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At least 253 records · Page 14Linked to original sources
PubMed2004

Complete MHC haplotype sequencing for common disease gene mapping.

The future systematic mapping of variants that confer susceptibility to common diseases requires the construction of a fully informative polymorphism map. Ideally, every base pair of the genome would be sequenced in many individuals. Here, we report 4.75 Mb of contiguous sequence for each of two common haplotypes of the major histocompatibility complex (MHC), to which susceptibility to >100 diseases has been mapped. The autoimmune disease-associated-haplotypes HLA-A3-B7-Cw7-DR15 and HLA-A1-B8-Cw7-DR3 were sequenced in their entirety through a bacterial artificial chromosome (BAC) cloning strategy using the consanguineous cell lines PGF and COX, respectively. The two sequences were annotated to encompass all described splice variants of expressed genes. We defined the complete variation content of the two haplotypes, revealing >18,000 variations between them. Average SNP densities ranged from less than one SNP per kilobase to >60. Acquisition of complete and accurate sequence data over polymorphic regions such as the MHC from large-insert cloned DNA provides a definitive resource for the construction of informative genetic maps, and avoids the limitation of chromosome regions that are refractory to PCR amplification.

Autoimmune Diseases↗
PubMed2026

CERTOMICS: trusted single-cell multiomics pipeline for high-resolution profiling of adoptive cellular immunotherapies.

SUMMARY: Adoptive cellular immunontherapies, such as chimeric antigen receptor (CAR) T cell therapy, have transformed cancer treatment, yet challenges such as resistance, relapse, and high costs limit their efficacy and accessibility. A comprehensive understanding of cellular heterogeneity and molecular profiles is essential to improve these therapies. Advanced single-cell multiomics technologies have the power to analyze the complex interactions between CAR-engineered cells, immune cells, and tumor cells. However, standardized single-cell multiomics computational pipelines specifically tailored to CAR-engineered cell products are lacking. Due to the synthetic nature of CAR transgenes, additional steps for reliable identification and characterization of CAR-positive cells are required but not included in existing data-processing workflows. To address this, we present CERTOMICS, a Nextflow-based, CAR-aware pipeline offering enhanced CERTainty in immunophenotyping and data interpretation, tailored for single-cell multiOMICSprofiling of adoptive cellular immunotherapies. The pipeline standardizes processing 10x Genomics single-cell multiomics data and integrates CAR-specific identification and quality control. Additionally, a curated repository of CAR construct sequences and annotation data is provided, serving as an extensible resource to support the analysis and development of CAR T cell therapies. AVAILABILITY AND IMPLEMENTATION: Detailed documentation of this pipeline, along with a resource on latest FDA-approved CAR therapies is available on our website: https://fraunhofer-izi.github.io/Living-Drugs-Wiki/. The data underlying this article are available on GitHub at https://github.com/fraunhofer-izi/CERTOMICS. The code is also published on Zenodo at https://doi.org/10.5281/zenodo.18709693.

Multiomics↗
PubMed2005

Molecular profile and partial functional analysis of novel endothelial cell-derived growth factors that regulate hematopoiesis.

Recent progress has been made in the identification of the osteoblastic cellular niche for hematopoietic stem cells (HSCs) within the bone marrow (BM). Attempts to identify the soluble factors that regulate HSC self-renewal have been less successful. We have demonstrated that primary human brain endothelial cells (HUBECs) support the ex vivo amplification of primitive human BM and cord blood cells capable of repopulating non-obese diabetic/severe combined immunodeficient repopulating (SCID) mice (SCID repopulating cells [SRCs]). In this study, we sought to characterize the soluble hematopoietic activity produced by HUBECs and to identify the growth factors secreted by HUBECs that contribute to this HSC-supportive effect. Extended noncontact HUBEC cultures supported an eight-fold increase in SRCs when combined with thrombopoietin, stem cell factor, and Flt-3 ligand compared with input CD34(+) cells or cytokines alone. Gene expression analysis of HUBEC biological replicates identified 65 differentially expressed, nonredundant transcripts without annotated hematopoietic activity. Gene ontology studies of the HUBEC transcriptome revealed a high concentration of genes encoding extracellular proteins with cell-cell signaling function. Functional analyses demonstrated that adrenomedullin, a vasodilatory hormone, synergized with stem cell factor and Flt-3 ligand to induce the proliferation of primitive human CD34(+)CD38(-)lin(-) cells and promoted the expansion of CD34(+) progenitors in culture. These data demonstrate the potential of primary HUBECs as a reservoir for the discovery of novel secreted proteins that regulate human hematopoiesis.

Adrenomedullin↗
PubMed2005

Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components.

