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PubMed1994

Evaluating library renovation at the University of Rochester Medical Center.

In 1987, the Edward G. Miner Library of the University of Rochester Medical Center completed the second major renovation in its 62-year history. The goal of the renovation was to provide additional space for the library's 3 main constituencies: its readers, its collections, and its staff. A case study of the renovation is presented, outlining processes and issues that shaped the final outcome. The success of the renovation is assessed in terms of project goals, design, and functionality. Changes made since 1987 to accommodate automation are also summarized. The conclusion suggests that library renovation be viewed as a continuing process, and proposes that principles of interface design are appropriate guidelines for evaluating library design across time.

Facility Design and Construction↗

PubMed1998

Solid-phase synthesis of a library of piperazinediones and diazepinediones via Kaiser oxime resin.

A combinatorial library of piperazinediones has been prepared by automated parallel solid-phase synthesis. The five-step reaction protocol makes use of Kaiser oxime resin, to enable cleavage from the polymeric support concomitant with an intramolecular displacement reaction, under very mild conditions. The methodology was also successfully extended to the preparation of the seven-membered ring homologs, diazepinediones.

Azepines↗

PubMed1999

The Brigham integrated computing system (BICS): advanced clinical systems in an academic hospital environment.

The Brigham integrated computing system (BICS) provides nearly all clinical, administrative, and financial computing services to Brigham and Women's Hospital, an academic tertiary-care hospital in Boston. The BICS clinical information system includes a very wide range of data and applications, including results review, longitudinal medical records, provider order entry, critical pathway management, operating-room dynamic scheduling, critical-event detection and altering, dynamic coverage lists, automated inpatient summaries, and an online reference library. BICS design emphasizes direct physician interaction and extensive clinical decision support. Impact studies have demonstrated significant value of the system in preventing adverse events and in saving costs, particularly for medications.

Computer Security↗

PubMed1996

Immobilized metal affinity chromatography of DNA.

Many of the most widely employed operations in molecular biology hinge upon the use of single-stranded DNA as a probe or template. Here we report a straightforward method by which to produce long single-stranded DNA molecules using the polymerase chain reaction (PCR) in combination with immobilized metal affinity chromatography (IMAC). We demonstrate that a tag consisting of six successive 6-histaminylpurine (H) residues (H6-tag) endows a DNA strand with selective retentivity onto a Ni2+-NTA-agarose chromatography matrix. The H6-tagged strand can then be eluted from the resin using 200 mM imidazole. Quantitative phosphorimaging analysis revealed that the PCR/IMAC procedure typically yields unmodified strands comprising >90% of the unbound DNA and H6-tagged strands comprising >95% of the bound fractions. DNA strands generated in this manner are shown to be excellent substrates for template-directed polymerization. The chemistry reported herein should facilitate a wide variety of operations in molecular biology, including automated DNA sequencing, hybridization screening of DNA libraries, assembly of gene cassettes, run-off transcription, site-directed mutagenesis and footprinting of protein-DNA complexes by template-directed interference footprinting.

Chromatography, Affinity↗

PubMed1999

Histidine placement in de novo-designed heme proteins.

The effects of histidine residue placement in a de novo-designed four-alpha-helix bundle are investigated by placement of histidine residues at coiled coil heptad a positions in two distinct heptads and at each position within a single heptad repeat of our prototype heme protein maquette, [H10H24]2 [[Ac-CGGGELWKL x HEELLKK x FEELLKL x HEERLKK x L-CONH2]2]2 composed of a generic (alpha-SS-alpha)2 peptide architecture. The heme to peptide stoichiometry of variants of [H10H24]2 with either or both histidines on each helix replaced with noncoordinating alanine residues ([H10A24]2, [A10H24]2, and [A10A24]2) demonstrates the obligate requirement of histidine for biologically significant heme affinity. Variants of [A10A24]2, [[Ac-CGGGELWKL x AEELLKK x FEELLKL x AEERLKK x L-CONH2]2]2, containing a single histidine per helix in positions 9 to 15 were evaluated to verify the design based on molecular modeling. The bis-histidine site formed between heptad positions a at 10 and 10' bound ferric hemes with the highest affinity, Kd1 and Kd2 values of 1.5 and 800 nM, respectively. Placement of histidine at position 11 (heptad position b) resulted in a protein that bound a single heme with moderate affinity, Kd1 of 9.5 microM, whereas the other peptides had no measurable apparent affinity for ferric heme with Kd1 values >200 microM. The bis-histidine ligation of heme to [H10A24]2 and [H11A24]2 was confirmed by electron paramagnetic resonance spectroscopy. The protein design rules derived from this study, together with the narrow tolerances revealed, are applicable for improving future heme protein designs, for analyzing the results of randomized heme protein combinatorial libraries, as well as for implementation in automated protein design.

Amino Acid Sequence↗

PubMed1992

DNA sequencing by primer walking with strings of contiguous hexamers.

When template DNA is saturated with a single-stranded DNA binding protein (SSB), strings of three or four contiguous hexanucleotides (hexamers) can cooperate through base-stacking interactions to prime DNA synthesis specifically from the 3' end of the string. Under the same conditions, priming by individual hexamers is suppressed. Strings of three of four hexamers representing more than 200 of the 4096 possible hexamers primed easily readable sequence ladders at more than 75 different sites in single-stranded or denatured double-stranded templates 6.4 kilobases to 40 kilobase pairs long, with a success rate of 60 to 90 percent. A synthesis of 1 micromole of hexamer supplies enough material for thousands of primings, so multiple libraries of all 4096 hexamers could be distributed at a reasonable cost. Such libraries would allow rapid and economical sequencing. Automating this strategy could increase the speed and efficiency of large-scale DNA sequencing by at least an order of magnitude.

Base Sequence↗

PubMed1999

Budding yeast as a screening tool for discovery of nucleoside analogs for use in HSV-1 TK suicide-gene therapy.

We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth. The method is based on the use of budding yeast (Saccharomyces cerevisiae) transformed with the HSV-1 TK gene on a multicopy plasmid. Eight nucleoside analogs (acyclovir, ganciclovir, penciclovir, lobucavir, brivudin, sorivudine, IVDU and ara-T), for which the cytostatic action against mammalian cells expressing the HSV-1 TK gene has been well documented, were studied for their inhibitory effect on the growth of yeast expressing the viral TK. These nucleoside analogs had little or no inhibitory effect on the growth of yeasts transformed with the empty vector, but inhibited to a significant extent the growth of yeast expressing the viral TK. Use of HSV-1 TK-expressing yeast allows quick screening in multi-well plate format for compounds with potential use in HSV-1 TK suicide gene therapy. The method may also be used as a tool to selectively suppress or arrest the growth of one population of yeast out of mixed yeast cell cultures.

Acyclovir↗

PubMed1993

Viral interleukin 10 is critical for the induction of B cell growth transformation by Epstein-Barr virus.

We have used an efficient cDNA subtraction library procedure to identify newly induced genes in human B lymphocytes infected for 6 h with Epstein-Barr virus (EBV). Among the genes identified by automated sequencing of a random subset of clones from this library, one coded the EBV BCRF1 open reading frame, which specifies the viral interleukin 10 gene (vIL-10). This molecule is highly homologous to human (h)IL-10 and was previously thought to represent a "late" viral gene expressed only during the lytic phase of virus replication. Using gene amplification by reverse transcriptase polymerase chain reaction of B cell RNA obtained at varying times after infection, we detected vIL-10 expression within a few hours of EBV infection, followed, 20-30 h later by expression of hIL-10. Expression of both genes continued beyond the initial transformation phase (5-10 d) and was present in all transformed cell lines tested. When added at the time of viral infection, antisense (but not sense) oligonucleotides for vIL-10 mRNA (cytosolic half-life, approximately 6 h) prevented subsequent B cell transformation. The antisense effect was highly specific, leaving the expression levels of other transformation-related genes intact. Addition of exogenous (h)IL-10 rescued the transformation process in antisense-treated cells. Our observations establish vIL-10 as a new latency gene with a directly transformation-prerequisite function.

