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At least 1,423 records · Page 79Linked to original sources
PubMed2004

Proteomics in postgenomic neuroscience: the end of the beginning.

Proteomics is complementary to genomic approaches anchored in DNA and RNA. Global characterization of proteins is providing new insights into general biological structures as well as synapses, receptor complexes and other neuronal and glial features. Current challenges for proteomics of the nervous system include problems relating to sample preparation, brain complexity, limited databases and informatics tools. The combination of proteomics with other global functional genomic approaches at the levels of genome and transcriptome, together with network biology, will provide important bridges between genes, physiology and pathology.

Animals↗
PubMed2005

Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.

Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.

Amino Acid Sequence↗
PubMed2005

Genome-wide estimation of transcript concentrations from spotted cDNA microarray data.

A method providing absolute transcript concentrations from spotted microarray intensity data is presented. Number of transcripts per microg total RNA, mRNA or per cell, are obtained for each gene, enabling comparisons of transcript levels within and between tissues. The method is based on Bayesian statistical modelling incorporating available information about the experiment from target preparation to image analysis, leading to realistically large confidence intervals for estimated concentrations. The method was validated in experiments using transcripts at known concentrations, showing accuracy and reproducibility of estimated concentrations, which were also in excellent agreement with results from quantitative real-time PCR. We determined the concentration for 10,157 genes in cervix cancers and a pool of cancer cell lines and found values in the range of 10(5)-10(10) transcripts per microg total RNA. The precision of our estimates was sufficiently high to detect significant concentration differences between two tumours and between different genes within the same tumour, comparisons that are not possible with standard intensity ratios. Our method can be used to explore the regulation of pathways and to develop individualized therapies, based on absolute transcript concentrations. It can be applied broadly, facilitating the construction of the transcriptome, continuously updating it by integrating future data.

Bayes Theorem↗
PubMed2004

Sperm-induced modification of the oviductal gene expression profile after natural insemination in mice.

In mammals, the physiological interaction between spermatozoa and oviductal epithelia involves intimate and specific contact between the two cell types. Spermatozoa may undergo stringent selection processes within the female reproductive tract before they meet and fertilize oocytes. The physiological basis of the sperm selection process is largely unknown. Here we tested the hypothesis that the oviduct has a recognition system for spermatozoa that can detect the arrival of spermatozoa in the oviduct after insemination, resulting in alterations of the oviductal transcriptome. We initially performed a global screening of the oviductal transcriptome in mice 1) at the time of estrus (mating) and 2) 6 h after mating. Transcriptional alterations in the oviduct after mating were attributed to the presence of spermatozoa in the oviduct after mating and also to changes in the hormonal environment as female mice underwent the transition from estrus to diestrus. To distinguish these possibilities, female mice were then mated with T145H mutant mice, which because of spermatogenic arrest, produce seminal plasma but no spermatozoa. Focusing on two molecules that in the first experiment were upregulated after mating, it was found that adrenomedullin and prostaglandin endoperoxidase synthase 2 transcripts were upregulated in the oviducts of mice only after mating with fertile males; those mated with T145H infertile males showed significantly less response. These results indicate that it is the arrival of spermatozoa in the oviduct that activates one or more signal transduction pathways and leads to changes in the oviductal transcriptome profiles.

Adrenomedullin↗
PubMed2002

Contributions of proteome profiling to the molecular analysis of cancer.

The proteome is the most functional compartment encoded for in the genome. Technologies for protein separation and quantitation, coupled with mass spectrometry for protein identification, have provided the means for proteome profiling of tumor cell lines and tissues that complement genomic and transcriptomic profiling. The application of established and novel proteomic technologies to the molecular analysis of cancer is reviewed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

The human pituitary proteome: the characterization of differentially expressed proteins in an adenoma compared to a control.

In order to clarify the basic molecular mechanisms that participate in the formation of human pituitary macroadenomas, this study, for the first time, describes the comparative proteomics between a pituitary adenoma tissue and a control tissue. A vertical, two-dimensional polyacrylamide gel electrophoresis system and PDQuest image analysis software were used to provide a high level of between-gel reproducibility and electrophoretic separation to accurately locate each differentially expressed protein. Mass spectrometry (MALDI-TOF and LC-ESI-Q-IT) and protein databases were used to characterize each differentially expressed protein. A total of 137 differential gel spots (37 increased spot volumes, 39 decreased, 19 new and 42 lost) were found when we compared an adenoma proteome to a control proteome. Seventy-one spots (20 increased, 27 decreased, 13 new, 11 lost), representing 39 differentially regulated proteins, were identified. Five differentially regulated proteins (prolactin, cellular retinoic acid-binding protein II, G-protein beta subunit 3, secretagogin and calreticulin) were also validated with results from a comparative transcriptomics study of pituitary adenomas and controls. The functional characteristics of these differentially expressed proteins provide a differential proteomic profile between a pituitary adenoma and a control.

Adenoma↗
PubMed2003

The path from molecular indicators of exposure to describing dynamic biological systems in an aquatic organism: microarrays and the fathead minnow.

The extent to which humans and wildlife are exposed to toxicants is an important focus of environmental research. This work has been directed toward the development of molecular indicators diagnostic for exposure to various stressors in freshwater fish. Research includes the discovery of genes, indicative of environmental exposure, in the Agency's long-established aquatic toxicological organism, the fathead minnow (Pimephales promelas). Novel cDNAs and coding sequences will be used in DNA microarray analyses for pattern identification of stressor-specific, differentially up- and down-regulated genes. The methods currently used to discover genes in this organism, for which few annotated nucleic acid sequences exist, are cDNA subtraction libraries, differential display, exploiting PCR primers for known genes of other members of the family Cyprinidae and use of degenerate PCR primers designed from regions of moderate protein homology. Single or multiple genes noted as being differentially expressed in microarray analyses will then be used in separate studies to measure bioavailable stressors in the laboratory and field. These analyses will be accomplished by quantitative RT-PCR. Moving from analysis of single gene exposures to the global state of the transcriptome offers possibilities that those genes identified by DNA microarray analyses might be critical components of dynamic biological systems and networks, wherein chemical stressors exert toxic effects through various modes of action. Additionally, the ability to discriminate bioavailability of stressors in complex environmental mixtures, and correlation with adverse effects downstream from these early molecular events, presents challenging new ground to be broken in the area of risk assessment.

Animals↗
PubMed2026

S100P as a Shared Biomarker in Inflammatory Bowel Disease, Colorectal Cancer, and Pancreatic Adenocarcinoma: An Integrated Transcriptomic Analysis.

Inflammatory bowel disease (IBD) is associated with an increased risk of colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD), yet the molecular features shared among these diseases remain incompletely understood. This study aimed to identify common genes and biological pathways associated with IBD, CRC, and PAAD through integrated transcriptomic analysis and experimental validation. Gene expression datasets for IBD, CRC, and PAAD were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases. Weighted gene co-expression network analysis and differential expression analysis were performed to identify disease-associated and shared genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (analyses were used to explore enriched biological functions and pathways. Immune cell infiltration was evaluated using Cell-type Identification by Estimating Relative Subsets of RNA Transcripts. Receiver operating characteristic analysis was performed to assess the diagnostic performance of common genes. Single-cell RNA sequencing analysis was conducted to examine the cellular distribution of S100P. In addition, the effects of S100P downregulation were evaluated in lipopolysaccharide (LPS)-stimulated colonic epithelial cells. A total of 162 disease-associated genes and four common genes were identified. Functional enrichment analyses indicated significant enrichment of immune- and inflammation-related pathways, including the interleukin-17 signaling pathway. Immune infiltration analysis revealed similar trends in several immune cell populations across IBD, CRC, and PAAD. Single-cell analysis showed elevated S100P expression in epithelial cells from all three diseases. Downregulation of S100P restored the proliferative capacity of LPS-stimulated colonic epithelial cells and reduced inflammatory cytokine expression. Integrated transcriptomic analysis identified S100P as a biomarker associated with IBD, CRC, and PAAD and highlighted shared immune-related features across these diseases.

Humans↗
PubMed2026

Long-read, high-coverage reference genome of the nymphalid butterfly Catonephele acontius (Nymphalidae: Biblidinae).

Catonephele acontius (Nymphalidae:Biblidinae:Epicalinii) is a butterfly species with a wide distribution across the Neotropics including the Amazon. Here, we present a long-read high-coverage reference genome for this species to serve as a genomic resource for future studies on Biblidinae butterflies, a group that is the subject of ongoing studies of seasonal adaptation under climate change. We used PacBio HiFi and IsoSeq reads to generate a highly contiguous and well-annotated reference genome. Five libraries were constructed, 4 using RNA from different tissues and 1 using high molecular weight (HMW) DNA from a wild-caught female. The DNA was sequenced using PacBio HiFi technology, and the RNA was sequenced using long read PacBio IsoSeq technology. About 20 Gb of raw HiFi data were generated and assembled to an initial size of 520.7 Mb (39 × homozygous coverage) in 90 contigs. The assembly was then polished and decontaminated into 40 contigs with an N50 of 19.927 Mb (BUSCO completeness: 99.0%; duplication: 0.5%; fragmentation: 0.7%; and missing: 0.3%). Final assembly size was 519.2 Mb. Repeats were annotated, showing that the genome consisted of 40.4% transposable elements. IsoSeq transcriptome data from antennae, leg, ovary, and digestive tissue was then used to structurally and functionally annotate gene models for the softmasked genome, uncovering ∼18,500 genes, with 70% of them given functional annotation. This reference assembly joins many published genomes in the Nymphalidae family but represents one of the first high-quality genomes from the Biblidinae subfamily. It provides a valuable resource to study the evolution of plastic and seasonal traits and will help investigate the genetic processes that may influence these species' responses to rapid climate change.

