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Joanne C Thomas, James R Young, Kathrin Schemann, Phaivanh Chankhamthong, Syseng Khounsy, Sonevilay Nampanya, Peter A Windsor, Russell D Bush
A study of operator knowledge, attitudes and practices (KAP) in red meat slaughter premise operations in northern Laos was conducted and compared with international best practice, to inform future industry development. The survey interviewed 68 (of 94 possible participants) employees from all ten commercial slaughter premises in six districts in three northern Laos provinces. This was supported by observations of slaughter premises, processes and the conduct of personnel. Descriptive analysis and linear regression modelling identified significant KAP predictor factors, and a gap analysis supported or rejected inferences from the generally low KAP scores for human and animal health, animal welfare, good manufacturing practices (GMP), work conditions and economics...
September 15, 2017: Tropical Animal Health and Production
Annika Cimdins, Roger Simm, Fengyang Li, Petra Lüthje, Kaisa Thorell, Åsa Sjöling, Annelie Brauner, Ute Römling
Agar plate-based biofilm of enterobacteria like Escherichia coli is characterized by expression of the extracellular matrix components amyloid curli and cellulose exopolysaccharide, which can be visually enhanced upon addition of the dye Congo Red, resulting in a red, dry, and rough (rdar) colony morphology. Expression of the rdar morphotype depends on the transcriptional regulator CsgD and occurs predominantly at ambient temperature in model strains. In contrast, commensal and pathogenic isolates frequently express the csgD-dependent rdar morphotype semi-constitutively, also at human host body temperature...
September 15, 2017: MicrobiologyOpen
Santosh Kumar, Stephen Spiro
The genome of the denitrifying bacterium Paracoccus denitrificans predicts the expression of a small heme-containing nitric oxide (NO) binding protein, H-NOX. The genome organization and prior work in other bacteria suggest that H-NOX interacts with a diguanylate cyclase that cyclizes GTP to make cyclic di-GMP (cdGMP). Since cdGMP frequently regulates attached growth as a biofilm, we first established conditions for biofilm development by P. denitrificans. We found that adhesion to a polystyrene surface is strongly stimulated by the addition of 10 mM Ca(2+) to rich media...
September 2017: MSphere
Liudmila Andreeva, Björn Hiller, Dirk Kostrewa, Charlotte Lässig, Carina C de Oliveira Mann, David Jan Drexler, Andreas Maiser, Moritz Gaidt, Heinrich Leonhardt, Veit Hornung, Karl-Peter Hopfner
Cytosolic DNA arising from intracellular pathogens triggers a powerful innate immune response. It is sensed by cyclic GMP-AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2'3'-cyclic-GMP-AMP (cGAMP). Endogenous nuclear or mitochondrial DNA can also be sensed by cGAS under certain conditions, resulting in sterile inflammation. The cGAS dimer binds two DNA ligands shorter than 20 base pairs side-by-side, but 20-base-pair DNA fails to activate cGAS in vivo and is a poor activator in vitro...
September 13, 2017: Nature
Tim Holm Jakobsen, Tim Tolker-Nielsen, Michael Givskov
The development of effective strategies to combat biofilm infections by means of either mechanical or chemical approaches could dramatically change today's treatment procedures for the benefit of thousands of patients. Remarkably, considering the increased focus on biofilms in general, there has still not been invented and/or developed any simple, efficient and reliable methods with which to "chemically" eradicate biofilm infections. This underlines the resilience of infective agents present as biofilms and it further emphasizes the insufficiency of today's approaches used to combat chronic infections...
September 13, 2017: International Journal of Molecular Sciences
Kristina Eriksson, Alexandra Svensson, Alon S Hait, Kerstin Schlüter, Petra Tunbäck, Inger Nordström, Leonid Padyukov, Jan-Åke Liljeqvist, Trine H Mogensen, Søren R Paludan
IFN-γ-inducible protein 16 (IFI16) is an immunological DNA sensor proposed to act in the cyclic GMP-AMP synthase-stimulator of IFN genes pathway. Because mice do not have a clear ortholog of IFI16, this system is not suitable for genetic studies of IFI16. In this study, we have compared the dependency on IFI16, cyclic GMP-AMP synthase, and stimulator of IFN genes for type I IFN induction by a panel of pathogenic bacteria and DNA viruses. The IFN response induced by HSV-2 was particularly dependent on IFI16...
September 11, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
Masako Uemura, Seiji Komeda
The series of tetrazolato-bridged complexes with the formula [{cis‑Pt(NH3)2}2(μ-OH)(μ-5-H-tetrazolato-N1,N2)](2+) (5-H-X) or [{cis‑Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)](n+) (R=H (5-H-Y), CH3 (1), CH2COOCH2CH3 (2), CH2COO(-) (3), n=2 (5-H-Y, 1, 2) or 1 (3)) are promising candidate complexes for formulation as next-generation platinum-based anticancer drugs that form multimodal bindings with DNA molecules. These multimodal bindings involve both non-covalent and covalent interactions, the latter of which are acknowledged to be essential for platinum-based drugs to exert their anticancer activity...
August 25, 2017: Journal of Inorganic Biochemistry
Elio Rossi, Sara Motta, Alessandro Aliverti, Federica Cossu, Louise Gourlay, Pierluigi Mauri, Paolo Landini
Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway, and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase...
September 11, 2017: Environmental Microbiology
Oleksandr Kondratov, Damien Marsic, Sean M Crosson, Hector R Mendez-Gomez, Oleksandr Moskalenko, Mario Mietzsch, Regine Heilbronn, Jonathan R Allison, Kari B Green, Mavis Agbandje-McKenna, Sergei Zolotukhin
The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors...
August 10, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
Livia Leoni, Sarika Vishnu Pawar, Giordano Rampioni
Pseudomonas aeruginosa infections are often difficult or impossible to treat, mainly due to its ability to form antibiotic-resistant biofilms. Since c-di-GMP signaling strongly influences P. aeruginosa biofilm development and sensitivity to antibiotics, it is considered a promising target for the development of anti-biofilm drugs and it is under intensive investigation. However, studying c-di-GMP signaling in P. aeruginosa is challenging, mainly due to (1) the multiplicity of enzymes involved in c-di-GMP metabolism, (2) the difficulty to extract and measure c-di-GMP intracellular levels by chemical methods, and (3) the lack of genetic tools specifically dedicated to this purpose...
2017: Methods in Molecular Biology
Julie Groizeleau, Jens Bo Andersen, Michael Givskov, Jens Berthelsen, Tim Tolker-Nielsen
Bacteria in the biofilm mode of growth cause numerous problematic infections due to their resistance to antimicrobials and the immune system. Because conventional antimicrobial compounds cannot efficiently eradicate biofilm infections, we urgently need new efficient anti-biofilm drugs. The secondary messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria, and it is assumed that drugs that lower the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle...
2017: Methods in Molecular Biology
S Rinaldo, G Giardina, F Mantoni, A Paiardini, Alessio Paone, Francesca Cutruzzolà
One of the most important signals involved in controlling biofilm formation is represented by the intracellular second messenger 3',5'-cyclic diguanylic acid (c-di-GMP). Since the pathways involved in c-di-GMP biosynthesis and breakdown are found only in bacteria, targeting c-di-GMP metabolism represents an attractive strategy for the development of biofilm-disrupting drugs. Here, we present the workflow required to perform a structure-based design of inhibitors of diguanylate cyclases, the enzymes responsible for c-di-GMP biosynthesis...
