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At least 631 records · Page 35Linked to original sources
PubMed2006

New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

Archaea↗
PubMed2006

Using DNA microarrays to identify library-independent markers for bacterial source tracking.

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.

Animals↗
PubMed2003

Induction of specific T-cell responses, opsonizing antibodies, and protection against Plasmodium chabaudi adami infection in mice vaccinated with genomic expression libraries expressed in targeted and secretory DNA vectors.

It has been proposed that a multivalent malaria vaccine is necessary to mimic the naturally acquired resistance to this disease observed in humans. A major experimental challenge is to identify the optimal components to be used in such a multivalent vaccine. Expression library immunization (ELI) is a method for screening genomes of a pathogen to identify novel combinations of vaccine sequences. Here we describe immune responses associated with, and the protective efficacy of, genomic Plasmodium chabaudi adami DS expression libraries constructed in VR1020 (secretory), monocyte chemotactic protein-3 (chemoattractant), and cytotoxic T lymphocyte antigen 4 (lymph node-targeting) DNA vaccine vectors. With splenocytes from vaccinated mice, specific T-cell responses, as well as gamma interferon and interleukin-4 production, were observed after stimulation with P. chabaudi adami-infected erythrocytes, demonstrating the specificity of genomic library vaccination for two of the three libraries constructed. Sera obtained from mice vaccinated with genomic libraries promoted the opsonization of P. chabaudi adami-infected erythrocytes by murine macrophages in vitro, further demonstrating the induction of malaria-specific immune responses following ELI. Over three vaccine trials using biolistic delivery of the three libraries, protection after lethal challenge with P. chabaudi adami DS ranged from 33 to 50%. These results show that protective epitopes or antigens are expressed within the libraries and that ELI induces responses specific to P. chabaudi adami malaria. This study further demonstrates that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

Abatacept↗
PubMed2004

Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi).

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey (Ateles geoffroyi). A total of 193 152 BAC clones were generated in this library. The average insert size of the BAC clones was estimated to be 184.6 kb with the small inserts (50-100 kb) accounting for less than 3% and the non-recombinant clones only 1.2%. Assuming a similar genome size with humans, the spider monkey BAC library has about 11x genome coverage. In addition, by end sequencing of randomly selected BAC clones, we generated 367 sequence tags for the library. When blasted against human genome, they showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the library. This black-handed spider monkey BAC library would serve as a valuable resource in comparative genomic study and large-scale genome sequencing of nonhuman primates.

Animals↗
PubMed2006

Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics.

Brachypodium is well suited as a model system for temperate grasses because of its compact genome and a range of biological features. In an effort to develop resources for genome research in this emerging model species, we constructed 2 bacterial artificial chromosome (BAC) libraries from an inbred diploid Brachypodium distachyon line, Bd21, using restriction enzymes HindIII and BamHI. A total of 73,728 clones (36,864 per BAC library) were picked and arrayed in 192,384-well plates. The average insert size for the BamHI and HindIII libraries is estimated to be 100 and 105 kb, respectively, and inserts of chloroplast origin account for 4.4% and 2.4%, respectively. The libraries individually represent 9.4- and 9.9-fold haploid genome equivalents with combined 19.3-fold genome coverage, based on a genome size of 355 Mb reported for the diploid Brachypodium, implying a 99.99% probability that any given specific sequence will be present in each library. Hybridization of the libraries with 8 starch biosynthesis genes was used to empirically evaluate this theoretical genome coverage; the frequency at which these genes were present in the library clones gave an estimated coverage of 11.6- and 19.6-fold genome equivalents. To obtain a first view of the sequence composition of the Brachypodium genome, 2185 BAC end sequences (BES) representing 1.3 Mb of random genomic sequence were compared with the NCBI GenBank database and the GIRI repeat database. Using a cutoff expectation value of E<10-10, only 3.3% of the BESs showed similarity to repetitive sequences in the existing database, whereas 40.0% had matches to the sequences in the EST database, suggesting that a considerable portion of the Brachypodium genome is likely transcribed. When the BESs were compared with individual EST databases, more matches hit wheat than maize, although their EST collections are of a similar size, further supporting the close relationship between Brachypodium and the Triticeae. Moreover, 122 BESs have significant matches to wheat ESTs mapped to individual chromosome bin positions. These BACs represent colinear regions containing the mapped wheat ESTs and would be useful in identifying additional markers for specific wheat chromosome regions.

Chromosomes, Artificial, Bacterial↗
PubMed2006

Quantitative comparison of EST libraries requires compensation for systematic biases in cDNA generation.

