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DNA chip technology ante portas.

The recent popularity of DNA chip technology has been fostered by the increasing demand for new diagnostic tools which allow the simultaneous analysis of large numbers of nucleic acid hybridization experiments in a timely fashion. The development of DNA chip-based assays has been strongly driven by modern approaches aiming at the comprehensive analysis of multiple gene mutations and expressed sequences. The broad range of current DNA chip applications include the detection of pathogens, the measurement of differences in the expression of genes between different cell populations, and the analysis of genomic alterations such as sequence and copy number alterations in disease-related genes and single nucleotide polymorphisms. We present an overview of the impact of DNA chip technology on the field of molecular medicine and discuss developments that can be expected in the near future.

Animals↗

Epidemiologic studies demonstrating increased morning incidence of sudden cardiac death.

To determine if sudden cardiac death shows a circadian variation, the mortality records of the Massachusetts population and the Framingham Heart Study were analyzed. The Massachusetts mortality data analysis revealed an increased morning incidence for out-of-hospital cardiac deaths similar to that previously described for nonfatal myocardial infarction. The records of the Framingham Heart Study allowed more comprehensive analysis of the time of sudden cardiac death. In this study, a prominent circadian variation was also identified in which frequency of sudden cardiac death increased sharply between 6 A.M. and 9 P.M.; distribution was fairly even throughout the rest of the day. This circadian pattern may be explained in part by physiologic changes that increase the likelihood of ventricular fibrillation or increase the risk of thrombosis in the morning hours. The possible role of the morning increase in physical and mental activity as a trigger of sudden cardiac death requires further investigation.

Circadian Rhythm↗

SAGE analysis to identify embryonic stem cell-predominant transcripts.

The Human Genome Consortium has successfully sequenced the entire human genome (http://www.genome.gov/11006945), but an unfinished goal remains the identification of specific genes responsible for unique cellular processes. With respect to embryonic stem (ES) cells, this includes the identification of factors that govern self-renewal and pluripotentiality. One technique that facilitates this last goal is serial analysis of gene expression (SAGE), a functional genomics technique that identifies and quantifies mRNA transcripts. This technique relies on the preparation and sequencing of complementary DNA concatemers to rapidly generate a comprehensive profile of gene expression within a cell, and unlike microarrays, it does not require prior knowledge of the genes to be assayed. Because SAGE is a sequence-based technique, it can be used to search for ES-restricted genes (i.e., markers) by sequence comparisons among stem cells, differentiated cells, and tissues. These markers can then be genetically manipulated to understand the molecular basis for stem cell biology to help define how transcriptional mechanisms distinguish ES cells from other, less-pluripotent cell types. SAGE is, thus, a powerful technique that permits a comprehensive analysis of mRNA abundance that can define, at a molecular level, fundamental characteristics of ES cells. In this chapter, we illustrate the basic principles of SAGE, describe a complete protocol for the generation of SAGE libraries, and show how this technique can be employed to analyze embryonic stem cells.

Animals↗

Epidemiology of neuroblastoma: analysis of a single institution.

BACKGROUND: Neuroblastoma is the fourth most frequent cancer among all pediatric neoplasms Epidemiological studies may shed more light on the disease and aid in improving treatment of patients with neuroblastoma. PATIENTS AND METHODS: Epidemiology data are presented for 333 children with neuroblastoma or ganglioneuroblastoma and 11 children with ganglioneuroma who were treated at the Institute of Mother and Child in Warsaw, Poland, from 1962 to 1996. RESULTS: Analysis of the stage of the disease, age, sex and survival of children with neuroblastoma demonstrated comparable distribution of good and intermediate stages versus the stages with a poor prognosis. CONCLUSION: Comprehensive analysis of epidemiological data on the stage of cancer, age, sex and survival in patients with neuroblastoma provides a basis for better prognosis of the disease and cost-efficient treatment.

Adolescent↗

The problems of the rational use of the medical staff in industrial health care complexes.

The awareness of resources scarcity in the occupational health care makes one analyze allocation in the selected institutions of occupational health service in Poland. The principal issue was to find out the differentiation in medical staff use and the costs involved. Such conclusions resulting from inputs and outputs evaluation are expected to be a basis for economic rationalization of resources allocation. An attempt to make such analysis has been made in Industrial Health Care Complexes (IHCCs) in Poland. Mainly, the costs of work of medical staff have been considered as an element of wider empirical study and comprehensive analysis of resources usage.