2017: Methods in Molecular Biology
Clement Opoku-Temeng, Herman O Sintim
Bacteria possess several signaling molecules that regulate distinct phenotypes. Cyclic di-GMP (c-di-GMP) has emerged as a ubiquitous second messenger that regulates bacterial virulence, cell cycle, motility, and biofilm formation. The link between c-di-GMP signaling and biofilm formation affords novel strategies for treatment of biofilm-associated infections, which is a major public health problem. The complex c-di-GMP signaling pathway creates a hurdle in the development of small molecule modulators. Nonetheless, some progress has been made in this regard and inhibitors of c-di-GMP metabolizing enzymes that affect biofilm formation and motility have been documented...
2017: Methods in Molecular Biology
Bruno Y Matsuyama, Petya V Krasteva, Marcos V A S Navarro
Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K a, K d) and stoichiometry of binding (n), as well as on changes in the Gibbs free energy (ΔG) and entropy (ΔS) along the interaction...
2017: Methods in Molecular Biology
Johann Peltier, Olga Soutourina
Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important signaling molecule for community behavior control, cell morphogenesis, and virulence in bacteria. In addition to protein effectors, this second messenger binds RNA molecules that act as riboswitches to control target gene expression. In this chapter, we describe a method for experimental validation of the functionality of c-di-GMP-responsive riboswitches and the analysis of c-di-GMP control of target gene expression by qRT-PCR and Northern blot...
2017: Methods in Molecular Biology
Benoît-Joseph Laventie, Timo Glatter, Urs Jenal
Capture compound technology coupled to mass spectrometry (CCMS) allows to biochemically identify ligand receptors. Using a c-di-GMP-specific Capture Compound, we adapted this method for the identification and characterization of c-di-GMP binding proteins in any bacterial species. Because in silico analysis often fails to predict novel c-di-GMP effectors, this universal method aims at better defining the cellular c-di-GMP network in a wide range of bacteria. CCMS was successfully applied in several bacterial species (Nesper et al...
2017: Methods in Molecular Biology
Jacob R Chambers, Karin Sauer
Cyclic di-GMP is an important regulatory messenger molecule that often directly interacts with proteins to alter function. It is therefore important to find potential c-di-GMP binding proteins and verify a direct interaction between them. Here, we describe a pull-down assay using biotinylated-c-di-GMP to capture a specific protein of interest followed by immunoblot analysis to determine relative protein abundance. This method also allows for addition of both specific and nonspecific competitors to determine specificity of c-di-GMP-protein binding...
2017: Methods in Molecular Biology
Claudine Baraquet, Caroline S Harwood
The transition of bacteria from a planktonic lifestyle to a collaborative, sessile biofilm lifestyle is a regulated process orchestrated by the intracellular second-messenger c-di-GMP (bis-(3'-5')-cyclic dimeric guanosine monophosphate). To modulate this transition, c-di-GMP acts at the transcriptional, posttranscriptional, and posttranslational levels. In this chapter, we describe a method to study of how a transcriptional regulator modulates gene expression in response to c-di-GMP binding. DNase I footprinting is a valuable tool for use in analyzing how regulatory proteins bind to DNA, the location of their binding sites or how c-di-GMP affects their binding to DNA...
2017: Methods in Molecular Biology
Jacob R Chambers, Karin Sauer
Modulation of signal transduction via binding of the secondary messenger molecule cyclic di-GMP to effector proteins is a near universal regulatory schema in bacteria. In particular, direct binding of c-di-GMP to transcriptional regulators has been shown to alter gene expression of a variety of processes. Here, we illustrate a pull-down-based DNA:protein binding reaction to determine the relative importance of c-di-GMP in the binding affinity of a target protein to specific DNA sequences. Specifically, the pull-down-based assay enables DNA binding to be analyzed with differing concentrations of c-di-GMP in the absence/presence of specific and nonspecific competitors...
2017: Methods in Molecular Biology
Barbara I Kazmierczak
Cyclic-di-GMP phosphodiesterases (PDEs) catalyze the hydrolysis of the bacterial second messenger c-di-GMP. This protocol describes a sensitive radioactive assay for PDE activity in which substrate and product can be quickly and easily separated by thin-layer chromatography.
2017: Methods in Molecular Biology
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