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Edge enhanced images can be produced on existing commercial computed tomographic equipment by a method called "digital unsharp masking" without any expense or computer software development. This technique permits display of anatomic areas having an extremely wide range of densities, while making edge detail more apparent.
BACKGROUND: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. METHODS: Nuclear DNA was stained in 14 microm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of x40 or x100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. RESULTS: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at x60 or x100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. CONCLUSIONS: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.
Accurate information on the relationships among individuals in a study is critical for valid linkage analysis. We extend the MLLR, EIBD, AIBS and IBS tests for detection of misspecified relationships to a broader range of relative pairs, and we improve the two-stage screening procedure for analyzing large data sets. We have developed software, PREST, which calculates the test statistics and performs the corresponding hypothesis tests for relationship misclassification in general outbred pedigrees. When a potential pedigree error is detected, our companion program, ALTERTEST, can be used to determine which relationships are compatible with the genotype data. Both programs are now freely available on the web.
BACKGROUND: There is increasing knowledge about the genetic basis of pancreatic cancer (PaCa). Tumor suppressor genes (TSGs; e.g. p53 and DPC4) and oncogenes (e.g. K-ras) have been shown to be involved in the development of PaCa. However, the extent of chromosomal changes (gains and losses) implicates that many more genes may be involved in the multistep progression of PaCa. Identification of these genes is essential for understanding the molecular events in the development of PaCa. METHODS: We assembled public and proprietary libraries of more than 4 million expressed sequence tags using newly developed software tools. RESULTS: We identified a total of 249 genes with specific expression patterns in normal and cancerous tissue of the pancreas. Of these, 27 genes were found to be preferentially expressed in normal tissue of the pancreas, while 222 genes showed significant upregulation of expression in PaCa. Of the 249 genes, 232 (93.2%) were found to represent known human genes or putative human homologues of genes characterized previously in other species, while 17 (6.8%) represent putative new genes. CONCLUSION: These genes may represent a valuable source to identify novel TSGs and oncogenes involved in the carcinogenesis of PaCa.
The new DIN ('Deutsche Industrie-Norm') 6875-1, which is currently being finalised, deals with quality assurance (QA) criteria and tests methods for linear accelerator and Gamma Knife stereotactic radiosurgery/radiotherapy including treatment planning, stereotactic frame and stereotactic imaging and a system test to check the whole chain of uncertainties. Our existing QA program, based on dedicated phantoms and test procedures, has been refined to fulfill the demands of this new DIN. The radiological and mechanical isocentre corresponded within 0.2 mm and the measured 50% isodose lines were in agreement with the calculated ones within less than 0.5 mm. The measured absorbed dose was within 3%. The resultant output factors measured for the 14-, 8- and 4-mm collimator helmet were 0.9870 +/- 0.0086, 0.9578 +/- 0.0057 and 0.8741 +/- 0.0202, respectively. For 170 consecutive tests, the mean geometrical accuracy was 0.48 +/- 0.23 mm. Besides QA phantoms and analysis software developed in-house, the use of commercially available tools facilitated the QA according to the DIN 6875-1 with which our results complied.
