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PubMed2005

Sal-Site: integrating new and existing ambystomatid salamander research and informational resources.

Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at http://www.ambystoma.org.

Animals↗
PubMed2006

Increasing recruitment to randomised trials: a review of randomised controlled trials.

BACKGROUND: Poor recruitment to randomised controlled trials (RCTs) is a widespread and important problem. With poor recruitment being such an important issue with respect to the conduct of randomised trials, a systematic review of controlled trials on recruitment methods was undertaken in order to identify strategies that are effective. METHODS: We searched the register of trials in Cochrane library from 1996 to end of 2004. We also searched Web of Science for 2004. Additional trials were identified from personal knowledge. Included studies had to use random allocation and participants had to be allocated to different methods of recruitment to a 'real' randomised trial. Trials that randomised participants to 'mock' trials and trials of recruitment to non-randomised studies (e.g., case control studies) were excluded. Information on the study design, intervention and control, and number of patients recruited was extracted by the 2 authors. RESULTS: We identified 14 papers describing 20 different interventions. Effective interventions included: telephone reminders; questionnaire inclusion; monetary incentives; using an 'open' rather than placebo design; and making trial materials culturally sensitive. CONCLUSION: Few trials have been undertaken to test interventions to improve trial recruitment. There is an urgent need for more RCTs of recruitment strategies.

Culture↗
PubMed2003

Synthesis and analysis of combinatorial libraries performed in an automated micro reactor system.

This paper presents the synthesis of combinatorial libraries performed on a single-channel glass micro reactor under hydrodynamic flow control. The experiments were carried out in a non-well based micro chip and consisted of the preparation of libraries of pyrazoles by means of a Knorr reaction of 1,3-dicarbonyl compounds with hydrazines. The aim of this work is to investigate the capabilities of an automated micro reactor based system to synthesise sequentially multiple analogue reactions. Small slugs of reactants were introduced automatically by an autosampler in a serpentine-etched glass chip. The mobility of the reagents and products was achieved using hydrodynamic driven flow. Reaction slug dilution and UV slug detection took place at the outlet. A sample of the slug was analysed by using an on-line LC-UV-MS system. The degree of conversion was quantified using the UV signal and comparing with standards of starting materials and final products. After the LC-UV-MS analysis, the automated system proceeds to inject the slugs to carry out the next reaction programmed. The results suggest that the micro reactor system is capable of repeating the process of injection, mixing and reaction in an automated manner as many times as required.

Automation↗
PubMed2001

Burkholderia pseudomallei-specific recombinant protein and its potential in the diagnosis of melioidosis.

Melioidosis is an important public health problem in Southeast Asia and Northern Australia. This disease is caused by the gram-negative bacilli, Burkholderia pseudomallei. Wide spectra of clinical manifestations are observed in melioidosis ranging from asymptomatic to septicemic infection. Although serodiagnostic methods of melioidosis have been improved significantly in recent years, a highly specific diagnostic test that can differentiate asymptomatic seropositive individuals and melioidosis patients remains to be the subject of current investigations. In this study, a B. pseudomallei-specific gene, pBps-1, expressing a novel 18.7 kDa recombinant protein was selected from genomic libraries of two B. pseudomallei virulent isolates by using pooled sera from septicemic melioidosis patients. Nucleotide sequence analysis demonstrated that this gene is unique and does not show substantial similarity with any known genes in the Genbank database. The Bps-1 recombinant protein was evaluated for its potential in serodiagnosis of melioidosis by Western blot analysis. A high degree of specificity was demonstrated using sera from healthy individuals in the endemic (98.5%) and non-endemic areas (100%), with moderate sensitivity (69.7%) in melioidosis patients. The study demonstrated that this approach can be used to obtain highly specific recombinant antigens such as that described in the present report. A combination of such antigens should provide materials for successful serodiagnosis of melioidosis in the endemic areas.

Antibodies, Bacterial↗
PubMed1992

Analysis of chromosome 21 yeast artificial chromosome (YAC) clones.

Chromosome 21 contains genes relevant to several important diseases. Yeast artificial chromosome (YAC) clones, because they span > 100 kbp, will provide attractive material for initiating searches for such genes. Twenty-two YAC clones, each of which maps to a region of potential relevance either to aspects of the Down syndrome phenotype or to one of the other chromosome 21-associated genetic diseases, have been analyzed in detail. Clones total approximately 6,000 kb and derive from all parts of the long arm. Rare restriction-site maps have been constructed for each clone and have been used to determine regional variations in clonability, methylation frequency, CpG island density, and CpG island frequency versus gene density. This information will be useful for the isolation and mapping of new genes to chromosome 21 and for walking in YAC libraries.

