The Role of Peptidyl Prolyl Isomerases in Aging and Vascular Diseases

Peptidyl prolyl isomerases (PPIases) are proteins belonging to the immunophilin family and are characterised by their cis-trans isomerization activity at the X-Pro peptide bond, in addition to their tetratricopeptide repeat (TPR) domain, important for interaction with the molecular chaperone, Hsp90. Due to this unique structure these proteins are able to facilitate protein-protein interactions which can impact significantly on a range of cellular processes such as cell signalling, differentiation, cell cycle progression, metabolic activity and apoptosis. Malfunction and/or dysregulation of most members of this class of proteins promotes cellular damage and tissue/organ failure, predisposing to ageing and age-related diseases. Many individual genes within the PPIase family are associated with several age-related diseases including cardiovascular diseases (CVDs), atherosclerosis, type II diabetes (T2D), chronic kidney disease (CDK), neurodegeneration, cancer and age-related macular degeneration (AMD), in addition to the ageing process itself. This review will focus on the different roles of PPIases, and their therapeutic/biomarker potential in these age-related vascular diseases.

Variables to be considered when assessing the photocatalytic destruction of bacterial pathogens

The current study sought to assess the importance of three common variables on the outcome of TiO₂ photocatalysis experiments with bacteria. Factors considered were (a) ability of test species to withstand osmotic pressure, (b) incubation period of agar plates used for colony counts following photocatalysis and (c) chemical nature of suspension medium used for bacteria and TiO₂. Staphylococcus aureus, Escherichia coli, Salmonella ser. Typhimurium and Pseudomonas aeruginosa were found to vary greatly in their ability to withstand osmotic pressure, raising the possibility that osmotic lysis may be contributing to loss of viability in some photocatalytic disinfection studies. Agar plate incubation time was also found to influence results, as bacteria treated with UV light only grew more slowly than those treated with a combination of UV and TiO_2. The chemical nature of the suspension medium used was found to have a particularly pronounced effect upon results. Greatest antibacterial activity was detected when aqueous sodium chloride solution was utilised, with ∼1 × 10⁶ CFU mL^-1 S. aureus being completely killed after 60 min. Moderate activity was observed when distilled water was employed with bacteria being killed after 2 h and 30 min, and no antibacterial activity at all was detected when aqueous tryptone solution was used. Interestingly, the antibacterial activity of UV light on its own appeared to be very much reduced in experiments where aqueous sodium chloride was employed instead of distilled water. 

Toluene dioxygenase-catalysed oxidation route to angular cis-monohydrodiols and other bioproducts from bacterial metabolism of 1,2-dihydrobenzocyclobutene and derivatives

A mutant strain (UV4) of the soil bacterium Pseudomonas putida, containing toluene dioxygenase, has been used in the metabolic oxidation of 1,2-dihydrobenzocyclobutene 12 dagger and the related substrates 1,2-dihydrobenzocyclobuten-1-ol 13 and biphenylene 33. Stable angular cis-monohydrodiol metabolites (1R,2S)-bicyclo[4.2.0]octa-3,5-diene-1,2 7, (1S,2S,8S)-bicyclo[4.2.0]octa-3,5-diene-1,2,8-triol 8 and biphenylene-cis-1,8b-diol 9, isolated from each of these substrates, have been structurally and stereochemically assigned. The structure, enantiopurity and absolute configuration of the other cis-diol metabolites, (2R,3S)-bicyclo[4.2.0]octa-1(6),4-diene-2,3-diol 14 and cis-1,2-dihydroxy-1,2-dihydrobenzocyclobutene 16, and the benzylic oxidation bioproducts, 1,2-dihydrobenzocyclobuten-1-ol 13, 1,2-dihydrobenzocyclobuten-1-one 15 and 2-hydroxy-1,2-dihydrobenzocyclobuten-1-one 17, obtained from 1,2-dihydrobenzocyclobutene and 1,2-dihydrobenzocyclobuten-1-ol, have been determined with the aid of chiral stationary-phase HPLC, NMR and CD spectroscopy, and stereochemical correlation. X-Ray crystallographic methods have been used in the determination of absolute configuration of the di-camphanates 27 (from diol 7) and 32 (from diol 9), and the di-MTPA ester 29 (from diol 14) of the corresponding cis-diol metabolites. The metabolic sequence involved in the formation of bioproducts derived from 1,2-dihydrobenzocyclobutene 12 has been investigated.

