The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland.

The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user's computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins, are preferentially expressed during lactation.

Flicker image comparison of 2-D gel images for putative protein identification using the 2DWG meta-database.

With the availability of two-dimensional (2-D) gel electrophoresis databases that have many characterized proteins, it may be possible to compare a researcher's gel images with those in relevant databases. This may lead to the putative identification of unknown protein spots in a researcher's gel with those characterized in a given database, saving the researcher time and money by suggesting monoclonal antibodies to try in confirming these identifications. We have developed two tools to help with this comparison: (1) Flicker, http:/(/)www.lecb.ncifcrf.gov/flicker/, a Java applet program running in the researcher's Web browser, to visually compare their gels against gels on the Internet; and (2) the 2DWG meta-database, http:/(/)www.lecb.ncifcrf.gov/2dwgDB /, a searchable database of locations of 2-D electrophoretic gel images found on the Internet. Recent additions to Flicker allow users to click on a protein spot in a gel that is linked to a federated 2D gel database, such as SWISS-2DPAGE, and have it retrieve a report from that Web database for that protein.

Exploratory data analysis groupware for qualitative and quantitative electrophoretic gel analysis over the Internet-WebGel.

Many scientists use quantitative measurements to compare the presence and amount, of various proteins and nucleotides among series of one- and two-dimensional (1-D and 2-D) electrophoretic gels. These gels are often scanned into digital image files. Gel spots are then quantified using stand-alone analysis software. However, as more research collaborations take place over the Internet, it has become useful to share intermediate quantitative data between researchers. This allows research group members to investigate their data and share their work in progress. We developed a World Wide Web group-accessible software system, WebGel, for interactively exploring qualitative and quantitative differences between electrophoretic gels. Such Internet databases are useful for publishing quantitative data and allow other researchers to explore the data with respect to their own research. Because intermediate results of one user may be shared with their collaborators using WebGel, this form of active data-sharing constitutes a groupware method for enhancing collaborative research. Quantitative and image gel data from a stand-alone gel image processing system are copied to a database accessible on the WebGel Web server. These data are then available for analysis by the WebGel database program residing on that server. Visualization is critical for better understanding of the data. WebGel helps organize labeled gel images into montages of corresponding spots as seen in these different gels. Various views of multiple gel images, including sets of spots, normalization spots, labeled spots, segmented gels, etc. may also be displayed. These displays are active and may be used for performing database operations directly on individual protein spots by simply clicking on them. Corresponding regions between sets of gels may be visually analyzed using Flicker-comparison (Electrophoresis 1997, 18, 122-140) as one of the WebGel methods for qualitative analysis. Quantitative exploratory data analysis can be performed by comparing protein concentration values between corresponding spots for multiple samples run in separate gels. These data are then used to generate reports on statistical differences between sets of gels (e.g., between different disease states such as benign or metastatic cancers, etc.). Using combined visual and quantitative methods, WebGel can help bridge the analysis of dissimilar gels which are difficult to analyze with stand-alone systems and can serve as a collaborative Internet tool in a groupware setting.

Nuclear shape and nuclear matrix protein composition in prostate and seminal vesicles.

OBJECTIVES: The nucleus controls cell function and behavior. The nuclear matrix determines internal nuclear changes. Two-dimensional gel electrophoresis is the reference standard for the analysis of nuclear matrix protein (NMP) composition. Differences in NMP composition should therefore be reflected by changes in nuclear shape. We investigated the differences in NMP composition and nuclear morphometry of the prostate and seminal vesicles. Both tissues are androgen-dependent sex accessory organs with completely different biologic behavior. METHODS: High-resolution two-dimensional gel electrophoresis and silver staining were used to evaluate NMP composition from histologically normal prostate and seminal vesicle epithelial cells. Nuclear morphometry, performed using a computer-assisted image analysis system, described the distribution, variability, and extremes of nuclear shape. RESULTS: NMP composition analysis demonstrated that both tissues have a similar NMP composition, and tissue-specific NMPs that were consistently present in all specimens of each tissue could not be demonstrated. Nuclear morphometry showed a significantly greater heterogeneity in nuclear shape in the seminal vesicles than in the prostate. CONCLUSIONS: The striking similarity of the NMP composition demonstrates the close biologic relationship between prostate and seminal vesicle tissue. The similar NMP composition does not correlate with the marked alterations in nuclear shape and structure between these tissues. Therefore, nuclear morphometry may depict differences in the functional state of a similar set of NMPs, shown by two-dimensional gel electrophoresis, which may be responsible for the different biologic behavior of these tissues.

Comparing two-dimensional electrophoretic gel images across the Internet.

Scientists around the world often work on similar data so the need to share results and compare data arises periodically. We describe a method of comparing two two-dimensional (2-D) protein gels of similar samples created in different laboratories to help identify or suggest protein spot identification. Now that 2-D gels and associated databases frequently appear on the Internet, this opens up the possibility of visually comparing one's own experimental 2-D gel image data with data from another gel in a remote Internet database. In general, there are a few ways to compare images: (i) slide one gel (autoradiograph or stained gel) over the other while back-illuminated, or (ii) build a 2-D gel computer database from both gels after scanning and analyzing these gels. These are impractical since in the first case the gel from the Internet database is not locally available. In the second, the costs of building a multi-gel database solely to answer the question of whether a spot is the same spot may be excessive if only a single visual comparison is needed. We describe a distributed gel comparison program (URL: http://www-lmmb.ncifcrf.gov/flicker) which runs on any World Wide Web (WWW) connected computer and is invoked from a Java-capable web browser. One gel image is read from any Internet 2-D gel database (e.g. SWISS-2DPAGE) and the other may reside on the investigator's computer. Images may be more easily compared by first applying spatial warping or other transforms interactively on the user's computer. First, regions of interest are "landmarked" with several corresponding points in each gel image, then one gel image is warped to the geometry of the other. As the two gels are rapidly alternated, or flickered, in the same window, the user can slide one gel past the other to visually align corresponding spots by matching local morphology. This flicker-comparison technique may be applied to analyzing other types of one-dimensional and 2-D biomedical images.

The 2DWG meta-database of two-dimensional electrophoretic gel images on the Internet.

The 2DWG meta-database is a searchable database of two-dimensional (2-D) electrophoretic gel images found on the Internet. A meta-database contains information about locating data in other databases - but not that data itself. This database was constructed because of a need for an enriched set of World Wide Web (WWW) locations (URLs) of 2-D gel images on the Internet. These gel images are used in conjunction with the National Cancer Institute (NCI) Flicker Server to manipulate and visually compare 2-D gel images across the Internet. User's gels may also be compared with those in the database. The 2DWG is organized as a spreadsheet table with each gel image being represented by a row sorted by tissue type. Data for each gel includes tissue type, species, cell-line, image URL, database URL, gel protocol, organization URL, image properties, map URL if it exists, etc. The 2DWG may be searched to find relevant subsets of gels. Searching is done using the dbEngine - a WWW database search engine which accesses selected rows of gels from the full 2DWG table. The 2DWG meta-database is accessible on the WWW at http://www-lecb.ncifcrf.gov/2dwgDB/ and the NCI Flicker server at http://www-lecb.ncifcrf.gov/flicker/

A World Wide Web (WWW) server database engine for an organelle database, MitoDat.

We describe a simple database search engine "dbEngine" which may be used to quickly create a searchable database on a World Wide Web (WWW) server. Data may be prepared from spreadsheet programs (such as Excel, etc.) or from tables exported from relationship database systems. This Common Gateway Interface (CGI-BIN) program is used with a WWW server such as available commercially, or from National Center for Supercomputer Algorithms (NCSA) or CERN. Its capabilities include: (i) searching records by combinations of terms connected with ANDs or ORs; (ii) returning search results as hypertext links to other WWW database servers; (iii) mapping lists of literature reference identifiers to the full references; (iv) creating bidirectional hypertext links between pictures and the database. DbEngine has been used to support the MitoDat database (Mendelian and non-Mendelian inheritance associated with the Mitochondrion) on the WWW.

