Development of detection techniques and diagnostics for airborne carbon nanoparticles.

We have recorded time-resolved LII signals from a laminar ethylene diffusion flame over a wide range of laser fluences at 532 nm. We have performed these experiments using an injection-seeded NdYAG laser with a pulse duration of 7 ns. The beam was spatially filtered and imaged into the flame to provide a homogeneous spatial profile. These data were used to aid in the development of a model, which will be used to test the validity of the LII technique under varying environmental conditions. The new model describes the heating of soot particles during the laser pulse and the subsequent cooling of the particles by radiative emission, sublimation, and conduction. The model additionally includes particle heating by oxidation, accounts for the likelihood of particle annealing, and incorporates a mechanism for nonthermal photodesorption, which is required for good agreement with our experimental results. In order to investigate the fast photodesorption mechanism in more detail, we have recorded LII temporal profiles using a regeneratively amplified Nd:YAG laser with a pulse duration of 70 ps to heat the particles and a streak camera with a temporal resolution of {approx}65 ps to collect the signal. Preliminary results confirm earlier indications of a fast mechanism leading to signal decay rates of much less than a nanosecond. Parameters to which the model is sensitive include the initial soot temperature, the temperature of the ambient gas, and the partial pressure of oxygen. In order to narrow the model uncertainties, we have developed a source of soot that allows us to determine and control these parameters. Soot produced by a burner is extracted, diluted, and cooled in a flow tube, which is equipped with a Scanning Mobility Particle Sizer (SMPS) for characterization of the aggregates

Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration

Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes [preprint]

Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery

WNT signaling enhances breast cancer cell motility and blockade of the WNT pathway by sFRP1 suppresses MDA-MB-231 xenograft growth

ABSTRACT: INTRODUCTION: In breast cancer deregulation of the WNT signaling pathway occurs by autocrine mechanisms. WNT ligands and Frizzled (FZD) receptors are coexpressed in primary breast tumors and cancer cell lines. Moreover, many breast tumors show hypermethylation of secreted Frizzled-related protein 1 (sFRP1)'s promoter region, causing low expression of this WNT antagonist. We have previously shown that the WNT pathway influences proliferation of breast cancer cell lines via activation of canonical signaling and epidermal growth factor receptor (EGFR) transactivation, and that interference with WNT signaling reduces proliferation. Here we examine the role of WNT signaling in breast tumor cell migration and on xenograft outgrowth. METHODS: The breast cancer cell line MDA-MB-231 was used to study WNT signaling. We examined the effects of activating or blocking the WNT pathway on cell motility by treatment with WNT ligands or by ectopic sFPR1 expression, respectively. The ability of sFRP1 expressing MDA-MB-231 cells to grow as xenografts was also tested. Microarray analyses were carried out to identify targets with roles in MDA-MB-231/sFRP1 tumor growth inhibition. RESULTS: We show that WNT stimulates the migratory ability of MDA-MB-231 cells. Furthermore, ectopic expression of sFRP1 in MDA-MB-231 cells blocks canonical WNT signaling and decreases their migratory potential. Moreover, the ability of MDA-MB-231/sFRP1 expressing cells to grow as xenografts in mammary glands and to form lung metastases is dramatically impaired. Microarray analyses led to the identification of two genes, CCND1 and CDKN1A, whose expression level is selectively altered in vivo in sFRP1 expressing tumors. The encoded proteins, Cyclin D1 and p21Cip1 were down- and up-regulated, respectively, in sFRP1 expressing tumors, suggesting that they are downstream mediators of WNT signaling. CONCLUSIONS: Our results show that the WNT pathway influences multiple biological properties of MDA-MB-231 breast cancer cells. WNT stimulates tumor cell motility; conversely sFRP1 mediated WNT pathway blockade reduces motility. Moreover, ectopic sFRP1 expression in MDA-MB-231 cells has a strong negative impact on tumor outgrowth and blocked lung metastases. These results suggest that interference with WNT signaling using sFRP1 to block the ligand-receptor interaction may be a valid therapeutic approach in breast cancer

Dvl2-Dependent Activation of Daam1 and RhoA Regulates Wnt5a-Induced Breast Cancer Cell Migration