Chromosome Mapping↗
PubMed1995

CpG island clones for chromosome 11p--a resource for mapping and gene identification.

A NotI end fragment library has been constructed for human Chromosome (Chr) 11p. Seventy-two clones were mapped to chromosomal subregions by use of somatic cell hybrids. The clones detect 44 different CpG islands, and we have isolated cosmid contigs for 36 of them. Extrapolation from the known 11p13 NotI restriction map suggests that every second CpG island from 11p containing a Not site is already represented in the clone collection. By sequence analysis all of the 11p13 clones exhibit typical features of CpG islands, and cross-species hybridization has been detected with at least one fragment in most cases. The cosmids serve as valuable linking clones for long-range restriction mapping. They also provide excellent starting material for transcript isolation procedures to identify genes on chromosome 11p associated with developmental anomalies and various tumor types. Several transcribed sequences have already been isolated with some of these clones.

Animals↗
PubMed2005

Changes in expansin activity and gene expression during ethylene-promoted leaflet abscission in Sambucus nigra.

During ethylene-promoted leaflet abscission in Sambucus nigra a 7-fold increase in expansin activity was detected specifically in tissues undergoing cell separation, whilst only low levels of activity were seen in adjacent non-abscising tissues. An RT-PCR strategy was used to amplify expansin fragments from a cDNA library generated from mRNA extracted from ethylene-treated leaflet abscission-zone tissue. Two different full-length expansin-encoding cDNAs were isolated with sizes of 1190 bp and 1169 bp and named SniExp2 and SniExp4, respectively. The deduced protein sequences of SniExp2 and SniExp4 exhibited 67% homology and comprised 256 and 249 amino acids, respectively. Both putative proteins contained signal sequences at their N terminal ends, suggesting that they were likely to encode secreted or transmembrane proteins. Northern analyses with probes specific for each gene revealed that transcripts for both of these cDNAs accumulated specifically in abscission zone tissues in response to ethylene treatment, with no expression being apparent in either freshly excised material or non-separating tissues. These data support a role for expansins in ethylene-stimulated abscission and provide evidence that specific family members contribute to the cell separation process that takes place during organ shedding.

Amino Acid Sequence↗
PubMed1988

Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin.

Cloned cDNAs encoding the precursor protein for motilin and a novel peptide, motilin-associated peptide, were isolated from a library derived from porcine intestinal mucosa mRNA. Nucleotide sequence analysis predicts a precursor protein of 119 amino acids including a signal peptide in direct linkage with the 22 amino acid sequence for motilin, and a 70 amino acid peptide of unknown function. The putative bioactive moieties are separated by Lys-Lys, dibasic residues that serve as substrates for cleavage by proteolytic maturation enzymes in many polyprotein precursors. While there is an abundant literature detailing a spectrum of tissues and cell types which express motilin like immunoreactivity, analysis of mRNA derived from many of these tissues suggests that the mRNA for the mucosal motilin precursor is only transcribed in this tissue. The nature of the immunoreactive material in the central nervous system and other peripheral tissues remains to be determined.

Animals↗
PubMed2001

Bridging the gap between proteins and nucleic acids: a metal-independent RNAseA mimic with two protein-like functionalities.

Two synthetically modified nucleoside triphosphate analogues (adenosine modified with an imidazole and uridine modified with a cationic amine) are enzymatically polymerized in tandem along a degenerate DNA library for the combinatorial selection of an RNAse A mimic. The selected activity is consistent with both electrostatic and general acid/base catalysis at physiological pH in the absence of divalent metal cations. The simultaneous use of two modified nucleotides to enrich the catalytic repertoire of DNA-based catalysts has never before been demonstrated and evidence of general acid/base catalysis at pH 7.4 for a DNAzyme has never been previously observed in the absence of a divalent metal cation or added cofactor. This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.

Adenosine Triphosphate↗
PubMed1995

Description of a 700-kb yeast artificial chromosome contig containing the BCL1 translocation breakpoint region at 11q13.

We screened two human yeast artificial chromosome (YAC) libraries by polymerase chain reaction (PCR) with oligonucleotides specific to the BCL1 major translocation breakpoint cluster region at 11q13. Five YACs were isolated. Two of them were chimeric. One of these and remaining three YACs were characterized by hybridization with various known 11q13 probes, Alu-PCR fingerprinting, in situ hybridization, and isolation of YAC ends. A map of this ca 700-kb YAC contig was obtained. This map was consistent with maps established from total human genomic DNA. Every YAC in this region was found unstable and gave rise to reproducibly deleted lineages. Analysis in detail of these deletions over many generations showed that more than a single sequence might be involved. The availability of cloned material will facilitate the search for the still elusive genetic elements responsible for amplifications, deletions and translocations observed at 11q13 in malignancies.

Base Sequence↗
PubMed1981

[Optimal exchange of resources and the administration of bibliographical services].

It is pointed out in this paper that an optimal exchange of resources requires a maximum availability of materials and services at minimum cost. The author is of the view that each activity of the biomedical information network will have to be subjected to critical scrutiny including a calculation of its costs. The overall plan will determine whether current activities can be maintained or expanded and new activities launched. The primary given to access to materials rather than to their possession is a step toward user-oriented networks, and therefore means that future development will be assessed in relation to the customer. The author is convinced that the recording and analysis of the use actually made of services by the user will generate many of the criteria that will govern some of the most important decisions to be taken in the future in connection with the network.

Information Services↗
PubMed2025

Extraction, Purification, and Next-Generation Sequencing (NGS) Analysis of DNA and RNA from Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue.

Formalin fixed paraffin embedded (FFPE) tissues have long been used for immunohistological analyses. FFPE tissues can be stored at room temperature for several years enabling analyses to be performed later. Ease of storage and transport makes these tissues an attractive source of biological material. However, formalin fixation results in chemical modifications of proteins and nucleic acids that poses a major challenge to any type of analysis. Recovery of nucleic acids for quantitative assays is rendered difficult due to degradation resulting from fixation and long-term storage, producing low usable yields. Extensive efforts in the last 20 years have led to significant improvements in use of FFPE tissues for DNA and RNA analyses and resulted in development of sensitive assays for a wide range of applications, including next-generation sequencing. In this chapter, we describe the optimization of methods for sequential extraction of DNA and RNA from FFPE tissue and subsequent preparation of DNA-seq and RNA-seq libraries for use with the Illumina platform using commercially available reagents/kits.

Paraffin Embedding↗
PubMed2005

Neutron analysis of spent fuel storage installation using parallel computing and advance discrete ordinates and Monte Carlo techniques.

In the United States, the Nuclear Waste Policy Act of 1982 mandated centralised storage of spent nuclear fuel by 1988. However, the Yucca Mountain project is currently scheduled to start accepting spent nuclear fuel in 2010. Since many nuclear power plants were only designed for -10 y of spent fuel pool storage, > 35 plants have been forced into alternate means of spent fuel storage. In order to continue operation and make room in spent fuel pools, nuclear generators are turning towards independent spent fuel storage installations (ISFSIs). Typical vertical concrete ISFSIs are -6.1 m high and 3.3 m in diameter. The inherently large system, and the presence of thick concrete shields result in difficulties for both Monte Carlo (MC) and discrete ordinates (SN) calculations. MC calculations require significant variance reduction and multiple runs to obtain a detailed dose distribution. SN models need a large number of spatial meshes to accurately model the geometry and high quadrature orders to reduce ray effects, therefore, requiring significant amounts of computer memory and time. The use of various differencing schemes is needed to account for radial heterogeneity in material cross sections and densities. Two P3, S12, discrete ordinate, PENTRAN (parallel environment neutral-particle TRANsport) models were analysed and different MC models compared. A multigroup MCNP model was developed for direct comparison to the SN models. The biased A3MCNP (automated adjoint accelerated MCNP) and unbiased (MCNP) continuous energy MC models were developed to assess the adequacy of the CASK multigroup (22 neutron, 18 gamma) cross sections. The PENTRAN SN results are in close agreement (5%) with the multigroup MC results; however, they differ by -20-30% from the continuous-energy MC predictions. This large difference can be attributed to the expected difference between multigroup and continuous energy cross sections, and the fact that the CASK library is based on the old ENDF/B-II library. Both MC and SN calculations were run in parallel on a BEOWULF PC-cluster (eight processors). Timing results indicate that the SN calculation yielded a detailed dose distribution at over 318,426 points in -164 h. Unbiased continuous energy MC required 214 h to calculate dose rates with a 1% relative error in only 18 regions on the surface of the cask. The biased A3MCNP calculations yields dose rates with -0.8% relative error in only 2.5 h on one processor. This study demonstrates that a parallel code, such as the 3-D parallel SN transport code, PENTRAN can solve a complex large problem, such as the storage cask, accurately and efficiently. Moreover, this calculation was performed on a relatively inexpensive PC-cluster. Possible inadequacies of the CASK cross section library still need to be evaluated.

Computer-Aided Design↗
PubMed2005

[Trends of electronic publishing in medicine and life sciences].

