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PubMed2005

Subject knowledge in the health sciences library: an online survey of Canadian academic health sciences librarians.

OBJECTIVES: This study investigated whether Canadian academic health sciences librarians found knowledge of the health sciences to be important and, if so, how they acquired and maintained this knowledge. METHODS: Data were gathered using a Web-based questionnaire made available to Canadian academic health sciences librarians. RESULTS: Respondents recognized the need for subject knowledge: 93.3% of respondents indicated that subject knowledge was "very important" or "somewhat important" to doing their job. However, few respondents felt that holding a degree in the health sciences was necessary. Respondents reported devoting on average more than 6 hours per week to continuing education through various means. Reading or browsing health sciences journals, visiting Websites, studying independently, and participating in professional associations were identified by the largest number of participants as the best ways to become and stay informed. CONCLUSIONS: Although more research needs to be done with a larger sample, subject knowledge continues to be important to Canadian academic health sciences librarians. Continuing education, rather than formal degree studies, is the method of choice for obtaining and maintaining this knowledge.

Canada↗

PubMed2006

Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries.

High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.

Combinatorial Chemistry Techniques↗

PubMedDate not supplied

High-throughput purification of single compounds and libraries.

The need for increasing productivity in medicinal chemistry and associated improvements in automated synthesis technologies for compound library production during the past few years have resulted in a major challenge for compound purification technology and its organization. To meet this challenge, we have recently set up three full-service chromatography units with the aid of in-house engineers, different HPLC suppliers, and several companies specializing in custom laboratory automation technologies. Our goal was to combine high-throughput purification with the high attention to detail which would be afforded by a dedicated purification service. The resulting final purification laboratory can purify up to 1000 compounds/week in amounts ranging from 5 to 300 mg, whereas the two service intermediate purification units take 100 samples per week from 0.3 to 100 g. The technologies consist of normal-phase and reversed-phase chromatography, robotic fraction pooling and reformatting, a bottling system, an automated external solvent supply and removal system, and a customized, high-capacity freeze-dryer. All work processes are linked by an electronic sample registration and tracking system.

Automation↗

PubMed2001

Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity.

Edwardsiella tarda is a Gram-negative bacterium that causes a systemic infection, edwardsiellosis, in fish. The virulence factors of this pathogen and its genetic determinants have not been systematically examined. In this study, TnphoA transposon mutagenesis was used to construct a library of 440 alkaline phosphatase (PhoA(+)) fusion mutants from a total of 400000 transconjugants derived from Ed. tarda PPD130/91. This library included genes for secreted and membrane-associated proteins normally involved in virulence. The library was screened for four virulence factors: siderophore production, motility, serum resistance and catalase production. Eight mutants deficient in one or more of these phenotypes were grouped into four classes. They were further characterized for their stimulation of reactive oxygen intermediate production by fish phagocytes, for their adhesion to and internalization into EPC (epithelioma papillosum of carp) cells, and for attenuation of virulence in blue gourami. Mutants 2A and 34 were highly attenuated in fish, with LD(50) values about 10 times higher than for the wild-type. These strains had mutations in the genes encoding arylsulfate sulfotransferase (mutant 2A) and a catalase precursor protein (mutant 34). One hyperinvasive/adhesive mutant and four pst mutants that were pleiotropic and slightly attenuated in fish were also isolated.

Alkaline Phosphatase↗

PubMed1988

The marketing audit: a new perspective on library services and products.

A marketing audit enables a library to look at audiences, services, and products with a structured approach. The audit can be used to assess operations and to provide a framework for ongoing decision making, evaluation, and long-range planning. An approach to the audit process is presented and its application is demonstrated in a case study featuring the American Hospital Association Resource Center.

American Hospital Association↗

PubMed1992

Identification of two distinct microtubule binding domains on recombinant rat MAP 1B.

