Extrauterine listeriosis in the gravid mouse influences embryonic growth and development

Gravid mice and other rodents inoculated with Listeria monocytogenes typically fail to clear an intrauterine infection and either succumb or expel their intrauterine contents. We took advantage of this property to investigate the effects of an extrauterine infection on parameters of pregnancy success. Pregnant mice were selected for our study if they showed no clinical signs of listeriosis following oral inoculation at 7.5 gestational days (gd), and had no detectable intrauterine colony forming units (cfu) at near term (18.5 gd). The range of oral doses employed was 10(6)-10(8) cfu per mouse for two listerial serotype strains (4nonb and 1/2a). At all doses, inoculation resulted in a decrease in average near-term (18.5 gd) fetal weight per litter compared to sham inoculated controls. Additionally, embryonic death (indicated by intrauterine resorptions) was exhibited by some inoculated mice but was absent in all sham inoculated animals. In parallel experiments designed to detect possible loss of placental function, gravid uteruses were examined histopathologically and microbiologically 96 h after oral inoculation. Placental lesions were associated with high (> 10(6)), but not low (< 10(2)) or absent intrauterine cfu. In vitro, mouse embryonic trophoblasts were indistinguishable from mouse enterocytes in terms of their sensitivity to listerial exposure. A model consistent with our observations is one in which products (host or bacterial) generated during an acute infection enter embryos transplacentally and influences embryonic survival and slows normal growth in utero

Prime Focus Spectrograph (PFS) for the Subaru Telescope: Overview, recent progress, and future perspectives

PFS (Prime Focus Spectrograph), a next generation facility instrument on the 8.2-meter Subaru Telescope, is a very wide-field, massively multiplexed, optical and near-infrared spectrograph. Exploiting the Subaru prime focus, 2394 reconfigurable fibers will be distributed over the 1.3 deg field of view. The spectrograph has been designed with 3 arms of blue, red, and near-infrared cameras to simultaneously observe spectra from 380nm to 1260nm in one exposure at a resolution of ~1.6-2.7A. An international collaboration is developing this instrument under the initiative of Kavli IPMU. The project is now going into the construction phase aiming at undertaking system integration in 2017-2018 and subsequently carrying out engineering operations in 2018-2019. This article gives an overview of the instrument, current project status and future paths forward.Comment: 17 pages, 10 figures. Proceeding of SPIE Astronomical Telescopes and Instrumentation 201

Polypeptide-grafted macroporous polyHIPE by surface-initiated N-Carboxyanhydride (NCA) polymerization as a platform for bioconjugation

A new class of functional macroporous monoliths from polymerized high internal phase emulsion (polyHIPE) with tunable surface functional groups was developed by direct polypeptide surface grafting. In the first step, amino-functional polyHIPEs were obtained by the addition of 4-vinylbenzyl or 4-vinylbenzylphthalimide to the styrenic emulsion and thermal radical polymerization. The obtained monoliths present the expected open-cell morphology and a high surface area. The incorporated amino group was successfully utilized to initiate the ring-opening polymer- ization of benzyl-L-glutamate N-carboxyanhydride (BLG NCA) and benzyloxycarbonyl-L-lysine (Lys(Z)) NCA, which resulted in a dense homogeneous coating of polypeptides throughout the internal polyHIPE surfaces as confirmed by SEM and FTIR analysis. The amount of polypeptide grafted to the polyHIPE surfaces could be modulated by varying the initial ratio of amino acid NCA to amino-functional polyHIPE. Subsequent removal of the polypeptide protecting groups yielded highly functional polyHIPE-g-poly(glutamic acid) and polyHIPE-g- poly(lysine). Both types of polypeptide-grafted monoliths responded to pH by changes in their hydrohilicity. The possibility to use the high density of function (−COOH or −NH2) for secondary reaction was demonstrated by the successful bioconjugation of enhanced green fluorescent protein (eGFP) and fluorescein isocyanate (FITC) on the polymer 3D-scaffold surface. The amount of eGFP and FITC conjugated to the polypeptide-grafted polyHIPE was significantly higher than to the amino- functional polyHIPE, signifying the advantage of polypeptide grafting to achieve highly functional polyHIPEs

Intervening with Urinary Tract Infections Using Anti-Adhesives Based on the Crystal Structure of the FimH–Oligomannose-3 Complex

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection

Type 1 Fimbriae, a Colonization Factor of Uropathogenic Escherichia coli, Are Controlled by the Metabolic Sensor CRP-cAMP

Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues

Use of Bacteriophage Ba1 To Identify Properties Associated with Bordetella avium Virulence

Bordetella avium causes bordetellosis, an upper respiratory disease of birds. Commercially raised turkeys are particularly susceptible. We report here on the use of a recently described B. avium bacteriophage, Ba1, as a tool for investigating the effects of lysogeny and phage resistance on virulence. We found that lysogeny had no effect on any of the in vivo or in vitro measurements of virulence we employed. However, two-thirds (six of nine) spontaneous phage-resistant mutants of our virulent laboratory strain, 197N, were attenuated. Phage resistance was associated, in all cases, with an inability of the mutants to bind phage. Further tests of the mutants revealed that all had increased sensitivities to surfactants, and increased amounts of incomplete (O-antigen-deficient) lipopolysaccharide (LPS) compared to 197N. Hot phenol-water-extracted 197N LPS inactivated phage in a specific and dose-dependent manner. Acid hydrolysis and removal of lipid A had little effect upon the ability of isolated LPS to inactivate Ba1, suggesting that the core region and possibly the O antigen were required for phage binding. All of the mutants, with one exception, were significantly more sensitive to naive turkey serum and, without exception, significantly less able to bind to tracheal rings in vitro than 197N. Interestingly, the three phage-resistant mutants that remained virulent appeared to be O antigen deficient and were among the mutants that were the most serum sensitive and least able to bind turkey tracheal rings in vitro. This observation allowed us to conclude that even severe defects in tracheal ring binding and serum resistance manifested in vitro were not necessarily indicative of attenuation and that complete LPS may not be required for virulence