Identification of dynamic structural motifs involved in peptidoglycan glycosyltransfer

We have determined the structure of a new form of the bifunctional peptidoglycan glycosyltransferase (GT)/transpeptidase penicillin-binding protein 2 from the pathogen Staphylococcus aureus. We observe several previously unstructured regions of the GT substrate-binding pockets, including a π-bulge in the outer helix that may be responsible for the conformational flexibility of active-site motifs required for transfer of product to the donor binding site during processive rounds of peptidoglycan polymerization. The identification of a β-hairpin in the usually unstructured region of the fold shares local structural homology to that of an exomuramidase, heightening comparisons between this biosynthetic enzyme and lytic peptidoglycan transglycosylases. This new form also shows remarkable interdomain flexibility, causing the linker region of the fold to project into the GT active site. This self-interaction may have significant consequences for the regulation of polymerization activity. The derived information is used to build a catalytic model of both donor and acceptor glycolipid substrates.     

Loss of muscleblind-like 1 promotes invasive mesenchyme formation in endocardial cushions by stimulating autocrine TGFbeta3

ABSTRACT: BACKGROUND: Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor beta (TGFbeta) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFbeta-dependent endocardial cell invasion. RESULTS: In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFbeta3 after it is no longer required to induce basal levels of invasion. TGFbeta3, but not TGFbeta2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFbeta3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFbeta3 production in the endocardium. More TGFbeta3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFbeta3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFbeta3 secretion, and early exposure to excess TGFbeta3 induces precocious invasion. MBNL1 expression precedes TGFbeta3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFbeta3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFbeta is necessary and sufficient to mediate this effect. CONCLUSIONS: Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFbeta3 production.     

Discovery of a novel series of selective HCN1 blockers

The discovery of a series of novel, potent, and selective blockers of the cyclic nucleotide-modulated channel HCN1 is disclosed. Here we report an SAR study around a series of selective blockers of the HCN1 channel. Utilization of a high-throughput VIPR assay led to the identification of a novel series of 2,2-disubstituted indane derivatives, which had moderate selectivity and potency at HCN1. Optimization of this hit led to the identification of the potent, 1,1-disubstituted cyclohexane HCN1 blocker, 2-ethoxy-N-((1-(4-isopropylpiperazin-1-yl)cyclohexyl)methyl)benzamide. The work leading to the discovery of this compound is described herein.     

Adiponectin Agonist ADP355 Attenuates CCl4-Induced Liver Fibrosis in Mice

Liver fibrosis is a growing global health problem characterized by excess deposition of fibrillar collagen, and activation of hepatic stellate cells (HSCs). Adiponectin is known to possess anti-fibrotic properties; however a high physiological concentration and multiple forms circulating in blood prohibit clinical use. Recently, an adiponectin-like small synthetic peptide agonist (ADP355: H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2) was synthesized for the treatment of murine breast cancer. The present study was designed to evaluate the efficacy of ADP355 as an anti-fibrotic agent in the in vivo carbon tetrachloride (CCl₄)-induced liver fibrosis model. Liver fibrosis was induced in eight-week old male C57BL/6J mice by CCl₄-gavage every other day for four weeks before injection of a nanoparticle-conjugated with ADP355 (nano-ADP355). Control gold nanoparticles and nano-ADP355 were administered by intraperitoneal injection for two weeks along with CCl₄-gavage. All mice were sacrificed after 6 weeks, and serum and liver tissue were collected for biochemical, histopathologic and molecular analyses. Biochemical studies suggested ADP355 treatment attenuates liver fibrosis, determined by reduction of serum aspartate aminotransferase (AST), alanine aminotransferase ALT) and hydroxyproline. Histopathology revealed chronic CCl₄-treatment results in significant fibrosis, while ADP355 treatment induced significantly reversed fibrosis. Key markers for fibrogenesis–α-smooth muscle actin (α-SMA), transforming growth factor-beta1 (TGF-β1), connective tissue growth factor (CTGF), and the tissue inhibitor of metalloproteinase I (TIMP1) were also markedly attenuated. Conversely, liver lysates from ADP355 treated mice increased phosphorylation of both endothelial nitric oxide synthase (eNOS) and AMPK while AKT phosphorylation was diminished. These findings suggest ADP355 is a potent anti-fibrotic agent that can be an effective intervention against liver fibrosis.     

Ants as egg predators of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Australian cotton crops

Abstract Helicoverpa armigera is a major pest of Australian cotton crops. To assess the impact of ant predation on H. armigera populations, the behaviour of four common ant taxa was observed in cotton crops in northern New South Wales over the 1999−2000 and 2001−02 seasons. Areas of cotton were artificially stocked with H. armigera eggs prior to observation. Pheidole spp. were the most frequently observed ants within the crop canopy in 1999−2000 and took the most H. armigera eggs. Iridomyrmex spp. were more frequently observed than Pheidole spp. in 2001−02 and also took some H. armigera eggs. Neither Paratrechina spp. nor Rhytidoponera metallica (Smith) took any H. armigera eggs, although both were seen in the crop canopy. Irrigation, cultivation and insecticide application disrupted foraging ants and limited their impact on H. armigera populations.     

Biological soil crust salvage for dryland restoration: an opportunity for natural resource restoration

Biocrusts' functional importance and vulnerability to disturbance have motivated consistent interest in biocrust restoration, as well as a recent increase in research to cultivate biocrusts in laboratory and greenhouse settings for use in ecological restoration. As part of a sustainable approach to developing biocrust restoration, we argue that a complementary step is to improve and accelerate methods for salvaging biocrusts that would otherwise be destroyed in a forthcoming disturbance. The increasing rate and scale of disturbance pressures in drylands where biocrusts flourish means that the supply of salvageable biocrust and demand for that material in restoration greatly exceed the present cultivable supply. In this article we describe the state of knowledge for biocrust salvage, present a simple set of steps for conducting a salvage harvest, discuss risks and benefits when considering using salvage, and suggest future research directions to facilitate scaling up biocrust restoration using salvaged material. A focus on the use of salvaged biocrust as a restoration source may prove an important step to improve ecological restoration in notoriously difficult to restore dryland ecosystems.     

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeY_Las was characterized as an endoribonuclease with activity on 3′ and 5′ termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeY_Las protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeY_Las. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeY_Las. Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.