B-Lymphocytes↗

PubMed1990

Automated evaluation of tryptic digest from recombinant human growth hormone using ultraviolet spectra and numeric peak information.

UV spectra were successfully employed in identifying peptide fragments from a tryptic digest of recombinant-DNA-derived human growth hormone (r-hGH). It was possible to distinguish very similar peptides utilizing a digital comparison of the UV spectra. An automated procedure was developed to generate a calibration library for the tryptic digest of a reference standard. The calibration library was then evaluated for reproducibility and selectivity and found to provide superior performance in correctly identifying ambiguous peaks as compared to the use of a conventional calibration table. Spectral match factors together with numerical information, derived from peak retention time, area and height, were used to arrive at a "peak score" descriptive of the similarity between standard and sample peaks. "Peak scores" could be combined to calculate a "sample score" indicative of overall similarity between an unknown and a standard. The scoring procedure was automated to generate a final report without operator intervention and successfully assigned appropriate scores to similar as well as dissimilar samples, e.g., native and oxidized r-hGH.

Algorithms↗

PubMed1995

Libraries of non-polymeric organic molecules.

New technology is emerging that permits the chemical synthesis of large numbers of different compounds simultaneously. Combinatorial chemistry is heavily dependent upon the adaptation of organic synthesis to solid supports and has necessitated the development of appropriate analytical and chemical approaches to both monitor solid-phase reactions and release finished compounds into solution. Considerable progress has recently been made in all of these areas. Small-molecule libraries of medicinally important chemical classes, such as 1,4-benzodiazepines, mercaptopropionyl amino acids, and peptidyl phosphonates, have recently been reported. Encoded combinatorial libraries of dihydrobenzopyran-based and acylpiperidine-based pharmacophores have yielded potent inhibitors of carbonic anhydrase. Automated instrumentation is growing in importance for the synthesis of small-molecule libraries.

Journal Article↗

PubMed1996

Mapping medical vocabularies to the Unified Medical Language System.

This paper presents our work in automated mapping of medical vocabularies to the National Library of Medicine's Unified Medical Language System (UMLS). We used the UMLS Knowledge Source (KS) tool to map terms from several sources to UMLS Metathesaurus concepts. We compared performance of the KS tools with our own Minimal Representable Units Method (MRUM). The KS tools were able to map terms from 13% to 54% of the time, depending on the term set and the KS options used. Our MRUM method mapped between 96% and 99% of the terms. Based on our experience, we believe that questions remain about the best method by which the UMLS can be used to achieve automated term translation.

Algorithms↗

PubMed1987

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.

Amino Acid Sequence↗

PubMed1997

Combinatorial protein design: strategies for screening protein libraries.

Powerful strategies for screening protein libraries further strengthen the arguments for applying 'irrational' approaches to understanding and designing new proteins. Developments during the past year include the application of functional complementation and automation to reduce screening loads, as well as the use of computerized data acquisition to characterize whole protein libraries rather than just selected individuals.

Automation↗

PubMed1999

A systematic comparison of protein structure classifications: SCOP, CATH and FSSP.

BACKGROUND: Several methods of structural classification have been developed to introduce some order to the large amount of data present in the Protein Data Bank. Such methods facilitate structural comparisons and provide a greater understanding of structure and function. The most widely used and comprehensive databases are SCOP, CATH and FSSP, which represent three unique methods of classifying protein structures: purely manual, a combination of manual and automated, and purely automated, respectively. In order to develop reliable template libraries and benchmarks for protein-fold recognition, a systematic comparison of these databases has been carried out to determine their overall agreement in classifying protein structures. RESULTS: Approximately two-thirds of the protein chains in each database are common to all three databases. Despite employing different methods, and basing their systems on different rules of protein structure and taxonomy, SCOP, CATH and FSSP agree on the majority of their classifications. Discrepancies and inconsistencies are accounted for by a small number of explanations. Other interesting features have been identified, and various differences between manual and automatic classification methods are presented. CONCLUSIONS: Using these databases requires an understanding of the rules upon which they are based; each method offers certain advantages depending on the biological requirements and knowledge of the user. The degree of discrepancy between the systems also has an impact on reliability of prediction methods that employ these schemes as benchmarks. To generate accurate fold templates for threading, we extract information from a consensus database, encompassing agreements between SCOP, CATH and FSSP.