Animals↗
PubMed2004

Microdissection and SAGE as a combined tool to reveal gene expression in ductal carcinoma in situ of the breast.

The interplay between cancer cells and the normal surrounding tissue is believed to influence the biological behavior of the tumor. However, the presence of multiple cell types within the prelevated tumor specimen may attenuate changes that occur specifically in the malignant cells within their microenvironment. To study gene expression of the malignant cells in situ, we used a new microdissection method to separate ductal carcinoma in situ (DCIS) cells from the surrounding stroma, immunological infiltrates, and endothelial cells. We applied an adapted microSAGE protocol, without total mRNA amplification, to study their gene expression profile. Three thousand two hundred one different transcripts were identified in a total of 29 534 observed tags. Of these unique tags, 88.3% matched known GenBank sequences and 11.7% represented unknown transcripts. As compared to a total DCIS SAGE library, microdissection combined with SAGE revealed additional genes expressed only in normal surrounding, probably stromal, cells and not or significantly less in DCIS tumor cells. This study demonstrates that microdissection can be combined with SAGE as a tool to study transcriptomes. This approach provides important new information on differential gene expression both in tumor cells and normal surrounding tissue. Several of the observed differences indeed disappear when the total tumor mass is analyzed.

Adult↗
PubMed2004

Over-representation of exonic splicing enhancers in human intronless genes suggests multiple functions in mRNA processing.

The human transcriptome is constituted of a great majority of intron-containing and a minority of intron-lacking mRNAs; given the different processing these transcripts undergo, they are expected to carry, intermingled with coding properties, very different editing information. Here we applied a computational approach to compare intronless and intron-containing coding sequences. Hexamer composition comparison allowed the definition of over- and under-represented motifs in intronless genes; surprisingly, experimental testing revealed that intron-lacking coding sequences are enriched rather than depleted in elements with splicing enhancement ability. Similarly, we show evidence that intronless transcripts display a significantly higher frequency of both shuttling and non-shuttling SR protein binding sites compared to intron-containing sequences. These observations suggest that SR proteins (and possibly other splicing factors) play a role in cellular processes distinct from splicing.

Base Sequence↗
PubMed2006

Novel urothelium specific gene expression identified by differential display reverse transcriptase-polymerase chain reaction.

PURPOSE: Understanding the molecular basis of differential gene expression among different tissues at various developmental stages and in neoplastic transformation is an important biological goal. The potential clinical applications of this improved understanding are more precise diagnosis of disease, prediction of prognosis, novel targeted therapies and prediction of response to therapy. MATERIALS AND METHODS: Differential display reverse transcriptase-polymerase chain reaction was used to compare gene expression in bovine urothelium to that in autologous lung, esophagus, liver and spleen. Products that appeared to have urothelial specific expression were sequenced and assessed for homology with known sequences. Ribonuclease protection assays were used to further confirm the expression pattern. RESULTS: A total of 32 discrete cDNAs were identified, including 3 products from genes known to be urothelium specific in their expression, 16 with significant homology to bovine, human or mouse expressed sequence tags and 5 with no sequence homology to any currently available sequence. Urothelium specific mRNA expression was confirmed for 3 genes by ribonuclease protection assays and one (Udd06) was further characterized as a urea transporter. CONCLUSIONS: The use of differential display reverse transcriptase-polymerase chain reaction and other complementary techniques for parallel gene expression analysis will permit the complete characterization of the urothelial transcriptome and help identify potential molecular targets for rationally targeted therapy.

Animals↗
PubMed2026

Host Genetic Regulation of NLRP3 Inflammasome Cytokines Reveals Immune and Vascular Pathways in HIV.

People with HIV exhibit elevated inflammation and cardiovascular risk despite antiretroviral therapy. To define the genetic architecture of inflammasome-associated inflammation, we performed whole-genome sequencing and quantified plasma IL-6, IL-1β, and IL-18 in 1,000 ART-suppressed PWH from the U.S. Military HIV Natural History Study. Genome-wide analyses identified 14 loci implicating antiviral defense (DDX17, DDX41, EEA1, BCL11A), lipid metabolism (ABCA1, ABCA12, ABCC1, AGMO), and vascular remodeling (KLHL29, RNF213, ETV1). Transcriptome-wide analyses across cardiovascular and immune tissues identified regulatory programs linking interferon signaling, immune activation, and vascular biology to circulating cytokine levels. Mendelian randomization analyses supported causal relationships between inflammasome-associated cytokines and vascular events. Functional integration with genome-wide CRISPR perturbation datasets in primary CD4+ T cells linked cytokine-associated loci to HIV antiviral pathways and cytokine regulatory networks. External validation in cohorts without HIV demonstrated pathway-level convergence despite limited variant-level overlap. These findings define genetic mechanisms linking inflammasome signaling, antiviral defense, and cardiovascular risk.

HIV↗
PubMed2007

Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis.

Differential stress/inflammatory responses were characterized at the mRNA and protein levels in mandibular lymph nodes (MLN) and oropharyngeal tonsils of European wild boars (Sus scrofa), naturally infected with Mycobacterium bovis. Suppression-subtractive hybridization combined with immunohistochemistry and/or quantitative real-time RT-PCR were used to identify and characterize abundant stress/inflammatory gene sequences differentially expressed in tuberculous (TB+) wild boars. Genes identified in MLN and tonsils corresponded to serum amyloid A, arginase I, osteopontin, lysozyme, annexin I, and heat shock proteins, respectively. Global protein patterns in MLN and tonsils were compared between TB+ and nontuberculous (TB-) boars by 2-DE and MALDI-TOF MS. Five proteins, including stress/inflammatory proteins annexin V, serum albumin, and apolipoprotein A1 were found at lower levels in MLN of TB+ boars. Manganese superoxide dismutase was found up-regulated in MLN of TB+ boars. Five proteins, including creatine kinase and MHC class II antigens were found up-regulated in tonsils of TB+ boars. These results demonstrated differential stress/inflammatory responses in wild boars naturally infected with M. bovis and suggest possible markers of tuberculosis in this species that may prove useful for future studies of host-pathogen interactions and for diagnostics and vaccine development.

Amino Acid Sequence↗
PubMed2004

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↗
PubMed2001

Using in silico transcriptomics to search for tumor-associated antigens for immunotherapy.

Immunotherapy approaches to fight cancer are based on the principle of mounting an immune response against a self-antigen expressed by the tumor cells. In order to reduce potential autoimmunity side-effects, the antigens used should be as tumor-specific as possible. A complementary approach to experimental tumor antigen discovery is to screen the human genome in silico, particularly the databases of "Expressed Sequence Tags" (ESTs), in search of tumor-specific and tumor-associated antigens. The public databases currently provide a massive amount of ESTs from several hundreds of cDNA tissue libraries, including tumoral tissues from various types. We describe a novel method of EST database screening that allows new potential tumor-associated genes to be efficiently selected. The resulting list of candidates is enriched in known genes, described as being expressed in tumor cells.

Antigens, Neoplasm↗
PubMed2002

Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach.

We studied differential gene expression in ipsilateral and contralateral bovine oviduct epithelial cells using a combination of subtracted cDNA libraries and cDNA array hybridization. Four Simmental heifers were synchronized and slaughtered 3.5 days after they entered standing heat. Epithelial cells were isolated from ipsilateral and contralateral oviducts. To identify genes that are differentially regulated in ipsilateral and contralateral epithelium, subtracted cDNA libraries were produced by suppression subtractive hybridization and analyzed by cDNA array hybridization. Sequencing of cDNAs showing differential expression levels in ipsilateral and contralateral epithelium revealed 35 different cDNAs, 30 of which matched genes with known functions and 5 of which matched genes without a known function. The majority of genes (n = 27) were expressed at a higher level in the ipsilateral oviduct, but for some genes (n = 8), mRNA abundance was higher in the contralateral oviduct. The regulated genes or their products include a variety of functional classes such as cell-surface proteins, cell-cell interaction proteins, members of signal transduction pathways, immune-related proteins, and enzymes. Identification of genes differentially regulated in ipsilateral and contralateral oviduct epithelial cells is the first step toward a systematic analysis of local mechanisms that regulate the function of the bovine oviduct epithelium in the postovulation period.

Animals↗