ABSTRACT
BACKGROUND: Human polyomaviruses (HPyV) infections cause mostly unapparent or mild primary infections, followed by lifelong nonpathogenic persistence. HPyV, and specifically JCPyV, are known to co-diverge with their host, implying a slow rate of viral evolution and a large timescale of virus/host co-existence. Recent bio-informatic reports showed a large level of peptide homology between JCPyV and the human proteome. In this study, the antibody response to PyV peptides is evaluated. METHODS: The in-silico analysis of the HPyV proteome was followed by peptide microarray serology. A HPyV-peptide microarray containing 4,284 peptides was designed and covered 10 polyomavirus proteomes. Plasma samples from 49 healthy subjects were tested against these peptides. RESULTS: In-silico analysis of all possible HPyV 5-mer amino acid sequences were compared to the human proteome, and 1,609 unique motifs are presented. Assuming a linear epitope being as small as a pentapeptide, on average 9.3% of the polyomavirus proteome is unique and could be recognized by the host as non-self. Small t Ag (stAg) contains a significantly higher percentage of unique pentapeptides. Experimental evidence for the presence of antibodies against HPyV 15-mer peptides in healthy subjects resulted in the following observations: i) antibody responses against stAg were significantly elevated, and against viral protein 2 (VP2) significantly reduced; and ii) there was a significant correlation between the increasing number of embedded unique HPyV penta-peptides and the increase in microarray fluorescent signal. CONCLUSION: The anti-peptide HPyV-antibodies in healthy subjects are preferably directed against the penta-peptide derived unique fraction of the viral proteome.
Subject(s)
Antibodies, Viral/blood , Antigens, Viral/immunology , Polyomavirus/immunology , Adult , Antigens, Viral/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Polyomavirus/genetics , Protein Array Analysis , Sequence Analysis, DNA , Seroepidemiologic Studies , Viral Proteins/genetics , Viral Proteins/immunology , Young AdultABSTRACT
Upon maturation, primary neuronal cultures form an interconnected network based on neurite outgrowth and synaptogenesis in which spontaneous electrical activity arises. Measurement of network activity allows quantification of neuronal health and maturation. A fluorescent indicator was used to monitor secondary calcium influxes after the occurrence of action potentials, allowing us to examine activity of hippocampal cultures via confocal live cell imaging. Subsequently, nuclear staining with DAPI allows accurate cell segmentation. To analyze the calcium recording in a robust, observer-independent manner, we implemented an automated image- and signal-processing algorithm and validated it against a visual, interactive procedure. Both methods yielded similar results on the emergence of synchronized activity and allowed robust quantitative measurement of acute and chronic modulation of drugs on network activity. Both the number of days in vitro (DIV) and neutralization of nerve growth factor (NGF) have a significant effect on synchronous burst frequency and correlation. Acute effects are demonstrated using 5-HT (serotonin) and ethylene glycol tetra-acetic acid. Automated analysis allowed measuring additional features, such as peak decay times and bursting frequency of individual neurons. Based on neuronal cell cultures in 96-well plates and accurate calcium recordings, the analysis method allows development of an integrated high-content screening assay. Because molecular biological techniques can be applied to assess the influence of genes on network activity, it is applicable for neurotoxicity or neurotrophics screening as well as development of in vitro disease models via, for example, pharmacologic manipulation or RNAi.
Subject(s)
Calcium Signaling , Drug Evaluation, Preclinical/methods , Nerve Net/drug effects , Neurotransmitter Agents/pharmacology , Action Potentials , Algorithms , Analysis of Variance , Animals , Cells, Cultured , Chelating Agents/pharmacology , Drug Discovery , Egtazic Acid/pharmacology , High-Throughput Screening Assays , Hippocampus/cytology , Mice , Neurons/drug effects , Neurons/metabolism , Optical Imaging , Primary Cell CultureABSTRACT
SNP arrays offer the opportunity to get a genome-wide view on copy number alterations and are increasingly used in oncology. DNA from formalin-fixed paraffin-embedded material (FFPE) is partially degraded which limits the application of those technologies for retrospective studies. We present the use of Affymetrix GeneChip SNP6.0 for identification of copy number alterations in fresh frozen (FF) and matched FFPE samples. Fifteen pairs of adenocarcinomas with both frozen and FFPE embedded material were analyzed. We present an optimization of the sample preparation and show the importance of correcting the measured intensities for fragment length and GC-content when using FFPE samples. The absence of GC content correction results in a chromosome specific "wave pattern" which may lead to the misclassification of genomic regions as being altered. The highest concordance between FFPE and matched FF were found in samples with the highest call rates. Nineteen of the 23 high level amplifications (83%) seen using FF samples were also detected in the corresponding FFPE material. For limiting the rate of "false positive" alterations, we have chosen a conservative False Discovery Rate (FDR). We observed better results using SNP probes than CNV probes for copy number analysis of FFPE material. This is the first report on the detection of copy number alterations in FFPE samples using Affymetrix GeneChip SNP6.0.
