Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology

Anterior knee pain from the evolutionary perspective

Background This paper describes the evolutionary changes in morphology and orientation of the PFJ using species present through our ancestry over 340 million years. Methods 37 specimens from the Devonian period to modern day were scanned using a 64-slice CT scanner. 3D geometries were created following routine segmentation and anatomical measurements taken from standardised bony landmarks. Results Findings are described according to gait strategy and age. The adoption of an upright bi-pedal stance caused a dramatic change in the loading of the PFJ which has subsequently led to changes in the arrangement of the PFJ. From Devonian to Miocene periods, our sprawling and climbing ancestors possessed a broad knee with a shallow, centrally located trochlea. A more rounded knee was present from the Paleolithic period onwards in erect and bipedal gait types (aspect ratio 0.93 vs 1.2 in late Devonian), with the PFJ being placed lateral to the midline compared to the medial position in quadrapeds. The depth of the trochlea groove was maximal in the Miocene period of the African ground apes with associated acute sulcus angles in Gorilla (117°) becoming more flattened towards the modern human (138°). Conclusions The evolving bipedal gait lead to anteriorisation of the patellofemoral joint, flattening of the trochlea sulcus, in a more lateral, dislocation prone arrangement. Ancestral developments might help explain the variety of presentations of anterior knee pain and patellofemoral instability

The Ursinus Weekly, April 29, 1976

Board announces promotions; Six granted tenure • S.F.A.R.C. discusses calendar reform • Egyptian Ambassador speaks on Middle East • Cub and Key holds reunion • Ec. Club meets • Whitians elect new members • U.C. professors attend national conferences • Editorial: Stewing students ask what\u27s cooking? • Letter to the editor: Correction on discussion of birth control policy • Wyeth show at Museum • Pi Gamma Mu accepts 22 students • Book Store robbery • Forum review: The Israeli stand • Mattress fun • U.S.D.A. open house • Zacharias\u27 inferno • Travelin\u27 9 delayed • Golfers swing • Track races • Baseball: Phillies • Baseball: Ursinus season report • ETS tests itself • Comparing college tuitions • PCO sponsors Vision Centerhttps://digitalcommons.ursinus.edu/weekly/1055/thumbnail.jp

Giant growth rate in nano-oxidation of p-silicon surfaces by using ethyl alcohol liquid bridges

We demonstrate that local oxidation nanolithography can be performed in liquid environments different from aqueous solutions with a significant improvement in the aspect ratio of the fabricated motives. Here, we perform a comparative study of noncontact atomic force microscopy oxidation experiments in water and ethyl alcohol. The growth rate of local oxides can be increased by almost an order of magnitude by using oxyanions from ethyl alcohol molecules. We propose that the enhanced growth rate is a consequence of the reduction of the trapped charges within the growing oxide. The present results open the possibility of using local oxidation nanolithography to directly fabricate vertical oxide structures while keeping lateral sizes in the nanometer range.This work was supported by the European Commission (MONA-LISA, G5RD-2000-00349).Peer reviewe

Aspirin Inhibits TGFβ2-Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells:Selective acetylation of K56 and K122 in histone H3

Posterior capsule opacification (PCO) is a complication after cataract surgery that can disrupt vision. The epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) in response to transforming growth factor β2 (TGFβ2) has been considered an obligatory mechanism for PCO. In this study, we tested the efficacy of aspirin in inhibiting the TGFβ2-mediated EMT of human LECs, LECs in human lens capsular bags, and lensectomized mice. In human LECs, the levels of the EMT markers α-smooth muscle actin (α-SMA) and fibronectin were drastically reduced by treatment with 2 mM aspirin. Aspirin also halted the EMT response of TGFβ2 when introduced after EMT initiation. In human capsular bags, treatment with 2 mM aspirin significantly suppressed posterior capsule wrinkling and the expression α-SMA in capsule-adherent LECs. The inhibition of TGFβ2-mediated EMT in human LECs was not dependent on Smad phosphorylation or MAPK and AKT-mediated signaling. We found that aspirin significantly increased the acetylation of K56 and K122 in histone H3 of human LECs. Chromatin immunoprecipitation assays using acetyl-H3K56 or acetyl-H3K122 antibody revealed that aspirin blocked the TGFβ2-induced acetylation of H3K56 and H3K122 at the promoter regions of ACTA2 and COL1A1. After lensectomy in mice, we observed an increase in the proliferation and α-SMA expression of the capsule-adherent LECs, which was ameliorated by aspirin administration through drinking water. Taken together, our results showed that aspirin inhibits TGFβ2-mediated EMT of LECs, possibly from epigenetic down-regulation of EMT-related genes

Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition

Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFbeta signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFbeta and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression

Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition

Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFbeta signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFbeta and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression

NGC 4314. IV. Photometry of Star Clusters with Hubble Space Telescope - History of Star Formation in the Vicinity of a Nuclear Ring

Using HST WFPC2 images, we have obtained U, B, V, I, and H-alpha photometry for 76 star clusters in the nuclear star-forming ring of the barred spiral galaxy NGC 4314. These clusters are likely associated with an inner Inner Lindblad Resonance, or IILR. The blue colors and H-alpha emission for most of these clusters imply very young ages of 1-15 Myr. Age estimates based on several reddening-free parameters indicate that the present epoch of star formation has lasted at least 30 Myr. By estimating the masses of stars in the clusters and comparing with the H-alpha luminosity, we conclude that a significant fraction of ongoing star formation in the nuclear ring of NGC 4314 occurs in clusters. The cluster masses identify these as young open clusters, not young globular clusters. Further out in the galaxy, just exterior to the ring of young stars, previous ground-based observations revealed two symmetric stellar spiral arms which may be associated with an outer Inner Lindblad Resonance, or OILR. With our HST data, we have revealed part of this structure and its colors in more detail. The spiral arm colors are consistent with stellar ages between 40 and 200 Myr. The age difference between the inner ring of young stars (IILR) and the larger oval-like feature containing the blue arms (OILR) supports an interpretation of the morphology of the nuclear region of NGC 4314 that requires a reservoir of gas that becomes more compact over time. We speculate that as the gas distribution becomes more centrally concentrated, it interacts with these two resonances. Each resonance triggers star formation, resulting in two distinct epochs of star formation.Comment: To appear in The Astronomical Journal, March 2002. For a version with higher quality figures, see http://clyde.as.utexas.edu/pub/galaxy/N4314NEW/AJPAPER/BenedictR7.ps.g

SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization