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Localized transfection on arrays of magnetic beads coated with PCR products.

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Animals↗

Internet in the workplace: censorship, liability, and freedom of speech.

Most hospital medical libraries are supported by private, corporate funds and thus fall under the corporation's policies in regard to discrimination, harassment, and sexual harassment. With the free flow of information available on the Internet and through e-mail, it is mandatory to create a corporate policy for appropriate use and review of materials. Access to "questionable" or inappropriate Internet sites is not a freedom of speech issue in a private corporation; it is a potential liability for the corporation, the library, and the librarian. It is also a misuse of company resources.

Florida↗

Synthesis of a 2, 4, 8-trisubstituted pyrimidino[5, 4-d]pyrimidine library via sequential SNAr reactions on solid-phase.

A solid-phase synthesis of 2, 4, 8-substituted pyrimidino[5, 4-d]pyrimidines involving three controlled S(N)Ar reactions has been developed. Exploration of different heterocyclic starting materials and resin-bound intermediates is highlighted. The preferred method starts with the treatment of resin-bound anilines with 2, 4, 8-trichloropyrimidino[5, 4-d]pyrimidine. This intermediate is subsequently treated with various amines in two steps to yield the final products. The scope of each diversity step was determined and a library of 16, 000 compounds was synthesized.

Combinatorial Chemistry Techniques↗

The nature of the use and users of the Midwest Regional Medical Library.

Using a 10 percent random sample of all requests submitted to the Midwest Regional Medical Library during 1968-1969, this study has revealed that physicians constitute the user group making the most requests, that physicians request periodical materials most, that 86.6 percent of materials requested were for periodicals, and that over one-third of materials requested was more than ten years old. It was found that 66.3 percent of requests were filled, that "Pathology, Diseases, and Treatment" was the subject category with the highest percentage of requests, and that this category and Materia Medica were the subjects accounting for over 50 percent of the requests from all users. The subject categories most frequently requested by physicians were found to be the same (Pathology, Diseases, and Treatment and Materia Medica) as for educators and researchers. The most requests from students were in the Materia Medica and "other" subject categories. These findings are significant for drawing up acquisition policy for both the John Crerar Library, which supplies the physical facilities for the MRML, and the borrowing library which submits the requests for users.

Chicago↗

Automated high-throughput liquid-liquid extraction for initial purification of combinatorial libraries.

An automated high-throughput liquid-liquid extraction (LLE) methodology has been developed and utilized for the initial purification of the combinatorial library samples containing unreacted amines and other water-soluble byproducts or impurities. Various extraction solvents were evaluated along with different extraction devices. The LLE method was automated using 96-well-format plates and a robotic liquid-handling workstation. In the optimized LLE method, crude combinatorial library samples were dissolved in a water-immiscible organic solvent, butyl acetate, and added to each well in a 96-well-format plate packed with an inert support material coated with hydrochloric acid. Separation occurs based on the partitioning of the compounds between two liquid phases. Product recovery, purity, and amine removal efficiency were determined by HPLC with and without precolumn derivatization. The automated method was successfully applied to the cleanup of some representative combinatorial library samples with greater than 98% amine removal and an average product purity of 90%. The application of the automated high-throughput LLE method should greatly reduce the labor, time, and cost associated with the purification of combinatorial libraries.

Amines↗

American medical literary firsts, 1700--1820, in the Countway Library.

A combination of two major collections, those of the Harvard Medical and the Boston Medical Libraries, took place at a formal dedication of the Francis A. Countway Library of Medicine in May of 1965. As a result of this unification the historian may now consult many significant primary source materials in one research collection in Boston. Many of these works are early imprints of medical Americana. This paper discusses twenty-four imprints, from 1700 to 1820, which were firsts of their kind in American medical literature. Presented are the first American medical publication, book, pharmacopeia, mortality statistics, anatomical illustrations, transactions of a medical society, medical journal, textbook of obstetrics, medical dictionary, textbook of medicine, and official pharmacopeia. Also discussed are the first works in this country on smallpox inoculation, scarlet fever, pleurisy, public health, medical education, medical ethics, the history of medicine, surgery, epidemiology, smallpox vaccination, dentistry, meningitis, and psychiatry. An exhibit of these items in the Countway Library is planned for the spring and summer of 1966.

History, 18th Century↗

Optical mapping of Plasmodium falciparum chromosome 2.

Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.

Animals↗

Development of rationally designed affinity-based drug delivery systems.