BACKGROUND: Modern nonfluoroscopic mapping systems construct 3D electroanatomic maps by tracking intracardiac catheters. They require specialized catheters and/or dedicated hardware. We developed a new method for electroanatomic mapping by merging detailed 3D models of the endocardial cavities with fluoroscopic images without the need for specialized hardware. This developmental work focused on the right atrium because of the difficulties in visualizing its anatomic landmarks in 3D with current approaches. METHODS AND RESULTS: Cardiac MRI images were acquired in 39 patients referred for radiofrequency catheter ablation using balanced steady state free-precession sequences. We optimized acquisition and developed software for construction of detailed 3D models, after contouring of endocardial cavities with cross-checking of different imaging planes. 3D models were then merged with biplane fluoroscopic images by methods for image calibration and registration implemented in a custom software application. The feasibility and accuracy of this merging process were determined in heart-cast experiments and electroanatomic mapping in patients. Right atrial dimensions and relevant anatomic landmarks could be identified and measured in all 3D models. Cephalocaudal, posteroanterior, and lateroseptal diameters were, respectively, 65+/-11, 54+/-11, and 57+/-9 mm; posterior isthmus length was 26+/-6 mm; Eustachian valve height was 5+/-5 mm; and coronary sinus ostium height and width were 16+/-3 and 12+/-3 mm, respectively (n=39). The average alignment error was 0.2+/-0.3 mm in heart casts (n=40) and 1.9 to 2.5 mm in patient experiments (n=9), ie, acceptable for clinical use. In 11 patients, reliable catheter positioning and projection of activation times resulted in 3D electroanatomic maps with an unprecedented level of anatomic detail, which assisted ablation. CONCLUSIONS: This new approach allows activation visualization in a highly detailed 3D anatomic environment without the need for a specialized nonfluoroscopic mapping system.
Peritrate (pentaerythritol tetranitrate), a nitrate coronary vasodilator, was capable of significantly increasing perfusion and function in ischemic heart muscle. The A2 image-processing computer with software developed by Burow was used to evaluate regional perfusion and segmental wall motion in six patients with ischemic areas in the myocardium. These image-processing techniques were satisfactory for evaluation of ischemic heart muscle.
Pediatricians in training and clinical practice may be eager to submit their first scientific publication by writing a letter to the editor, case report, review article, or short clinical series. Unfortunately, the would-be author encounters a literary developmental delay when facing the blank page or computer screen. This article is dedicated to the novice author and offers practical tips on writing the first manuscript. Authors are encouraged to draw from their extensive experience writing other materials and to approach the task in a systematic way. Computer technology and software developments should facilitate the process.
Analytic approaches, including the structural equation model (autoregressive panel model), hierarchical linear model, latent growth curve model, survival/event history analysis, latent transition model, and time-series analysis (interrupted time series, multivariate time-series analysis) are discussed for their applicability to data of different structures and their utility in evaluating temporal effects of treatment. Methods are illustrated by presenting applications of the various approaches in previous studies examining temporal patterns of treatment effects. Recent advancements in these longitudinal modeling approaches and the accompanying computer software development offer tremendous flexibility in examining long-term treatment effects through longitudinal data with varying numbers and intervals of assessment and types of measures. A multimethod assessment will contribute to a more complete understanding of the complex phenomena of the long-term courses of substance use and its treatment.
BACKGROUND: Our understanding of both the importance of local control for survival of patients with unresectable lung cancer and the inadequacy of conventional radiation therapy (RT) to provide this local control has undergone marked changes in the past 2 decades. METHODS: A review was conducted of recent studies and meta-analyses in the literature that have convincingly demonstrated the value of thoracic irradiation in increasing long-term survival in patients with both small-cell lung cancer and non-small-cell lung cancer (NSCLC). RESULTS: Large cooperative trials have shown long-term local control of only approximately 10% for NSCLC using conventionally planned radiation to doses of 60-64 Gy either as a single modality or when preceded by induction chemotherapy. Concurrent chemotherapy may modestly improve local control at the cost of greater acute esophageal toxicity. Simple escalation of radiation dose is limited by the tolerance of normal intrathoracic organs. Recent developments in anatomic and functional imaging, computerized RT planning, and RT delivery, as well as a reassessment of the appropriate target volumes for RT in the context of combined modality therapy, provide the capability to better conform regions of high dose to the target volume and test the hypothesis that increases in tumor dose will improve local control and survival. CONCLUSIONS: Encouraging phase II data have been reported from single institutions using individually developed software and hardware. The availability of commercial tools for planning and delivering such conformal treatment will allow prospective assessment of the true value of these technologies in the management of patients with lung cancer.
