Pseudomonas aeruginosa in the Cystic Fibrosis Lung.

Cystic fibrosis is a common genetically inherited, multisystem disorder caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an apically situated anion channel. In the lung, lack of CFTR leads to airway surface dehydration, mucociliary clearance failure and an acidic pH in which innate defence molecules are rendered ineffective. Infection occurs early in life, with P. aeruginosa dominating by adolescence. The characteristic features of the CF airway highlighted above encourage persistence of infection, but P. aeruginosa also possess an array of mechanisms with which they attack host defences and render themselves protected from antimicrobials. Early eradication is usually successful, but this is usually transient. Chronic infection is manifest by biofilm formation which is resistant to treatment. Outcomes for people with CF have improved greatly in the last few decades, but particularly so with the recent advent of small molecule CFTR modulators. However, despite impressive efficacy on lung function and exacerbation frequency, most people with chronic infection remain with their pathogens. There is an active pipeline of new treatments including anti-biofilm and anti-quorum sensing molecules and non-drug approaches such as bacteriophage. Studies are reviewed and challenges for future drug development considered.

Maximal oxygen consumption and oxygen uptake efficiency in adolescent males.

BACKGROUND/OBJECTIVE: Measures of oxygen uptake efficiency (OUE) have been used to evaluate cardiorespiratory fitness (CRF) in adolescents unable to perform maximal exercise. The oxygen uptake efficiency slope (OUES) and oxygen uptake efficiency plateau (OUEP) have been proposed as surrogates for maximal oxygen consumption (V̇O2max). We assessed the validity of the OUES and OUEP as predictors of V̇O2max in healthy male adolescents. METHODS: Sixty-three healthy male adolescents aged 15.40 ± 0.34 years underwent an incremental treadmill test to determine V̇O2max, OUES and OUEP. OUE throughout the test was assessed by dividing each V̇O2 value by the corresponding minute ventilation (V̇E) value. OUEP was determined as the 90 s average highest consecutive values for OUE. OUES was determined using data up to the ventilatory threshold (VT) by calculating the slope of the linear relation between V̇O2 and the logarithm of V̇E. RESULTS: Limits of agreement for V̇O2max predicted by OUES (±13.3 mL kg-1.min-1) and OUEP (±16.7 mL kg-1.min-1) relative to V̇O2max were wide and a magnitude bias was found for OUES and OUEP as predictors of V̇O2max (p < 0.001). CONCLUSION: The OUES and OUEP do not accurately predict V̇O2max in male adolescents and should not replace V̇O2max when assessing CRF in this population.

Circulating angiogenic cell response to sprint interval and continuous exercise.

INTRODUCTION: Although commonly understood as immune cells, certain T lymphocyte and monocyte subsets have angiogenic potential, contributing to blood vessel growth and repair. These cells are highly exercise responsive and may contribute to the cardiovascular benefits seen with exercise. PURPOSE: To compare the effects of a single bout of continuous (CONTEX) and sprint interval exercise (SPRINT) on circulating angiogenic cells (CAC) in healthy recreationally active adults. METHODS: Twelve participants (aged 29 ± 2 years, BMI 25.5 ± 0.9 kg m- 2, [Formula: see text]peak 44.3 ± 1.8 ml kg- 1 min- 1; mean ± SEM) participated in the study. Participants completed a 45-min bout of CONTEX at 70% peak oxygen uptake and 6 × 20 s sprints on a cycle ergometer, in a counterbalanced design. Blood was sampled pre-, post-, 2 h and 24 h post-exercise for quantification of CAC subsets by whole blood flow cytometric analysis. Angiogenic T lymphocytes (TANG) and angiogenic Tie2-expressing monocytes (TEM) were identified by the expression of CD31 and Tie2, respectively. RESULTS: Circulating (cells µL- 1) CD3+CD31+ TANG increased immediately post-exercise in both trials (p < 0.05), with a significantly greater increase (p < 0.05) following SPRINT (+ 57%) compared to CONTEX (+ 14%). Exercise increased (p < 0.05) the expression of the chemokine receptor CXCR4 on TANG at 24 h. Tie2-expressing classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes and circulating CD34+CD45dim progenitor cells were higher post-exercise in SPRINT, but unchanged in CONTEX. All post-exercise increases in SPRINT were back to pre-exercise levels at 2 h and 24 h. CONCLUSION: Acute exercise transiently increases circulating TANG, TEM and progenitor cells with greater increases evident following very high intensity sprint exercise than following prolonged continuous paced endurance exercise.

Neutral genomic microevolution of a recently emerged pathogen, Salmonella enterica serovar Agona.

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks.

A human 3'UTR clone collection to study post-transcriptional gene regulation.

