Biomedical subjects

Lixin Zhou

Publications and source records attributed to Lixin Zhou.

8 recordsLinked to original sources

PubMed2006

Cutaneous gene expression by DNA microarray in murine sclerodermatous graft-versus-host disease, a model for human scleroderma.

The molecular mechanisms governing skin fibrosis in murine sclerodermatous graft-versus-host disease (Scl GVHD) are not known. We used Affymetrix DNA microarrays representing >14,000 mouse genes to characterize global gene expression in skin during development of Scl GVHD in lethally irradiated BALB/c (H-2d) mice transplanted with B10.D2 (H-2d) bone marrow and spleen cells. These mice develop skin thickening, whereas control mice transplanted with syngeneic BALB/c (H-2d) bone marrow and spleen cells do not develop disease. We found consistent differences between mice with Scl GVHD and controls in cytokine messenger RNAs (mRNAs) for both Th1-like (IFN-gamma) and Th2-like (IL-6, Il-10, and IL-13) inflammatory patterns. mRNAs for chemokines CCL2, CCL5, CCL17, IFN-gamma inducible chemokines (CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC), and for growth factors such as platelet-derived growth factor-c, connective tissue growth factor, fibroblast growth factor 1, epidermal growth factor, nerve growth factor-beta, vascular endothelial growth factor (VEGF)-alpha, and VEGF-beta were elevated, similar to human scleroderma. mRNAs for cell adhesion molecules, such as L-selectin (selectin lymphocyte), P-selectin (selectin platelet), E-selectin (selectin endothelium), and vascular cell adhesion molecule 1, were also upregulated. In separate experiments, we confirmed the increased synthesis of IFN-gamma and IL-2, unchanged IL-10, and absence of tumor necrosis factor-alpha, and IL-4 proteins by flow cytometry of isolated skin T cells. These constellations of immunologic changes provide a "fingerprint" for fibrosing autoimmune disease. They are useful to understand the pathogenesis of Scl GVHD, to identify markers for early diagnosis of disease, and to devise more effective strategies for intervention in early scleroderma and Scl GVHD.

Animals↗

PubMed2006

Human embryonic stem cells have a unique epigenetic signature.

Human embryonic stem (hES) cells originate during an embryonic period of active epigenetic remodeling. DNA methylation patterns are likely to be critical for their self-renewal and pluripotence. We compared the DNA methylation status of 1536 CpG sites (from 371 genes) in 14 independently isolated hES cell lines with five other cell types: 24 cancer cell lines, four adult stem cell populations, four lymphoblastoid cell lines, five normal human tissues, and an embryonal carcinoma cell line. We found that the DNA methylation profile clearly distinguished the hES cells from all of the other cell types. A subset of 49 CpG sites from 40 genes contributed most to the differences among cell types. Another set of 25 sites from 23 genes distinguished hES cells from normal differentiated cells and can be used as biomarkers to monitor differentiation. Our results indicate that hES cells have a unique epigenetic signature that may contribute to their developmental potential.

Cell Differentiation↗

PubMed2006

Two-dimensional transcriptome profiling: identification of messenger RNA isoform signatures in prostate cancer from archived paraffin-embedded cancer specimens.

The expression of specific mRNA isoforms may uniquely reflect the biological state of a cell because it reflects the integrated outcome of both transcriptional and posttranscriptional regulation. In this study, we constructed a splicing array to examine approximately 1,500 mRNA isoforms from a panel of genes previously implicated in prostate cancer and identified a large number of cell type-specific mRNA isoforms. We also developed a novel "two-dimensional" profiling strategy to simultaneously quantify changes in splicing and transcript abundance; the results revealed extensive covariation between transcription and splicing in prostate cancer cells. Taking advantage of the ability of our technology to analyze RNA from formalin-fixed, paraffin-embedded tissues, we derived a specific set of mRNA isoform biomarkers for prostate cancer using independent panels of tissue samples for feature selection and cross-analysis. A number of cancer-specific splicing switch events were further validated by laser capture microdissection. Quantitative changes in transcription/RNA stability and qualitative differences in splicing ratio may thus be combined to characterize tumorigenic programs and signature mRNA isoforms may serve as unique biomarkers for tumor diagnosis and prognosis.

Aged↗

PubMed2006

High-throughput DNA methylation profiling using universal bead arrays.

We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

Base Sequence↗

PubMed2006

Whole-genome genotyping.