A set of partially overlapping cDNA clones covering 9 kb of continuous sequence encoding the high molecular weight microtubule-associated protein (MAP) 1B, was isolated from a rat brain library in lambda gt11. The protein encoded was immunoreactive with monoclonal antibodies raised against calf MAP 1B, rat MAP 1X, and rat MAP 5, as shown by immunoblotting. Using Northern blot analysis, it was shown that the level of MAP 1B mRNA increased dramatically upon nerve growth factor-induced PC12 cell differentiation. The expression of polypeptides encoded by cDNA constructs, in conjunction with microtubule binding assays, revealed two separate microtubule binding domains, corresponding to sequences at the 5' and 3' end of the mRNA. As shown by DNA sequencing, the binding domain encoded by 5' terminal sequences consisted of the basic repeat motif KKEE(I/V), previously identified in mouse MAP 1B (Noble, M., S. A. Lewis, N. J. Cowan, J. Cell Biol. 109, 3367-3376 (1989)). The second binding domain, too, was found to be basic, but without any apparent repeat structure. It is concluded that single proteolytically unprocessed MAP 1B molecules would have the potential to function as microtubule cross-linkers.

Amino Acid Sequence↗

PubMed1999

Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

Animals↗

PubMed2006

Serial analysis of gene expression in the hippocampus of patients with mesial temporal lobe epilepsy.

Hippocampal sclerosis constitutes the most frequent neuropathological finding in patients with medically intractable mesial temporal lobe epilepsy. Serial analysis of gene expression was used to get a global view of the gene profile in human hippocampus in control condition and in epileptic condition associated with hippocampal sclerosis. Libraries were generated from control hippocampus, obtained by rapid autopsy, and from hippocampal surgical specimens of patients with mesial temporal lobe epilepsy and the classical pattern of hippocampal sclerosis. More than 50,000 tags were analyzed (28,282, control hippocampus; 25,953, hippocampal sclerosis) resulting in 9206 (control hippocampus) and 9599 (hippocampal sclerosis) unique tags (genes), each representing a specific mRNA transcript. Comparison of the two libraries resulted in the identification of 143 transcripts that were differentially expressed. These genes belong to a variety of functional classes, including basic metabolism, transcription regulation, protein synthesis and degradation, signal transduction, structural proteins, regeneration and synaptic plasticity and genes of unknown identity of function. The database generated by this study provides an extensive inventory of genes expressed in human control hippocampus, identifies new high-abundant genes associated with altered hippocampal morphology in patients with mesial temporal lobe epilepsy and serves as a reference for future studies aimed at detecting hippocampal transcriptional responses under various pathological conditions.

Base Sequence↗

PubMed2005

Characterization and RH mapping of bovine microsatellites generated from a microdissected BTA20-specific DNA library.

Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.

Animals↗

PubMed2005

Chemical library screen for novel inhibitors of Kaposi's sarcoma-associated herpesvirus processive DNA synthesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and certain lymphoproliferative disorders. The role of KSHV lytic replication has been implicated in the tumor pathogenesis. A highly specific molecular complex formed by the KSHV DNA polymerase (POL8) and processivity factor (PF8) is indispensable for lytic viral DNA synthesis and may serve as an excellent molecular anti-KSHV target. The majority of conventional nucleoside-based anti-herpetic DNA synthesis inhibitors require intracellular phosphorylation/activation before they can exert inhibitory activity as competitive substrates for viral DNA polymerases. Novel and more potent inhibitors of KSHV DNA synthesis may be discovered through POL8/PF8-targeted high throughput screening (HTS) of small molecule chemical libraries. We developed a microplate-based KSHV POL8/PF8-mediated DNA synthesis inhibition assay suitable for HTS and screened the NCI Diversity Set that comprised 1992 synthetic compounds. Twenty-eight compounds exhibited greater than 50% inhibition. The inhibitory activity was confirmed for 25 of the 26 hit compounds available for further testing, with the 50% inhibitory concentrations ranging from 0.12+/-0.07 microM (mean+/-S.D.) to 10.83+/-4.19 microM. Eighteen of the confirmed active compounds efficiently blocked KSHV processive DNA synthesis in vitro. One of the hit compounds, NSC 373989, a pyrimidoquinoline analog, was shown to dose-dependently reduce the levels of KSHV virion production and KSHV DNA in lytically induced KSHV-infected BCBL-1 cells, suggesting that the compound blocked lytic KSHV DNA synthesis. HTS for KSHV POL8/PF8 inhibitors is feasible and may lead to discovery of novel non-nucleoside KSHV DNA synthesis inhibitors.

