Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1^‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l ‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1^‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.     

Dihydro-pyrano[2,3-b]pyridines and tetrahydro-1,8-naphthyridines as CB1 receptor inverse agonists: Synthesis,SAR and biological evaluation

Synthesis and structure–activity relationships of cannabinoid-1 receptor (CB1R) inverse agonists based on dihydro-pyrano[2,3-b] pyridine and tetrahydro-1,8-naphtyridine scaffolds are presented. Rat food intake and pharmacokinetic evaluation of 13g, 13i, 13k and 17a revealed these compounds to be highly efficacious orally active modulators of CB1R.     

Peptide Functionalised Gold Nanoparticles: Effect of Loading on Aggregation and Proteolysis

By designing and coupling two functional peptides, CKAFKRK and C(KAFKRK)₃ in differing ratios to the surface of gold nanoparticles (GNPs), we evaluated the effect of loading on aggregation and proteolysis. Transmission electron microscopy images of the functionalised GNPs indicated a direct relationship between the degree of aggregation of the particles and the extent of peptide loading: The greater the percentage of the C(KAFKRK)₃ peptide, the greater the dispersion (less aggregation) of the peptide-capped GNPs. The functionalised GNPs were subjected to trypsin digestion over increasing time periods and it was found that the peptides were cleaved at the site of Lys and Arg. The extent of cleavage was analysed by mass spectrometry. The results indicate that the rate of enzymatic degradation was directly proportional to the extent of loading, such that the greater percentage of the C(KAFKRK)₃ peptide, the greater the rate and efficiency of the cleavage. These results could be attributed to the different peptide distribution of the particles and the entropy of the peptides with varying peptide ratios.     

Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia brains

Claudins (Cls) are a multigene family of transmembrane proteins with different tissue distribution, which have an essential role in the formation and sealing capacity of tight junctions (TJs). At the level of the blood–brain barrier (BBB), TJs are the main molecular structures which separate the neuronal milieu from the circulatory space, by a restriction of the paracellular flow of water, ions and larger molecules into the brain. Different studies suggested recently significant BBB alterations in both vascular and degenerative dementia types. In a previous study we found in Alzheimer’s disease (AD) and vascular dementia (VaD) brains an altered expression of occludin, a molecular partner of Cls in the TJs structure. Therefore in this study, using an immunohistochemical approach, we investigated the expression of Cl family proteins (Cl‐2, Cl‐5 and Cl‐11) in frontal cortex of aged control, AD and VaD brains. To estimate the number of Cl‐expressing cells, we applied a random systematic sampling and the unbiased optical fractionator method. We found selected neurons, astrocytes, oligodendrocytes and endothelial cells expressing Cl‐2, Cl‐5 and Cl‐11 at detectable levels in all cases studied. We report a significant increase in ratio of neurons expressing Cl‐2, Cl‐5 and Cl‐11 in both AD and VaD as compared to aged controls. The ratio of astrocytes expressing Cl‐2 and Cl‐11 was significantly higher in AD and VaD as compared to aged controls. The ratio of oligodendrocytes expressing Cl‐11 was significantly higher in AD and the ratio of oligodendrocytes expressing Cl‐2 was significantly higher in VaD as compared to aged controls. Within the cerebral cortex, Cls were selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings suggest a new function of Cl family proteins which might be linked to response to cellular stress.     

The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection

Gastrointestinal (GI) anthrax, caused by the bacterial infection of Bacillus anthracis, posts a significant bioterrorism threat by its relatively high mortality rate in humans. Different from inhalational anthrax by the route of infection, accumulating evidence indicates the bypass of vegetative bacteria across GI epithelium is required to initiate GI anthrax. Previously, we reported that purified anthrolysin O (ALO), instead of tripartite anthrax edema and lethal toxins, is capable of disrupting gut epithelial tight junctions and barrier function in cultured cells. Here, we show that ALO can disrupt intestinal tissue barrier function in an ex vivo mouse model. To explore the effects of ALO in a cell culture model of B. anthracis infection, we showed that anthrax bacteria can effectively reduce the monolayer integrity of human Caco-2 brush-border expressor (C2BBE) cells based on the reduced transepithelial resistance and the increased leakage of fluorescent dye. This disruption is likely caused by tight junction dysfunction observed by the reorganization of the tight junction protein occludin. Consequently, we observe significant passage of vegetative anthrax bacteria across C2BBE cells. This barrier disruption and bacterial crossover requires ALO since ALO-deficient B. anthracis strains fail to induce monolayer dysfunction and allow the passage of anthrax bacteria. Together these findings point to a pivotal role for ALO within the establishment of GI anthrax infection and the initial bypass of the epithelial barrier.     

Developmental regulation of group I metabotropic glutamate receptors in the premature brain and their protective role in a rodent model of periventricular leukomalacia

Cerebral white matter injury in premature infants, known as periventricular leukomalacia (PVL), is common after hypoxia-ischemia (HI). While ionotropic glutamate receptors (iGluRs) can mediate immature white matter injury, we have previously shown that excitotoxic injury to premyelinating oligodendrocytes (preOLs) in vitro can be attenuated by group I metabotropic glutamate receptor (mGluR) agonists. Thus, we evaluated mGluR expression in developing white matter in rat and human brain, and tested the protective efficacy of a central nervous system (CNS)-penetrating mGluR agonist on injury to developing oligodendrocytes (OLs) in vivo. Group I mGluRs (mGluR1 and mGluR5) were strongly expressed on OLs in neonatal rodent cerebral white matter throughout normal development, with highest expression early in development on preOLs. Specifically at P6, mGluR1 and mGLuR5 were most highly expressed on GalC-positive OLs compared to neurons, axons, astrocytes and microglia. Systemic administration of (1S,3R) 1-aminocyclopentane-trans-1,3,-dicarboxylic acid (ACPD) significantly attenuated the loss of myelin basic protein in the white matter following HI in P6 rats. Assessment of postmortem human tissue showed both mGluR1 and mGluR5 localized on immature OLs in white matter throughout development, with mGluR5 highest in the preterm period. These data indicate group I mGluRs are highly expressed on OLs during the peak period of vulnerability to HI and modulation of mGluRs is protective in a rodent model of PVL. Group I mGluRs may represent important therapeutic targets for protection from HI-mediated white matter injury.     

The dynamic nature of contact networks in infectious disease epidemiology

Although contact network models have yielded important insights into infectious disease transmission and control throughout the last decade, researchers have just begun to explore the dynamic nature of contact patterns and their epidemiological significance. Most network models have assumed that contacts are static through time. Developing more realistic models of the social interactions that underlie the spread of infectious diseases thus remains an important challenge for both data gatherers and modelers. In this article, we review some recent data-driven and process-driven approaches that capture the dynamics of human contact, and discuss future challenges for the field.     

Effects of polymer molecular weight on the size, activity, and stability of PEG-functionalized trypsin

Polymer conjugation increases an enzyme's circulation time and stability for use as a therapeutic agent, but this attachment indubitably affects its properties. Covalent attachment of multiple polyethylene glycol chains with sizes of either 2, 5, 10, or 20 kDa increases the molecular weight and hydrodynamic radius of the model enzyme trypsin. The sizes of these polymer-enzyme conjugates are increased to be within the recommended limits for PDEPT applications. The T(d) increases from 49 to 60 °C to expand the enzyme's workable range of conditions. This functionalization with PEG polymers of varying lengths maintains trypsin's enzymatic activity. Conjugate activities are 79-120% that of native trypsin at room temperature and 221-432% that of trypsin at 37 °C.     

Direct cellular lysis/protein extraction protocol for soil metaproteomics

We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.