BACKGROUND: Publicly accessible EST libraries contain valuable information that can be utilized for studies of tissue-specific gene expression and processing of individual genes. This information is, however, confounded by multiple systematic effects arising from the procedures used to generate these libraries. RESULTS: We used alignment of ESTs against a reference set of transcripts to estimate the size distributions of the cDNA inserts and sampled mRNA transcripts in individual EST libraries and show how these measurements can be used to inform quantitative comparisons of libraries. While significant attention has been paid to the effects of normalization and substraction, we also find significant biases in transcript sampling introduced by the combined procedures of reverse transcription and selection of cDNA clones for sequencing. Using examples drawn from studies of mRNA 3'-processing (cleavage and polyadenylation), we demonstrate effects of the transcript sampling bias, and provide a method for identifying libraries that can be safely compared without bias. All data sets, supplemental data, and software are available at our supplemental web site. CONCLUSION: The biases we characterize in the transcript sampling of EST libraries represent a significant and heretofore under-appreciated source of false positive candidates for tissue-, cell type-, or developmental stage-specific activity or processing of genes. Uncorrected, quantitative comparison of dissimilar EST libraries will likely result in the identification of statistically significant, but biologically meaningless changes.

Algorithms↗
PubMed2001

Quality assessment of combinatorial pooled libraries using mass spectrometry.

Parallel synthesis techniques aim to prepare collections of single compounds which, once tested, can easily be identified by their sole location in the synthesic array. On the other hand, true combinatorial chemistry produces libraries of compounds as mixtures of variable size which require a deconvolution procedure for identification of the active hits or leads. In the latter case, analytical methods are crucial for the success of the strategy and mass spectrometry plays a major role. If the goal is to identify all the library components, including expected products as well as by-products, various mass spectrometric techniques may be necessary. Library components can be separated according to their mass by increasing mass resolution or by their elution time by coupling liquid chromatography and mass spectrometry. The efficiency of such separation techniques are discussed as a function of the size and the degeneracy of the library. Library members possess common structural features which impart similar fragmentation patterns after ionization in the gas phase. This feature can be exploited by tandem mass spectrometry to specifically detect subfamilies of products. Examples of precursor ion scans, product ion scans and constant neutral loss scans will be shown that facilitate partial characterization of libraries. To solve the difficult problem of the quantitative analysis of libraries, i.e., to evaluate their equimolarity, the use of an evaporative light scattering detector (ELSD) or a chemiluminescent nitrogen detector (CLND) is suggested as more appropriate.

Chromatography, Liquid↗
PubMed2004

The different strategies for designing GPCR and kinase targeted libraries.

In recent years the trend in combinatorial library design has shifted to include target class focusing along with diversity and drug-likeness criteria. In this manuscript we review the computational tools available for target class library design and highlight the areas where they have proven useful in our work. The protein kinase family is used to illustrated structure-based target class focused library design, and the G-protein coupled receptor (GPCR) family is used to illustrate ligand-based target class focused library design. Most of the tools discussed are those designed for libraries targeted to a single protein and are simply applied "brute-force" to a large number of targets within the family. The tools that have proven to be the most useful in our work are those that can extract trends from the computational data such as docking and clustering or data mining large amounts of structure activity or high throughput screening data. Finally, areas where improvements are needed in the computational tools available for target class focusing are highlighted. These areas include tools to extract the relevant patterns from all available information for a family of targets, tools to efficiently apply models for all targets in the family rather than just a small subset, mining tools to extract the relevant information from the computational absorption, distribution, metabolism, excretion and toxicity (ADMET) and targeting data, and tools to allow interactive exploration of the virtual space around a library to facilitate the selection of the library that best suits the needs of the design team.

Animals↗
PubMed2006

A gradient descent algorithm for minimizing amino acid coupling reactions when synthesizing cyclic-peptide libraries.

Combinatorial chemistry has become an invaluable tool in medicinal chemistry for the identification of new drug leads. For example, libraries of predetermined sequences and head-to-tail cyclized peptides are routinely synthesized in our laboratory using the IRORI approach. Such libraries are used as molecular toolkits that enable the development of pharmacophores that define activity and specificity at receptor targets. These libraries can be quite large and difficult to handle, due to physical and chemical constraints imposed by their size. Therefore, smaller sub-libraries are often targeted for synthesis. The number of coupling reactions required can be greatly reduced if the peptides having common amino acids are grouped into the same sub-library (batching). This paper describes a schedule optimizer to minimize the number of coupling reactions by rotating and aligning sequences while simultaneously batching. The gradient descent method thereby reduces the number of coupling reactions required for synthesizing cyclic peptide libraries. We show that the algorithm results in a 75% reduction in the number of coupling reactions for a typical cyclic peptide library.

Algorithms↗
PubMed2006

Design and characterization of a functional library for NMR screening against novel protein targets.

In the past few years, NMR has been extensively utilized as a screening tool for drug discovery using various types of compound libraries. The designs of NMR specific chemical libraries that utilize a fragment-based approach based on drug-like characteristics have been previously reported. In this article, a new type of compound library will be described that focuses on aiding in the functional annotation of novel proteins that have been identified from various ongoing genomics efforts. The NMR functional chemical library is comprised of small molecules with known biological activity such as: co-factors, inhibitors, metabolites and substrates. This functional library was developed through an extensive manual effort of mining several databases based on known ligand interactions with protein systems. In order to increase the efficiency of screening the NMR functional library, the compounds are screened as mixtures of 3-4 compounds that avoids the need to deconvolute positive hits by maintaining a unique NMR resonance and function for each compound in the mixture. The functional library has been used in the identification of general biological function of hypothetical proteins identified from the Protein Structure Initiative.

Binding Sites↗
PubMed2004

Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes.

AIM: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs) cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor. METHODS: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs. Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effects in vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofluorescence, cell cycle analysis and MTT assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX). The positive clones were sequenced and analyzed by bio-informatics. RESULTS: The primary cDNA library consisted of 2 x 10(6) recombinants. Thirty-six positive clones were obtained from 6 x 10(5) independent clones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other 15 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70, GRP58, GAPDH, SSB, S100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, S100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries. CONCLUSION: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs, the functional genes associated with tumor endothelium or angiogenesis were identified. The modified SEREX, xenogeneic functional serum screening, was demonstrated to be effective for isolation and identification of antigen genes of tumor endothelium, and also for other tumor cell antigen genes. These antigen genes obtained in this study could be a valuable resource for basic and clinical studies of tumor angiogenesis, thus facilitating the development of anti- angiogenesis targeting therapy of tumors.

Animals↗
PubMed2001

Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer.

AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain kappa and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and kappa gained by RT-PCR were about 650 bp. Fd and kappa PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 10(6). The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

Antibodies↗
PubMed2003

Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma.

AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database. METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used. RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed. CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.

DNA, Complementary↗
PubMed2003

cDNA suppression subtraction library for screening down-regulated genes in gastric carcinoma.

AIM: To establish cDNA suppression subtraction library with a high subtraction efficiency by counterpart normal gastric mucosa mixture mRNA subtracting gastric cancer cells mixture mRNA for screening down-regulated genes in gastric carcinoma. METHODS: RNA of gastric cancer tissues and counterpart normal gastric mucosa were respectively isolated in five patients with gastric cancer, and their mRNA was purified. cDNA suppression subtraction library was established by counterpart normal gastric mucosa mixture mRNA (tester) subtracting gastric cancer tissues mixture mRNA (driver) of five patients with gastric carcinoma. The library plasmids were transformed into competent bacteria DH5a after ligation of the library cDNA fragments with T vectors. Library plasmids were extracted after picking colonies and shaking bacteria overnight. Its subtraction efficiency was confirmed by PCR and reverse hybridization of a nylon filter onto which the colonies of bacteria were transferred with probes of reverse transcription products cDNA of gastric cancer tissues mRNA and counterpart normal gastric mucosa mRNA labeled with alpha- (32)P dCTP. RESULTS: mRNA purified from total RNA of gastric cancer tissues and counterpart normal gastric mucosa in five patients with gastric carcinoma revealed a good quality. cDNA suppression subtraction library constructed for screening down-regulated genes in gastric carcinoma represented a high subtraction efficiency. 86 % of differential expression in down-regulated genes between counterpart normal gastric mucosa and gastric carcinoma was confirmed. CONCLUSION: cDNA suppression subtraction library with a high subtraction efficiency for screening down-regulated genes in gastric carcinoma is successfully established.

Cloning, Molecular↗
PubMed1997

From vision to reality--managing change in the provision of library and information services to nurses, midwives, health visitors and PAMs: (professions allied to medicine) a case study of the North Thames experience with the Inner London Consortium.

One of the North Thames' pioneering consortia, the Inner London Consortium (ILC) is a complex body which includes NHS Trusts with teaching hospital university connections, community-based Trusts and general hospital acute Trusts. Within the consortium there are 12,000 trained nurses, midwives, health visitors and other professional staff working in the professions allied to medicine (PAMs), all of whom require access to and provision of appropriate library information services. In 1994, taking into account experiences elsewhere in the Region and nationally, it became clear that library issues were complex and would become acute with the move of nursing libraries from ILC Trust sites over a very short timescale. A report on the issues commissioned by the Consortium recommended that a library project, which built on existing NHS Trust PGMDE funded library resources and moved these to a multidisciplinary base to serve the consortium membership, be implemented. The objective of providing access to library information services for nurses and PAMs was achieved. Successes that emerged from the implementation included: The registration in Trust libraries of almost 12 000 new members within the initial 6-month monitoring period. The development of service level agreements and standards for the delivery of services to these new user groups. This paper describes the processes behind these significant and complex changes.

Allied Health Personnel↗
PubMed1998

Rapid characterization of combinatorial libraries using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The relatively new field of combinatorial chemistry has enabled researchers to create large mixtures of compounds that can be screened for leads in developing potential drug candidates. The new synthetic method has also created a need for better procedures to analyze the complex mixtures that are generated. The immediate goal in most cases is to verify the synthetic procedure and to determine the purity and completeness of the library sample before binding studies are initiated. We report here a method to rapidly characterize small-molecule combinatorial libraries in solution. All combinatorial library samples were synthesized by combining a core molecule bearing two acid chloride functionalities with various amino acids to generate libraries of 36, 78 and 120 components. Using electrospray ionization fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) we were able to identify 70-80% of the library components. All samples were analyzed as mixtures by direct infusion without chromatographic separation. Furthermore, nominally isobaric components could be resolved and identified through exact mass assignments without tandem mass spectrometery. ESI-FTICR-MS is a rapid and convenient tool for the characterization of small-molecule libraries. The method is especially useful for the analysis of larger libraries that contain many nominally isobaric components and impurities.

Chemistry, Pharmaceutical↗
PubMed1998

Combinatorial libraries: studies in molecular recognition.

In recent years, combinatorial libraries have become a major tool for drug discovery and drug development. Along the way, one potential use of combinatorial chemistry libraries almost been neglected: the basic study of intermolecular interactions. Especially "one-bead-one-structure" libraries can be a powerful means for the discovery of ligands to synthetic receptors and vice versa. Encoded combinatorial libraries have been used to disclose ligands for well designed macrocyclic host molecules and to elucidate their specificities for peptide sequences. These studies led via receptors with more flexibility to simple host molecules without elaborate design that are accessible to combinatorial synthesis. These findings open a realm of possibilities and applications. An intriguing one is the development of chemical sensors for analytes that are otherwise hard or only unspecifically detected. Furthermore, such libraries and the techniques that were developed to handle them have been used to find new catalysts and enzyme mimics. In this review we put the emphasis on studies involving "one-bead-one-structure" libraries. We will review the techniques to generate them, to encode and analyze them, and to assay them. We will describe their past usage and the intriguing results of these studies and point out interesting new applications of such libraries for the study of non-covalent intermolecular interactions.

Chemistry↗
PubMed1999

[Construction of human combinatorial immunoglobulin library containing HBsAb on the surface of phage].

OBJECTIVE: To construct a human recombinant immunoglobulin library containing HBsAb. METHODS: The mRNA isolated from peripheral blood lymphocytes of two blood donors with HBsAb were reversely transcribed to the first strand cDNA with oligo-(dT) primer. Human immunoglobulin kappa light chains and heavy chains Fd genes were amplified respectively by PCR with designed oligo nucleotide primers and the first strand cDNA templates. The kappa light chain genes were first cloned into PComb3Hss vector to construct a human recombinant light chain library. The heavy chain genes were subsequently inserted into the corresponding sites of kappa-PComb3Hss plasmid to generate a combinatorial kappa-Fd-PComb3Hss plasmid. The plasmid transformed into E.Coli.XLI-Blue and the E.Coli.XLI-Blue infected by help phage. RESULTS: The constructed light chains library size was 1.5 x 10(5), and the recombinant frequency of kappa chain genes was 57% and the human combinatorial Fab immunoglobulin library was 5 x 10(5) members and the recombinant frequency of Fab genes was 50%. Finally, the titer of phages in the phage antibody library containing HBsAb was 5 x 10(14) CFU/ml. CONCLUSION: Our work in constructing human immunoglobulin combinatorial library containing HBsAb will be beneficial to further study on selecting Fab phage antibodies of HBsAg from the library.

Hepatitis B Antibodies↗