A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research

Endogenous TNF alpha orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

This work was supported by funds from Arthritis Research UK (19913 to M.-B.V.) and the Wellcome Trust (098291/Z/12/Z to S.N.). S. A. was supported by a QMUL Principal’s Award PhD Studentship

Genetic and epigenetic changes in the common 1p36 deletion in neuroblastoma tumours

Chromosome 1p is frequently deleted in neuroblastoma (NB) tumours. The commonly deleted region has been narrowed down by loss of heterozygosity studies undertaken by different groups. Based on earlier mapping data, we have focused on a region on 1p36 (chr1: 7 765 595–11 019 814) and performed an analysis of 30 genes by exploring features such as epigenetic regulation, that is DNA methylation and histone deacetylation, mutations at the DNA level and mRNA expression. Treatment of NB cell lines with the histone deacetylase inhibitor trichostatin A led to increased gene transcription of four of the 30 genes, ERRFI1 (MIG-6), PIK3CD, RBP7 (CRBPIV) and CASZ1, indicating that these genes could be affected by epigenetic downregulation in NBs. Two patients with nonsynonymous mutations in the PIK3CD gene were detected. One patient harboured three variations in the same exon, and p.R188W. The other patient had the variation p.M655I. In addition, synonymous variations and one variation in an intronic sequence were also found. The mRNA expression of this gene is downregulated in unfavourable, compared to favourable, NBs. One nonsynonymous mutation was also identified in the ERRFI1 gene, p.N343S, and one synonymous. None of the variations above were found in healthy control individuals. In conclusion, of the 30 genes analysed, the PIK3CD gene stands out as one of the most interesting for further studies of NB development and progression

HLA-DR+ leukocytes acquire CD1 antigens in embryonic and fetal human skin and contain functional antigen-presenting cells

Adequate numbers and functional maturity are needed for leukocytes to exhibit a protective role in host defense. During intrauterine life, the skin immune system has to acquire these prerequisites to protect the newborn from infection in the hostile external environment after birth. We investigated the quantitative, phenotypic, and functional development of skin leukocytes and analyzed the factors controlling their proliferation and trafficking during skin development. We show that CD45+ leukocytes are scattered in embryonic human skin and that their numbers continuously increase as the developing skin generates an environment that promotes proliferation of skin resident leukocytes as well as the influx of leukocytes from the circulation. We also found that CD45+HLA-DRhighCD1c+ dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor β1 production precedes Langerin and CD1a expression on CD45+CD1c+ Langerhans cell (LC) precursors. Functionally, embryonic antigen-presenting cells (APCs) are able to phagocytose antigen, to up-regulate costimulatory molecules upon culture, and to efficiently stimulate T cells in a mixed lymphocyte reaction. Collectively, our data provide insight into skin DC biology and the mechanisms through which skin DCs presumably populate the skin during development

Aberrant Expression of Proteins Involved in Signal Transduction and DNA Repair Pathways in Lung Cancer and Their Association with Clinical Parameters

Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues.We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA) assay. The results showed that 18 molecules were significantly different (p<0.05) by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery.Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers

Myoferlin Depletion in Breast Cancer Cells Promotes Mesenchymal to Epithelial Shape Change and Stalls Invasion

Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET). These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin) and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNAMYOF transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF

Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner

Differences in the pattern and regulation of mineral deposition in human cell lines of osteogenic and non-osteogenic origin

Bone marrow-derived mesenchymal stem cells (MSCs) are widely used as a cellular model of bone formation, and can mineralize in vitro in response to osteogenic medium (OM). It is unclear, however, whether this property is specific to cells of mesenchymal origin. We analysed the OM response in 3 non-osteogenic lines, HEK293, HeLa and NTera, compared to MSCs. Whereas HEK293 cells failed to respond to OM conditions, the 2 carcinoma-derived lines NTera and HeLa deposited a calcium phosphate mineral comparable to that present in MSC cultures. However, unlike MSCs, HeLa and NTera cultures did so in the absence of dexamethasone. This discrepancy was confirmed, as bone morphogenetic protein inhibition obliterated the OM response in MSCs but not in HeLa or NTera, indicating that these 2 models can deposit mineral through a mechanism independent of established dexamethasone or bone morphogenetic protein signalling

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

International audienceBACKGROUND: Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis. METHOLOGY/PRINCIPAL FINDINGS: We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21-66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process. CONCLUSIONS/SIGNIFICANCE: Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis