enzyme–inhibitor complex, but not the native enzyme. These new peptides served as secondary inhibitors that enhanced the inhibition of the enzyme together with the primary peptides. Without the primary peptides, the secondary effect peptides had little effect on the enzyme activity. Conversely, we also selected peptides that recovered the activities of inhibited enzyme–peptide complex. The selection of cooperative peptide pairs will provide a versatile toolkit for modulating enzyme functions, which may potentially be applied to drug discovery and biocatalysis.
IgE antibodies serve as the gatekeeper for the release of mediators from sensitized (IgE positive) mast cells and basophils following a relevant allergen exposure which can lead to an immediate-type hypersensitivity (allergic) reaction. Purified recombinant and native allergens were combined in the 1990s with state of the art chip technology to establish the first microarray-based IgE antibody assay. Triplicate spots to over 100 allergenic molecules are immobilized on an amine-activated glass slide to form a single panel multi-allergosorbent assay. Human antibodies, typically of the IgE and IgG isotypes, specific for one or many allergens bind to their respective allergen(s) on the chip. Following removal of unbound serum proteins, bound IgE antibody is detected with a fluorophore-labeled anti-human IgE reagent. The fluorescent profile from the completed slide provides a sensitization profile of an allergic patient which can identify IgE antibodies that bind to structurally similar (cross-reactive) allergen families versus molecules that are unique to a single allergen specificity. Despite its ability to rapidly analyze many IgE antibody specificities in a single simple assay format, the chip-based microarray remains less analytically sensitive and quantitative than its singleplex assay counterpart (ImmunoCAP, Immulite). Microgram per mL quantities of allergen-specific IgG antibody can also complete with nanogram per mL quantities of specific IgE for limited allergen binding sites on the chip. Microarray assays, while not used in clinical immunology laboratories for routine patient IgE antibody testing, will remain an excellent research tool for defining sensitization profiles of populations in epidemiological studies.
The application of DNA microarray assay (DMA) has entered a new era owing to recent innovations in omics technologies. This review summarizes recent applications of DMA-based gene expression profiling by focusing on the screening and characterizationof traditional Chinese medicine. First, herbs, mushrooms, and dietary plants analyzed by DMA along with their effective components and their biological/physiological effects are summarized and discussed by examining their comprehensive list and a list of representative effective chemicals. Second, the mechanisms of action of traditional Chinese medicine are summarized by examining the genes and pathways responsible for the action, the cell functions involved in the action, and the activities found by DMA (silent estrogens). Third, applications of DMA for traditional Chinese medicine are discussed by examining reported examples and new protocols for its use in quality control. Further innovations in the signaling pathway based evaluation of beneficial effects and the assessment of potential risks of traditional Chinese medicine are expected, just as are observed in other closely related fields, such as the therapeutic, environmental, nutritional, and pharmacological fields.
Cronobacter (previously known as Enterobacter sakazakii) is a genus of Gram-negative, facultatively anaerobic, oxidase-negative, catalase-positive, rod-shaped bacteria of the family Enterobacteriaceae. These organisms cause a variety of illnesses such as meningitis, necrotizing enterocolitis, and septicemia in neonates and infants, and urinary tract, wound, abscesses or surgical site infections, septicemia, and pneumonia in adults. The total gene content of 379 strains of Cronobacter spp. and taxonomically-related isolates was determined using a recently reported DNA microarray. The Cronobacter microarray as a genotyping tool gives the global food safety community a rapid method to identify and capture the total genomic content of outbreak isolates for food safety, environmental, and clinical surveillance purposes. It was able to differentiate the seven Cronobacter species from one another and from non-Cronobacter species. The microarray was also able to cluster strains within each species into well-defined subgroups. These results also support previous studies on the phylogenic separation of species members of the genus and clearly highlight the evolutionary sequence divergence among each species of the genus compared to phylogenetically-related species. This review extends these studies and illustrates how the microarray can also be used as an investigational tool to mine genomic data sets from strains. Three case studies describing the use of the microarray are shown and include: (1) the determination of allelic differences among Cronobacter sakazakii strains possessing the virulence plasmid pESA3; (2) mining of malonate and myo-inositol alleles among subspecies of Cronobacter dublinensis strains to determine subspecies identity; and (3) lastly using the microarray to demonstrate sequence divergence and phylogenetic relatedness trends for 13 outer-membrane protein alleles among 240 Cronobacter and phylogenetically-related strains. The goal of this review is to describe microarrays as a robust tool for genomics research of this assorted and important genus, a criterion toward the development of future preventative measures to eliminate this foodborne pathogen from the global food supply.
The editors of Microarrays would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2015. [...]
Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC)-optimized variant (MB314). MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (Physcomitrella patens) to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), and Aleuria aurantia lectin (AAL) were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (FcγRIII; receptor for the constant region of an antibody)-whose affinity is mediated through core fucosylation-and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.
Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.
Borrelia burgdorferi, the spirochetal agent of Lyme disease, is maintained in nature in a cycle involving a tick vector and a mammalian host. Adaptation to the diverse conditions of temperature, pH, oxygen tension and nutrient availability in these two environments requires the precise orchestration of gene expression. Over 25 microarray analyses relating to B. burgdorferi genomics and transcriptomics have been published. The majority of these studies has explored the global transcriptome under a variety of conditions and has contributed substantially to the current understanding of B. burgdorferi transcriptional regulation. In this review, we present a summary of these studies with particular focus on those that helped define the roles of transcriptional regulators in modulating gene expression in the tick and mammalian milieus. By performing comparative analysis of results derived from the published microarray expression profiling studies, we identified composite gene lists comprising differentially expressed genes in these two environments. Further, we explored the overlap between the regulatory circuits that function during the tick and mammalian phases of the enzootic cycle. Taken together, the data indicate that there is interplay among the distinct signaling pathways that function in feeding ticks and during adaptation to growth in the mammal.
The papers published in this Special Issue "SNP arrays" (Single Nucleotide Polymorphism Arrays) focus on several perspectives associated with arrays of this type. The range of papers vary from a case report to reviews, thereby targeting wider audiences working in this field. The research focus of SNP arrays is often human cancers but this Issue expands that focus to include areas such as rare conditions, animal breeding and bioinformatics tools. Given the limited scope, the spectrum of papers is nothing short of remarkable and even from a technical point of view these papers will contribute to the field at a general level. Three of the papers published in this Special Issue focus on the use of various SNP array approaches in the analysis of three different cancer types. Two of the papers concentrate on two very different rare conditions, applying the SNP arrays slightly differently. Finally, two other papers evaluate the use of the SNP arrays in the context of genetic analysis of livestock. The findings reported in these papers help to close gaps in the current literature and also to give guidelines for future applications of SNP arrays.
Genome-wide association studies of schizophrenia encompassing the major histocompatibility locus (MHC) were highly significant following genome-wide correction. This broad region implicates many genes including the MHC complex class II. Within this interval we examined the expression of two MHC II genes (HLA-DPA1 and HLA-DRB1) in brain from individual subjects with schizophrenia (SZ), bipolar disorder (BD), major depressive disorder (MDD), and controls by differential gene expression methods. A third MHC II mRNA, CD74, was studied outside of the MHC II locus, as it interacts within the same immune complex. Exon microarrays were performed in anterior cingulate cortex (ACC) in BD compared to controls, and both HLA-DPA1 and CD74 were decreased in expression in BD. The expression of HLA-DPA1 and CD74 were both reduced in hippocampus, amygdala, and dorsolateral prefrontal cortex regions in SZ and BD compared to controls by specific qPCR assay. We found several novel HLA-DPA1 mRNA variants spanning HLA-DPA1 exons 2-3-4 as suggested by exon microarrays. The intronic rs9277341 SNP was a significant cis expression quantitative trait locus (eQTL) that was associated with the total expression of HLA-DPA1 in five brain regions. A biomarker study of MHC II mRNAs was conducted in SZ, BD, MDD, and control lymphoblastic cell lines (LCL) by qPCR assay of 87 subjects. There was significantly decreased expression of HLA-DPA1 and CD74 in BD, and trends for reductions in SZ in LCLs. The discovery of multiple splicing variants in brain for HLA-DPA1 is important as the HLA-DPA1 gene is highly conserved, there are no reported splicing variants, and the functions in brain are unknown. Future work on the function and localization of MHC Class II proteins in brain will help to understand the role of alterations in neuropsychiatric disorders. The HLA-DPA1 eQTL is located within a large linkage disequilibrium block that has an irrefutable association with schizophrenia. Future tests in a larger cohort are needed to determine the significance of this eQTL association with schizophrenia. Our findings support the long-held hypothesis that alterations in immune function are associated with the pathophysiology of psychiatric disorders.
As recognised by the National Institutes of Health (NIH) Precision Medicine Initiative (PMI), microarray technology currently provides a rapid, inexpensive means of identifying large numbers of known genomic variants or gene transcripts in experimental and clinical settings. However new generation sequencing techniques are now being introduced in many clinical genetic contexts, particularly where novel mutations are involved. While these methods can be valuable for screening a restricted set of genes for known or novel mutations, implementation of whole genome sequencing in clinical practice continues to present challenges. Even very accurate high-throughput methods with small error rates can generate large numbers of false negative or false positive errors due to the high numbers of simultaneous readings. Additional validation is likely to be required for safe use of any such methods in clinical settings. Custom-designed arrays can offer advantages for screening for common, known mutations and, in this context, may currently be better suited for accredited, quality-controlled clinical genetic screening services, as illustrated by their successful application in several large-scale pre-emptive pharmacogenomics programs now underway. Excessive, inappropriate use of next-generation sequencing may waste scarce research funds and other resources. Microarrays presently remain the technology of choice in applications that require fast, cost-effective genome-wide screening of variants of known importance, particularly for large sample sizes. This commentary considers some of the applications where microarrays continue to offer advantages over next-generation sequencing technologies.
Antibody microarrays have emerged as an important tool within proteomics, enabling multiplexed protein expression profiling in both health and disease. The design and performance of antibody microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies and the solid surfaces plays a central role. In this study, we have taken on the first comprehensive view and evaluated the overall impact of solid surfaces on the recombinant antibody microarray design. The results clearly demonstrated the importance of the surface-antibody interaction and showed the effect of the solid supports on the printing process, the array format of planar arrays (slide- and well-based), the assay performance (spot features, reproducibility, specificity and sensitivity) and assay processing (degree of automation). In the end, two high-end recombinant antibody microarray technology platforms were designed, based on slide-based (black polymer) and well-based (clear polymer) arrays, paving the way for future large-scale protein expression profiling efforts.
Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40(®). This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStamp(TM) bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStamp(TM) were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays.
BACKGROUND:The tumor microenvironment participates in the regulation of tumor progression and influences treatment sensitivity. In breast cancer, it also may play a role in determining the fate of non-invasive lesions such as ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive diseases, which is aggressively treated despite its indolent nature in many patients since no biomarkers are available to predict the progression of DCIS to invasive disease. In vitro models of stromal activation by breast tumor cells might provide clues as to specific stromal genes crucial for the transition from DCIS to invasive disease.METHODS:normal human dermal fibroblasts (NHDF) were treated under serum-free conditions with cell culture media conditioned by breast cancer cell lines (SkBr3, MDA-MB-468, T47D) for 72 h and subjected to gene expression profiling with Illumina platform.RESULTS:TGM2, coding for a tissue transglutaminase, was identified as candidate gene for stromal activation. In public transcriptomic datasets of invasive breast tumors TGM2 expression proved to provide prognostic information. Conversely, its role as an early biosensor of tumor invasiveness needs to be further investigated by in situ analyses.CONCLUSION:Stromal TGM2 might probably be associated with precancerous evolution at earlier stages compared to DCIS.
Cell fate decisions like apoptosis are heterogeneously implemented within a cell population and, consequently, the population response is recognized as sum of many individual dynamic events. Here, we report on the use of micro-patterned single-cell arrays for real-time tracking of nanoparticle-induced (NP) cell death in sets of thousands of cells in parallel. Annexin (pSIVA) and propidium iodide (PI), two fluorescent indicators of apoptosis, are simultaneously monitored after exposure to functionalized polystyrene (PS − NH 2) nanobeads as a model system. We find that the distribution of Annexin onset times shifts to later times and broadens as a function of decreasing NP dose. We discuss the mean time-to-death as a function of dose, and show how the EC 50 value depends both on dose and time of measurement. In addition, the correlations between the early and late apoptotic markers indicate a systematic shift from apoptotic towards necrotic cell death during the course of the experiment. Thus, our work demonstrates the potential of array-based single cell cytometry for kinetic analysis of signaling cascades in a high-throughput format.
Data obtained from expression microarrays enables deeper understanding of the molecular signatures of infectious diseases. It provides rapid and accurate information on how infections affect the clustering of gene expression profiles, pathways and networks that are transcriptionally active during various infection states compared to conventional diagnostic methods, which primarily focus on single genes or proteins. Thus, microarray technologies offer advantages in understanding host-parasite interactions associated with filarial infections. More importantly, the use of these technologies can aid diagnostics and helps translate current genomic research into effective treatment and interventions for filarial infections. Studying immune responses via microarray following infection can yield insight into genetic pathways and networks that can have a profound influence on the development of anti-parasitic vaccines.
High-throughput microarray technologies have long been a source of data for a wide range of biomedical investigations. Over the decades, variants have been developed and sophistication of measurements has improved, with generated data providing both valuable insight and considerable analytical challenge. The cost-effectiveness of microarrays, as well as their fundamental applicability, made them a first choice for much early genomic research and efforts to improve accessibility, quality and interpretation have continued unabated. In recent years, however, the emergence of new generations of sequencing methods and, importantly, reduction of costs, has seen a preferred shift in much genomic research to the use of sequence data, both less ‘noisy’ and, arguably, with species information more directly targeted and easily interpreted. Nevertheless, new microarray data are still being generated and, together with their considerable legacy, can offer a complementary perspective on biological systems and disease pathogenesis. The challenge now is to exploit novel methods for enhancing and combining these data with those generated by alternative high-throughput techniques, such as sequencing, to provide added value. Augmentation and integration of microarray data and the new horizons this opens up, provide the theme for the papers in this Special Issue.
