“Targeting particular mRNAs for degradation is a fascinating approach to achieve gene silencing. Here we describe a new gene silencing tool exploiting a cell-penetrating, nucleic-acid hydrolyzing, single-domain antibody of the light-chain variable domain, 3D8 VL. We generated a synthetic library of 3D8 VL on the yeast surface by randomizing residues located in one of two beta-sheets. Using 18-bp single-stranded
nucleic acids as target substrates, including the human Her2/neu-targeting sequence, we selected 3D8 VL variants that had similar to 100-1000-fold Dibutyryl-cAMP higher affinity and similar to 2-5-fold greater selective hydrolyzing activity for target substrates than for off targets. 3D8 VL variants efficiently penetrated into living cells to be accumulated in the cytosol and selectively decreased the amount of target sequence-carrying mRNAs as well as the proteins encoded
by these mRNAs with minimal effects on off-target genes. In particular, one 3D8 VL variant targeting the Her2 sequence showed Crenigacestat clinical trial more efficient downregulation of Her2 expression than a small-interfering RNA targeting the same Her2 sequence, resulting in apoptotic cell death of Her2-overexpressing breast cancer cells. Our results demonstrate that cell-penetrating 3D8 VL variants with sequence-selective, nucleic-acid-hydrolyzing activity can selectively degrade target mRNAs in the cytosol, providing a new gene silencing tool mediated by antibody.”
“Objective-To determine diagnostic accuracy of using erythrocyte indices and polychromasia to identify regenerative anemia in dogs.\n\nDesign-Retrospective and prospective cross-sectional study.\n\nAnimals-4,521 anemic dogs.\n\nProcedures-CBC results
obtained between July 2002 and July 2008 by use of an automated laser-based flow cytometric hematology analyzer from dogs with Hct values <= 35% were retrieved. Sensitivity, specificity, accuracy, and predictive values of using erythrocyte indices and polychromasia to identify regeneration were determined, with a reticulocyte count > 65,000 reticulocytes/mu L considered the gold standard. Similarly, 134 blood BMS-777607 order samples from anemic dogs were analyzed prospectively with an in-house electrical impedance analyzer.\n\nResults-Of 4,387 dogs with samples analyzed retrospectively, 1,426 (32.5%) had regenerative anemia. Of these, 168 (11.8%) had macrocytic hypochromic anemia. High mean cell volume and low mean cell hemoglobin concentration had low sensitivity (11%), high specificity (98%), and moderate accuracy (70%) when used to identify regenerative anemia. Use of polychromasia alone had an accuracy of 77%, and use of polychromasia combined with a high RBC distribution width (RDW) had an accuracy of 79%. Results obtained with the in-house analyzer were similar.\n\nConclusions and Clinical Relevance-Results suggested that most regenerative anemias in dogs were not macrocytic hypochromic.