Exogenous glycosaminoglycans coat damaged bladder surfaces in experimentally damaged mouse bladder

BACKGROUND: Interstital cystitis is often treated with exogenous glycosaminoglycans such as heparin, chondroitin sulphate (Uracyst), hyaluronate (Cystistat) or the semi-synthetic pentosan polysulphate (Elmiron). The mechanism of action is presumed to be due to a coating of the bladder surface to replace the normally present chondroitin sulphate and heparan sulphate lost as a result of the disease. This study used fluorescent labelled chondroitin sulphate to track the distribution of glycosaminoglycans administered intravesically to mouse bladder that had been damaged on the surface. METHODS: The surfaces of mouse bladders were damaged by 3 mechanisms – trypsin, 10 mM HCl, and protamine sulphate. Texas Red-labeled chondroitin sulphate was instilled into the bladders of animals with damaged bladders and controls instilled only with saline. Bladders were harvested, frozen, and sectioned for examination by fluorescence. RESULTS: The normal mouse bladder bound a very thin layer of the labelled chondroitin sulphate on the luminal surface. Trypsin- and HCl-damaged bladders bound the labelled chondroitin sulphate extensively on the surface with little penetration into the bladder muscle. Protamine produced less overt damage, and much less labelling was seen, presumably due to loss of the label as it complexed with the protamine intercalated into the bladder surface. CONCLUSION: Glycosaminoglycan administered intravesically does bind to damaged bladder. Given that the changes seen following bladder damage resemble those seen naturally in interstitial cystitis, the mechanisms proposed for the action of these agents is consistent with a coating of damaged bladder

Short-Term Starvation of Immune Deficient Drosophila Improves Survival to Gram-Negative Bacterial Infections

Background: Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1receptor-nuclear factor kappa B (TIR-NF-kB) pathway. Since the TIR-NF-kB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions. Methodology/Principal Findings: We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-kB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-kB protein Dif, usually needed for responses against Gram-positive bacteria. Conclusions/Significance: Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natura

Potential conservation of circadian clock proteins in the phylum Nematoda as revealed by bioinformatic searches

Although several circadian rhythms have been described in C. elegans, its molecular clock remains elusive. In this work we employed a novel bioinformatic approach, applying probabilistic methodologies, to search for circadian clock proteins of several of the best studied circadian model organisms of different taxa (Mus musculus, Drosophila melanogaster, Neurospora crassa, Arabidopsis thaliana and Synechoccocus elongatus) in the proteomes of C. elegans and other members of the phylum Nematoda. With this approach we found that the Nematoda contain proteins most related to the core and accessory proteins of the insect and mammalian clocks, which provide new insights into the nematode clock and the evolution of the circadian system.Fil: Romanowski, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; ArgentinaFil: Garavaglia, Matías Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Goya, María Eugenia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Golombek, Diego Andres. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

C. elegans SWAN-1 Binds to EGL-9 and Regulates HIF-1-Mediated Resistance to the Bacterial Pathogen Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a nearly ubiquitous human pathogen, and infections can be lethal to patients with impaired respiratory and immune systems. Prior studies have established that strong loss-of-function mutations in the egl-9 gene protect the nematode C. elegans from P. aeruginosa PAO1 fast killing. EGL-9 inhibits the HIF-1 transcription factor via two pathways. First, EGL-9 is the enzyme that targets HIF-1 for oxygen-dependent degradation via the VHL-1 E3 ligase. Second, EGL-9 inhibits HIF-1-mediated gene expression through a VHL-1-independent mechanism. Here, we show that a loss-of-function mutation in hif-1 suppresses P. aeruginosa PAO1 resistance in egl-9 mutants. Importantly, we find stabilization of HIF-1 protein is not sufficient to protect C. elegans from P. aeruginosa PAO1 fast killing. However, mutations that inhibit both EGL-9 pathways result in higher levels of HIF-1 activity and confer resistance to the pathogen. Using forward genetic screens, we identify additional mutations that confer resistance to P. aeruginosa. In genetic backgrounds that stabilize C. elegans HIF-1 protein, loss-of-function mutations in swan-1 increase the expression of hypoxia response genes and protect C. elegans from P. aeruginosa fast killing. SWAN-1 is an evolutionarily conserved WD-repeat protein belonging to the AN11 family. Yeast two-hybrid and co-immunoprecipitation assays show that EGL-9 forms a complex with SWAN-1. Additionally, we present genetic evidence that the DYRK kinase MBK-1 acts downstream of SWAN-1 to promote HIF-1-mediated transcription and to increase resistance to P. aeruginosa. These data support a model in which SWAN-1, MBK-1 and EGL-9 regulate HIF-1 transcriptional activity and modulate resistance to P. aeruginosa PAO1 fast killing

ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

<p>Abstract</p> <p>Background</p> <p>Zinc Finger Nucleases (ZFNs) have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN) method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms.</p> <p>Description</p> <p>ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; <it>S. cerevisiae, C. reinhardtii, A. thaliana</it>, <it>D. melanogaster, D. rerio, C. elegans</it>, and <it>H. sapiens </it>and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s). Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence). Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter) "confidence" scores that estimate the likelihood that a chosen ZFN target site will function <it>in vivo</it>. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the effectiveness of a given ZFN in creating double-stranded breaks.</p> <p>Conclusions</p> <p>ZFNGenome provides a user-friendly interface that allows researchers to access resources and information regarding genomic target sites for engineered ZFNs in seven model organisms. This genome-wide database of potential ZFN target sites should greatly facilitate the utilization of ZFNs in both basic and clinical research.</p> <p>ZFNGenome is freely available at: <url>http://bindr.gdcb.iastate.edu/ZFNGenome</url> or at the Zinc Finger Consortium website: <url>http://www.zincfingers.org/</url>.</p

MRI of Arterial Flow Reserve in Patients with Intermittent Claudication: Feasibility and Initial Experience

Objectives: The aim of this work was to develop a MRI method to determine arterial flow reserve in patients with intermittent claudication and to investigate whether this method can discriminate between patients and healthy control subjects. Methods: Ten consecutive patients with intermittent claudication and 10 healthy control subjects were included. All subjects underwent vector cardiography triggered quantitative 2D cine MR phase-contrast imaging to obtain flow waveforms of the popliteal artery at rest and during reactive hyperemia. Resting flow, maximum hyperemic flow and absolute flow reserve were determined and compared between the two groups by two independent MRI readers. Also, interreader reproducibility of flow measures was reported. Results: Resting flow was lower in patients compared to controls (4.961.6 and 11.163.2 mL/s in patients and controls, respectively (p,0.01)). Maximum hyperemic flow was 7.362.9 and 16.463.2 mL/s (p,0.01) and the absolute flow reserve was 2.461.6 and 5.361.3 mL/s (p,0.01), respectively in patients and controls. The interreader coefficient of variation was below 10 % for all measures in both patients and controls. Conclusions: Quantitative 2D MR cine phase-contrast imaging is a promising method to determine flow reserve measures in patients with peripheral arterial disease and can be helpful to discriminate patients with intermittent claudication fro

Histone H3 Localizes to the Centromeric DNA in Budding Yeast

During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA

Age-Dependent Maturation of Toll-Like Receptor-Mediated Cytokine Responses in Gambian Infants

The global burden of neonatal and infant mortality due to infection is staggering, particularly in resource-poor settings. Early childhood vaccination is one of the major interventions that can reduce this burden, but there are specific limitations to inducing effective immunity in early life, including impaired neonatal leukocyte production of Th1-polarizing cytokines to many stimuli. Characterizing the ontogeny of Toll-like receptor (TLR)-mediated innate immune responses in infants may shed light on susceptibility to infection in this vulnerable age group, and provide insights into TLR agonists as candidate adjuvants for improved neonatal vaccines. As little is known about the leukocyte responses of infants in resource-poor settings, we characterized production of Th1-, Th2-, and anti-inflammatory- cytokines in response to agonists of TLRs 1-9 in whole blood from 120 Gambian infants ranging from newborns (cord blood) to 12 months of age. Most of the TLR agonists induced TNFα, IL-1β, IL-6, and IL-10 in cord blood. The greatest TNFα responses were observed for TLR4, -5, and -8 agonists, the highest being the thiazoloquinoline CLO75 (TLR7/8) that also uniquely induced cord blood IFNγ production. For most agonists, TLR-mediated TNFα and IFNγ responses increased from birth to 1 month of age. TLR8 agonists also induced the greatest production of the Th1-polarizing cytokines TNFα and IFNγ throughout the first year of life, although the relative responses to the single TLR8 agonist and the combined TLR7/8 agonist changed with age. In contrast, IL-1β, IL-6, and IL-10 responses to most agonists were robust at birth and remained stable through 12 months of age. These observations provide fresh insights into the ontogeny of innate immunity in African children, and may inform development of age-specific adjuvanted vaccine formulations important for global health

Metabolism before, during and after anaesthesia in colic and healthy horses

<p>Abstract</p> <p>Background</p> <p>Many colic horses are compromised due to the disease state and from hours of starvation and sometimes long trailer rides. This could influence their muscle energy reserves and affect the horses' ability to recover. The principal aim was to follow metabolic parameter before, during, and up to 7 days after anaesthesia in healthy horses and in horses undergoing abdominal surgery due to colic.</p> <p>Methods</p> <p>20 healthy horses given anaesthesia alone and 20 colic horses subjected to emergency abdominal surgery were anaesthetised for a mean of 228 minutes and 183 minutes respectively. Blood for analysis of haematology, electrolytes, cortisol, creatine kinase (CK), free fatty acids (FFA), glycerol, glucose and lactate was sampled before, during, and up to 7 days after anaesthesia. Arterial and venous blood gases were obtained before, during and up to 8 hours after recovery. Gluteal muscle biopsy specimens for biochemical analysis of muscle metabolites were obtained at start and end of anaesthesia and 1 h and 1 day after recovery.</p> <p>Results</p> <p>Plasma cortisol, FFA, glycerol, glucose, lactate and CK were elevated and serum phosphate and potassium were lower in colic horses before anaesthesia. Muscle adenosine triphosphate (ATP) content was low in several colic horses. Anaesthesia and surgery resulted in a decrease in plasma FFA and glycerol in colic horses whereas levels increased in healthy horses. During anaesthesia muscle and plasma lactate and plasma phosphate increased in both groups. In the colic horses plasma lactate increased further after recovery. Plasma FFA and glycerol increased 8 h after standing in the colic horses. In both groups, plasma concentrations of CK increased and serum phosphate decreased post-anaesthesia. On Day 7 most parameters were not different between groups. Colic horses lost on average 8% of their initial weight. Eleven colic horses completed the study.</p> <p>Conclusion</p> <p>Colic horses entered anaesthesia with altered metabolism and in a negative oxygen balance. Muscle oxygenation was insufficient during anaesthesia in both groups, although to a lesser extent in the healthy horses. The post-anaesthetic period was associated with increased lipolysis and weight loss in the colic horses, indicating a negative energy balance during the first week post-operatively.</p

Differential Effect of TLR2 and TLR4 on the Immune Response after Immunization with a Vaccine against Neisseria meningitidis or Bordetella pertussis

Neisseria meningitidis and Bordetella pertussis are Gram-negative bacterial pathogens that can cause serious diseases in humans. N. meningitidis outer membrane vesicle (OMV) vaccines and whole cell pertussis vaccines have been successfully used in humans to control infections with these pathogens. The mechanisms behind their effectiveness are poorly defined. Here we investigated the role of Toll-like receptor (TLR) 2 and TLR4 in the induction of immune responses in mice after immunization with these vaccines. Innate and adaptive immune responses were compared between wild type mice and mice deficient in TLR2, TLR4, or TRIF. TRIF-deficient and TLR4-deficient mice showed impaired immunity after immunization. In contrast, immune responses were not lower in TLR2−/− mice but tended even to be higher after immunization. Together our data demonstrate that TLR4 activation contributes to the immunogenicity of the N. meningitidis OMV vaccine and the whole cell pertussis vaccine, but that TLR2 activation is not required