High-throughput screening (HTS) is used in modern drug discovery to screen hundreds of thousands to millions of compounds on selected protein targets. It is an industrial-scale process relying on sophisticated automation and state-of-the-art detection technologies. Quality control (QC) is an integral part of the process and is used to ensure good quality data and mini mize assay variability while maintaining assay sensitivity. The authors describe new QC methods and show numerous real examples from their biologist-friendly Stat Server HTS application, a custom-developed software tool built from the commercially available S-PLUS and Stat Server statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables. It allows users to visualize HTS data and examine assay performance during the HTS campaign to quickly react to or avoid quality problems.
High-throughput screening (HTS) plays a central role in modern drug discovery, allowing the rapid screening of large compound collections against a variety of putative drug targets. HTS is an industrial-scale process, relying on sophisticated automation, control, and state-of-the art detection technologies to organize, test, and measure hundreds of thousands to millions of compounds in nano- to microliter volumes. Despite this high technology, hit selection for HTS is still typically done using simple data analysis and basic statistical methods. The authors discuss in this article some shortcomings of these methods and present alternatives based on modern methods of statistical data analysis. Most important, they describe and show numerous real examples from the biologist-friendly Stat Server HTS application (SHS), a custom-developed software tool built on the commercially available S-PLUS and StatServer statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables.
This article presents the use of digital image processing using hue, saturation, and intensity measurements as a technique for the color analysis of chronic wounds on the skin. An adaptive spline technique was used to segment the wound boundary in the images of venous leg ulcers. This technique was further used to approximate the position of venous leg ulcers. The amount of slough within the wound site was quantified using the software developed and was compared with a grading system based on visual inspection by an experienced clinician, and the results were compared by deriving Kappa (K) statistic. There was moderate agreement over all grades between the computer and clinician. At lower grades 1 and 2, there was excellent agreement. The results from this preliminary study suggest that this analytical technique has the potential to image process chronic skin wounds.
BACKGROUND: In order to take full advantage of the newly available public human genome sequence data and associated annotations, biologists require visualization tools ("genome browsers") that can accommodate the high frequency of alternative splicing in human genes and other complexities. RESULTS: In this article, we describe visualization techniques for presenting human genomic sequence data and annotations in an interactive, graphical format. These techniques include: one-dimensional, semantic zooming to show sequence data alongside gene structures; color-coding exons to indicate frame of translation; adjustable, moveable tiers to permit easier inspection of a genomic scene; and display of protein annotations alongside gene structures to show how alternative splicing impacts protein structure and function. These techniques are illustrated using examples from two genome browser applications: the Neomorphic GeneViewer annotation tool and ProtAnnot, a prototype viewer which shows protein annotations in the context of genomic sequence. CONCLUSION: By presenting techniques for visualizing genomic data, we hope to provide interested software developers with a guide to what features are most likely to meet the needs of biologists as they seek to make sense of the rapidly expanding body of public genomic data and annotations.
BACKGROUND: Computer programs for the generation of multiple sequence alignments such as "Clustal W" allow detection of regions that are most conserved among many sequence variants. However, even for regions that are equally conserved, their potential utility as hybridization targets varies. Mismatches in sequence variants are more disruptive in some duplexes than in others. Additionally, the propensity for self-interactions amongst oligonucleotides targeting conserved regions differs and the structure of target regions themselves can also influence hybridization efficiency. There is a need to develop software that will employ thermodynamic selection criteria for finding optimal hybridization targets in related sequences. RESULTS: A new scheme and new software for optimal detection of oligonucleotide hybridization targets common to families of aligned sequences is suggested and applied to aligned sequence variants of the complete HIV-1 genome. The scheme employs sequential filtering procedures with experimentally determined thermodynamic cut off points: 1) creation of a consensus sequence of RNA or DNA from aligned sequence variants with specification of the lengths of fragments to be used as oligonucleotide targets in the analyses; 2) selection of DNA oligonucleotides that have pairing potential, greater than a defined threshold, with all variants of aligned RNA sequences; 3) elimination of DNA oligonucleotides that have self-pairing potentials for intra- and inter-molecular interactions greater than defined thresholds. This scheme has been applied to the HIV-1 genome with experimentally determined thermodynamic cut off points. Theoretically optimal RNA target regions for consensus oligonucleotides were found. They can be further used for improvement of oligo-probe based HIV detection techniques. CONCLUSIONS: A selection scheme with thermodynamic thresholds and software is presented in this study. The package can be used for any purpose where there is a need to design optimal consensus oligonucleotides capable of interacting efficiently with hybridization targets common to families of aligned RNA or DNA sequences. Our thermodynamic approach can be helpful in designing consensus oligonucleotides with consistently high affinity to target variants in evolutionary related genes or genomes.
BACKGROUND: Genome databases contain diverse kinds of information, including gene annotations and nucleotide and amino acid sequences. It is not easy to integrate such information for genomic study. There are few tools for integrated analyses of genomic data, therefore, we developed software that enables users to handle, manipulate, and analyze genome data with a variety of sequence analysis programs. RESULTS: The G-InforBIO system is a novel tool for genome data management and sequence analysis. The system can import genome data encoded as eXtensible Markup Language documents as formatted text documents, including annotations and sequences, from DNA Data Bank of Japan and GenBank encoded as flat files. The genome database is constructed automatically after importing, and the database can be exported as documents formatted with eXtensible Markup Language or tab-deliminated text. Users can retrieve data from the database by keyword searches, edit annotation data of genes, and process data with G-InforBIO. In addition, information in the G-InforBIO database can be analyzed seamlessly with nine different software programs, including programs for clustering and homology analyses. CONCLUSION: The G-InforBIO system simplifies genome analyses by integrating several available software programs to allow efficient handling and manipulation of genome data. G-InforBIO is freely available from the download site.
BACKGROUND: When organismal phylogenies based on sequences of single marker genes are poorly resolved, a logical approach is to add more markers, on the assumption that weak but congruent phylogenetic signal will be reinforced in such multigene trees. Such approaches are valid only when the several markers indeed have identical phylogenies, an issue which many multigene methods (such as the use of concatenated gene sequences or the assembly of supertrees) do not directly address. Indeed, even when the true history is a mixture of vertical descent for some genes and lateral gene transfer (LGT) for others, such methods produce unique topologies. RESULTS: We have developed software that aims to extract evidence for vertical and lateral inheritance from a set of gene trees compared against an arbitrary reference tree. This evidence is then displayed as a synthesis showing support over the tree for vertical inheritance, overlaid with explicit lateral gene transfer (LGT) events inferred to have occurred over the history of the tree. Like splits-tree methods, one can thus identify nodes at which conflict occurs. Additionally one can make reasonable inferences about vertical and lateral signal, assigning putative donors and recipients. CONCLUSION: A tool such as ours can serve to explore the reticulated dimensionality of molecular evolution, by dissecting vertical and lateral inheritance at high resolution. By this, we mean that individual nodes can be examined not only for congruence, but also for coherence in light of LGT. We assert that our tools will facilitate the comparison of phylogenetic trees, and the interpretation of conflicting data.
The goal of this study is to evaluate, compare, and contrast several standard and new linkage analysis methods. First, we compare a recently proposed confidence set approach with MAPMAKER/SIBS. Then, we evaluate a new Bayesian approach that accounts for heterogeneity. Finally, the newly developed software SIMPLE is compared with GENEHUNTER. We apply these methods to several replicates of the Genetic Analysis Workshop 13 simulated data to assess their ability to detect the high blood pressure genes on chromosome 21, whose positions were known to us prior to the analyses. In contrast to the standard methods, most of the new approaches are able to identify at least one of the disease genes in all the replicates considered.