The Distributed Annotation System

Background  

Currently, most genome annotation is curated by centralized groups with limited resources. Efforts to share annotations transparently among multiple groups have not yet been satisfactory.     

Chemoreceptors of Escherichia coli CFT073 Play Redundant Roles in Chemotaxis toward Urine

Community-acquired urinary tract infections (UTIs) are commonly caused by uropathogenic Escherichia coli (UPEC). We hypothesize that chemotaxis toward ligands present in urine could direct UPEC into and up the urinary tract. Wild-type E. coli CFT073 and chemoreceptor mutants with tsr, tar, or aer deletions were tested for chemotaxis toward human urine in the capillary tube assay. Wild-type CFT073 was attracted toward urine, and Tsr and Tar were the chemoreceptors mainly responsible for mediating this response. The individual components of urine including L-amino acids, D-amino acids and various organic compounds were also tested in the capillary assay with wild-type CFT073. Our results indicate that CFT073 is attracted toward some L- amino acids and possibly toward some D-amino acids but not other common compounds found in urine such as urea, creatinine and glucuronic acid. In the murine model of UTI, the loss of any two chemoreceptors did not affect the ability of the bacteria to compete with the wild-type strain. Our data suggest that the presence of any strong attractant and its associated chemoreceptor might be sufficient for colonization of the urinary tract and that amino acids are the main chemoattractants for E. coli strain CFT073 in this niche.     

Synthesis and evaluation of novel tricyclic benzo[4.5]cyclohepta[1.2]pyridine derivatives as NMDA/NR2B antagonists

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure–activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K⁺ channel. Preferred compounds were subsequently evaluated for selectivity in an α₁-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Prothrombinase assembly and S1 site occupation restore the catalytic activity of FXa impaired by mutation at the sodium-binding site

Two loop segments (183-189 and 221-225) in the protease domain of factor Xa contribute to the formation of a Na(+)-binding site. Studies with factor Xa indicate that binding of a single Na(+) ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr(225) with Pro diminishes sensitivity to Na(+). Using full-length factor Xa(Y225P), the allosteric relationship between the Na(+) site and other structural determinants in factor Xa and prothrombinase was investigated. Direct binding and kinetic measurements with probes that target the S1 specificity pocket indicate that assembly of the mutant in prothrombinase corrected the impaired binding of these probes observed with free factor Xa(Y225P). This appears to result from the apparent allosteric linkage between the factor Va, S1, and Na(+)-binding sites, since binding of the cofactor to membrane-bound factor Xa(Y225P) enhances binding at the S1 site and vice versa. Additional studies revealed that the internal salt bridge (Ile(16)-Asp(194)) of factor Xa(Y225P) is partially destabilized, a process that is reversible upon occupation of the S1 site. The data establish that alterations at the factor Xa Na(+)-binding site shift the zymogen-protease equilibrium to a more zymogen-like state, and as a consequence binding of S1-directed probes and factor Va are adversely affected. Therefore, the zymogen-like characteristics of factor Xa(Y225P) have allowed for the apparent allosteric linkage between the S1, factor Va, and Na(+) sites to become evident and has provided insight into the structural transitions which accompany the conversion of factor X to factor Xa.     

A New Species of Glypheoid Lobster, Pseudoglyphea Foersteri (Decapoda: Astacidea: Mecochiridae) from the Lower Jurassic (Pliensbachian) of Raasay, Inner Hebrides, UK

Mecochirid lobsters assigned to the genus Pseudoglyphea Oppel, 1861 have previously been recorded from several localities in Europe. In this paper Pseudoglyphea foersteri sp. nov. is described from the Lower Jurassic of Raasay, Inner Hebrides, Scotland, providing the first evidence of a vagile benthic predator/scavenger in the Scalpa Sandstone Formation. Re-examination of the systematic placement of the genus supports allying the Mecochiridae with the Glypheidae within the Astacidea, not the Palinura as traditionally done.     

MIF-1 and its peptidomimetic analogs attenuate haloperidol-induced vacuous chewing movements and modulate apomorphine-induced rotational behavior in 6-hydroxydopamine-lesioned rats

Two melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1) also known as L-prolyl-L-leucyl-glycinamide (PLG) peptidomimetic analogs, 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]-amino]-3-(butyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (A) and 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]amino]-3-(benzyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (B), were evaluated for their ability to modulate dopaminergic activity by measuring apomorphine-induced rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats, and haloperidol (HP)-induced vacuous chewing movements (VCMs) in rats; animal models of Parkinson's disease (PD) and human tardive dyskinesia (TD), respectively. In the 6-OHDA model, both analogs were found to potentiate the contralateral rotational behavior induced by apomorphine dose-dependently and with approximately the same potency. Furthermore, each analog was able to significantly attenuate HP-induced VCMs with almost equal efficacy. The potency and efficacy of these analogs were significantly greater than their parent compound, PLG. These results suggest that both analogs can modulate dopaminergic activity in vivo, likely by the same mechanisms recruited by PLG previously reported.     

Lecithostaphylus retroflexus (Molin, 1859) (Zoogonidae) and Tergestia acanthocephala (Stossich, 1887) (Fellodistomidae) (Digenea) from the epipelagic teleost Belone belone (L.) in the western Mediterranean

Numerous individuals of the poorly known species Lecithostaphylus retroflexus (Zoogonidae) and Tergestia acanthocephala (Fellodistomidae) have been recovered from the teleost fish Belone belone gracilis from off the Scandola Nature Reserve, Western Mediterranean. They are redescribed, incorporating previously undescribed features: for L. retroflexus, a post-oral ring, a bipartite seminal vesicle, the shape of the excretory vesicle, the subterminal excretory pore and the flask-shaped gland-cells associated with the distinctly pedunculate ventral sucker; and for T. acanthocephala, the intestinal bifurcation in the forebody, necessitating its return to the genus Tergestia from Theledera. Additionally, T. acanthocephala is compared with T. laticollis from various species of Trachurus from the same geographical area.     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

Rapid Isolation of Extracellular Vesicles from Cell Culture and Biological Fluids Using a Synthetic Peptide with Specific Affinity for Heat Shock Proteins

Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.