BACTERIAL OXIDATION OF BENZOCYCLOALKENES TO YIELD MONOL, DIOL AND TRIOL METABOLITES

Benzylic monooxygenation of benzocycloalkenes, 2-4, by enzymes in intact cultures of Pseudomonas putida UV4 yielded exclusively the [R] enantiomers, 6-8, and the derived ketones 10-12; by contrast, biotransformation of benzocyclobutene, 1, yielded both monooxygenation (5 and 9), dioxygenation (13, 14 and 15), and trioxygenation (16) products.

Appointment keeping for medical review among patients with selected chronic diseases in an urban area of Uganda

Introduction: proper management of chronic diseases is important for prevention of disease complications and yet some patients miss appointments for medical review thereby missing the opportunity for proper monitoring of their disease conditions. There is limited information on missed appointments among chronic disease patients in resource limited settings. This study aimed to determine the prevalence of missed appointments for medical review and associated factors among chronic disease patients in an urban area of Uganda.

Methods: patients or caregivers of children with chronic diseases were identified as they bought medicines from a community pharmacy. They were visited at home to access their medical documents and those whose chronic disease status was ascertained were enrolled. The data was collected using: questionnaires, review of medical documents, and in-depth interviews with chronic disease patients.

Results: the prevalence of missed appointments was 42% (95%CI=35-49%). The factors associated with missed appointments were: monthly income ?30US Dollars (OR=2.56, CI=1.25–5.26), affording less than half of prescribed drugs (OR=3.92, CI=1.64–9.40), not experiencing adverse events (OR=2.66, CI=1.26–5.61), not sure if treatment helps (OR=2.84, CI=1.047.77), not having a medicines administration schedule (OR=6.77, CI=2.11–21.68), and increasing number of drugs (OR=0.72, CI=0.53–0.98). Conclusion: patients missed appointments mainly due to: financial and health system barriers, conflicting commitments with appointments, and perceptions of the disease condition. Patients should be supported with accessible and affordable health services.

Clinical trial designs for rare diseases: Studies developed and discussed by the International Rare Cancers Initiative

BACKGROUND: The past three decades have seen rapid improvements in the diagnosis and treatment of most cancers and the most important contributor has been research. Progress in rare cancers has been slower, not least because of the challenges of undertaking research.

SETTINGS: The International Rare Cancers Initiative (IRCI) is a partnership which aims to stimulate and facilitate the development of international clinical trials for patients with rare cancers. It is focused on interventional--usually randomized--clinical trials with the clear goal of improving outcomes for patients. The key challenges are organisational and methodological. A multi-disciplinary workshop to review the methods used in ICRI portfolio trials was held in Amsterdam in September 2013. Other as-yet unrealised methods were also discussed.

RESULTS: The IRCI trials are each presented to exemplify possible approaches to designing credible trials in rare cancers. Researchers may consider these for use in future trials and understand the choices made for each design.

INTERPRETATION: Trials can be designed using a wide array of possibilities. There is no 'one size fits all' solution. In order to make progress in the rare diseases, decisions to change practice will have to be based on less direct evidence from clinical trials than in more common diseases.

Interfacial self-assembly of a bacterial hydrophobin

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded β-sheet. This form then self-assembles into a well-ordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

Bdellovibrio predation in the presence of decoys:Three-way bacterial interactions revealed by mathematical and experimental analyses

Bdellovibrio bacteriovorus is a small, gram-negative, motile bacterium that preys upon other gram-negative bacteria, including several known human pathogens. Its predation efficiency is usually studied in pure cultures containing solely B. bacteriovorus and a suitable prey. However, in natural environments, as well as in any possible biomedical uses as an antimicrobial, Bdellovibrio is predatory in the presence of diverse decoys, including live nonsusceptible bacteria, eukaryotic cells, and cell debris. Here we gathered and mathematically modeled data from three-member cultures containing predator, prey, and nonsusceptible bacterial decoys. Specifically, we studied the rate of predation of planktonic late-log-phase Escherichia coli S17-1 prey by B. bacteriovorus HD100, both in the presence and in the absence of Bacillus subtilis nonsporulating strain 671, which acted as a live bacterial decoy. Interestingly, we found that although addition of the live Bacillus decoy did decrease the rate of Bdellovibrio predation in liquid cultures, this addition also resulted in a partially compensatory enhancement of the availability of prey for predation. This effect resulted in a higher final yield of Bdellovibrio than would be predicted for a simple inert decoy. Our mathematical model accounts for both negative and positive effects of predator-prey-decoy interactions in the closed batch environment. In addition, it informs considerations for predator dosing in any future therapeutic applications and sheds some light on considerations for modeling the massively complex interactions of real mixed bacterial populations in nature.

Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.