Two-dimensional protein electrophoresis and multiple hypothesis testing to detect potential serum protein biomarkers in children with fetal alcohol syndrome.

Fetal alcohol syndrome (FAS) surveillance and intervention efforts are hampered by the lack of a specific biochemical test for diagnosis of the syndrome. Based on the hypothesis that abnormalities in growth and development (key features of FAS) involve altered protein metabolism, we analyzed serum proteins by two-dimensional gel electrophoresis and image analysis to search for potential protein biomarkers of FAS. Serum samples from 12 participants in whom FAS had been diagnosed and 8 sex- and age-matched participants whose mothers did not consume alcohol were analyzed in duplicate to determine whether the integrated intensities of matched proteins are significantly altered in children with FAS. Multiple hypothesis testing on 34 of the gels consisting of more than 1700 spots per gel revealed 21 proteins that we classified as potential protein biomarkers of FAS on the basis of significant t-test differences at p < 0.02. We classified 8 of the proteins as candidate biomarkers on the basis of significant concentration differences between case and control subjects at p < 0.01. One of the proteins is clearly an isoform of retinol binding protein; two appear in the area of the gel where alcohol dehydrogenase is expected to appear; one appears to be an isoform of alpha-1-antitrypsin; three appear to be isoforms of the beta-chain of haptoglobin; three may be forms of immunoglobulin light chains; and several others have not been associated with known proteins. No single protein differentiated all case subjects from control subjects, but stepwise canonical discriminant analyses revealed four groups of spots that distinguished between FAS case and control subjects with no misclassifications.(ABSTRACT TRUNCATED AT 250 WORDS)

The protein disease database of human body fluids: I. Rationale for the development of this database.

We are developing a relational database to facilitate quantitative and qualitative comparisons of proteins in human body fluids in normal and disease states. For decades researchers and clinicians have been studying proteins in body fluids such as serum, plasma, cerebrospinal fluid and urine. Currently, most clinicians evaluate only a few specific proteins in a body fluid such as plasma when they suspect that a patient has a disease. Now, however, high resolution two-dimensional protein electrophoresis allows the simultaneous evaluation of 1,500 to 3,000 proteins in complex solutions, such as the body fluids. This and other high resolution methods have encouraged us to collect the clinical data for the body fluid proteins into an easily accessed database. For this reason, it has been constructed on the Internet World Wide Web (WWW) under the title Protein Disease Database (PDD). In addition, this database will provide a linkage between the disease-associated protein alterations and images of the appropriate proteins on high-resolution electrophoretic gels of the body fluids. This effort requires the normalization of data to account for variations in methods of measurement. Initial efforts in the establishment of the PDD have been concentrated on alterations in the acute-phase proteins in individuals with acute and chronic diseases. Even at this early stage in the development of our database, it has proven to be useful as we have found that there appear to be several common acute-phase protein alterations in the plasma and cerebrospinal fluid from patients with Alzheimer's disease, schizophrenia and major depression. Our goal is to provide access to the PDD so that systematic correlations and relationships between disease states can be examined and extended.

The Protein Disease Database of human body fluids: II. Computer methods and data issues.

The Protein Disease Database (PDD) is a relational database of proteins and diseases. With this database it is possible to screen for quantitative protein abnormalities associated with disease states. These quantitative relationships use data drawn from the peer-reviewed biomedical literature. Assays may also include those observed in high-resolution electrophoretic gels that offer the potential to quantitate many proteins in a single test as well as data gathered by enzymatic or immunologic assays. We are using the Internet World Wide Web (WWW) and the Web browser paradigm as an access method for wide distribution and querying of the Protein Disease Database. The WWW hypertext transfer protocol and its Common Gateway Interface make it possible to build powerful graphical user interfaces that can support easy-to-use data retrieval using query specification forms or images. The details of these interactions are totally transparent to the users of these forms. Using a client-server SQL relational database, user query access, initial data entry and database maintenance are all performed over the Internet with a Web browser. We discuss the underlying design issues, mapping mechanisms and assumptions that we used in constructing the system, data entry, access to the database server, security, and synthesis of derived two-dimensional gel image maps and hypertext documents resulting from SQL database searches.

