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High density peptide microarrays. In situ synthesis and applications.

The technologies enabling the creation of large scale, miniaturized peptide or protein microarrays are emerging. The focuses of this review are the synthesis and applications of peptide and peptidomimetic microarrays, especially the light directed parallel synthesis of individually addressable high density peptide microarrays using a novel photogenerated reagent chemistry and digital photolithography (Gao et al., 1998, J. Am. Chem. Soc. 120, 12698; Pellois et al. 2002, Nat. Biotechnol. 20, 922). Concepts related to the synthesis are discussed, such as the reactions of photogenerated acids in the deprotection step of peptide synthesis or oligonucleotide synthesis, and the applications of high density peptide chips in antibody binding assays are discussed. Peptide chips provide versatile tools for probing antigen-antibody, protein-protein, peptide-ligand interactions and are basic components for miniaturization, automation, and system integration in research and clinical diagnosis applications.

Peptide Library↗

JAtlasView: a Java atlas-viewer for browsing biomedical 3D images and atlases.

BACKGROUND: Many three-dimensional (3D) images are routinely collected in biomedical research and a number of digital atlases with associated anatomical and other information have been published. A number of tools are available for viewing this data ranging from commercial visualization packages to freely available, typically system architecture dependent, solutions. Here we discuss an atlas viewer implemented to run on any workstation using the architecture neutral Java programming language. RESULTS: We report the development of a freely available Java based viewer for 3D image data, descibe the structure and functionality of the viewer and how automated tools can be developed to manage the Java Native Interface code. The viewer allows arbitrary re-sectioning of the data and interactive browsing through the volume. With appropriately formatted data, for example as provided for the Electronic Atlas of the Developing Human Brain, a 3D surface view and anatomical browsing is available. The interface is developed in Java with Java3D providing the 3D rendering. For efficiency the image data is manipulated using the Woolz image-processing library provided as a dynamically linked module for each machine architecture. CONCLUSION: We conclude that Java provides an appropriate environment for efficient development of these tools and techniques exist to allow computationally efficient image-processing libraries to be integrated relatively easily.

Anatomy↗

Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes.

Colored chromosome staining patterns, termed chromosomal 'bar codes' (CBCs), were obtained on human chromosomes by fluorescence in situ hybridization (FISH) with pools of Alu-PCR products from YAC clones containing human DNA inserts ranging from 100 kbp to 1 Mbp. In contrast to conventional G- or R-bands, the chromosomal position, extent, individual color and relative signal intensity of each 'bar' could be modified depending on probe selection and labeling procedures. Alu-PCR amplification products were generated from 31 YAC clones which mapped to 37 different chromosome bands. For multiple color FISH, Alu-PCR amplification products from various clones were either biotinylated or labeled with digoxigenin. Probes from up to twenty YAC clones were used simultaneously to produce CBCs on selected human chromosomes. Evaluation using a cooled CCD camera and digital image analysis confirmed the high reproducibility of the bars from one metaphase spread to another. Combinatorial FISH with mixtures of whole chromosomes paint probes was applied to paint seven chromosomes simultaneously in different colors along with a set of YAC clones which map to these chromosomes. We discuss the potential to construct analytical chromosomal bar codes adapted to particular needs of cytogenetic investigations and automated image analysis.

Chromosome Banding↗

Continuing quality improvement procedures for a clinical PACS.

The University of California at San Francisco (USCF) Department of Radiology currently has a clinically operational picture archiving and communication system (PACS) that is thirty-five percent filmless, with the goal of becoming seventy-five percent filmless within the year. The design and implementation of the clinical PACS has been a collaborative effort between an academic research laboratory and a commercial vendor partner. Images are digitally acquired from three computed radiography (CR) scanners, five computed tomography (CT) scanners, five magnetic resonance (MR) imagers, three digital fluoroscopic rooms, an ultrasound mini-PACS and a nuclear medicine mini-PACS. The DICOM (Digital Imaging and Communications in Medicine) standard communications protocol and image format is adhered to throughout the PACS. Images are archived in hierarchical staged fashion, on a RAID (redundant array of inexpensive disks) and on magneto-optical disk jukeboxes. The clinical PACS uses an object-oriented Oracle SQL (systems query language) database, and interfaces to the Radiology Information System using the HL7 (Health Languages 7) standard. Components are networked using a combination of switched and fast ethernet, and ATM (asynchronous transfer mode), all over fiber optics. The wide area network links six UCSF sites in San Francisco. A combination of high and medium resolution dual-monitor display stations have been placed throughout the Department of Radiology, the Emergency Department (ED) and Intensive Care Units (ICU). A continuing quality improvement (CQI) committee has been formed to facilitate the PACS installation and training, workflow modifications, quality assurance and clinical acceptance. This committee includes radiologists at all levels (resident, fellow, attending), radiology technologists, film library personnel, ED and ICU clinician end-users, and PACS team members. The CQI committee has proved vital in the creation of new management procedures, providing a means for user feedback and education, and contributing to the overall acceptance of, and user satisfaction with the system. Well developed CQI procedures have been essential to the successful clinical operation of the PACS as UCSF Radiology moves toward a filmless department.

Computer Communication Networks↗

From genomes to vaccines for leishmaniasis.

A total of 2183 clones derived from four life cycle stage-specific, spliced-leader cDNA libraries of Leishmania major LV39 Neal strain were randomly picked and sequenced to generate expressed sequence tags (ESTs). Then 1094 unique genes were identified, with 18.2% having BLAST hits with known genes/proteins and 81.8% failing to match genes currently deposited in public databases. Approximately 250 unique genes were obtained from a lesion-derived amastigote complementary DNA (cDNA) library, the form of the parasite that is infective to the mammalian host. Polymerase chain reaction (PCR)-amplified ESTs were spotted onto glass slides, and DNA microarray used to identify a further approx 100 unique cDNAs highly expressed in amastigotes. One hundred unique, randomly selected amastigote-expressed genes were PCR amplified to exclude the 5'-spliced leader, and the full-length genes subcloned into the TOPO-TA cloning vector. The genes were sequence-verified, excised using restriction enzyme digestion, and cloned upstream of the eukaryotic cytomegalovirus promoter into the expression vector pcDNA3. Expression plasmids were sequence-verified and large-scale, endotoxin-free plasmids prepared. Then 100 microg of each expression plasmid (DNA vaccine) was delivered subcutaneously to the rump of susceptible BALB/c mice, the mice boosted 4 wk later and then challenged 2 wk post-boost with 2 x 10(6) L. major LV39 parasites to the hind footpad. Infection was monitored on a weekly basis by measuring footpad depth with digital calipers. Protection was scored by comparing the footpad depth of mice receiving empty vector DNA to those immunized with DNA containing L. major amastigote-expressed genes.

Animals↗

Quality management, certification and rating of health information on the Net with MedCERTAIN: using a medPICS/RDF/XML metadata structure for implementing eHealth ethics and creating trust globally.

MedCERTAIN (MedPICS Certification and Rating of Trustworthy Health Information on the Net, http://www.medcertain.org/) is a recently launched international project funded under the European Union's (EU) "Action Plan for safer use of the Internet". It provides a technical infrastructure and a conceptual basis for an international system of "quality seals", ratings and self-labelling of Internet health information, with the final aim to establish a global "trustmark" for networked health information. Digital "quality seals" are evaluative metadata (using standards such as PICS=Platform for Internet Content Selection, now being replaced by RDF/XML) assigned by trusted third-party raters. The project also enables and encourages self-labelling with descriptive metainformation by web authors. Together these measures will help consumers as well as professionals to identify high-quality information on the Internet. MedCERTAIN establishes a fully functional demonstrator for a self- and third-party rating system enabling consumers and professionals to filter harmful health information and to positively identify and select high quality information. We aim to provide a trustmark system which allows citizens to place greater confidence in networked information, to encourage health information providers to follow best practices guidelines such as the Washington eHealth Code of Ethics, to provide effective feedback and law enforcement channels to handle user complaints, and to stimulate medical societies to develop standard for patient information. The project further proposes and identifies standards for interoperability of rating and description services (such as libraries or national health portals) and fosters a worldwide collaboration to guide consumers to high-quality information on the web.

Certification↗

Alu and L1 sequence distributions in Xq24-q28 and their comparative utility in YAC contig assembly and verification.

The contents of Alu- and L1-containing TaqI restriction fragments were assessed by Southern blot analyses across YAC contigs already assembled by other means and localized within Xq24-q28. Fingerprinting patterns of YACs in contigs were concordant, and using software based on that of M. V. Olson et al. (1986, Proc. Natl. Acad. Sci. USA 83: 7826) to analyze digitized data on fragment sizes, fingerprinting itself could establish matches among about 40% of a test group of 435 YACs. At 100-kb resolution, both repetitive elements were found throughout the region, with no apparent enrichment of Alu or L1 in DNA of G compared to that found in R bands. However, consistent with a random overall distribution, delimited regions of up to 100 kb contained clusters of repetitive elements. The local concentrations may help to account for the reported differential hybridization of Alu and L1 probes to segments of metaphase chromosomes.

Chromosome Mapping↗

Functional expression of recombinant G-protein-coupled receptors monitored by video imaging of pigment movement in melanophores.

Xenopus laevis melanophores are capable of functionally expressing recombinant receptors which couple via G-proteins to adenylate cyclase or phospholipase C (PLC). Receptor-mediated stimulation of either of these enzymes causes dispersion of melanosomes while receptor stimulation leading to inhibition of adenylate cyclase induces their aggregation. Translocation of melanosomes within thousands of individual pigment cells was simultaneously tracked by capturing gray scale video images before and after receptor activation. Digital subtraction of poststimulation from prestimulation images was performed on a microcomputer, generating bitplane images containing pixels with nonzero gray scale values wherever melanosome movement had occurred. Movement in both centripetal and centrifugal directions was detectable. The assay was tested using four receptors: a human beta 2-adrenergic receptor which stimulates adenylate cyclase, murine substance P and bombesin receptors which stimulate PLC, and a human D2 dopamine receptor which inhibits adenylate cyclase. Based on melanosome translocation following application of ligands, expression of functional receptors could be consistently detected in melanophores which received only single copies of a plasmid encoding any of the four receptors. By imaging fields containing up to 11,000 melanophores, the presence of a plasmid coding for a receptor could be detected when its frequency was one per 10,000 plasmids transfected. Combining receptor-mediated pigment translocation in melanophores with the rapid data-handling ability of video technology should provide a bioassay useful for investigating the function of G-protein-coupled receptors and for screening cDNA libraries for clones encoding new receptors.

Animals↗

Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities.

Pax genes have been shown to play important roles in mammalian development and organogenesis. Pax9, a member of this transcription factor family, is expressed in somites, pharyngeal pouches, mesenchyme involved in craniofacial, tooth, and limb development, as well as other sites during mouse embryogenesis. To analyze its function in vivo, we generated Pax9 deficient mice and show that Pax9 is essential for the development of a variety of organs and skeletal elements. Homozygous Pax9-mutant mice die shortly after birth, most likely as a consequence of a cleft secondary palate. They lack a thymus, parathyroid glands, and ultimobranchial bodies, organs which are derived from the pharyngeal pouches. In all limbs, a supernumerary preaxial digit is formed, but the flexor of the hindlimb toes is missing. Furthermore, craniofacial and visceral skeletogenesis is disturbed, and all teeth are absent. In Pax9-deficient embryos tooth development is arrested at the bud stage. At this stage, Pax9 is required for the mesenchymal expression of Bmp4, Msx1, and Lef1, suggesting a role for Pax9 in the establishment of the inductive capacity of the tooth mesenchyme. In summary, our analysis shows that Pax9 is a key regulator during the development of a wide range of organ primordia.

Animals↗

Journey Mapping of the Patient Experience from Diagnosis to End of Life in Lung Cancer: A Qualitative Meta-Synthesis.

OBJECTIVES: This study aimed to systematically synthesize the lived experiences and journey narratives of lung cancer patients across disease stages, and identify key tasks and pain points during the disease course through patient journey mapping, providing evidence for comprehensive disease management throughout the patient journey. METHODS: Ten databases, including PubMed, Embase, Web of Science, Scopus, PsycINFO, CINAHL, Cochrane Library, CNKI, Wanfang, and SinoMed, were systematically searched, with a search period from database inception to August 15, 2025. The JBI Critical Appraisal Tool for qualitative studies was used to evaluate the quality of studies, and the results were integrated using a meta-aggregative approach. RESULTS: Thirteen studies were included. Based on the patient journey mapping, the lung cancer patient journey comprises four potential stages: evaluation and diagnosis, initial treatment, maintenance therapy, and end-of-life. A total of 30 themes emerged within three dimensions: tasks, emotions, and pain points. Each dimension of each stage consists of 2-3 themes. CONCLUSION: The journey of lung cancer patients is protracted and complex, characterized by stage-specific needs and challenges. Future management strategies should be tailored to these distinct phases, providing precision supportive care to optimize treatment outcomes and enhance patients' quality of life. IMPLICATIONS FOR NURSING PRACTICE: This Patient Journey Map integrates routine clinical pathways with patients' lived experiences across each stage, revealing stage-specific challenges and providing targets for tailored nursing interventions. The framework promotes multidisciplinary, digitally enabled supportive care and indicates the importance of including patients' social circles to enhance patient-centered outcomes.

Humans↗

Arrhythmia database for algorithm testing: surface leads plus intracardiac leads for validation.

