Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Next-generation tissue microarray (ngTMA) increases the quality of biomarker studies: an example using CD3, CD8, and CD45RO in the tumor microenvironment of six different solid tumor types

Background Tissue microarray (TMA) technology revolutionized the investigation of potential biomarkers from paraffin-embedded tissues. However, conventional TMA construction is laborious, time-consuming and imprecise. Next-generation tissue microarrays (ngTMA) combine histological expertise with digital pathology and automated tissue microarraying. The aim of this study was to test the feasibility of ngTMA for the investigation of biomarkers within the tumor microenvironment (tumor center and invasion front) of six tumor types, using CD3, CD8 and CD45RO as an example. Methods Ten cases each of malignant melanoma, lung, breast, gastric, prostate and colorectal cancers were reviewed. The most representative H&E slide was scanned and uploaded onto a digital slide management platform. Slides were viewed and seven TMA annotations of 1 mm in diameter were placed directly onto the digital slide. Different colors were used to identify the exact regions in normal tissue (n = 1), tumor center (n = 2), tumor front (n = 2), and tumor microenvironment at invasion front (n = 2) for subsequent punching. Donor blocks were loaded into an automated tissue microarrayer. Images of the donor block were superimposed with annotated digital slides. Exact annotated regions were punched out of each donor block and transferred into a TMA block. 420 tissue cores created two ngTMA blocks. H&E staining and immunohistochemistry for CD3, CD8 and CD45RO were performed. Results All 60 slides were scanned automatically (total time < 10 hours), uploaded and viewed. Annotation time was 1 hour. The 60 donor blocks were loaded into the tissue microarrayer, simultaneously. Alignment of donor block images and digital slides was possible in less than 2 minutes/case. Automated punching of tissue cores and transfer took 12 seconds/core. Total ngTMA construction time was 1.4 hours. Stains for H&E and CD3, CD8 and CD45RO highlighted the precision with which ngTMA could capture regions of tumor-stroma interaction of each cancer and the T-lymphocytic immune reaction within the tumor microenvironment. Conclusion Based on a manual selection criteria, ngTMA is able to precisely capture histological zones or cell types of interest in a precise and accurate way, aiding the pathological study of the tumor microenvironment. This approach would be advantageous for visualizing proteins, DNA, mRNA and microRNAs in specific cell types using in situ hybridization techniques.

A next-generation tissue microarray (ngTMA) protocol for biomarker studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a 'donor' block into a 'recipient' block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 'recipient' blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

CD47 protein expression in acute myeloid leukemia: A tissue microarray-based analysis

Binding of CD47 to signal regulatory protein alpha (SIRPα), an inhibitory receptor, negatively regulates phagocytosis. In acute myeloid leukemia (AML), CD47 is overexpressed on peripheral blasts and leukemia stem cells and inversely correlates with survival. Aim of the study was to investigate the correlation between CD47 protein expression by immunohistochemistry (IHC) in a bone marrow (BM) tissue microarray (TMA) and clinical outcome in AML patients. CD47 staining on BM leukemia blasts was scored semi-quantitatively and correlated with clinical parameters and known prognostic factors in AML. Low (scores 0-2) and high (score 3) CD47 protein expression were observed in 75% and 25% of AML patients. CD47 expression significantly correlated with percentage BM blast infiltration and peripheral blood blasts. Moreover, high CD47 expression was associated with nucleophosmin (NPM1) gene mutations. In contrast, CD47 expression did not significantly correlate with overall or progression free survival or response to therapy. In summary, a BM TMA permits rapid and reproducible semi-quantitative analysis of CD47 protein expression by IHC. While CD47 expression on circulating AML blasts has been shown to be a negative prognostic marker for a very defined population of AML patients with NK AML, CD47 expression on AML BM blasts is not.

Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

BACKGROUND AND OBJECTIVE Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. MATERIAL AND METHODS Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. RESULTS The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. CONCLUSION The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

Salivary Pellicle vs Whole Saliva: The Response of Oral Fibroblasts Based on a Genome-Wide Microarray.

PURPOSE Whole saliva comprises components of the salivary pellicle that spontaneously forms on surfaces of implants and teeth. However, there are no studies that functionally link the salivary pellicle with a possible change in gene expression. MATERIALS AND METHODS This study examined the genetic response of oral fibroblasts exposed to the salivary pellicle and whole saliva. Oral fibroblasts were seeded onto a salivary pellicle and the respective untreated surface. Oral fibroblasts were also exposed to freshly harvested sterile-filtered whole saliva. A genome-wide microarray of oral fibroblasts was performed, followed by gene ontology screening with DAVID functional annotation clustering, KEGG pathway analysis, and the STRING functional protein association network. RESULTS Exposure of oral fibroblasts to saliva caused 61 genes to be differentially expressed (P < .05). Gene ontology screening assigned the respective genes into 262 biologic processes, 3 cellular components, 13 molecular functions, and 7 pathways. Most remarkable was the enrichment in the inflammatory response. None of the genes regulated by whole saliva was significantly changed when cells were placed onto a salivary pellicle. CONCLUSION The salivary pellicle per se does not provoke a significant inflammatory response of oral fibroblasts in vitro, whereas sterile-filtered whole saliva does produce a strong inflammatory response.

Possible involvement of plant ABC transporters in cadmium detoxification: a cDNA sub-microarray approach

As a nontolerant plant to a large number of toxic compounds, Arabidopsis thaliana is a suitable model to study regulation of genes involved in response to heavy metals. Using a cDNA-microarray approach, we identified some ABC transporters that are differentially regulated after cadmium treatments, making them putative candidates for being involved in Cd sequestration and redistribution in plants. Regarding yeast and fission yeast, in which Cd is able to form complexes either with glutathione (GSH) or phytochelatins (PC) subsequently transported into vacuoles via ABC transporters, it is also very likely that some plant ABC transporters are able to transport GS2–Cd or PC–Cd complexes into subcellular compartments or outside of the cell. The characterization of such transporters is of great interest for developing molecular biology approaches in phytoremediation.