Databases, Factual↗

PubMed2001

Solid-phase synthesis of peptidomimetic inhibitors for the hepatitis C virus NS3 protease.

The NS3 serine protease enzyme of the hepatitis C virus (HCV) is essential for viral replication. Short peptides mimicking the N-terminal substrate cleavage products of the NS3 protease are known to act as weak inhibitors of the enzyme and have been used as templates for the design of peptidomimetic inhibitors. Automated solid-phase synthesis of a small library of compounds based on such a peptidomimetic scaffold has led to the identification of potent and highly selective inhibitors of the NS3 protease enzyme.

Combinatorial Chemistry Techniques↗

PubMed1999

Organic-Fluorous Phase Switches: A Fluorous Amine Scavenger for Purification in Solution Phase Parallel Synthesis.

The synthesis of the fluorous amine scavenger [(C(6)F(13)CH(2)CH(2))(3)SiCH(2)CH(2)CH(2)](2)NH and its successful application in the automated solution phase parallel synthesis of a urea library are described. Ureas were made by robotic synthesis from organic amines and excess isocyanates. The amine scavenger reacts with excess isocyanate, and the fluorous tag serves to solubilize the resulting adduct in the fluorous phase so it can be removed by fluorous-organic extraction. Organic urea products are isolated in high yields and purities after liquid-liquid extraction. Preliminary biological evaluation shows that several of the ureas have ion channel modulation abilities. In contrast to polymer and ionic quenching methods, the fluorous quench works whether the product is soluble or insoluble in the reaction medium, and ionizable functional groups are tolerated in the products.

Journal Article↗

PubMed2001

The preparation of a series of nitrostilbene ester compounds using micro reactor technology.

The synthesis of stilbene esters using Wittig chemistry has been used to illustrate the generic diversity micro reactors offer in terms of chemical control and rapid method development. The micro reactor consisted of a 'T' design based on channel geometries 200 microns wide and 100 microns deep, etched into borosilicate glass and sealed with a borosilicate top plate using a thermal bonding technique. The movement of the reagent and products was achieved using electroosmotic flow (EOF), assisted by the incorporation of micro porous silica frits within the micro-channels to allow accurate solution control. To optimise the operating conditions methyl 4-formylbenzoate, premixed with sodium methoxide, was reacted with 2-nitrobenzyl-triphenylphosphonium bromide in dry degassed MeOH using flow conditions for both reagents of 0.40 microL min-1 for 20 min. A product yield of 70% (2:1 reaction stoichiometry with the aldehyde in excess) was obtained representing a 10% increase compared with the traditional batch synthesis. To demonstrate the capability of micro reactors to perform atom efficient synthesis a series of experiments based on an injection methodology (optimised to 30 s) were performed in the micro reactor at 1:1 stoichiometry resulting in a yield of 59%. Finally, the capability of micro reactors to perform a series of analogue reactions was investigated. The yields for a further three aldehydes indicated that the technology will be suitable for the development of automated device to support the generation of combinatorial libraries and rapid high throughput synthetic methods.

Journal Article↗

PubMed1994

Navigating the Brookhaven Protein Data Bank.

The Protein Data Bank maintained at Brookhaven National Laboratories has expanded to the point where even experienced users have difficulty understanding, exploring, and exploiting it. This paper describes a text file, an annotation of the Protein Data Bank, which helps users find information on related files and structures. The most recent version of this file includes information on homologous structures, including both sequence homology and structural homology. This file is in ASCII format and is available electronically. It is easy to search locally on any type of computer, using an editor or a pattern-matching program, such as grep.

Base Sequence↗