Animals↗

PubMed2006

Cough and aspiration of food and liquids due to oral-pharyngeal dysphagia: ACCP evidence-based clinical practice guidelines.

BACKGROUND: Cough may be an indicator of aspiration due to oral-pharyngeal dysphagia. METHODS: Relevant literature was identified by searching the Communication Sciences and Disorders Dome, the Cumulative Index to Nursing and Allied Health Literature, the Educational Resource Information Center, Health & Psychosocial Instruments, the American Psychological Association, and the National Library of Medicine databases from 1965 to 2004 using the terms "deglutition," "aspiration," and "cough." RESULTS: Aspiration was observed on radiologic evaluation in over one third of acute stroke patients and in >40% of patients undergoing cervical spine surgery. Cough while eating may indicate aspiration, but aspiration may be clinically silent. Subjective patient and caregiver reports of cough while eating are useful in identifying patients who are at risk for aspiration. Objective measures of voluntary cough and tussigenic challenges to inhaled irritants are under investigation to determine their capacity to predict the risk for aspiration and subsequent pneumonia. The treatment of dysphagic patients by a multidisciplinary team, including early evaluation by a speech-language pathologist, is associated with improved outcomes. Effective clinical interventions such as the use of compensatory swallowing strategies and the alteration of food consistencies can be based on the results of instrumental swallowing studies. The efficacy of swallowing exercises and electrical muscle stimulation is under study. Surgical interventions may be considered in selected patients, but studies proving efficacy are generally lacking. CONCLUSIONS: Patients who are at risk for aspiration can be identified, and appropriate interventions can reduce its associated morbidity.

Cough↗

PubMed2000

[Vilnius Medical Association: the first half-century (1805-1850)].

Vilnius Medical Association, oldest scientific medical association in Poland and in the Russian Empire, was founded for the purposes of self-development in 1805 on the initiative of an Austrian professor of pathology and detailed therapy, Józef Frank (1771-1842). From its beginnings, the association acted for the good of society and science, independently of political conditions and with varying intensity. Apart from doctors, it also included pharmacists who contributed towards raising the standard of pharmacy in Lithuania. The first half-century of its existence constitutes the most fruitful period of activity as it was supported by professors of the then Medical Department of Vilnius University. At that time the Association established its regulations, founded the Library, and published "Pamietnik Wileńskiego Towarzystwa Lekarskiego" (Journal of Vilnius Medical Association, 1818 and 1821), "Pamietnik Farmaceutyczny" (Pharmaceutical Journal, 1820 and 1821), "Dziennik Medycyny Chirurgii i Farmacji" (Journal of Medicine, Surgery and Pharmacy, 1820, 1823, and 1830), as well as "Collectanea Medico Chirurgica", 1838. It also began gathering and analysing data for medical statistics, as well as developing Polish medical and chemical nomenclature. The Association re-organised shipments of medicines from pharmacies. It endeavoured to raise the standard of medical knowledge in the Vilnius area, combated epidemics of plague, cholera, scarlet fever, smallpox, typhus, diphteria, dysentery and monitored occurrences of plica. It also established the first Institute of Vaccination against Smallpox in Vilnius (1809); carried out scientific research on health resorts at Druskienniki, Birsztany and Stokliszki; promoted hygiene and examined the health of the inhabitants of Vilnius and the surrounding area as well as endorsed authority decisions related to the location of temporary military hospitals. At its meetings, members of the Association discussed their own medical cases as well as methods of treatment, presented the most recent reports in the medical literature, combated obsolete theories and scientific systems and established cooperation with other scientific associations in the country as well as abroad. World famous scientists were also members of the Vilnius Medical Association.

History, 19th Century↗

PubMed1998

Reversion of the tyrosine ochre strain Escherichia coli WU3610 under starvation conditions depends on a new gene tas.

