Electrospinning of ultra-thin polymer fibers

The electrospinning technique was used to spin ultra-thin fibers from several polymer/solvent systems. The diameter of the electrospun fibers ranged from 16 nm to 2 μm. The morphology of these fibers was investigated with an atomic force microscope (AFM) and an optical microscope. Polyethylene oxide) (PEO) dissolved in water or chloroform was studied in greater detail. PEO fibers spun from aqueous solution show a “beads on a string” morphology. An AFM study showed that the surface of these fibers is highly ordered. The “beads on a string” morphology can be avoided if PEO is spun from solution in chloroform; the resulting fibers show a lamellar morphology. Polyvinylalcohol (PVA) dissolved in water and cellulose acetate dissolved in acetone were additional polymer/solvent systems which were investigated. Furthermore, the electrospinning process was studied: different experimental lay-outs were tested, electrostatic fields were simulated, and voltage - current characteristics of the electrospinning process were recorded

Sema3E/Plexin-D1 Mediated Epithelial-to-Mesenchymal Transition in Ovarian Endometrioid Cancer

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration

Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

BACKGROUND:The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS:Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines"), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE:Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation

SLUG/SNAI2 and Tumor Necrosis Factor Generate Breast Cells With CD44+/CD24- Phenotype

<p>Abstract</p> <p>Background</p> <p>Breast cancer cells with CD44+/CD24- cell surface marker expression profile are proposed as cancer stem cells (CSCs). Normal breast epithelial cells that are CD44+/CD24- express higher levels of stem/progenitor cell associated genes. We, amongst others, have shown that cancer cells that have undergone epithelial to mesenchymal transition (EMT) display the CD44+/CD24- phenotype. However, whether all genes that induce EMT confer the CD44+/CD24- phenotype is unknown. We hypothesized that only a subset of genes associated with EMT generates CD44+/CD24- cells.</p> <p>Methods</p> <p>MCF-10A breast epithelial cells, a subpopulation of which spontaneously acquire the CD44+/CD24- phenotype, were used to identify genes that are differentially expressed in CD44+/CD24- and CD44-/CD24+ cells. Ingenuity pathway analysis was performed to identify signaling networks that linked differentially expressed genes. Two EMT-associated genes elevated in CD44+/CD24- cells, SLUG and Gli-2, were overexpressed in the CD44-/CD24+ subpopulation of MCF-10A cells and MCF-7 cells, which are CD44-/CD24+. Flow cytometry and mammosphere assays were used to assess cell surface markers and stem cell-like properties, respectively.</p> <p>Results</p> <p>Two thousand thirty five genes were differentially expressed (p < 0.001, fold change ≥ 2) between the CD44+/CD24- and CD44-/CD24+ subpopulations of MCF-10A. Thirty-two EMT-associated genes including SLUG, Gli-2, ZEB-1, and ZEB-2 were expressed at higher levels in CD44+/CD24- cells. These EMT-associated genes participate in signaling networks comprising TGFβ, NF-κB, and human chorionic gonadotropin. Treatment with tumor necrosis factor (TNF), which induces NF-κB and represses E-cadherin, or overexpression of SLUG in CD44-/CD24+ MCF-10A cells, gave rise to a subpopulation of CD44+/CD24- cells. Overexpression of constitutively active p65 subunit of NF-κB in MCF-10A resulted in a dramatic shift to the CD44+/CD24+ phenotype. SLUG overexpression in MCF-7 cells generated CD44+/CD24+ cells with enhanced mammosphere forming ability. In contrast, Gli-2 failed to alter CD44 and CD24 expression.</p> <p>Conclusions</p> <p>EMT-mediated generation of CD44+/CD24- or CD44+/CD24+ cells depends on the genes that induce or are associated with EMT. Our studies reveal a role for TNF in altering the phenotype of breast CSC. Additionally, the CD44+/CD24+ phenotype, in the context of SLUG overexpression, can be associated with breast CSC "stemness" behavior based on mammosphere forming ability.</p

The Liberation of Embryonic Stem Cells

Mouse embryonic stem (ES) cells are defined by their capacity to self-renew and their ability to differentiate into all adult tissues including the germ line. Along with efficient clonal propagation, these properties have made them an unparalleled tool for manipulation of the mouse genome. Traditionally, mouse ES (mES) cells have been isolated and cultured in complex, poorly defined conditions that only permit efficient derivation from the 129 mouse strain; genuine ES cells have not been isolated from another species in these conditions. Recently, use of small molecule inhibitors of glycogen synthase kinase 3 (Gsk3) and the Fgf-MAPK signaling cascade has permitted efficient derivation of ES cells from all tested mouse strains. Subsequently, the first verified ES cells were established from a non-mouse species, Rattus norvegicus. Here, we summarize the advances in our understanding of the signaling pathways regulating mES cell self-renewal that led to the first derivation of rat ES cells and highlight the new opportunities presented for transgenic modeling on diverse genetic backgrounds. We also comment on the implications of this work for our understanding of pluripotent stem cells across mammalian species

PAI-1 and functional blockade of SNAI1 in breast cancer cell migration

12 pages, 5 figures.-- PMID: 19055748 [PubMed].-- et al.[Introduction]: Snail, a family of transcriptional repressors implicated in cell movement, has been correlated with tumour invasion. The Plasminogen Activation (PA) system, including urokinase plasminogen activator (uPA), its receptor and its inhibitor, plasminogen activator inhibitor type 1(PAI-1), also plays a key role in cancer invasion and metastasis, either through proteolytic degradation or by non-proteolytic modulation of cell adhesion and migration. Thus, Snail and the PA system are both over-expressed in cancer and influence this process. In this study we aimed to determine if the activity of SNAI1 (a member of the Snail family) is correlated with expression of the PA system components and how this correlation can influence tumoural cell migration.[Methods]: We compared the invasive breast cancer cell-line MDA-MB-231 expressing SNAI1 (MDA-mock) with its derived clone expressing a dominant-negative form of SNAI1 (SNAI1-DN). Expression of PA system mRNAs was analysed by cDNA microarrays and real-time quantitative RT-PCR. Wound healing assays were used to determine cell migration. PAI-1 distribution was assessed by immunostaining.[Results]: We demonstrated by both cDNA microarrays and realtime quantitative RT-PCR that the functional blockade of SNAI1 induces a significant decrease of PAI-1 and uPA transcripts. After performing an in vitro wound-healing assay, we observed that SNAI1-DN cells migrate more slowly than MDA-mock cells and in a more collective manner. The blockade of SNAI1 activity resulted in the redistribution of PAI-1 in SNAI1-DN cells decorating large lamellipodia, which are commonly found structures in these cells.[Conclusions]: In the absence of functional SNAI1, the expression of PAI-1 transcripts is decreased, although the protein is redistributed at the leading edge of migrating cells in a manner comparable with that seen in normal epithelial cells.This work was supported by the CNRS ACI Program "Complexité du vivant" (grant # 050009DR11) and by the Evry Genopole grant "Aide à l'acquisition d'équipement semi-lourd" 2007 and 2008.Peer reviewe

Acute mountain sickness.

Acute mountain sickness (AMS) is a clinical syndrome occurring in otherwise healthy normal individuals who ascend rapidly to high altitude. Symptoms develop over a period ofa few hours or days. The usual symptoms include headache, anorexia, nausea, vomiting, lethargy, unsteadiness of gait, undue dyspnoea on moderate exertion and interrupted sleep. AMS is unrelated to physical fitness, sex or age except that young children over two years of age are unduly susceptible. One of the striking features ofAMS is the wide variation in individual susceptibility which is to some extent consistent. Some subjects never experience symptoms at any altitude while others have repeated attacks on ascending to quite modest altitudes. Rapid ascent to altitudes of 2500 to 3000m will produce symptoms in some subjects while after ascent over 23 days to 5000m most subjects will be affected, some to a marked degree. In general, the more rapid the ascent, the higher the altitude reached and the greater the physical exertion involved, the more severe AMS will be. Ifthe subjects stay at the altitude reached there is a tendency for acclimatization to occur and symptoms to remit over 1-7 days