Cost Control↗

Somatic microindels: analysis in mouse soma and comparison with the human germline.

Microindels, defined as mutations that result in a colocalized microinsertion and microdeletion with a net gain or loss of between 1 and 50 nucleotides, may be an important contributor to cancer. We report the first comprehensive analysis of somatic microindels. Our large database of mutations in the lacI transgene of Big Blue((R)) mice contains 0.5% microindels, 2.8% pure microinsertions, and 11.5% pure microdeletions. There appears to be no age, gender, or tissue-type specificity in the frequency of microindels. Of the independent somatic mutations that result in a net in-frame insertion or deletion, microindels are responsible for 13% of protein expansions and 6% of protein contractions. These in-frame microindels may play a crucial role in oncogenesis and evolution via "protein tinkering" (i.e., modest expansion or contraction of proteins). Four characteristics suggest that microindels are caused by unique mechanisms, not just simple combinations of the same mechanisms that cause pure microinsertions and pure microdeletions. First, microinsertions and microdeletions commonly occur at hotspots, but none of the 30 microindels are recurrent. Second, the sizes of the deletions and insertions in microindels are larger and more varied than in pure microdeletions and pure microinsertions. Third, microinsertions overwhelmingly repeat the adjacent base (97%) while the insertions in microindels do so only infrequently (17%). Fourth, analysis of the sequence contexts of microindels is consistent with unique mechanisms including recruitment of translesion DNA synthesis polymerases. The mouse somatic microindels have characteristics similar to those of human germline microindels, consistent with similar causative mechanisms in mouse and human, and in soma and germline.

Age Factors↗

Analysis of binding reactions by fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) is now widely used to investigate binding interactions in live cells. Although various idealized solutions have been identified for the reaction-diffusion equations that govern FRAP, there has been no comprehensive analysis or systematic approach to serve as a guide for extracting binding information from an arbitrary FRAP curve. Here we present a complete solution to the FRAP reaction-diffusion equations for either single or multiple independent binding interactions, and then relate our solution to the various idealized cases. This yields a coherent approach to extract binding information from FRAP data which we have applied to the question of transcription factor mobility in the nucleus. We show that within the nucleus, the glucocorticoid receptor is transiently bound to a single state, with each molecule binding on average 65 sites per second. This rapid sampling is likely to be important in finding a specific promoter target sequence. Further we show that this predominant binding state is not the nuclear matrix, as some studies have suggested. We illustrate how our analysis provides several self-consistency checks on a FRAP fit. We also define constraints on what can be estimated from FRAP data, show that diffusion should play a key role in many FRAP recoveries, and provide tools to test its contribution. Overall our approach establishes a more general framework to assess the role of diffusion, the number of binding states, and the binding constants underlying a FRAP recovery.

Algorithms↗

Prevalent expression of fibroblast growth factor (FGF) receptors and FGF2 in human tumor cell lines.

Basic fibroblast growth factor (FGF2) has potent mitogenic and angiogenic activities that have been implicated in tumor development and malignant progression. The biological effects of FGF2 and other members of the FGF ligand family are mediated by 4 transmembrane tyrosine kinase receptors (FGFRs). To better understand the roles of FGFRs in cancer, the expression of FGF2 and each of the 4 FGFRs was assessed by RNase protection analysis of 60 human tumor cell lines, representing 9 tumor types. Expression of at least one FGFR isoform was detected in 90% and FGF2 mRNA in 35% of the cell lines. Our comprehensive analysis of FGF2 and FGFR expression in human tumor cell lines provides evidence that FGF signaling pathways are active in a majority of human tumor cell lines, and lends support to the development of anti-tumor strategies that target FGFRs.

Fibroblast Growth Factor 2↗

Proteomic analysis of Acinetobacter lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry.

The MS/MS analysis by Electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) was applied to identify proteins in proteome analysis of bacteria whose genomes are not known. The protein identification by ESI-Q-TOF MS was performed sequentially by database search and then de novo sequencing using MS/MS spectra. Soil bacteria having unanalyzed genome, Acinetobacter lwoffii K24 is an aniline degrading bacterium. In this report, we present the results of a comparison between the proteome profile of A. lwoffii K24 cultured in aniline- or succinate-containing media. Protein analysis was performed using two-dimensional gel electrophoresis (2-DE) with pH 3-10 immobilized pH gradient (IPG) strips followed by ESI-Q-TOF MS. More than 780 protein spots were detected by 2-DE from the soluble proteome. Forty-eight of these proteins were expressed exclusively in aniline cultured bacteria, and 81 proteins increased and 162 proteins decreased in aniline-cultured versus succinate cultured A. lwoffii K24. Internal amino acid sequences of 43 major protein spots were successfully determined by ESI-Q-TOF MS to try to identify the bacterial proteins responding to aniline culture condition. Since the A. lwoffii K24 genome is not yet sequenced, many proteins were found to be hypothetical. Comparative proteome analysis of the insoluble protein fractions showed that one novel protein that was strongly induced by succinate-cultured A. lwoffii K24 was repressed under aniline culture conditions. These results suggest that comprehensive analysis of bacterial proteomes by 2-DE and amino acid sequence analysis by ESI-Q-TOF MS is useful for understanding induced novel proteins of biodegrading bacteria.

Acinetobacter↗

An analysis of the concept of countertransference.

A knowledge of countertransference is a necessary step in using it therapeutically in the nurse-patient relationship. However, this concept, which has its roots in psychoanalytic theory, has been applied to nurses and nursing situations in an unhelpful and ambiguous manner. In fact, the term is often used to denigrate nurses without providing clarity as to what the components of countertransference are and how they might be understood and used therapeutically. As such, it is necessary to delineate the meaning of countertransference in its entirely as a concept suitable for nursing practice. Positive countertransference, negative countertransference, and empathy are the composite parts of the concept of countertransference. Through concept analysis, by using methods outlined by Rodgers (1989), Journal of Advanced Nursing, 14, 330-335, it is suggested that countertransference is an inescapable factor in the nurse-patient relationship. Furthermore, a comprehensive analysis of the concept of countertransference will assist nurses in recognizing it when it occurs and may enhance the use of the understanding gained to the benefit of the nurse-patient relationship.

Adult↗

Clonal breadth of the HIV-1-specific T-cell receptor repertoire in vivo as determined by subtractive analysis.

OBJECTIVE: Although the epitopic breadth of HIV-1-specific CD8 T lymphocyte (CTL) responses has been described, the T cell receptor (TCR) diversity of virus-specific cells remains poorly defined. DESIGN AND METHODS: To address this issue, we applied a novel technique for subtractive analysis of the HIV-1-specific CTL repertoire, combining specific deletion of peptide-specific cells by 5-fluorouracil with TCR spectratyping to identify clonal breadth of CTL recognizing individual peptides. RESULTS: Comprehensive analysis of an infected individual reveals that nine identified HIV-1-specific responses are comprised of at least 38 distinct T-cell clones (ranging from two to 10 distinct clones per epitope). CONCLUSION: Given the potentially crucial role of T-cell receptor breadth for viral recognition and avoidance of escape, this subepitopic analysis of CTL may offer an important measure of cellular immunity for pathogenesis and vaccine studies.

CD8-Positive T-Lymphocytes↗

13C-methacetin breath test parameter S for liver diseases diagnosis.

The mechanism of 13C-methacetin breath test is set forth clearly with the analysis of pharmacokinetics mode, and the measuring method of 13C-methacetin breath test and its clinical applications in the diagnosis of liver diseases are reported in detail. On the basis of comprehensive analysis of the clinical test data, the advanced diagnostic parameter S is of important significance for the application and development of breath test.

Acetamides↗

Analysis of protein domain families in Caenorhabditis elegans.

The Caenorhabditis elegans genome sequencing project has completed over half of this nematode's 100-Mb genome. Proteins predicted in the finished sequence have been compiled and released in the data-base Wormpep. Presented here is a comprehensive analysis of protein domain families in Wormpep 11, which comprises 7299 proteins. The relative abundance of common protein domain families was counted by comparing all Wormpep proteins to the Pfam collection of protein families, which is based on recognition by hidden Markov models. This analysis also identified a number of previously unannotated domains. To investigate new apparently nematode-specific protein families, Wormpep was clustered into domain families on the basis of sequence similarity using the Domainer program. The largest clusters that lacked clear homology to proteins outside Nematoda were analyzed in further detail, after which some could be assigned a putative function. We compared all proteins in Wormpep 11 to proteins in the human, Saccharomyces cerevisiae, and Haemophilus influenzae genomes. Among the results are the estimation that over two-thirds of the currently known human proteins are likely to have a homologue in the whole C. elegans genome and that a significant number of proteins are well conserved between C. elegans and H. influenzae, that are not found in S. cerevisiae.