Many drug delivery systems have been developed to provide sustained release of proteins in vivo. However, the ability to predict and control the rate of release from delivery systems is still a challenge. Toward this goal, we screened a random drug-binding peptide library (12 amino acids) to identify peptides of varying (i.e. low, moderate, and high) affinity for a model polysaccharide drug (heparin). Peptide domains of varying affinity for heparin identified from the library were synthesized using standard solid phase chemistry. A mathematical model of drug release from a biomaterial scaffold containing drug-binding peptide domains identified from the library was developed. This model describes the binding kinetics of drugs to the peptides, the diffusion of free drug, and the kinetics of enzymatic matrix degradation. The effect of the ratio of binding sites to drug, the effect of varying the binding kinetics and the rate of enzymatic matrix degradation on the rate of drug release was examined. The in vitro release of the model drug from scaffold containing the peptide drug-binding domains was measured. The ability of this system to deliver and modulate the biological activity of protein drugs was also assessed using nerve growth factor (NGF) in a chick dorsal root ganglia (DRG) neurite extension model. These studies demonstrate that our rational approach to drug delivery system design can be used to control drug release from tissue-engineered scaffolds and may be useful for promoting tissue regeneration in vivo.

Affinity Labels↗

Protein disulphide isomerase of Ostertagia ostertagi: an excretory-secretory product of L4 and adult worms?

A pepstatin A-agarose column was used in an attempt to purify a previously described antibody-degrading aspartyl proteinase from excretory-secretory material from the L4 and the adult stages of the bovine abomasal nematode Ostertagia ostertagi. However, no aspartyl proteinase activity was detected in the eluted fractions (L4Pepst and AdPepst). Screening of cDNA libraries with polyclonal antibodies raised against L4Pepst and AdPepst showed that a protein disulphide isomerase (Ost-PDI2) was present in both antigen fractions. This multifunctional enzyme was detected in extracts of L3, L4 and adult parasites and, interestingly, also in excretory-secretory material of L4 and adult O. ostertagi. By immunohistochemistry, the Ost-PDI2 enzyme was localised in some parts of the hypodermis of L4 and adult worms and in the intestinal cells of all three parasitic life stages. Two-dimensional Western blot analysis indicated that Ost-PDI2 is recognised by calves during a natural O. ostertagi infection, which suggests that Ost-PDI2 could be used for immunological control of ostertagiosis.

Animals↗

Evaluation of monograph selection in a health sciences library.

This article reports on an evaluation of recent monograph selections in a small academic health sciences library. Actual use of each new book was determined from date-due slips. Data were analyzed by broad subject, discipline, and number of uses. The startling result was that more than 60% of recent selections had been used little or not at all. To determine factors affecting use, the author examined aggregate data, used intuition, and assessed the raw data in more detail. Recommendations made to management were approved and implemented. The study bolstered confidence that it is possible to select materials with the highest potential for use and to depend on remote access for other needed works. It is suggested that other health sciences libraries undertake such studies and question the need to strive for comprehensive collections.

Book Selection↗

Outcomes of cytopathology studies presented at national pathology meetings.

The purpose of this study was to determine what factors influence the final publication status of cytopathology studies presented at national meetings. Abstracts involving cytopathology material were obtained from the following journals: Modern Pathology (volume 11, 1998), Acta Cytologica (volume 42, 1998), and the American Journal of Clinical Pathology (volumes 109 and 110, 1998). Using the National Library of Medicine Website, each abstract was searched by author and topic to determine if the study was published as a peer-reviewed article. The following parameters were evaluated: meeting where the abstract was presented, type of institution where the research was based, type of material used in the study, and application of ancillary techniques used in the study. The subsequent published articles were evaluated for journal and time to publication. Out of 257 studies presented in 1998, 85 (33%) were published in peer-reviewed journals by May 2000. The majority of papers were published in Diagnostic Cytopathology (n = 21), Acta Cytologica (n = 15), and Cancer (n = 18). The mean time for publication was 12.8 mo. The highest percentage of published studies was presented at the United States and Canadian Academy of Pathology (USCAP) meeting (50% of presented abstracts), followed by American Society of Cytopathology (ASC) (28%) and American Society of Clinical Pathologists (ASCP) (17%) meetings. Ancillary techniques were applied in 40 of 85 (47%) published studies, 27 of 85 (32%) articles focused on morphology, and 18 of 85 (21%) papers covered other topics (e.g., quality assurance (QA), cost, and role of cytology). In nonpublished studies (n = 172), special techniques were the main focus in 40%, morphology in 25%, and other topics in 35% of abstracts. The great majority (97%) of published studies were from academic institutions. Gynecological and nongynecological material were roughly equally covered in published and nonpublished studies. Only a relatively small percentage of presented studies was finalized in the form of peer-reviewed articles. Type of material and application of ancillary techniques do not significantly influence the outcome.

Congresses as Topic↗

Analysis of one year's circulation at the Downstate Medical Center Library.

A survey of the circulation of books and journals at the Downstate Medical Center Library was conducted, based on cancelled circulation cards accumulated during a one-year period. Analysis of the results shows the frequency of use of various materials by several groups of borrowers and brings out important differences between circulation of books and that of journals. One of the results was the compilation of a list of most frequently used journals. The findings are graphically represented by several tables and charts.

Libraries, Medical↗

Expression cloning of neurotrophic factors using Xenopus oocytes.

We have explored the potential for cloning novel neurotrophic factor cDNAs via assay of neurotrophic activities following expression in Xenopus oocytes. In this report, we describe the successful application of the method to tract rat ciliary neurotrophic factor (CNTF) activity from mRNA purified from cultured cells and from mRNA synthesized by in vitro transcription of a cDNA library. Rat C6 glioma cells, which had been previously shown to have CNTF-like activity (Westermann et al., 1988), were used as source material. We tested protein extracts of C6 cells using an in vitro assay of primary neurons from the chick ciliary ganglion (CCG assay) and detected a CNTF-like activity. RNA isolated from C6 cells was shown to direct the synthesis of the activity following microinjection into Xenopus oocytes and one-step fractionation of Xenopus extract. C6 mRNA was size-fractionated, and fractions encoding CNTF-like activity were cloned into a lambda phage vector at a site distal to a T7 promoter. Synthetic RNA transcribed from total library DNA was injected into Xenopus oocytes, and a CNTF-like activity in the oocyte extract was detected by the CCG assay. Further fractionation of library clones narrowed the presence of the clone encoding the CNTF-like activity to a pool containing 20,000 members. The presence of a full-length CNTF cDNA clone in this pool and partial clones in other pools was confirmed by Polymerase Chain Reaction (PCR) using oligonucleotides from the rabbit CNTF cDNA (Lin et al., 1989) as primers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Human monoclonal Fab fragments recovered from a combinatorial library bind specifically to the platelet HPA-1a alloantigen on glycoprotein IIb-IIIa.

BACKGROUND AND OBJECTIVES: Certain clinical conditions are related to the presence of platelet-specific alloantibodies in the patient's serum. We studied the molecular diversity of HPA-1a antibodies to analyze some peculiarities of this antibody response. MATERIALS AND METHODS: Human antibody Fab fragments that bind to the platelet alloantigen HPA-1a on glycoprotein IIb-IIIa (GPIIbIIIa) were generated by using a recombinant phage display system. We established an immunoglobulin G1, kappa combinatorial library from the peripheral blood lymphocytes of a person undergoing a severe posttransfusion purpura. RESULTS: Characterization of Fab clones selected from the fifth round of antigen-specific panning of this library demonstrates a highly specific reactivity to the HPA-1a alloantigen. The nucleotide sequence analysis of representative HPA-la-specific clones reveals at least 3 distinct V1 and 3VH gene segments that present an extensive degree of mutation as demonstrated by comparison of gene usage and homologies to the nearest germline genes. CONCLUSIONS: These human HPA-la-specific Fab reagents should allow us to better understand the molecular mechanism involved in HPA-la alloimmunization.

Amino Acid Sequence↗

Antibiotics for preterm labour with intact membranes.

BACKGROUND: There may be a link between infection and preterm birth. OBJECTIVES: Since rupture of the membranes is an important factor in the progression of preterm labour, it is important to see if antibiotics are of any benefit prior to membrane rupture. The objective of this review was to assess the effects of antibiotics administered to women in preterm labour with intact membranes, on maternal and neonatal outcomes. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group trials register and reference lists of articles. We contacted experts in the field. SELECTION CRITERIA: Randomised trials which compared antibiotic treatment with placebo or no treatment for women in preterm labour (between 20 and 36 weeks gestation) with intact membranes. DATA COLLECTION AND ANALYSIS: Trial quality was assessed and data were extracted independently by two reviewers. Study authors were contacted for missing data. MAIN RESULTS: Meta-analysis of the ten included trials demonstrated a statistically significant prolongation of pregnancy associated with the use of antibiotics (5. 4 days, 95% confidence interval (CI) 0.9- 9.8 days). Antibiotic treatment was also associated with a statistically significant reduction in maternal infection (odds ratio (OR) 0.59, 95% CI 0.36, 0.97) and neonatal necrotising enterocolitis (OR 0.33, 95% CI 0.13, 0.88). No statistically significant effect was detected on the neonatal outcomes of respiratory distress syndrome and neonatal sepsis, although there was a trend towards the latter (OR 0.67, 95% CI 0.42, 1.07). An increase in perinatal mortality was observed in the group receiving antibiotics (OR 3.36, 95% CI 1.21, 9.32). REVIEWER'S CONCLUSIONS: Although a prolongation in time to delivery and a trend towards a reduction in neonatal sepsis was detected, this review fails to demonstrate a clear overall benefit from antibiotic treatment for preterm labour with intact membranes on neonatal outcomes and raises concerns about increased perinatal mortality for those who received antibiotics. This treatment cannot therefore be currently recommended for routine practice. Further research is required to identify a subgroup of women (and their babies) who are more likely to experience benefit from antibiotic treatment for preterm labour prior to membrane rupture, and to identify which antibiotic or combination of antibiotics is most effective. The results of the ongoing ORACLE trial should help resolve some of the uncertainties revealed by this review.

Anti-Bacterial Agents↗

Use of click chemistry to define the substrate specificity of Leishmania beta-1,2-mannosyltransferases.

Leishmania spp. are human pathogens that utilize a novel beta-1,2-mannan as their major carbohydrate reserve material. We describe a new approach that combines traditional substrate-modification methods and "click chemistry" to assemble a library of modified substrates that were used to qualitatively define the substrate tolerance of the Leishmania beta-1,2-mannosyltransferases responsible for beta-1,2-mannan biosynthesis. The library was assembled by using the highly selective copper(I)-catalysed cycloaddition reaction of azides and alkynes to couple an assortment of azide- and alkyne-functionalized small molecules with complementary alkyne- and azide-functionalized mannose derivatives. All mannose derivatives with alpha-orientated substituents on the anomeric carbon were found to act as substrates when incubated with a Leishmania mexicana particulate fraction containing GDP-mannose. In contrast, 6-substituted mannose derivatives were not substrates. Representative products formed from the library compounds were analysed by mass spectrometry, methylation linkage analysis and beta-mannosidase digestions and showed extension with up to four beta-1,2-linked mannosyl residues. This work provides insights into the substrate specificity of this new class of glycosyltransferases that can be applied to the development of highly specific tools and inhibitors for their study.

Alkynes↗

Double minutes in the papillary thyroid cancer cell line PTC-1113A.

The cell line PTC-1113A was established from a metastasizing recurrent papillary thyroid cancer. The cell line was growing as monolayer and showed a complex karyotype with chromosome numbers ranging from 30 to 140/metaphase. A proportion of metaphases contained double minutes and/or pulverized chromosomes. Extrachromosomal DNA seemed to originate from a B-group chromosome. A chromosome 4 painting probe hybridized to extrachromosomal material, representing double minutes (dmin) and possibly minutes. In addition, fluorescence in situ hybridization (FISH) with the chromosome 4 library detected a translocation chromosome and a pulverized chromosome originating from chromosome 4. PTC-1113A is, to our knowledge, the single papillary thyroid cancer cell line demonstrating evidence of gene amplification.

Aged↗

Transcriptome of axenic liver stages of Plasmodium yoelii.

Plasmodium liver stages or early exo-eythrocytic forms (EEFs) contain antigens that are essential for achieving sterile, protective immunity against malaria. Yet, attempts at identifying these antigens have been hampered by the challenge of obtaining large numbers of purified EEFs, uncontaminated with hepatocyte material. Using a recently described system for producing axenically cultured EEFs from Plasmodium yoelii, we have constructed a cDNA library and generated 1453 expressed sequence tags (ESTs) resulting in 652 unique transcripts. Analysis of the library provides insight into processes required for the initiation and development of Plasmodium liver stages, such as protein degradation, cell cycle progression and nutrient transport. Analysis of the gene expression profile of liver stages, as revealed by this library, suggests that liver stages represent a shift from "sporozoite-like" to "blood-stage-like". This is the first study of the transcriptional repertoire of Plasmodium liver stages.

Animals↗