When 3 x 10(8) bacteria of the Escherichia coli tyrA14(oc) leu308(am) strain WU3610 are plated on glucose salts agar supplemented with leucine only, colonies of slow-growing Tyr+ suppressor mutants begin to appear after about a week and increase in numbers roughly linearly with time thereafter (stationary phase or starvation-associated mutation). From a library constructed from two of these mutants, a clone was obtained that suppressed the tyrosine requirement of WU3610 when present on a multicopy plasmid. The activity was identified to an open reading frame we call tas, the sequence for which has homology with a variety of known genes with aldo-keto reductase activity. The activity of tas complements the prephenate dehydrogenase dysfunction of tyrA14 (the chorismate mutase activity of tyrA possibly being still functional). A strain deleted for tas showed no spontaneous mutation under starvation conditions. Whereas neither tas+ nor tas bacteria showed any increase in viable or total count when plated under conditions of tyrosine starvation at 3 x 10(8) cells per plate, at lower density (approximately 10(7) per plate) tas+ but not tas bacteria showed considerable residual growth. We suggest that the single copy of tas present in WU3610 allows cryptic cell or DNA turnover under conditions of tyrosine starvation and that this is an essential prerequisite for starvation-associated mutation in this system. The target gene for mutation is not tas, although an increase in the expression of this gene, for example, resulting from a suppressor mutation affecting supercoiling, could be responsible for the slow-growing Tyr+ phenotype.

Alcohol Oxidoreductases↗

PubMed2003

Pseudouridylation (Psi) of U2 snRNA in S. cerevisiae is catalyzed by an RNA-independent mechanism.

Pseudouridylation of snRNAs in vertebrates is guided by small nucleolar/Cajal body-specific RNAs (sno/scaRNAs). We developed an in vitro system using cell extracts and single site-radiolabeled U2 snRNAs to study pseudouridylation in Saccharomyces cerevisiae. Micrococcal nuclease-treated cell extracts are fully competent to catalyze U2 pseudouridylation, suggesting an RNA-independent process. A pseudouridylase activity for Psi(35) within yeast U2 is identified via a screen of an S.cerevisiae GST-ORF protein library. This activity is associated with YOR243c ORF, which has not previously been assigned function. When the GST-YOR243c protein is expressed in Escherichia coli, pseudouridylation activity is comparable to that expressed in S.cerevisiae, demonstrating that this protein (designated Pus7) alone can catalyze Psi(35) formation in U2. Both in vitro and in vivo analyses using wild-type and pus7-Delta strains show that Pus7 is indispensable for Psi(35) formation in U2. Using site-specific radiolabeled U2 and U2 fragments, we show that Pus7 activity is specific for Psi(35) and that the U2 stem- loop II region is essential for the pseudouridylation reaction. A BLAST search revealed Pus7 homologs in various organisms.

Amino Acid Sequence↗

PubMed2000

Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races.

Several diazotrophic species of Azoarcus spp. occur as endophytes in the pioneer plant Kallar grass. The purpose of this study was to screen Asian wild rice and cultivated Oryza sativa varieties for natural association with these endophytes. Populations of culturable diazotrophs in surface-sterilized roots were characterized by 16S rDNA sequence analysis, and Azoarcus species were identified by genomic fingerprints. A. indigens and Azoarcus sp. group C were detected only rarely, whereas Azoarcus sp. group D occurred frequently in samples of flooded plants: in 75% of wild rice, 80% of land races of O. sativa from Nepal and 33% of modern cultivars from Nepal and Italy. The putatively endophytic populations of diazotrophs differed with the rice genotype. The diversity of cultured diazotrophs was significantly lower in wild rice species than in modern cultivars. In Oryza officinalis (from Nepal) and O. minuta (from the Philippines), Azoarcus sp. group D were the predominant diazotrophic putative endophytes in roots. In contrast, their number was significantly lower in modern cultivars of O. sativa, whereas numbers and diversity of other diazotrophs, such as Azospirillum spp., Klebsiella sp., Sphingomonas paucimobilis, Burkholderia sp. and Azorhizobium caulinodans, were increased. In land races of O. sativa, the diazotrophic diversity was equally high; however, Azoarcus sp. was found in high apparent numbers. Similar differences in populations were also observed in a culture-independent approach comparing a wild rice (O. officinalis) and a modern-type O. sativa plant: in clone libraries of root-associated nitrogenase (nifH) gene fragments, the diazotrophic diversity was lower in the wild rice species. New lineages of nifH genes were detected, e.g. one deeply branching cluster within the anf (iron) nitrogenases. Our studies demonstrate that the natural host range of Azoarcus spp. extends to rice, wild rice species and old varieties being preferred over modern cultivars.