Amino Acid Sequence↗

Postmortem cardiac troponin T levels in the blood and pericardial fluid. Part 1. Analysis with special regard to traumatic causes of death.

In forensic pathology, previous studies have suggested the possible application of cardiac troponins in the diagnosis of myocardial infarction. However, there appears to be insufficient practical data on other causes of death. The present study was a comprehensive analysis of the cardiac, peripheral blood and pericardial levels of cardiac troponin T (cTnT) in serial medicolegal autopsy cases (n = 405) with a survival time <24 h and within 48 h postmortem to assess the validity of investigating myocardial damage with special regard to traumatic causes of death. These included blunt and sharp instrument injury (n = 122 and 21, respectively), asphyxiation (n = 35), drowning (n = 27), fire fatalities (n = 94), hyperthermia (n = 13), hypothermia (n = 6), fatal methamphetamine (MA) abuse (n = 12) and carbon monoxide (CO) poisoning (n = 5) in comparison with myocardial infarction (MI, n = 57) and cerebrovascular diseases (n = 13). Cases within 12h postmortem usually showed lower cardiac and pericardial cTnT levels than did those of longer postmortem time of 12-48 h. In the early postmortem period of <12 h, significantly elevated serum cTnT levels were observed for hyperthermia. Thereafter, fatal MA abuse, CO poisoning and MI cases also showed higher levels. However, cTnT remained at lower levels for hypothermia and drowning. The elevation of cTnT was associated with the pathology of advanced myocardial damage involving swelling and liquefactive necrosis. The above-mentioned differences were the smallest for peripheral blood. These findings suggest that elevations in postmortem serum and pericardial cTnT levels depend on the severity of myocardial damage at the time of death and are related to the pathological findings, although postmortem interference should be taken into consideration.

Adolescent↗

Array2BIO: from microarray expression data to functional annotation of co-regulated genes.

BACKGROUND: There are several isolated tools for partial analysis of microarray expression data. To provide an integrative, easy-to-use and automated toolkit for the analysis of Affymetrix microarray expression data we have developed Array2BIO, an application that couples several analytical methods into a single web based utility. RESULTS: Array2BIO converts raw intensities into probe expression values, automatically maps those to genes, and subsequently identifies groups of co-expressed genes using two complementary approaches: (1) comparative analysis of signal versus control and (2) clustering analysis of gene expression across different conditions. The identified genes are assigned to functional categories based on Gene Ontology classification and KEGG protein interaction pathways. Array2BIO reliably handles low-expressor genes and provides a set of statistical methods for quantifying expression levels, including Benjamini-Hochberg and Bonferroni multiple testing corrections. An automated interface with the ECR Browser provides evolutionary conservation analysis for the identified gene loci while the interconnection with Crème allows prediction of gene regulatory elements that underlie observed expression patterns. CONCLUSION: We have developed Array2BIO - a web based tool for rapid comprehensive analysis of Affymetrix microarray expression data, which also allows users to link expression data to Dcode.org comparative genomics tools and integrates a system for translating co-expression data into mechanisms of gene co-regulation. Array2BIO is publicly available at http://array2bio.dcode.org.

Algorithms↗

Identification of Freezing-Responsive microRNAs and Their Targets in Chinese Jujube by Small RNA and Degradome Sequencing.

The jujube tree fruit remains a primary fruit in northern China, yet its geographical distribution and yield are significantly constrained by freezing stress during winter. Numerous studies have highlighted the pivotal regulatory function of microRNAs (miRNAs) in plant responses to low-temperature stress. Nevertheless, the specific miRNAs involved in the response to low temperatures and their associated gene networks in Ziziphus jujuba Mill are not well understood. In this investigation, we utilized high-throughput sequencing to analyze small RNA libraries from branches subjected to temperatures of 4 &#xb0;C and -30 &#xb0;C. Our analysis identified a total of 342 miRNAs, comprising 123 known miRNAs and 219 novel miRNAs. The differential expression analysis revealed that under low-temperature conditions, 177 miRNAs underwent significant changes. Among them, specific upregulation of miR319 in the less cold-resistant variety and miR6483 in sensitive variety was observed. By employing degradome sequencing, we identified a total of 1551 target genes corresponding to 3059 unique miRNA target interaction pairs involving 299 miRNAs. Functional analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that these target genes are primarily associated with transcriptional regulation, metabolic pathways, and genetic information processing. Through a comprehensive analysis, we pinpointed 11 genes corresponding to 9 miRNAs that are implicated in jujube tree cold stress, and 7 target genes of 7 miRNAs were confirmed by 5'-RACE analysis. These miRNAs are likely to exert crucial regulatory functions in the context of jujube tree cold stress. This study is the first to systematically identify miRNAs and their target genes in the response of Ziziphus jujuba Mill to low-temperature stress, which provides important resources for in-depth analysis of the molecular mechanism of jujube tree cold resistance and for cold-resistant breeding.

Ziziphus↗

Characterization of arterial hemodynamics in rats with established hypertension.

In long-term hypertension, hemodynamic alterations are associated with functional and structural changes in the cardiovascular system. The technique of arterial impedance spectral analysis has long been used to characterize the arterial hemodynamics of steady and pulsatile components in human hypertension. Although rats with spontaneous hypertension (SHR) are the most common model for studies of primary hypertension, characterization of complete hemodynamic parameters has not been accomplished in this animal model. In the present experiment, aortic flow and pressure waves were recorded in anesthetized, open-chest and ventilated rats. Arterial impedance spectral analysis was employed to obtain steady and pulsatile hemodynamic parameters. A total of 26 SHRs (22-24 wk) and 22 age-matched normotensive WKYs was used. The purpose was to provide a comprehensive analysis of arterial hemodynamics in rats with established hypertension. The SHR had higher arterial pressure (50% increase over the control) than WKY. The total peripheral resistance was elevated by 57%. Stroke volume was decreased by 23% and heart rate increased by 18% without a significant change in cardiac output. The characteristic impedance was increased by 43%, while arterial compliance was decreased by 52%. There were also big rises in ventricular work and wave reflection. The results provide quantitative analysis of the alteration in arterial hemodynamics of steady and pulsatile components in rats with established hypertension. In long-term hypertension, the hemodynamics reflect functional abnormalities in the resistance and Windkessel vessels.

Animals↗

NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases.

NRPS-PKS is web-based software for analysing large multi-enzymatic, multi-domain megasynthases that are involved in the biosynthesis of pharmaceutically important natural products such as cyclosporin, rifamycin and erythromycin. NRPS-PKS has been developed based on a comprehensive analysis of the sequence and structural features of several experimentally characterized biosynthetic gene clusters. The results of these analyses have been organized as four integrated searchable databases for elucidating domain organization and substrate specificity of nonribosomal peptide synthetases and three types of polyketide synthases. These databases work as the backend of NRPS-PKS and provide the knowledge base for predicting domain organization and substrate specificity of uncharacterized NRPS/PKS clusters. Benchmarking on a large set of biosynthetic gene clusters has demonstrated that, apart from correct identification of NRPS and PKS domains, NRPS-PKS can also predict specificities of adenylation and acyltransferase domains with reasonably high accuracy. These features of NRPS-PKS make it a valuable resource for identification of natural products biosynthesized by NRPS/PKS gene clusters found in newly sequenced genomes. The training and test sets of gene clusters included in NRPS-PKS correlate information on 307 open reading frames, 2223 functional protein domains, 68 starter/extender precursors and their specific recognition motifs, and also the chemical structure of 101 natural products from four different families. NRPS-PKS is a unique resource which provides a user-friendly interface for correlating chemical structures of natural products with the domains and modules in the corresponding nonribosomal peptide synthetases or polyketide synthases. It also provides guidelines for domain/module swapping as well as site-directed mutagenesis experiments to engineer biosynthesis of novel natural products. NRPS-PKS can be accessed at http://www.nii.res.in/nrps-pks.html.

Databases, Protein↗