Scientific publication in the electronic media is gaining popularity in academic libraries, research institutions and commercial organizations. The electronic journal may shorten the processes of writing and publication, decrease publication and distribution costs, and enable access from any location in the world. Electronic publications have unique advantages: it is possible to search them, to create hyperlinks to references and footnotes, as well as to information on the web and to include graphics and photographs at a very low cost. Audio, video and tri-dimensional images may also be included. Electronic publishing may also speed up review and publication processes and enable the writer to receive immediate feedback through the web. However, in spite of the advantages, there are certain points that must be considered: accessibility to previously published material is not guaranteed as databases are not always stable and coverage may change without notice. In addition, the price that commercial publishers charge for their services may be very high or be subject to the purchase of a packaged deal that may include unwanted databases. Many issues of copyright and the use of published material are not yet finalized. In this review we discuss the advantages and disadvantages of the electronic scientific publication, the feasibility of keeping appropriate quality and peer-review process, the stability and accessibility of databases managed by the publishers and the acceptance of the electronic format by scientists and clinicians.

Biological Science Disciplines↗
PubMed1990

A computerized simulator for critical-care training: new technology for medical education.

A patient simulator has been developed for training, certification, modeling, and demonstrating problems in the management of critical-care patients. The Critical Care & Hemodynamic Monitoring Training System consists of a personal computer, software, and a replica of a human torso designed to enable students to practice critical-care medicine. The computer displays patient histories, laboratory results, treatment options, patient responses, and a real-time cardiac monitor. The torso apparatus is used to practice insertion of a hemodynamic monitoring catheter; the cardiac monitor displays catheter pressure readings as the catheter is advanced into the heart and also pulmonary artery and wedge position. Special screen calculators in the program may be used to determine hemodynamic, respiratory, ventilatory, and renal function indices. In contrast to previously described simulators and computer oriented instructional programs, this system contains no inherent data base. Instead, authors build a library of informative cases by using the hardware and software tools provided. Individual "modules" of patient information are authored, and these are transparently linked as a student undertakes management of a patient. Although this system is a technologic achievement, determination of its usefulness as an instructional tool or certification aid must come from broader use and controlled studies.

Clinical Competence↗
PubMed2003

[Acute poisoning by chemical warfare agent: sulfur mustard].

OBJECTIVE: To review story, mechanism of action, clinical and therapeutic bases of a sulfur mustard poisoning, by accidental, terrorism or war exposure. DATA SOURCES: References were obtained from computerised bibliographic research (Medline), from personnel data (academic memoir, documents under approbation of the National Defense Office) and from the Library of Military Medical Service. DATA SYNTHESIS: Sulfur mustard is a chemical warfare agent with peace time results: leak, accidental handling, acts of terrorism. Sulfur mustard is a vesicant agent, an organochlorine agent, who alkylate DNA. Under liquid or gas form its main target are skin and lungs. Clinical effects are like burns with loss of immunity, with respiratory failure, ophthalmic, gastrointestinal and haematological signs. The last studies have improved knowledge about the mechanism of action, detection, protection and treatment. Methods for determination of sulfur mustard are based on gas chromatographic method and mass spectrometry. During sulfur mustard contamination the first priorities of treatment are to remove victims from the contaminated place and to initiate decontamination. Emergency workers and materials must take protection to avoid secondary contamination of emergency unit. With treatment of vital functions and respiratory failure, the new ways of treatment are about N-acetyl cysteine for lung injury, poly (ADP-ribose) polymerase inhibitors, calmodulin antagonists and Ca(++) chelators. Interactions between sulfur mustard and anaesthetic agents are not well known and are based on clinical observations. CONCLUSION: Emergency care unit can be confronted with sulfur mustard during accidental contamination or acts of terrorism. First and most efficacy priorities of treatment are to remove and to decontaminate victims. New means of detection and treatment are studied since several years but are not still appropriate to human victims or mass treatment.

Alkylating Agents↗
PubMed1993

Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA).

A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region. One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1991

Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli.

Bacterial expression systems can greatly facilitate protein engineering of antibodies. We have developed a system for high-level expression of antibodies, antibody fragments, or hybrid antibodies with novel effector functions in the periplasm of Escherichia coli. From 5 ml of cells, a simple extraction yields sufficient material for SDS-gel electrophoresis, detection and characterization of hapten binding. To demonstrate our system, heavy-chain variable regions and lambda 1 light chains of a mouse anti-NP antibody were synthesized as hybrid proteins with a bacterial signal peptide (Omp F). Each chain is secreted into the periplasm where processing (cleavage of the signal peptide), folding and heterodimer association take place. Periplasmic proteins are released by cold osmotic shock, and hapten-binding activity is easily detected without further manipulation. The ease of genetic engineering in this system will facilitate the production of immunoglobulin derivatives designed for specific applications, and expression of these molecules in a native state will allow the rapid screening of combinatorial libraries and the results of mutagenesis.

Animals↗