Asia↗

PubMed2005

Effects of varied pH, growth rate and temperature using controlled fermentation and batch culture on matrix assisted laser desorption/ionization whole cell protein fingerprints.

Rapid identification of microorganisms using matrix assisted laser desorption/ionization (MALDI) is a rapidly growing area of research due to the minimal sample preparation, speed of analysis and broad applicability of the technique. This approach relies on expressed biochemical markers, often proteins, to identify microorganisms. Therefore, variations in culture conditions that affect protein expression may limit the ability of MALDI-MS to correctly identify an organism. We have expanded our efforts to investigate the effects of culture conditions on MALDI-MS signatures to specifically examine the effects of pH, growth rate and temperature. Continuous cultures maintained in bioreactors were used to maintain specific growth rates and pH for E. coli HB 101. Despite measurable morphological differences between growth conditions, the MALDI-MS data associated each culture with the appropriate library entry (E. coli HB 101 generated using batch culture on a LB media), independent of pH or growth rate. The lone exception was for a biofilm sample collected from one of the reactors which had no appreciable degree of association with the correct library entry. Within the data set for planktonic organisms, variations in growth rate created the largest variation between fingerprints. The effect of varying growth temperature on Y. enterocolitica was also examined. While the anticipated effects on phenotype were observed, the MALDI-MS technique provided the proper identification.

Bacterial Proteins↗

PubMed2004

Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities.

Two decades of culture-independent studies have confirmed that microbial communities represent the most complex and concentrated pool of phylogenetic diversity on the planet. There remains a need for innovative molecular tools that can further our knowledge of microbial diversity and its functional implications. We present the method and application of serial analysis of ribosomal sequence tags (SARST) as a novel tool for elucidating complex microbial communities, such as those found in soils and sediments. Serial analysis of ribosomal sequence tags uses a series of enzymatic reactions to amplify and ligate ribosomal sequence tags (RSTs) from bacterial small subunit rRNA gene (SSU rDNA) V1-regions into concatemers that are cloned and sequenced. This approach offers a significant increase in throughput over traditional SSU rDNA clone libraries, as up to 20 RSTs are obtained from each sequencing reaction. To test SARST and measure the bias associated with this approach, RST libraries were prepared from a defined mixture of pure cultures and from duplicate arctic soil DNA samples. The actual RST distribution reflected the theoretical composition of the original defined mixture. Data from duplicate soil libraries (1345 and 1217 RSTs, with 525 and 505 unique RSTs, respectively) indicated that replication provides a strongly correlated RST profile (r(2) = 0.80) and division-level distribution of RSTs (r(2) = 0.99). Using sequence data from abundant soil RSTs, we designed specific primers that successfully amplified a larger portion of the SSU rDNA for further phylogenetic analysis. These results suggest that SARST is a powerful approach for reproducible high-throughput profiling of microbial diversity amenable to medical, industrial or environmental microbiology applications.

Bacteria↗

PubMed1994

Cloning of a cDNA coding for the acetylcholine receptor alpha-subunit from a thymoma associated with myasthenia [correction of myastenia] gravis.

To investigate the role of the acetylcholine receptor (AchR) in the pathogenesis of paraneoplastic Myasthenia gravis (MG), we screened a cDNA library of a MG-associated thymoma with a DNA oligonucleotide coding for aa 371-378, i.e. for part of the very immunogenic cytoplasmatic epitope (VICE-alpha, aa 373-380) of the human AChR alpha-subunit. We isolated two cDNA clones. Analysis of these clones has identified an open reading frame of 1371 bp, coding for the AChR alpha-subunit. No point mutation, insertion or deletion could be detected. Since the thymoma did not contain thymic myoid cells, which normally express AChR, the origin of the AChR transcripts must be the tumor cells itself. These findings confirm former results, where AChR alpha-subunit sequences from MG-thymomas were amplified by PCR.

Base Sequence↗