Ann Arbor Electrogram Libraries (AAEL) is a database of over 500 electrocardiographic recordings made during clinical electrophysiology studies over a period of 15 years. These data are used by university researchers and cardiac device research and development laboratories to develop and test arrhythmia detection algorithms. The AAEL library is in use by all major implantable cardioverter defibrillator manufacturers as well as several Automated External Defibrillator developers. In 1996, the USFDA Center for Devices and Radiological Health licensed a portion of Volumes I for proposed device testing. The data consist of 7 channels of signals: surface leads I, III, V1, an intra-atrial high right atrium (bipolar), an intra-atrial high right atrium (unipolar), an intraventricular right ventricular apex (bipolar), and intraventricular right ventricular apex (unipolar) from the distal catheter electrode. Data were recorded under careful engineering quality control continuously (30 min-1.5 hr) on FM magnetic tape at a tape speed of 3.75 in/s after signal amplification at a filter setting of 1-500 Hz. Amplifier gain and filter settings were held constant during the entire recording procedure and a 1 mV calibration signal was entered as a reference at the time of recording. All patient recordings contain a baseline sinus rhythm, and subsequently induced ventricular tachycardia and/or ventricular fibrillation, or less frequently, supraventricular tachycardia, atrial tachycardia, and/or atrial fibrillation. Data are typically made available in digitized format (1,000 Hz); digital or FM recordings are also available. AAEL recordings are the only widespread intracardiac test files in the industry.

Arrhythmias, Cardiac↗

miniSAGE: gene expression profiling using serial analysis of gene expression from 1 microg total RNA.

The use of serial analysis of gene expression (SAGE) to determine gene expression profiles is increasing because the technique can provide absolute transcript numbers in a digital format and identify new genes. We developed a miniSAGE technique, which uses only 1 microg total RNA and reduces the amount of the starting material by 250- to 500-fold. Unlike the other modified SAGE methods, the miniSAGE technique does not require the additional PCR amplifications. The additional PCR amplifications potentially introduce bias and compromise the quantitative aspects of the SAGE method. Three key modifications in the miniSAGE technique are: (i) using the phase lock gel (PLG, Eppendorf) to increase the recovery and the purity of DNA material after each phenol extraction step; (ii) reducing the amount of linkers in the ligation, thereby minimizing their interference with SAGE ditag amplification and increasing the SAGE ditag yield; and (iii) employing the mRNA capture kit (Boehringer Mannheim) to allow the first five steps: mRNA isolation, cDNA synthesis, enzyme cleavage of cDNA, binding of the cleaved biotin-cDNA to the streptavidin-magnetic beads, ligating linkers to the bound cDNA, and the release of cDNA tags to occur within one tube to significantly reduce the loss of material between successive steps. Two fibroblast SAGE libraries have been successfully prepared. The preliminary analysis of 3838 tags from one library demonstrated a typical fibroblast gene expression pattern. This miniSAGE technique will permit a broader application of SAGE.

Biopsy↗

Characterization of 954 bovine full-CDS cDNA sequences.

BACKGROUND: Genome assemblies rely on the existence of transcript sequence to stitch together contigs, verify assembly of whole genome shotgun reads, and annotate genes. Functional genomics studies also rely on transcript sequence to create expression microarrays or interpret digital tag data produced by methods such as Serial Analysis of Gene Expression (SAGE). Transcript sequence can be predicted based on reconstruction from overlapping expressed sequence tags (EST) that are obtained by single-pass sequencing of random cDNA clones, but these reconstructions are prone to errors caused by alternative splice forms, transcripts from gene families with related sequences, and expressed pseudogenes. These errors confound genome assembly and annotation. The most useful transcript sequences are derived by complete insert sequencing of clones containing the entire length, or at least the full protein coding sequence (CDS) portion, of the source mRNA. While the bovine genome sequencing initiative is nearing completion, there is currently a paucity of bovine full-CDS mRNA and protein sequence data to support bovine genome assembly and functional genomics studies. Consequently, the production of high-quality bovine full-CDS cDNA sequences will enhance the bovine genome assembly and functional studies of bovine genes and gene products. The goal of this investigation was to identify and characterize the full-CDS sequences of bovine transcripts from clones identified in non-full-length enriched cDNA libraries. In contrast to several recent full-length cDNA investigations, these full-CDS cDNAs were selected, sequenced, and annotated without the benefit of the target organism's genomic sequence, by using comparison of bovine EST sequence to existing human mRNA to identify likely full-CDS clones for full-length insert cDNA (FLIC) sequencing. RESULTS: The predicted bovine protein lengths, 5' UTR lengths, and Kozak consensus sequences from 954 bovine FLIC sequences (bFLICs; average length 1713 nt, representing 762 distinct loci) are all consistent with previously sequenced mammalian full-length transcripts. CONCLUSION: In most cases, the bFLICs span the entire CDS of the genes, providing the basis for creating predicted bovine protein sequences to support proteomics and comparative evolutionary research as well as functional genomics and genome annotation. The results demonstrate the utility of the comparative approach in obtaining predicted protein sequences in other species.