We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of interest are captured by specific hybridization of amplified gDNA to 50-mer probe arrays. After target capture, SNPs are genotyped on the array by a primer extension reaction in the presence of hapten-labeled nucleotides. The resultant signal is amplified during staining and the array is read out on a high-resolution confocal scanner. We have employed our high-density BeadChips supporting up to 288,000 bead types to create an array that can query over 100,000 SNPs using the Infinium assay. In addition, we have developed an automated BeadChip processing platform using Tecan's GenePaint slide processing system. Hybridization, washing, array-based primer extension, and staining are performed directly in Tecan's capillary gap Te-Flow chambers. This automation process increases assay robustness and throughput greatly while enabling laboratory information management system control of sample tracking.

Animals↗

PubMed2006

Transduced monocyte/macrophages targeted to murine skin by UV light.

We have selectively targeted monocyte/macrophages overexpressing an immunomodulatory molecule, latency-associated peptide (LAP), a naturally occurring antagonist for transforming growth factor-beta1, to murine skin utilizing UV light to produce a cutaneous influx of transduced monocyte/macrophages. Bone marrow (BM) cells from BALB/c mice were transduced in vitro with a retroviral construct containing green fluorescent protein (GFP) for detection and human LAP (hLAP) as a test molecule. The transduced BM cells were then cultured in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) to produce differentiation to monocyte/macrophages. More than 80% of transduced BM cells were CD11b-positive and MOMA-positive by fluorescence-activated cell-sorter analysis and secreted LAP by ELISA after 10 days of culture in granulocyte-macrophage colony-stimulating factor (GM-CSF). Transduced monocyte/macrophages containing either GFP or hLAP-GFP were then injected intravenously into BALB/c mice. One-half of recipients in each group were exposed to UVB (72 mJ) to induce monocyte/macrophage infiltration into skin. Recipients were sacrificed 60 h after UV irradiation. We found transduced cutaneous macrophages expressing GFP by examining with fluorescence microscopy frozen skin sections of recipient mice immunostained with antibodies to GFP and to macrophage marker F4/80. We identified hLAP sequences by polymerase chain reaction (PCR) of total DNA in recipient blood and UV-irradiated skin but not in unirradiated skin. LAP sequences were also detected at much lower levels in other organs (lung, spleen, and liver) by PCR. Therefore, we have shown that genetically altered monocytic cells can be injected intravenously and targeted to mouse skin by UV exposure. This macrophage-based gene-transfer method may be a potentially useful immunotherapeutic approach for delivering monocyte/macrophage-derived products to skin.

Animals↗

PubMed2004

ERR-10: a new repressor in transcriptional signaling activation of estrogen receptor-alpha.

Estrogen receptor-alpha (ER-alpha) is a nuclear transcriptional factor that is part of the nuclear receptor superfamily. In this study, we isolated and identified a new LXXLL-containing protein that interacts with the ER-alpha via a yeast two-hybrid assay. We have termed this protein estrogen receptor repressor-10 (ERR-10). The ERR-10 cDNA is predicted to encode a polypeptide of 94 amino acids, with a molecular mass of about 10 kDa. Although the ERR-10 mRNA transcript is expressed in a wide range of normal human tissues, higher expression levels are found in endocrinal tissues relative to other tissues. We have demonstrated, through immunoprecipitation, Western blot and GST pull-down assays, that ERR-10 associates with ER-alpha. Moreover, ERR-10 decreased 17beta-estrodial-induced activation of ER-alpha transcriptional activity in transient transfection assays of mammalian cells. The ERR-10 N-terminus, which resembles two LXXLL motifs, is essential for ER-alpha binding and repression activity. Estrogen modulation of estrogen-responsive gene expression was markedly blocked by ERR-10. These results suggest that ERR-10 is a novel mediator in ER transcriptional activation.

Amino Acid Motifs↗

PubMed2004

Decoding randomly ordered DNA arrays.

We have developed a simple and efficient algorithm to identify each member of a large collection of DNA-linked objects through the use of hybridization, and have applied it to the manufacture of randomly assembled arrays of beads in wells. Once the algorithm has been used to determine the identity of each bead, the microarray can be used in a wide variety of applications, including single nucleotide polymorphism genotyping and gene expression profiling. The algorithm requires only a few labels and several sequential hybridizations to identify thousands of different DNA sequences with great accuracy. We have decoded tens of thousands of arrays, each with 1520 sequences represented at approximately 30-fold redundancy by up to approximately 50,000 beads, with a median error rate of <1 x 10(-4) per bead. The approach makes use of error checking codes and provides, for the first time, a direct functional quality control of every element of each array that is manufactured. The algorithm can be applied to any spatially fixed collection of objects or molecules that are associated with specific DNA sequences.

Algorithms↗