Epithelial-Mesenchymal Transition in Cancer: Parallels Between Normal Development and Tumor Progression

From the earliest stages of embryonic development, cells of epithelial and mesenchymal origin contribute to the structure and function of developing organs. However, these phenotypes are not always permanent, and instead, under the appropriate conditions, epithelial and mesenchymal cells convert between these two phenotypes. These processes, termed Epithelial-Mesenchymal Transition (EMT), or the reverse Mesenchymal-Epithelial Transition (MET), are required for complex body patterning and morphogenesis. In addition, epithelial plasticity and the acquisition of invasive properties without the full commitment to a mesenchymal phenotype are critical in development, particularly during branching morphogenesis in the mammary gland. Recent work in cancer has identified an analogous plasticity of cellular phenotypes whereby epithelial cancer cells acquire mesenchymal features that permit escape from the primary tumor. Because local invasion is thought to be a necessary first step in metastatic dissemination, EMT and epithelial plasticity are hypothesized to contribute to tumor progression. Similarities between developmental and oncogenic EMT have led to the identification of common contributing pathways, suggesting that the reactivation of developmental pathways in breast and other cancers contributes to tumor progression. For example, developmental EMT regulators including Snail/Slug, Twist, Six1, and Cripto, along with developmental signaling pathways including TGF-β and Wnt/β-catenin, are misexpressed in breast cancer and correlate with poor clinical outcomes. This review focuses on the parallels between epithelial plasticity/EMT in the mammary gland and other organs during development, and on a selection of developmental EMT regulators that are misexpressed specifically during breast cancer

An NF-κB and Slug Regulatory Loop Active in Early Vertebrate Mesoderm

BACKGROUND: In both Drosophila and the mouse, the zinc finger transcription factor Snail is required for mesoderm formation; its vertebrate paralog Slug (Snai2) appears to be required for neural crest formation in the chick and the clawed frog Xenopus laevis. Both Slug and Snail act to induce epithelial to mesenchymal transition (EMT) and to suppress apoptosis. METHODOLOGY & PRINCIPLE FINDINGS: Morpholino-based loss of function studies indicate that Slug is required for the normal expression of both mesodermal and neural crest markers in X. laevis. Both phenotypes are rescued by injection of RNA encoding the anti-apoptotic protein Bcl-xL; Bcl-xL's effects are dependent upon IκB kinase-mediated activation of the bipartite transcription factor NF-κB. NF-κB, in turn, directly up-regulates levels of Slug and Snail RNAs. Slug indirectly up-regulates levels of RNAs encoding the NF-κB subunit proteins RelA, Rel2, and Rel3, and directly down-regulates levels of the pro-apopotic Caspase-9 RNA. CONCLUSIONS/SIGNIFICANCE: These studies reveal a Slug/Snail–NF-κB regulatory circuit, analogous to that present in the early Drosophila embryo, active during mesodermal formation in Xenopus. This is a regulatory interaction of significance both in development and in the course of inflammatory and metastatic disease

Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity

The epithelial-to-mesenchymal transition (EMT) is a critical developmental process that has recently come to the forefront of cancer biology. In breast carcinomas, acquisition of a mesenchymal-like phenotype that is reminiscent of an EMT, termed oncogenic EMT, is associated with pro-metastatic properties, including increased motility, invasion, anoikis resistance, immunosuppression and cancer stem cell characteristics. This oncogenic EMT is a consequence of cellular plasticity, which allows for interconversion between epithelial and mesenchymal-like states, and is thought to enable tumor cells not only to escape from the primary tumor, but also to colonize a secondary site. Indeed, the plasticity of cancer cells may explain the range of pro-metastatic traits conferred by oncogenic EMT, such as the recently described link between EMT and cancer stem cells and/or therapeutic resistance. Continued research into this relationship will be critical in developing drugs that block mechanisms of breast cancer progression, ultimately improving patient outcomes