The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were identified in four independent analyses; 479 of these proteins showed evidence of differential expression after exposure of P. gingivalis to either conditioned epithelial cell growth medium or control conditions: i.e., they were only detected under one set of conditions. Moreover, 276 genes annotated as hypothetical were found to encode expressed proteins. Among the proteins up-regulated in the presence of epithelial cell components were a homolog of the internalin proteins of Listeria monocytogenes and subunits of the ATP-dependent Clp protease complex. Insertional inactivation of clpP, encoding the Clp proteolytic subunit, resulted in approximately a 50% reduction in invasion of P. gingivalis. These results suggest that adaptation to an epithelial cell environment induces a major shift in the expressed proteome of the organism. Furthermore, ClpP, that is up-regulated in this environment, is required for optimal invasive activity of P. gingivalis.

Bacterial Adhesion↗
PubMed2004

Pharmacogenomic identification of targets for adjuvant therapy with the topoisomerase poison camptothecin.

The response of tumor cells to the unusual form of DNA damage caused by topoisomerase poisons such as camptothecin (CPT) is poorly understood, and knowledge regarding which drugs can be effectively combined with CPT is lacking. To better understand the response of tumor cells to CPT and to identify potential targets for adjuvant therapy, we examined global changes in mRNA abundance in HeLa cells after CPT treatment using Affymetrix U133A GeneChips, which include all annotated human genes (22,283 probe sets). Statistical analysis of the data using a Bayesian/Cyber t test and a modified Benjamini and Hochberg correction for multiple hypotheses testing identified 188 probe sets that are induced and 495 that are repressed 8 h after CPT treatment at a False Discovery Rate of <0.05 and a minimum 3-fold change. This pharmacogenomic approach led us to identify two pathways that are CPT induced: (a) the epidermal growth factor receptor; and (b) nuclear factor-kappaB-regulated antiapoptotic factors. Experiments using HeLa cells in our lab and prior animal model studies performed elsewhere confirm that inhibitors of these respective pathways super-additively enhance CPT's cytotoxicity, suggesting their potential as targets for adjuvant therapy with CPT.

Antineoplastic Agents, Phytogenic↗
PubMed2005

CLIP: a method for identifying protein-RNA interaction sites in living cells.

Nucleic-acid binding proteins constitute nearly one-fourth of all functionally annotated human genes. Genome-wide analysis of protein-nucleic acid contacts has not yet been performed for most of these proteins, restricting attempts to establish a comprehensive understanding of protein function. UV cross-linking is a method typically used to determine the position of direct interactions between proteins and nucleic acids. We have developed the cross-linking and immunoprecipitation assay, which exploits the covalent protein-nucleic acid cross-linking to stringently purify a specific protein-RNA complex using immunoprecipitation followed by SDS-PAGE separation. In this way, the vast majority of non-specific contaminating RNA, which can bind to co-immunoprecipitated proteins or beads, can be removed. Here, we present an improved protocol that performs RNA linker ligation before the SDS-PAGE step, and describe its application to the specific purification and amplification of RNA ligands of Nova in neurons.

Animals↗
PubMed2006

A comparative study of cells in inflammation, EAE and MS using biomedical literature data mining.

Biomedical literature and database annotations, available in electronic forms, contain a vast amount of knowledge resulting from global research. Users, attempting to utilize the current state-of-the-art research results are frequently overwhelmed by the volume of such information, making it difficult and time-consuming to locate the relevant knowledge. Literature mining, data mining, and domain specific knowledge integration techniques can be effectively used to provide a user-centric view of the information in a real-world biological problem setting. Bioinformatics tools that are based on real-world problems can provide varying levels of information content, bridging the gap between biomedical and bioinformatics research. We have developed a user-centric bioinformatics research tool, called BioMap, that can provide a customized, adaptive view of the information and knowledge space. BioMap was validated by using inflammatory diseases as a problem domain to identify and elucidate the associations among cells and cellular components involved in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). The BioMap system was able to demonstrate the associations between cells directly excavated from biomedical literature for inflammation, EAE and MS. These association graphs followed the scale-free network behavior (average gamma = 2.1) that are commonly found in biological networks.

Animals↗
PubMed2006

Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease models.

We present a digital atlas system that allows mapping of molecular expression patterns at cellular resolution through large series of histological sections. Using this system, we have mapped the distribution of a distinct marker, encoded by the LacZ reporter gene driven by the tetracycline-responsive prion protein promoter in double transgenic mice. The purpose is to evaluate the suitability of this promoter mouse line for targeting genes of interest to specific brain regions, essential for construction of inducible transgenic disease models. Following processing to visualize the promoter expression, sections were counterstained to simultaneously display cytoarchitectonics. High-resolution mosaic images covering entire coronal sections were collected through the mouse brain at intervals of 200 microm. A web-based application provides access to a customized virtual microscopy tool for viewing and navigation within and across the section images. For each section image, the nearest section in a standard atlas is defined, and annotations of key structures and regions inserted. Putative categorization of labeled cells was performed with use of distribution patterns, followed by cell size and shape, as parameters that were compared to legacy data. Among the ensuing results were expression of this promoter in putative glial cells in the cerebellum (and not in Purkinje cells), in putative glial cells in the substantia nigra, in pallidal glial cells or interneurons, and in distinct cell layers and regions of the hippocampus. The study serves as a precursor for a database resource allowing evaluation of the suitability of different promoter mouse lines for generating disease models.

Animals↗
PubMed2006

Identification of up-regulated genes after complete spinal cord transection in adult rats.

Spinal cord injury (SCI) initiates a cascade of events and these responses to injury are likely to be mediated and reflected by changes in mRNA concentrations. As a step towards understanding the complex mechanisms underlying repair and regeneration after SCI, the gene expression pattern was examined 4.5 days after complete transection at T8-9 level of rat spinal cord. Improved subtractive hybridization was used to establish a subtracted cDNA library using cDNAs from normal rat spinal cord as driver and cDNAs from injured spinal cord as tester. By expressed sequence tag (EST) sequencing, we obtained 73 EST fragments from this library, representing 40 differentially expressed genes. Among them, 32 were known genes and 8 were novel genes. Functions of all annotated genes were scattered in almost every important field of cell life such as DNA repair, detoxification, mRNA quality control, cell cycle control, and signaling, which reflected the complexity of SCI and regeneration. Then we verified subtraction results with semiquantitative RT-PCR for eight genes. These analyses confirmed, to a large extent, that the subtraction results accurately reflected the molecular changes occurring at 4.5 days post-SCI. The current study identified a number of genes that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using improved subtractive hybridization may lead to a better understanding of molecular mechanisms responsible for repair and regeneration after SCI.

Animals↗
PubMed2004

A preliminary investigation of the Nostoc punctiforme proteome.

Nostoc punctiforme ATCC 29133 is a filamentous terrestrial cyanobacterium (prokaryote) that expresses several different phenotypes in response to environmental cues. When grown in nitrogen-deficient media the most abundant proteins in addition to phycobiliproteins were superoxide dismutase, ATP synthase, and peptidyl-prolyl cis-trans isomerases. A methylated peptide from an akinete marker protein was also identified, suggesting that methylation could potentially play a regulatory role through signaling. C-phycocyanin alpha-chain was methylated at the C-terminal end of the protein and tandem mass spectrometric data also identified peptides that were deamidated. Since a significant number of putative polyketide/non-ribosomal peptide synthase genes are present in the annotated genome, an analysis of a methanolic extract of whole cells was also performed, and a series of nostopeptolides were identified.

Amino Acid Sequence↗
PubMed2002

Escherichia coli K-12 undergoes adaptive evolution to achieve in silico predicted optimal growth.

Annotated genome sequences can be used to reconstruct whole-cell metabolic networks. These metabolic networks can be modelled and analysed (computed) to study complex biological functions. In particular, constraints-based in silico models have been used to calculate optimal growth rates on common carbon substrates, and the results were found to be consistent with experimental data under many but not all conditions. Optimal biological functions are acquired through an evolutionary process. Thus, incorrect predictions of in silico models based on optimal performance criteria may be due to incomplete adaptive evolution under the conditions examined. Escherichia coli K-12 MG1655 grows sub-optimally on glycerol as the sole carbon source. Here we show that when placed under growth selection pressure, the growth rate of E. coli on glycerol reproducibly evolved over 40 days, or about 700 generations, from a sub-optimal value to the optimal growth rate predicted from a whole-cell in silico model. These results open the possibility of using adaptive evolution of entire metabolic networks to realize metabolic states that have been determined a priori based on in silico analysis.

Acetic Acid↗
PubMed2005

Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions.

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.

Amino Acid Sequence↗
PubMed2006

Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets.

Using a unique microarray platform for cytosine methylation profiling, the DNA methylation landscape of the human genome was monitored at more than 21,000 sites, including 79% of the annotated transcriptional start sites (TSS). Analysis of an oligodendroglioma derived cell line LN-18 revealed more than 4000 methylated TSS. The gene-centric analysis indicated a complex pattern of DNA methylation exists along each autosome, with a trend of increasing density approaching the telomeres. Remarkably, 2% of CpG islands (CGI) were densely methylated, and 17% had significant levels of 5 mC, whether or not they corresponded to a TSS. Substantial independent verification, obtained from 95 loci, suggested that this approach is capable of large scale detection of cytosine methylation with an accuracy approaching 90%. In addition, we detected large genomic domains that are also susceptible to DNA methylation reinforced inactivation, such as the HOX cluster on chromosome 7 (CH7). Extrapolation from the data suggests that more than 2000 genomic loci may be susceptible to methylation and associated inactivation, and most have yet to be identified. Finally, we report six new targets of epigenetic inactivation (IRX3, WNT10A, WNT6, RARalpha, BMP7 and ZGPAT). These targets displayed cell line and tumor specific differential methylation when compared with normal brain samples, suggesting they may have utility as biomarkers. Uniquely, hypermethylation of the CGI within an IRX3 exon was correlated with over-expression of IRX3 in tumor tissues and cell lines relative to normal brain samples.

Brain↗
PubMed2003

eVOC: a controlled vocabulary for unifying gene expression data.

Expression data contribute significantly to the biological value of the sequenced human genome, providing extensive information about gene structure and the pattern of gene expression. ESTs, together with SAGE libraries and microarray experiment information, provide a broad and rich view of the transcriptome. However, it is difficult to perform large-scale expression mining of the data generated by these diverse experimental approaches. Not only is the data stored in disparate locations, but there is frequent ambiguity in the meaning of terms used to describe the source of the material used in the experiment. Untangling semantic differences between the data provided by different resources is therefore largely reliant on the domain knowledge of a human expert. We present here eVOC, a system which associates labelled target cDNAs for microarray experiments, or cDNA libraries and their associated transcripts with controlled terms in a set of hierarchical vocabularies. eVOC consists of four orthogonal controlled vocabularies suitable for describing the domains of human gene expression data including Anatomical System, Cell Type, Pathology and Developmental Stage. We have curated and annotated 7016 cDNA libraries represented in dbEST, as well as 104 SAGE libraries,with expression information,and provide this as an integrated, public resource that allows the linking of transcripts and libraries with expression terms. Both the vocabularies and the vocabulary-annotated libraries can be retrieved from http://www.sanbi.ac.za/evoc/. Several groups are involved in developing this resource with the aim of unifying transcript expression information.

Animals↗
PubMed2005

A method for automated detection of gene expression required for the establishment of a digital transcriptome-wide gene expression atlas.

Acquiring information about the expression of a gene in different cell populations and tissues can provide key insight into the function of the gene. A high-throughput in situ hybridization (ISH) method was recently developed for rapid and reproducible acquisition of gene expression patterns in serial tissue sections at cellular resolution. Characterizing and analysing expression patterns on thousands of sections requires efficient methods for locating cells and estimating the level of expression in each cell. Such cellular quantification is an essential step in both annotating and quantitatively comparing high-throughput ISH results. Here we describe a novel automated and efficient methodology for performing this quantification on postnatal mouse brain.

Animals↗
PubMed2006

Head and neck squamous cell carcinoma transcriptome analysis by comprehensive validated differential display.

Head and neck squamous cell carcinoma (HNSCC) is common worldwide and is associated with a poor rate of survival. Identification of new markers and therapeutic targets, and understanding the complex transformation process, will require a comprehensive description of genome expression, that can only be achieved by combining different methodologies. We report here the HNSCC transcriptome that was determined by exhaustive differential display (DD) analysis coupled with validation by different methods on the same patient samples. The resulting 820 nonredundant sequences were analysed by high throughput bioinformatics analysis. Human proteins were identified for 73% (596) of the DD sequences. A large proportion (>50%) of the remaining unassigned sequences match ESTs (expressed sequence tags) from human tumours. For the functionally annotated proteins, there is significant enrichment for relevant biological processes, including cell motility, protein biosynthesis, stress and immune responses, cell death, cell cycle, cell proliferation and/or maintenance and transport. Three of the novel proteins (TMEM16A, PHLDB2 and ARHGAP21) were analysed further to show that they have the potential to be developed as therapeutic targets.

Amino Acid Sequence↗
PubMed2004

A genome wide screening approach for membrane-targeted proteins.

Membrane-associated proteins are critical for intra- and intercellular communication. Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue. Here we describe a modification of the yeast Ras recruitment system to this end and designate the modified approach the Ras membrane trap (RMT). A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome. 112 gene products were identified in the screen of which 79 lack assignable homologs in available data bases. Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested. Thus, genome annotation using currently available tools is likely to underpredict representation of membrane-associated gene products. The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes. Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks and indeed from representatives of all major invertebrate phyla.

Animals↗
PubMed2005

Targeting malaria parasite proteins to the erythrocyte.

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.

Amino Acid Sequence↗