An efficient disk based data structure for rapid searching of quantitative two-dimensional gel databases.

Fast access of two-dimensional (2-D) gel quantitative databases is important for rapid searching for protein differences between sets of 2-D gels from an experiment. The GELLAB-II system organizes corresponding spots from the gels in the database into reference or "Rspot" sets. These Rspot numeric names index fixed regions in the paged composite gel database file. This is adequate for an existing database, but has several problems. (i) Building the initial database requires guessing how much disk space to pre-allocate for each corresponding spot (i.e. spots from different gels). If it ever runs out of pre-allocated space during this process, it must expand the size of each corresponding set of spots copying the old database data into the new in-place on the disk. (ii) When adding new gels or editing the database, if a new spot is created, the system may also go into this expansion mode. The time spent and wasted disk space can be appreciable--depending on the size of the database (order of 100 gel database). (iii) Because each set of corresponding spots is the same size, we waste space in most spot sets since they do not require the additional space a few spot sets require which contain additional fragmented spots. We present a new low-level disk object-based structure and algorithm, paged indexed buckets (PIB), which optimizes disk space usage while having similar retrieval speed to the original method.

A fast spot segmentation algorithm for two-dimensional gel electrophoresis analysis.

An important issue in the automation of two-dimensional gel electrophoresis image analysis is the detection and quantification of protein spots. A spot segmentation algorithm must detect, define the extent of, and measure the integrated density of spots under a wide variety of actual gel image conditions. Besides these functions, the algorithm must be memory efficient to be able to process very large gel images and do this in a reasonable amount of computation time on low-cost computers, such as workstations and personal computers. We have developed a fast spot segmentation algorithm, extending the GELLAB-II segmenter, which extracts spots in a single raster scanning pass through the gel image. The performance analysis of the algorithm will be given in the paper as well as a discussion of the algorithm.

ECGF and heparin determine differentiation of cloned cerebral endothelial cells in vitro.

Protein expression patterns of morphologically different cloned capillary endothelial cells from porcine and murine brain cortices were examined. Type I cells, grown in medium containing heparin and endothelial cell growth factor (ECGF), exhibited a polygonal, cobblestone appearance and appeared to replicate the cells of the blood-brain barrier endothelium. Type II cells, grown in medium without heparin and ECGF, were elongated and appeared to replicate capillaries in central nervous system tissue. Cells of both phenotypes stained positive by the specific endothelial cell marker Bandeiraea simplicifolia lectin. The expression of alpha smooth-muscle actin (mRNA and protein) was taken as a marker for type II cells. By use of 2-D gel images and the GELLAB II system, a data base was created revealing that two proteins (90 kDa, pI 5.1, and 35 kDa, pI 5.7) were exclusively expressed in type I cells. Furthermore, the synergistic action of ECGF and heparin in respect to the phenotypic determination of cerebral endothelial cells was demonstrated.

Xconf: a network-based image conferencing system.

People often need to get together to share and discuss small amounts of image and textual data, but this is difficult when they are not located in the same place. One solution to this problem is Xconf, a multimedia computer conferencing groupware tool using existing national and international networks (the Internet). Simultaneous conferencing supports real-time interaction between multiple remote computer displays. Xconf multimedia may include conversational text, images, pointers to objects in images, and group execution of programs. Conferencing may take place with or without images. Interaction is tightly coupled with all users aware of global changes to the shared session and alternatively, individuals may monitor a specific subgroup of other users to concentrate on what they are discussing. Collaborative groups who have access to both computer networks and networked based X-Window System graphics displays can participate in a conference. Xconf is an X-Window "client" program which provides multimedia conferencing support for a group of X-Window displays. Because it is centralized, no software other than the standard X-Window System is required on any of the participants display systems. Key data structures and algorithms for image conferencing are present.

Splitting merged spots in two-dimensional polyacrylamide gel electrophoresis gel images.

