bluuebunny/crossref_metadata_2025_split huggingface dataset:
Predicting mutually exclusive spliced exons based on exon length, splice site and reading frame conservation, and exon sequence homology
Authors: Pillmann Holger , Hatje Klas , Odronitz Florian , Hammesfahr Björn , Kollmar Martin 
Published: July 2011

Abstract

Background
Alternative splicing of pre-mature RNA is an important process eukaryotes utilize to increase their repertoire of different protein products. Several types of different alternative splice forms exist including exon skipping, differential splicing of exons at their 3'- or 5'-end, intron retention, and mutually exclusive splicing. The latter term is used for clusters of internal exons that are spliced in a mutually exclusive manner.

Results
We have implemented an extension to the WebScipio software to search for mutually exclusive exons. Here, the search is based on the precondition that mutually exclusive exons encode regions of the same structural part of the protein product. This precondition provides restrictions to the search for candidate exons concerning their length, splice site conservation and reading frame preservation, and overall homology. Mutually exclusive exons that are not homologous and not of about the same length will not be found. Using the new algorithm, mutually exclusive exons in several example genes, a dynein heavy chain, a muscle myosin heavy chain, and Dscam were correctly identified. In addition, the algorithm was applied to the whole Drosophila melanogaster X chromosome and the results were compared to the Flybase annotation and an ab initio prediction. Clusters of mutually exclusive exons might be subsequent to each other and might encode dozens of exons.

Conclusions
This is the first implementation of an automatic search for mutually exclusive exons in eukaryotes. Exons are predicted and reconstructed in the same run providing the complete gene structure for the protein query of interest. WebScipio offers high quality gene structure figures with the clusters of mutually exclusive exons colour-coded, and several analysis tools for further manual inspection. The genome scale analysis of all genes of the Drosophila melanogaster X chromosome showed that WebScipio is able to find all but two of the 28 annotated mutually exclusive spliced exons and predicts 39 new candidate exons. Thus, WebScipio should be able to identify mutually exclusive spliced exons in any query sequence from any species with a very high probability. WebScipio is freely available to academics at http://www.webscipio.org.
Detection of Fused Genes in Eukaryotic Genomes using Gene deFuser: Analysis of the Tetrahymena thermophila genome
Authors: Salim Hannah MW , Koire Amanda M , Stover Nicholas A , Cavalcanti Andre RO 
Published: July 2011

Abstract

Background
Fused genes are important sources of data for studies of evolution and protein function. To date no service has been made available online to aid in the large-scale identification of fused genes in sequenced genomes. We have developed a program, Gene deFuser, that analyzes uploaded protein sequence files for characteristics of gene fusion events and presents the results in a convenient web interface.

Results
To test the ability of this software to detect fusions on a genome-wide scale, we analyzed the 24,725 gene models predicted for the ciliated protozoan Tetrahymena thermophila. Gene deFuser detected members of eight of the nine families of gene fusions known or predicted in this species and identified nineteen new families of fused genes, each containing between one and twelve members. In addition to these genuine fusions, Gene deFuser also detected a particular type of gene misannotation, in which two independent genes were predicted as a single transcript by gene annotation tools. Twenty-nine of the artifacts detected by Gene deFuser in the initial annotation have been corrected in subsequent versions, with a total of 25 annotation artifacts (about 1/3 of the total fusions identified) remaining in the most recent annotation.

Conclusions
The newly identified Tetrahymena fusions belong to classes of genes involved in processes such as phospholipid synthesis, nuclear export, and surface antigen generation. These results highlight the potential of Gene deFuser to reveal a large number of novel fused genes in evolutionarily isolated organisms. Gene deFuser may also prove useful as an ancillary tool for detecting fusion artifacts during gene model annotation.
Isolation of mouse mesenchymal stem cells with normal ploidy from bone marrows by reducing oxidative stress in combination with extracellular matrix
Authors: Fan Guokuan , Wen Lai , Li Minshu , Li Chao , Luo Benping , Wang Fang , Zhou Lingjun , Liu Lin 
Published: July 2011

Abstract

Background
Isolation of mouse MSCs (mMSCs) with normal ploidy from bone marrow remains challenging. mMSCs isolated under 20% O2 are frequently contaminated by overgrown hematopoietic cells, and could also be especially vulnerable to oxidative damage, resulting in chromosomal instability. Culture under low oxygen or extracellular matrix (ECM) improves proliferation of MSCs in several species. We tested the hypothesis that culture under low oxygen in combination with ECM prepared from mouse embryonic fibroblast (MEF-ECM) could be used to purify proliferative mMSCs, and to reduce oxidative damage and maintain their chromosomal stability.

Results
Optimization of culture conditions under 20% O2 resulted in immortalization of mMSCs, showing extensive chromosome abnormalities, consistent with previous studies. In contrast, culture under low oxygen (2% O2) improved proliferation of mMSCs and reduced oxidative damage, such that mMSCs were purified simply by plating at low density under 2% O2. MEF-ECM reduced oxidative damage and enhanced proliferation of mMSCs. However, these isolated mMSCs still exhibited high frequency of chromosome abnormalities, suggesting that low oxygen or in combination with MEF-ECM was insufficient to fully protect mMSCs from oxidative damage. Notably, antioxidants (alpha -phenyl-t-butyl nitrone (PBN) and N-acetylcysteine (NAC)) further reduced DNA damage and chromosomal abnormalities, and increased proliferation of mMSCs. mMSCs isolated by the combination method were successfully used to generate induced pluripotent stem (iPS) cells by ectopic expression of Oct4, Sox2, Klf4 and c-Myc.

Conclusions
We have developed a technique that allows to reduce the number of karyotypic abnormalities for isolation of primary mMSCs and for limited culture period by combination of low oxygen, MEF-ECM, antioxidants and low density plating strategy. The effectiveness of the new combination method is demonstrated by successful generation of iPS cells from the isolated mMSCs. However, a culture system for mMSCs still is needed to prevent all the anomalies, especially after a long-term culture period.
Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems
Authors: Lauter Gilbert , Söll Iris , Hauptmann Giselbert 
Published: July 2011

Abstract

Background
Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round.

Results
We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated.

Conclusion
Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.
RASOnD - A comprehensive resource and search tool for RAS superfamily oncogenes from various species
Authors: Kulsum Umay , Singh Vishwadeep , Sharma Sujata , Srinivasan A , Singh Tej P , Kaur Punit 
Published: July 2011

Abstract

Background
The Ras superfamily plays an important role in the control of cell signalling and division. Mutations in the Ras genes convert them into active oncogenes. The Ras oncogenes form a major thrust of global cancer research as they are involved in the development and progression of tumors. This has resulted in the exponential growth of data on Ras superfamily across different public databases and in literature. However, no dedicated public resource is currently available for data mining and analysis on this family. The present database was developed to facilitate straightforward accession, retrieval and analysis of information available on Ras oncogenes from one particular site.

Description
We have developed the RAS Oncogene Database (RASOnD) as a comprehensive knowledgebase that provides integrated and curated information on a single platform for oncogenes of Ras superfamily. RASOnD encompasses exhaustive genomics and proteomics data existing across diverse publicly accessible databases. This resource presently includes overall 199,046 entries from 101 different species. It provides a search tool to generate information about their nucleotide and amino acid sequences, single nucleotide polymorphisms, chromosome positions, orthologies, motifs, structures, related pathways and associated diseases. We have implemented a number of user-friendly search interfaces and sequence analysis tools. At present the user can (i) browse the data (ii) search any field through a simple or advance search interface and (iii) perform a BLAST search and subsequently CLUSTALW multiple sequence alignment by selecting sequences of Ras oncogenes. The Generic gene browser, GBrowse, JMOL for structural visualization and TREEVIEW for phylograms have been integrated for clear perception of retrieved data. External links to related databases have been included in RASOnD.

Conclusions
This database is a resource and search tool dedicated to Ras oncogenes. It has utility to cancer biologists and cell molecular biologists as it is a ready source for research, identification and elucidation of the role of these oncogenes. The data generated can be used for understanding the relationship between the Ras oncogenes and their association with cancer. The database updated monthly is freely accessible online at http://202.141.47.181/rasond/ and http://www.aiims.edu/RAS.html.
Generating libraries of iTol2-end insertions at BAC ends using loxP and lox511 Tn10 transposons
Authors: Shakes Leighcraft A , Abe Gembu , Eltayeb Mugtaba A , Wolf Hope M , Kawakami Koichi , Chatterjee Pradeep K 
Published: July 2011

Abstract

Background
Bacterial Artificial Chromosomes (BACs) have been widely used as transgenes in vertebrate model systems such as mice and zebrafish, for a variety of studies. BAC transgenesis has been a powerful tool to study the function of the genome, and gene regulation by distal cis- regulatory elements. Recently, BAC transgenesis in both mice and zebrafish was further facilitated by development of the transposon-mediated method using the Tol2 element. Tol2 ends, in the inverted orientation and flanking a 1 kb spacer DNA (iTol2), were introduced into the BAC DNA within the bacterial host using recombination of homologous sequences. Here we describe experiments designed to determine if a simpler and more flexible system could modify BACs so that they would be suitable for transgenesis into zebrafish or mouse embryos using the Tol2 transposase.

Results
A new technique was developed to introduce recognition sequences for the Tol2 transposase into BACs in E. coli using the Tn10 transposon vector system. We constructed pTnloxP-iTol2kan and pTnlox511-iTol2kan to introduce the loxP or lox511 site and iTol2 cassette, containing the Tol2 cis-sequences in the inverted orientation, into BACs that have loxP and lox511 sites flanking genomic DNA inserts by Tn10-mediated transposition. The procedure enables rapid generation of a large collection of BACs ready for transgenesis with the iTol2 cassette at the new end of a progressively truncated genomic insert via lox-Cre recombination. The iTol2 ends are efficiently recognized by the Tol2 transposase, and the BACs readily integrate into zebrafish chromosomes.

Conclusion
The new technology described here can rapidly introduce iTol2 ends at a BAC end of choice, and simultaneously generate a large collection of BACs with progressive deletions of the genomic DNA from that end in a single experiment. This procedure should be applicable to a wider variety of BACs containing lox sites flanking the genomic DNA insert, including those with sequence repeats. The libraries of iTol2 inserted BACs with truncations from an end should facilitate studies on the impact of distal cis-regulatory sequences on gene function, as well as standard BAC transgenesis with precisely trimmed genes in zebrafish or mouse embryos using Tol2 transposition.
Gene discovery by genome-wide CDS re-prediction and microarray-based transcriptional analysis in phytopathogen Xanthomonas campestris
Authors: Zhou Lian , Vorhölter Frank-Jörg , He Yong-Qiang , Jiang Bo-Le , Tang Ji-Liang , Xu Yuquan , Pühler Alfred , He Ya-Wen 
Published: July 2011

Abstract

Background
One of the major tasks of the post-genomic era is "reading" genomic sequences in order to extract all the biological information contained in them. Although a wide variety of techniques is used to solve the gene finding problem and a number of prokaryotic gene-finding software are available, gene recognition in bacteria is far from being always straightforward.

Results
This study reported a thorough search for new CDS in the two published Xcc genomes. In the first, putative CDSs encoded in the two genomes were re-predicted using three gene finders, resulting in the identification of 2850 putative new CDSs. In the second, similarity searching was conducted and 278 CDSs were found to have homologs in other bacterial species. In the third, oligonucleotide microarray and RT-PCR analysis identified 147 CDSs with detectable mRNA transcripts. Finally, in-frame deletion and subsequent phenotype analysis of confirmed that Xcc_CDS002 encoding a novel SIR2-like domain protein is involved in virulence and Xcc_CDS1553 encoding a ArsR family transcription factor is involved in arsenate resistance.

Conclusions
Despite sophisticated approaches available for genome annotation, many cellular transcripts have remained unidentified so far in Xcc genomes. Through a combined strategy involving bioinformatic, postgenomic and genetic approaches, a reliable list of 306 new CDSs was identified and a more thorough understanding of some cellular processes was gained.
Comparative analysis of two complete Corynebacterium ulcerans genomes and detection of candidate virulence factors
Authors: Trost Eva , Al-Dilaimi Arwa , Papavasiliou Panagiotis , Schneider Jessica , Viehoever Prisca , Burkovski Andreas , Soares Siomar C , Almeida Sintia S , Dorella Fernanda A , Miyoshi Anderson , Azevedo Vasco , Schneider Maria P , Silva Artur , Santos Cíntia S , Santos Louisy S , Sabbadini Priscila , Dias Alexandre A , Hirata Raphael , Mattos-Guaraldi Ana L , Tauch Andreas 
Published: July 2011

AbstractBackgroundCorynebacterium ulceranshas been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated withC. ulceransappear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of twoC. ulceransstrains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics:C. ulcerans809 was initially isolated from an elderly woman with fatal pulmonary infection andC. ulceransBR-AD22 was recovered from a nasal sample of an asymptomatic dog.ResultsThe circular chromosome ofC. ulcerans809 has a total size of 2,502,095 bp and encodes 2,182 predicted proteins, whereas the genome ofC. ulceransBR-AD22 is 104,279 bp larger and comprises 2,338 protein-coding regions. The minor difference in size of the two genomes is mainly caused by additional prophage-like elements in theC. ulceransBR-AD22 chromosome. Both genomes show a highly similar order of orthologous coding regions; and both strains share a common set of 2,076 genes, demonstrating their very close relationship. A screening for prominent virulence factors revealed the presence of phospholipase D (Pld), neuraminidase H (NanH), endoglycosidase E (EndoE), and subunits of adhesive pili of the SpaDEF type that are encoded in bothC. ulceransgenomes. Therbpgene coding for a putative ribosome-binding protein with striking structural similarity to Shiga-like toxins was additionally detected in the genome of the human isolateC. ulcerans809.ConclusionsThe molecular data deduced from the complete genome sequences provides considerable knowledge of virulence factors inC. ulceransthat is increasingly recognized as an emerging pathogen. This bacterium is apparently equipped with a broad and varying set of virulence factors, including a novel type of a ribosome-binding protein. Whether the respective protein contributes to the severity of human infections (and a fatal outcome) remains to be elucidated by genetic experiments with defined bacterial mutants and host model systems.
Phenotypic and genotypic characterization of Stenotrophomonas maltophiliaisolates from patients with cystic fibrosis: Genome diversity, biofilm formation, and virulence
Authors: Pompilio Arianna , Pomponio Stefano , Crocetta Valentina , Gherardi Giovanni , Verginelli Fabio , Fiscarelli Ersilia , Dicuonzo Giordano , Savini Vincenzo , D'Antonio Domenico , Di Bonaventura Giovanni 
Published: July 2011

AbstractBackgroundStenotrophomonas maltophiliais emerging as one of the most frequently found bacteria in cystic fibrosis (CF) patients. In the present study, phenotypic and genotypic traits of a set of 98 isolates ofS. maltophiliaobtained from clinical (CF and non-CF patients) and environmental sources were comparatively evaluated.ResultsS. maltophiliaexhibited a high level of genomic diversity in both CF and non-CF group, thus possibly allowing this bacterium to expand its pathogenic potentials. Strains sharing the same pulsotype infected different patients, thus likely indicating the occurrence of clonal spread or acquisition by a common source. CF isolates differed greatly in some phenotypic traits among each other and also when compared with non-CF isolates, demonstrating increased mean generation time and susceptibility to oxidative stress, but reduced ability in forming biofilm. Furthermore, in CF isolates flagella- and type IV pili-based motilities were critical for biofilm development, although not required for its initiation. Sequential isogenic strains isolated from the same CF patient displayed heterogeneity in biofilm and other phenotypic traits during the course of chronic infection. CF and non-CF isolates showed comparable virulence in a mouse model of lung infection.ConclusionsOverall, the phenotypic differences observed between CF and non-CF isolates may imply different selective conditions and persistence (adaptation) mechanisms in a hostile and heterogeneous environment such as CF lung. Molecular elucidation of these mechanisms will be essential to better understand the selective adaptation in CF airways in order to design improved strategies useful to counteract and eradicateS. maltophiliainfection.
Methodological reporting of randomized controlled trials in major hepato-gastroenterology journals in 2008 and 1998: a comparative study
Authors: Wang Ji-Lin , Sun Tian-Tian , Lin Yan-Wei , Lu Rong , Fang Jing-Yuan 
Published: July 2011

Abstract

Background
It was still unclear whether the methodological reporting quality of randomized controlled trials (RCTs) in major hepato-gastroenterology journals improved after the Consolidated Standards of Reporting Trials (CONSORT) Statement was revised in 2001.

Methods
RCTs in five major hepato-gastroenterology journals published in 1998 or 2008 were retrieved from MEDLINE using a high sensitivity search method and their reporting quality of methodological details were evaluated based on the CONSORT Statement and Cochrane Handbook for Systematic Reviews of interventions. Changes of the methodological reporting quality between 2008 and 1998 were calculated by risk ratios with 95% confidence intervals.

Results
A total of 107 RCTs published in 2008 and 99 RCTs published in 1998 were found. Compared to those in 1998, the proportion of RCTs that reported sequence generation (RR, 5.70; 95%CI 3.11-10.42), allocation concealment (RR, 4.08; 95%CI 2.25-7.39), sample size calculation (RR, 3.83; 95%CI 2.10-6.98), incomplete outecome data addressed (RR, 1.81; 95%CI, 1.03-3.17), intention-to-treat analyses (RR, 3.04; 95%CI 1.72-5.39) increased in 2008. Blinding and intent-to-treat analysis were reported better in multi-center trials than in single-center trials. The reporting of allocation concealment and blinding were better in industry-sponsored trials than in public-funded trials. Compared with historical studies, the methodological reporting quality improved with time.

Conclusion
Although the reporting of several important methodological aspects improved in 2008 compared with those published in 1998, which may indicate the researchers had increased awareness of and compliance with the revised CONSORT statement, some items were still reported badly. There is much room for future improvement.