5' Untranslated Regions↗

Internet patient records: new techniques.

BACKGROUND: The ease by which the Internet is able to distribute information to geographically-distant users on a wide variety of computers makes it an obvious candidate for a technological solution for electronic patient record systems. Indeed, second-generation Internet technologies such as the ones described in this article--XML (eXtensible Markup Language), XSL (eXtensible Style Language), DOM (Document Object Model), CSS (Cascading Style Sheet), JavaScript, and JavaBeans--may significantly reduce the complexity of the development of distributed healthcare systems. OBJECTIVE: The demonstration of an experimental Electronic Patient Record (EPR) system built from those technologies that can support viewing of medical imaging exams and graphically-rich clinical reporting tools, while conforming to the newly emerging XML standard for digital documents. In particular, we aim to promote rapid prototyping of new reports by clinical specialists. METHODS: We have built a prototype EPR client, InfoDOM, that runs in both the popular web browsers. In this second version it receives each EPR as an XML record served via the secure SSL (Secure Socket Layer) protocol. JavaBean software components manipulate the XML to store it and then to transform it into a variety of useful clinical views. First a web page summary for the patient is produced. From that web page other JavaBeans can be launched. In particular, we have developed a medical imaging exam Viewer and a clinical Reporter bean parameterized appropriately for the particular patient and exam in question. Both present particular views of the XML data. The Viewer reads image sequences from a patient-specified network URL on a PACS (Picture Archiving and Communications System) server and presents them in a user-controllable animated sequence, while the Reporter provides a configurable anatomical map of the site of the pathology, from which individual "reportlets" can be launched. The specification of these reportlets is achieved using standard HTML forms and thus may conceivably be authored by clinical specialists. A generic JavaScript library has been written that allows the seamless incorporation of such contributions into the InfoDOM client. In conjunction with another JavaBean, that library renders graphically-enhanced reporting tools that read and write content to and from the XML data-structure, ready for resubmission to the EPR server. RESULTS: We demonstrate the InfoDOM experimental EPR system that is currently being adapted for test-bed use in three hospitals in Cagliari, Italy. For this we are working with specialists in neurology, radiology, and epilepsy. CONCLUSIONS: Early indications are that the rapid prototyping of reports afforded by our EPR system can assist communication between clinical specialists and our system developers. We are now experimenting with new technologies that may provide services to the kind of XML EPR client described here.

Internet↗

Managing knowledge and technology to foster innovation at the Ohio State University Medical Center.

Biomedical knowledge is expanding at an unprecedented rate-one that is unlikely to slow anytime in the future. While the volume and scope of this new knowledge poses significant organizational challenges, it creates tremendous opportunities to release and direct its power to the service of significant goals. The authors explain how the Center for Knowledge Management at The Ohio State University Medical Center, created during the academic year 2003-04, is doing just that by integrating numerous resource-intensive, technology-based initiatives-including personnel, services and infrastructure, digital repositories, data sets, mobile computing devices, high-tech patient simulators, computerized testing, and interactive multimedia-in a way that enables the center to provide information tailored to the needs of students, faculty and staff on the medical center campus and its surrounding health sciences colleges. The authors discuss how discovering, applying, and sharing new knowledge, information assets, and technologies in this way is a collaborative process. This process creates open-ended opportunities for innovation and a roadmap for working toward seamless integration, synergy, and substantial enhancement of the academic medical center's research, educational, and clinical mission areas.

Academic Medical Centers↗

Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase.

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.

Directed Molecular Evolution↗

Evolution of robustness in digital organisms.

We study the evolution of robustness in digital organisms adapting to a high mutation rate. As genomes adjust to the harsh mutational environment, the mean effect of single mutations decreases, up until the point where a sizable fraction (up to 30% in many cases) of the mutations are neutral. We correlate the changes in robustness along the line of descent to changes in directional epistasis, and find that increased robustness is achieved by moving from antagonistic epistasis between mutations towards codes where mutations are, on average, independent. We interpret this recoding as a breakup of linkage between vital sections of the genome, up to the point where instructions are maximally independent of each other. While such a recoding often requires sacrificing some replication speed, it is the best strategy for withstanding high rates of mutation.

Biological Evolution↗

Cloning differentially expressed mRNAs.

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

Antiviral Agents↗