BACKGROUND: 3'untranslated regions (3'UTRs) are poorly understood portions of eukaryotic mRNAs essential for post-transcriptional gene regulation. Sequence elements in 3'UTRs can be target sites for regulatory molecules such as RNA binding proteins and microRNAs (miRNAs), and these interactions can exert significant control on gene networks. However, many such interactions remain uncharacterized due to a lack of high-throughput (HT) tools to study 3'UTR biology. HT cloning efforts such as the human ORFeome exemplify the potential benefits of genomic repositories for studying human disease, especially in relation to the discovery of biomarkers and targets for therapeutic agents. Currently there are no publicly available human 3'UTR libraries. To address this we have prepared the first version of the human 3'UTRome (h3'UTRome v1) library. The h3'UTRome is produced to a single high quality standard using the same recombinational cloning technology used for the human ORFeome, enabling universal operating methods and high throughput experimentation. The library is thoroughly sequenced and annotated with simple online access to information, and made publically available through gene repositories at low cost to all scientists with minimal restriction. RESULTS: The first release of the h3'UTRome library comprises 1,461 human 3'UTRs cloned into Gateway® entry vectors, ready for downstream analyses. It contains 3'UTRs for 985 transcription factors, 156 kinases, 171 RNA binding proteins, and 186 other genes involved in gene regulation and in disease. We demonstrate the feasibility of the h3'UTRome library by screening a panel of 87 3'UTRs for targeting by two miRNAs: let-7c, which is implicated in tumorigenesis, and miR-221, which is implicated in atherosclerosis and heart disease. The panel is enriched with genes involved in the RAS signaling pathway, putative novel targets for the two miRNAs, as well as genes implicated in tumorigenesis and heart disease. CONCLUSIONS: The h3'UTRome v1 library is a modular resource that can be utilized for high-throughput screens to identify regulatory interactions between trans-acting factors and 3'UTRs, Importantly, the library can be customized based on the specifications of the researcher, allowing the systematic study of human 3'UTR biology.

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation.

Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in ß-catenin expression. ß-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6(-/-)), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects.

Thrombomodulin (TM) is a 557-amino acid protein with a broad cell and tissue distribution consistent with its wide-ranging physiological roles. When expressed on the lumenal surface of vascular endothelial cells in both large vessels and capillaries, its primary function is to mediate endothelial thromboresistance. The complete integral membrane-bound protein form displays five distinct functional domains, although shorter soluble (functional) variants comprising the extracellular domains have also been reported in fluids such as serum and urine. TM-mediated binding of thrombin is known to enhance the specificity of the latter serine protease toward both protein C and thrombin activatable fibrinolysis inhibitor (TAFI), increasing their proteolytic activation rate by almost three orders of magnitude with concomitant anticoagulant, antifibrinolytic, and anti-inflammatory benefits to the vascular wall. Recent years have seen an abundance of research into the cellular mechanisms governing endothelial TM production, processing, and regulation (including flow-mediated mechanoregulation)--from transcriptional and posttranscriptional (miRNA) regulation of TM gene expression, to posttranslational processing and release of the expressed protein--facilitating greater exploitation of its therapeutic potential. The goal of the present paper is to comprehensively review the endothelial/TM system from these regulatory perspectives and draw some fresh conclusions. This paper will conclude with a timely examination of the current status of TM's growing therapeutic appeal, from novel strategies to improve the clinical efficacy of recombinant TM analogs for resolution of vascular disorders such as disseminated intravascular coagulation (DIC), to an examination of the complex pleiotropic relationship between statin treatment and TM expression.

Dynamics of protein folding: probing the kinetic network of folding-unfolding transitions with experiment and theory.

The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been achieved owing to the rapidly growing interdisciplinary interest and significant progress in both experimental techniques and theoretical methods. Improvements in the experimental time resolution led to determination of the timescales of the important elementary events in folding, such as formation of secondary structure and tertiary contacts. Sensitive single molecule methods made possible probing the distributions of the unfolded and folded states and following the folding reaction of individual protein molecules. Discovery of proteins that fold in microseconds opened the possibility of atomic-level theoretical simulations of folding and their direct comparisons with experimental data, as well as of direct experimental observation of the barrier-less folding transition. The ultra-fast folding also brought new questions, concerning the intrinsic limits of the folding rates and experimental signatures of barrier-less "downhill" folding. These problems will require novel approaches for even more detailed experimental investigations of the folding dynamics as well as for the analysis of the folding kinetic data. For theoretical simulations of folding, a main challenge is how to extract the relevant information from overwhelmingly detailed atomistic trajectories. New theoretical methods have been devised to allow a systematic approach towards a quantitative analysis of the kinetic network of folding-unfolding transitions between various configuration states of a protein, revealing the transition states and the associated folding pathways at multiple levels, from atomistic to coarse-grained representations. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Communicable diseases in humanitarian operations and disasters.

Military organisations have battled communicable disease for millennia. They have pioneered disease prevention from the Crusades to the World Wars and continue to do so today. Predeployment vaccinations and chemoprophylaxis are effective in preventing communicable disease, as is reliable vector destruction and bite prevention, especially in the era of multidrug resistant organisms. These measures are unlikely to be fully possible in disasters, but reactive vaccination and efforts to reduce exposure to communicable disease should be a priority. Communicable diseases can be challenging to diagnose-the UK Defence Medical Services have become familiar with tools such as multiplex PCR and mass spectrometry. These have the potential to accurately identify organisms and sensitivity patterns in austere environments. Management of communicable diseases depends on accurate diagnosis and has a largely well-established evidence base but can be limited by a lack of resources and skills in an austere setting, therefore telemedicine can assist diagnosis and treatment of infections by projecting specialist skill. Systems such as EpiNATO2 are useful in monitoring diseases and identifying trends in order to establish control measures. Many of these tools and techniques are effective in austere environments and offer learning opportunities for those providing care in similar settings. Further research is ongoing into diagnostic tools as well as remote management.

Effect of a brief dietary counselling intervention on emergency department cardiac chest pain presentations.

Introduction: 15% of all presentations to our emergency department last year were chest pain related. This presented an opportunity to evaluate the impact of a brief physician counselling intervention on patient-reported changes in cardio-protective foodstuff intake. Methods: This is a prospective non-randomised before and after comparison study without controls, conducted between an emergency department presentation and a scheduled follow-up visit at a cardiac diagnostics department. Participants were recruited between February and March 2021. The selected dietary components for inclusion after review of the literature were green leafy vegetables, other coloured vegetables, wholegrains, legumes and fruits. A food frequency questionnaire was completed by patients before and after a physician counselling intervention aided by a dietary infographic. Additionally, using the transtheoretical model for health behaviour change, we assessed each patient's evolution during the study. Results: 38 patients were recruited. For patients with total baseline consumptions of five or fewer per day, there was an increase in cardioprotective foodstuff intakes (z=-2.784 p<0.005 effect size 0.39). Corresponding to this, there was a participant shift observed towards the action and maintenance phases of behaviour change from the contemplation and preparation phases. Discussion: We demonstrated a statistically significant change with moderate effect size using a simple infographic, coupled with brief physician counselling, to promote increased intake of cardioprotective foodstuffs by patients with poor baseline intakes (<5 cardio-protective foods per day) and known modifiable risk factors for ischaemic heart disease. Conclusion: Diet is one arm in the prevention of cardiovascular disease that is often neglected by physicians. This study found that a brief dietary counselling intervention applied in an emergency department setting, administered by non-nutritionists can have a role in changing patient dietary behaviour.

Diversity and prevalence of type VI secretion system effectors in clinical Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa is an opportunistic pathogen and a major driver of morbidity and mortality in people with Cystic Fibrosis (CF). The Type VI secretion system (T6SS) is a molecular nanomachine that translocates effectors across the bacterial membrane into target cells or the extracellular environment enabling intermicrobial interaction. P. aeruginosa encodes three T6SS clusters, the H1-, H2- and H3-T6SS, and numerous orphan islands. Genetic diversity of T6SS-associated effectors in P. aeruginosa has been noted in reference strains but has yet to be explored in clinical isolates. Here, we perform a comprehensive bioinformatic analysis of the pangenome and T6SS effector genes in 52 high-quality clinical P. aeruginosa genomes isolated from CF patients and housed in the Personalised Approach to P. aeruginosa strain repository. We confirm that the clinical CF isolate pangenome is open and principally made up of accessory and unique genes that may provide strain-specific advantages. We observed genetic variability in some effector/immunity encoding genes and show that several well-characterised vgrG and PAAR islands are absent from numerous isolates. Our analysis shows clear evidence of disruption to T6SS genomic loci through transposon, prophage, and mobile genetic element insertions. We identified an orphan vgrG island in P. aeruginosa strain PAK and five clinical isolates using in silico analysis which we denote vgrG7, predicting a gene within this cluster to encode a Tle2 lipase family effector. Close comparison of T6SS loci in clinical isolates compared to reference P. aeruginosa strain PAO1 revealed the presence of genes encoding eight new T6SS effectors with the following putative functions: cytidine deaminase, lipase, metallopeptidase, NADase, and pyocin. Finally, the prevalence of characterised and putative T6SS effectors were assessed in 532 publicly available P. aeruginosa genomes, which suggests the existence of accessory effectors. Our in silico study of the P. aeruginosa T6SS exposes a level of genetic diversity at T6SS genomic loci not seen to date within P. aeruginosa, particularly in CF isolates. As understanding the effector repertoire is key to identifying the targets of T6SSs and its efficacy, this comprehensive analysis provides a path for future experimental characterisation of these mediators of intermicrobial competition and host manipulation.

Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin against Cystic Fibrosis Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonize and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate antipseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers, potentiating the activities of antibiotics when given in combination, restoring and/or enhancing antibiotic efficacy. Growth, viability, MIC determinations, and synergy analysis showed that GA improved the efficacy of tobramycin (TOB) against reference strains of P. aeruginosa, reducing TOB MICs and synergizing with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the MIC50 of TOB for viable cells from 1.69 mg/L (95% confidence interval [CI], 0.26 to 8.97) to 0.62 mg/L (95% CI, 0.15 to 3.94; P = 0.002) and the MIC90 for viable cells from 7.00 mg/L (95% CI, 1.18 to 26.50) to 2.20 mg/L (95% CI, 0.99 to 15.03; P = 0.001), compared to results with TOB only. Investigation of mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarization of cytoplasmic membranes, and permeabilization of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three mechanisms. IMPORTANCE The antimicrobial resistance crisis urgently requires solutions to the lost efficacy of antibiotics. The repurposing of drugs already in clinical use, with strong safety profiles, as antibiotic adjuvants to restore the efficacy of antibiotics is an important avenue to alleviating the resistance crisis. This research shows that a clinically used drug from outside infection treatment, glatiramer acetate, reduces the concentration of tobramycin required to be effective in treating Pseudomonas aeruginosa, based on analyses of both reference and clinical respiratory isolates from people with cystic fibrosis. The two agents acted synergistically against P. aeruginosa, being more effective combined in vitro than predicted for their combination. As a peptide drug, glatiramer acetate functions similarly to many antimicrobial peptides, interacting with and disrupting the P. aeruginosa cell wall and permeabilizing bacterial cells, thereby allowing tobramycin to work. Our findings demonstrate that glatiramer acetate is a strong candidate for repurposing as an antibiotic resistance breaker of pathogenic P. aeruginosa.

Stabilization of brain microvascular endothelial barrier function by shear stress involves VE-cadherin signaling leading to modulation of pTyr-occludin levels.

Blood-brain barrier (BBB) regulation involves the coordinated interaction of intercellular adherens and tight junctions in response to stimuli. One such stimulus, shear stress, has been shown to upregulate brain microvascular endothelial cell (BMvEC) barrier function, although our knowledge of the signaling mechanisms involved is limited. In this article, we examined the hypothesis that VE-cadherin can transmit shear signals to tight junction occludin with consequences for pTyr-occludin and barrier function. In initial studies, chronic shear enhanced membrane localization of ZO-1 and claudin-5, decreased pTyr-occludin (in part via a dephostatin-sensitive mechanism), and reduced BMvEC permeability, with flow reduction in pre-sheared BMvECs having converse effects. In further studies, VE-cadherin inhibition (VE-cad ΔEXD) blocked shear-induced Rac1 activation, pTyr-occludin reduction, and barrier upregulation, consistent with an upstream role for VE-cadherin in transmitting shear signals to tight junctions through Rac1. As VE-cadherin is known to mediate Rac1 activation via Tiam1 recruitment, we subsequently confirmed that Tiam1 inhibition (Tiam1-C580) could elicit effects similar to VE-cad ΔEXD. Finally, the observed attenuation of shear-induced changes in pTyr-occludin level and barrier phenotype following Rac1 inhibition (NSC23766, T17N) establishes a downstream role for Rac1 in this pathway. In summary, we describe for the first time in BMvECs a role for VE-cadherin in the transmission of physiological shear signals to tight junction occludin through engagement of Tiam1/Rac1 leading to barrier stabilization. A downstream role is also strongly indicated for a protein tyrosine phosphatase in pTyr-occludin modulation. Importantly, these findings suggest an important route of inter-junctional signaling cross-talk during BBB response to flow.

Revival of Seeliger's historical 'Special Listeria Culture Collection'.

The recent evolution of bacterial species can be elucidated with the aid of large historical strain collections. Unfortunately, information for some of these strain collections is not publicly available, or only in a format which is not readily digitized. The form of storage of traditional collection often requires considerable space and microbiological access to the individual strains can be time consuming. One such historical strain collection was assembled by Professor H.P. Seeliger, the so-called 'Special Listeria Culture Collection' (SLCC). The SLCC contains over 6000 Listeria strains which had been isolated between 1921 and 1987. The information on the properties of the strains was hand written or typed, primarily in German, and the stabs and lyophils used for storage were not ordered. Here we present a description of this strain collection after resuscitation and digitalization. Data were transcribed into a relational database and the revived bacterial strains were stored in a robotically friendly format, where the location of each tube is stored in a database. We resuscitated 4404 Listeria strains from the SLCC, and summarize their properties as well as making the detailed strain information publicly available. This digital information and the revival of the SLCC will facilitate historical analyses of the phylogeography of Listeria.