The Dishevelled (Dvl) and Dishevelled-associated activator of morphogenesis 1 (Daam1) pathway triggered by Wnt5a regulates cellular polarity during development and tissue homoeostasis. However, Wnt5a signaling in breast cancer progression remains poorly defined.We showed here that Wnt5a activated Dvl2, Daam1 and RhoA, and promoted migration of breast cancer cells, which was, however, abolished by Secreted Frizzled-related protein 2 (sFRP2) pretreatment. Dominant negative Dvl2 mutants or Dvl2 siRNA significantly decreased Wnt5a-induced Daam1/RhoA activation and cell migration. Ectopic expression of N-Daam1, a dominant negative mutant, or Daam1 siRNA remarkably inhibited Wnt5a-induced RhoA activation, stress fiber formation and cell migration. Ectopic expression of dominant negative RhoA (N19) or C3 exoenzyme transferase, a Rho inhibitor, decreased Wnt5a-induced stress fiber formation and cell migration.Taken together, we demonstrated for the first time that Wnt5a promotes breast cancer cell migration via Dvl2/Daam1/RhoA

Ground Water Chemistry Changes before Major Earthquakes and Possible Effects on Animals

Prior to major earthquakes many changes in the environment have been documented. Though often subtle and fleeting, these changes are noticeable at the land surface, in water, in the air, and in the ionosphere. Key to understanding these diverse pre-earthquake phenomena has been the discovery that, when tectonic stresses build up in the Earth’s crust, highly mobile electronic charge carriers are activated. These charge carriers are defect electrons on the oxygen anion sublattice of silicate minerals, known as positive holes, chemically equivalent to O− in a matrix of O2−. They are remarkable inasmuch as they can flow out of the stressed rock volume and spread into the surrounding unstressed rocks. Travelling fast and far the positive holes cause a range of follow-on reactions when they arrive at the Earth’s surface, where they cause air ionization, injecting massive amounts of primarily positive air ions into the lower atmosphere. When they arrive at the rock-water interface, they act as •O radicals, oxidizing water to hydrogen peroxide. Other reactions at the rock-water interface include the oxidation or partial oxidation of dissolved organic compounds, leading to changes of their fluorescence spectra. Some compounds thus formed may be irritants or toxins to certain species of animals. Common toads, Bufo bufo, were observed to exhibit a highly unusual behavior prior to a M6.3 earthquake that hit L’Aquila, Italy, on April 06, 2009: a few days before the seismic event the toads suddenly disappeared from their breeding site in a small lake about 75 km from the epicenter and did not return until after the aftershock series. In this paper we discuss potential changes in groundwater chemistry prior to seismic events and their possible effects on animals

Ror2 Enhances Polarity and Directional Migration of Primordial Germ Cells

The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell

CD146 expression is associated with a poor prognosis in human breast tumors and with enhanced motility in breast cancer cell lines.

INTRODUCTION: Metastasis is a complex process involving loss of adhesion, migration, invasion and proliferation of cancer cells. Cell adhesion molecules play a pivotal role in this phenomenon by regulating cell-cell and cell-matrix interactions. CD146 (MCAM) is associated with an advanced tumor stage in melanoma, prostate cancer and ovarian cancer. Studies of CD146 expression and function in breast cancer remain scarce except for a report concluding that CD146 could act as a tumor suppressor in breast carcinogenesis. METHODS: To resolve these apparent discrepancies in the role of CD146 in tumor cells, we looked at the association of CD146 expression with histoclinical features in human primary breast cancers using DNA and tissue microarrays. By flow cytometry, we characterized CD146 expression on different breast cancer cell lines. Using siRNA or shRNA technology, we studied functional consequences of CD146 downmodulation of MDA-MB-231 cells in migration assays. Wild-type, mock-transfected and downmodulated transfected cells were profiled using whole-genome DNA microarrays to identify genes whose expression was modified by CD146 downregulation. RESULTS: Microarray studies revealed the association of higher levels of CD146 with histoclinical features that belong to the basal cluster of human tumors. Expression of CD146 protein on epithelial cells was detected in a small subset of cancers with histoclinical features of basal tumors. CD146+ cell lines displayed a mesenchymal phenotype. Downmodulation of CD146 expression in the MDA-MB-231 cell line resulted in downmodulation of vimentin, as well as of a set of genes that include both genes associated with a poor prognosis in a variety of cancers and genes known to promote cell motility. In vitro functional assays revealed decreased migration abilities associated with decreased CD146 expression. CONCLUSIONS: In addition to its expression in the vascular compartment, CD146 is expressed on a subset of epithelial cells in malignant breast. CD146 may directly or indirectly contribute to tumor aggressiveness by promoting malignant cell motility. Changes in molecular signatures following downmodulation of CD146 expression suggest that CD146 downmodulation is associated with the reversal of several biological characteristics associated with epithelial to mesenchymal transition, and the phenomenon associated with the metastatic process.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are