We describe a heuristic computer algorithm using boundary analysis for improving spot finding and spot quantitation of large saturated or near-saturated spots in two-dimensional polyacrylamide electrophoresis gels. This spot quantitation is done using spot segmentation, which consists of spot finding and subsequent quantification steps. Occasionally, clusters of large saturated spots may become merged during spot finding. To correct this, the merged spots must be cut apart before quantitation. It is generally obvious from viewing the merged spot's border where they should be cut--at opposing saddlepoints (concavities in the boundary). The algorithm uses an analysis of the missegmented spot's boundary when a saturated spot is detected. If a near-saturated spot is larger than a given size, the spot segmenter program attempts to merge saturated fragments. When merging occurs, the segmenter program analyses the boundary to see if the spot should be split. The new algorithm first finds all robust concavities and then tries to match complementary ones. These paired concavities are then used to guide cutting of the missegmented spot into two or more separate spot regions. Finally, control is returned to the segmenter program to reprocess the data as a set of smaller separated spots.

Comparison of the Bio Image Visage 2000 and the GELLAB-II two-dimensional electrophoresis image analysis systems.

To compare the Visage 2000 analysis system (Bio Image, Ann Arbor, MI, USA) with the GELLAB-II analysis system (National Cancer Institute, Frederick, MD, USA), we used each to perform image analysis of the same 29 silver-stained two-dimensional electrophoresis (2DE) gel image files from a study of urinary proteins in metal recovery plant workers who had confirmed body burdens of cadmium. Visage, aided by interactive analysis, detected an average of 890 +/- 177.6 spots per gel, or a total of 25,800 spots, whereas GELLAB-II detected 1971 +/- 198.5 spots per gel, or a total of 57,160 (a 222% increase over the Visage system), without operator intervention. Visage automatically quantified 52.5% (13,556) of the spots; 47.2% (12,173), consisting mostly of larger spots, had to be quantified interactively with an image editor, and 0.3% (71) were not quantified. GELLAB-II automatically quantified all detected spots. After we interactively assigned the maximum allowed number of landmarks (30 for Visage and 52 for GELLAB-II), we found that Visage matched 657 +/- 211.2 spots per gel, and GELLAB-II matched all detected spots and also extrapolated an average of 1269 virtual spots per gel. Plots of densities from the two systems on selected spots showed excellent agreement, and both systems showed high correlation between their measurements of the beta-2-microglobulin spot densities and an independent radioimmunoassay quantification of the original urine samples. By comparing the regression of the densities of all spots with urinary cadmium (UCD) levels, we found that several of the same detected spots from each system were highly correlated. The densities of four acidic proteins with relative molecular weights of approximately 112,000 Da (as quantified by GELLAB-II but not by Visage) were highly correlated with UCD concentrations. These proteins are new candidate biomarkers of cadmium toxicity. We compared the estimated labor costs of using each system to analyse a hypothetical 20-sample (60 gels) 2DE study and found that GELLAB-II was six times less expensive to use than Visage, primarily because of the operator time required to do interactive error correction with the Visage system.

Automatic detection of noisy spots in two-dimensional Southern blots. International Electrophoresis Society Meeting, Washington DC, March 16-19, 1991.

One of the problems of automatically quantitating 2D DNA gels, is clearly detecting spots visualized by Southern hybridization blots using DNA probes (Rogan et al. in this conference). Spots appear noisy due to multiple transfer steps. If one applies standard 2D PAGE protein gel spot segmentation methods, spots fragment due to highly textured image noise and a weak radioautograph signal and thus are poorly detected. We have observed that these spots are all of a minimum size. Therefore an image processing filter which both takes minimum spot size into account and has immunity to image texture-type noise should be able to reliably detect this class of spots. The 'Busse' Laplacian filter used in the GELLAB-II system, is a modification of the standard (1-21) digital approximation of the Laplacian. In the Busse Laplacian, the sampling interval is n pixels (n greater than 1) instead of 1. In addition, 3 x 3 averaged 'super pixel' values are used instead of single pixels for each element of the Laplacian convolution. This gives the needed noise immunity by filtering out the high spatial frequency image noise while preserving the low spatial frequency character of the spots. We have used this filter successfully on 2D DNA Southern blot image data.

Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons.

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-1 and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification.