Cyclic-di-GMP Signaling in the Borrelia Spirochetes

Lyme disease is the most common tick-borne disease in North America, with approximately 35,000 cases reported to the Centers for Disease Control in 2008. The genome of its causative agent, Borrelia burgdorferi, encodes for a set of genes involved in the metabolism and regulatory activities of the second messenger nucleotide, cyclic-di-GMP (c-di-GMP). Rrp1 is a response regulatory-diguanylate cyclase, and its regulatory capability is likely mediated via production of c-di-GMP, as it lacks a DNA-binding domain. One known class of c-di-GMP effector/binding proteins are those that harbor a PIlZ domain. The genome of B. burgdorferi strain 5A4 encodes for one chromosomally-carried PilZ domain, which we have designated PlzA. Additionally, certain B. burgdorferi strains encode for a second PilZ domain-containing protein (PlzB) which is plasmid-carried. Both PlzA and PlzB were found to bind specifically to c-di-GMP, and c-di-GMP binding by PlzA was found to be dependant upon arginine residues in the c-di-GMP binding region. Additionally, expression of PlzA was found to be upregulated by tick feeding and was constitutive in the mammalian host. We next constructed two deletion/allelic exchange mutants – one with the targeted deletion of PlzA, and on ethat replaced PlzA with PlzB in a strain lacking the plzB gene. Our studies demonstrated that ΔplzA was deficient in motility and was also non-infectious in the mouse model of B. burgdorferi infection. Additionally, this strain remained viable in larval Ixodes ticks. Also, B31-plzB KI was deficient in motility, as well as infectivity, demonstrating that PlzB is unable to complement for functions fo PlzA in vitro and in vivo and that it may play other roles in the biology of B. burgdorferi strains carrying the plzB gene. These studies represent the first identification of a c-di-GMP binding protein in any spirochete, but also represent the first demonstration of the importance of PilZ domain proteins in a spirochetal system. We additionally examined the effects of c-di-GMP synthesis and breakdown in the related bacterium, B. hermsii, a causative agent of tick-borne relapsing fever (TBRF). Deletion mutants in Rrp1 (B. hermsii’s sole diguanylate cyclase) and PdeA (B. hermsii’s only EAL domain-containing phosphodiesterase) were created. These strains were analyzed in order to determine: 1) the effect(s) of the losse of Rrp1/PdeA on intracellular spirochete c-di-GMP levels, and 2) the effects of Rrp1/PdeA on the establishment of murine infection and on gross motility/chemotaxis. It was demonstrated that c-di-GMP accumulates intracellularly in the cells lacking PdeA. Additionally, spirochetes were shown to chemotax towards N-acetyl-glucosamine (NAG) and they did not form soft agar swarms. In contrast, cells lacking Rrp1 did not accumulate detectable levels of c-di-GMP, demonstrated a reduced ability to chemotax towards NAG, and swarmed on soft agar in a fashion indistinguishable from wild type. Despite these differences in phenotype, both mutant strains display an attenuated murine infectivity. These results indicate that c-di-GMP is indeed important in the TBRF spirochete, B. hermsii and this vital second messenger plays key roles in virulence, motility, and chemotaxis. These studies also pave the way for future investigation of B. hermsii through use of targeted genetic manipulation

Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents.

Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. Importance: Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural host intestinal contents. These analyses demonstrated that (i) ETX processing in host intestinal contents occurs in an ordered, stepwise fashion, (ii) processing of prototoxin by host intestinal contents results in higher-molecular-mass material and 3 distinct ~27-kDa ETX species, and (iii) serine proteases, such as trypsin, chymotrypsin, and other proteases, including carboxypeptidases, play a role in the activation of ETX by intestinal contents. These studies provide new insights into the activation and processing of ETX and demonstrate that this process is more complicated than previously appreciated

Complements of tori and Klein bottles in the 4-sphere that have hyperbolic structure

Many noncompact hyperbolic 3-manifolds are topologically complements of links in the 3-sphere. Generalizing to dimension 4, we construct a dozen examples of noncompact hyperbolic 4-manifolds, all of which are topologically complements of varying numbers of tori and Klein bottles in the 4-sphere. Finite covers of some of those manifolds are then shown to be complements of tori and Klein bottles in other simply-connected closed 4-manifolds. All the examples are based on a construction of Ratcliffe and Tschantz, who produced 1171 noncompact hyperbolic 4-manifolds of minimal volume. Our examples are finite covers of some of those manifolds.Comment: Published by Algebraic and Geometric Topology at http://www.maths.warwick.ac.uk/agt/AGTVol5/agt-5-41.abs.htm

Reliability analysis of a structural ceramic combustion chamber

The Weibull modulus, fracture toughness and thermal properties of a silicon nitride material used to make a gas turbine combustor were experimentally measured. The location and nature of failure origins resulting from bend tests were determined with fractographic analysis. The measured Weibull parameters were used along with thermal and stress analysis to determine failure probabilities of the combustor with the CARES design code. The effect of data censoring, FEM mesh refinement, and fracture criterion were considered in the analysis

Alternative approaches to tax risk and tax avoidance: analysis of a face-to-face corporate survey

This paper analyzes the results of a survey of views of large businesses regarding recent UK Government initiatives aimed at modifying taxpayer behaviour and tackling what is perceived by the tax authorities acting on behalf of Government to be unacceptable/aggressive tax avoidance. Specifically, it examines the views of tax directors obtained from face-to-face interviews conducted in spring 2008 with representatives of 30 corporate groups regarding alternative approaches to tax risk and tax avoidance. The paper first describes the experiences and opinions of large business representatives with respect to the Risk Rating Approach, a key feature of the HMRC links with large business programme (Varney Programme), as well as the status of relationships between HMRC and large business more generally. It next considers the respondents’ views on the practical implications of two developing legislative approaches – targeted anti-avoidance rules (TAARs) and principles-based legislation (PBL) – and how these approaches impact upon and are influenced by relationships between HMRC and large businesses

Journal Article – Psychiatry in the Army Air Forces & The Services of the Military Mental Hygiene Unit

Two articles published in the July 1943 issue of the American Journal of Psychiatry that discuss the Special Training Unit Program. The titles are, Psychiatry in the Army Air Forces and The Services of the Military Mental Hygiene Unit.https://scholarworks.moreheadstate.edu/stu_1210th_fort_ontario/1056/thumbnail.jp

Spreading primitive groups of diagonal type do not exist

The synchronisation hierarchy of finite permutation groups consists of classes of groups lying between 2-transitive groups and primitive groups. This includes the class of spreading groups, which are defined in terms of sets and multisets of permuted points, and which are known to be primitive of almost simple, affine or diagonal type. In this paper, we prove that in fact no spreading group of diagonal type exists. As part of our proof, we show that all non-abelian finite simple groups, other than six sporadic groups, have a transitive action in which a proper normal subgroup of a point stabiliser is supplemented by all corresponding two-point stabilisers.Comment: 10 pages. Version 2 resolves the Monster group case of Theorem 1.3, with the aid of a result drawn to our attention by Prof. Tim Burnes