Small-Molecule Targeting of RNA Polymerase I Activates a Conserved Transcription Elongation Checkpoint

Summary Inhibition of RNA polymerase I (Pol I) is a promising strategy for modern cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme, but how this exceptional response is enforced is not known. Here, we define key elements requisite for the response. We show that Pol I preinitiation factors and polymerase subunits (e.g., RPA135) are required for BMH-21-mediated degradation of RPA194. We further find that Pol I inhibition and induced degradation by BMH-21 are conserved in yeast. Genetic analyses demonstrate that mutations that induce transcription elongation defects in Pol I result in hypersensitivity to BMH-21. Using a fully reconstituted Pol I transcription assay, we show that BMH-21 directly impairs transcription elongation by Pol I, resulting in long-lived polymerase pausing. These studies define a conserved regulatory checkpoint that monitors Pol I transcription and is activated by therapeutic intervention.Peer reviewe

P424Short-term ACE Inhibition upregulates cardiac expression of SERCA2a and protects against ventricular arrhythmias in healthy rats

Introduction: Chronic angiotensin converting enzyme inhibitor (ACEIs) treatment can suppress arrhythmogenesis. To examine whether the effect is more immediate and independent of suppression of pathological remodelling, we tested the antiarrhythmic effect of short-term ACE inhibition in healthy normotensive rats. Methods and results: Wistar rats were administered with enalaprilat (ENA, i.p., 5 mg/kg every 12 h) or vehicle (CON) for two weeks. Cellular shortening was measured in isolated, electrically paced cardiomyocytes. Standard 12-lead electrocardiography was performed and, hearts of anesthetized open-chest rats were subjected to 6-min ischemia followed by 10-minute reperfusion to examine susceptibility to ventricular arrhythmias. Expressions of calcium regulating proteins (SERCA2a, cardiac sarco/endoplasmic reticulum Ca2+-ATPase; CSQ, calsequestrin; TRD, triadin; PLB, phospholamban; FKBP12.6, FK506-binding protein) were measured by Western blot and mRNA levels of L-type calcium channel (Cacna1c), ryanodine receptor (Ryr2) and potassium channels Kcnh2 and Kcnq1 were measured by qRT-PCR. ENA decreased systolic as well as diastolic blood pressure (by 20%, and by 31%, respectively, for both P<0.05) but enhanced shortening of cardiomyocytes at basal conditions (by 34%, P<0.05) and under beta-adrenergic stimulation (by 73%, P<0.05). Enalaprilat shortened QTc interval duration (CON: 78±1 ms vs. ENA: 72±2 ms; P<0.05) and significantly decreased the total duration of ventricular fibrillations (VF) and the number of VF episodes (P<0.05). Reduction in arrhythmogenesis was associated with a pronounced upregulation of SERCA2a and increased Cacna1c mRNA levels. Conclusion: Short-term ACEI treatment can provide protection against I/R injury-induced ventricular arrhythmias in healthy myocardium and this effect is associated with increased SERCA2a expression. CON ENA Calcium regulating proteins SERCA2a 100±20 304±13* CSQ 100±6 105±7 TRD 100±16 117±10 PLB 100±9 109±16 FKBP12 100±12 93±

Biofilm formation on enteral feeding tubes by Cronobacter sakazakii, Salmonella serovars and other Enterobacteriaceae

WHO (2007) recommended that to reduce microbial risks, powdered infant formula should be reconstituted with water at temperatures >70 °C, and that such feeds should be used within 2 h of preparation. However, this recommendation does not consider the use of enteral feeding tubes which can be in place for more than 48 h and can be loci for bacterial attachment. This study determined the extent to which 29 strains of Cronobacter sakazakii, Salmonella serovars, other Enterobacteriaceae and Acinetobacter spp. can adhere and grow on enteral feeding tubes composed of polyvinyl chloride and polyurethane. The study also included silver-impregnated tubing which was expected to have antibacterial activity. Bacterial biofilm formation by members of the Enterobacteriaceae was ca. 105-106 cfu/cm after 24 h. Negligible biofilm was detected for Acinetobacter gensp. 13; ca. 10 cfu/cm, whereas Cr. sakazakii strain ATCC 12868 had the highest biofilm cell density of 107 cfu/cm. Biofilm formation did not correlate with capsule production, and was not inhibited on silver-impregnated tubing. Bacteria grew in the tube lumen to cell densities of 107 cfu/ml within 8 h, and 109 cfu/ml within 24 h. It is plausible that in vivo the biofilm will both inoculate subsequent routine feeds and as the biofilm ages, clumps of cells will be shed which may survive passage through the neonate's stomach. Therefore biofilm formation on enteral feeding tubes constitutes a risk factor for susceptible neonates

Capsular profiling of the Cronobacter genus and the association of specific Cronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and necrotizing enterocolitis

Background: Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. Methods: This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen. Results: Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell+] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections. Conclusion: This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes

Early Blockade of CB1 Receptors Ameliorates Schizophrenia-like Alterations in the Neurodevelopmental MAM Model of Schizophrenia

In agreement with the neurodevelopmental hypothesis of schizophrenia, prenatal exposure of Sprague-Dawley rats to the antimitotic agent methylazoxymethanol acetate (MAM) at gestational day 17 produces long-lasting behavioral alterations such as social withdrawal and cognitive impairment in adulthood, mimicking a schizophrenia-like phenotype. These abnormalities were preceded at neonatal age both by the delayed appearance of neonatal reflexes, an index of impaired brain maturation, and by higher 2-arachidonoylglycerol (2-AG) brain levels. Schizophrenia-like deficits were reversed by early treatment [from postnatal day (PND) 2 to PND 8] with the CB1 antagonist/inverse agonist AM251 (0.5 mg/kg/day). By contrast, early CB1 blockade affected the behavioral performance of control rats which was paralleled by enhanced 2-AG content in the prefrontal cortex (PFC). These results suggest that prenatal MAM insult leads to premorbid anomalies at neonatal age via altered tone of the endocannabinoid system, which may be considered as an early marker preceding the development of schizophrenia-like alterations in adulthood

Synchronization modulation increases transepithelial potentials in MDCK monolayers through Na/K pumps

Peer reviewedPublisher PD

Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium

PURPOSE: To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. METHODS: The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6). Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway. Reporter mice, mosaic for expression of the gene encoding β-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo. RESULTS: Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage. Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation. Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh. Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium. CONCLUSIONS: The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands

Control of Patterns of Corneal Innervation by Pax6

PURPOSE: Corneal nerves play essential roles in maintaining the ocular surface through provision of neurotrophic support, but genetic control of corneal innervation is poorly understood. The possibility of a neurotrophic failure in ocular surface disease associated with heterozygosity at the Pax6 locus (aniridia-related keratopathy [ARK]) was investigated. METHODS: Patterns of corneal innervation were studied during development and aging in mice with different Pax6 dosages and in chimeras. Immunohistochemistry and ELISA-based assays were used to determine the molecular basis of defects seen in Pax6 mutants, and wound healing assays were performed. RESULTS: In adults, the Pax6(+/−) epithelium was less densely innervated than the wild-type epithelium, and radial projection of epithelial nerves was disrupted. Neurotrophic support of the corneal epithelium appeared normal. Directed nerve projection correlated with patterns of epithelial cell migration in adult wild-types, but innervation defects observed in Pax6(+/−) mice were not fully corrected in wound healing or chimeric models where directed epithelial migration was restored. CONCLUSIONS: Pax6 dosage nonautonomously controls robust directed radial projection of corneal neurons, and the guidance cues for growth cone guidance are not solely dependent on directed epithelial migration. There is little evidence that ARK represents neurotrophic keratitis