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.

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.

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.

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.

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.

A dry immersion model of microgravity modulates platelet phenotype, miRNA signature, and circulating plasma protein biomarker profile.

Ground based research modalities of microgravity have been proposed as innovative methods to investigate the aetiology of chronic age-related conditions such as cardiovascular disease. Dry Immersion (DI), has been effectively used to interrogate the sequelae of physical inactivity (PI) and microgravity on multiple physiological systems. Herein we look at the causa et effectus of 3-day DI on platelet phenotype, and correlate with both miRomic and circulating biomarker expression. The miRomic profile of platelets is reflective of phenotype, which itself is sensitive and malleable to the exposome, undergoing responsive transitions in order to fulfil platelets role in thrombosis and haemostasis. Heterogeneous platelet subpopulations circulate at any given time, with varying degrees of sensitivity to activation. Employing a DI model, we investigate the effect of acute PI on platelet function in 12 healthy males. 3-day DI resulted in a significant increase in platelet count, plateletcrit, platelet adhesion, aggregation, and a modest elevation of platelet reactivity index (PRI). We identified 15 protein biomarkers and 22 miRNA whose expression levels were altered after DI. A 3-day DI model of microgravity/physical inactivity induced a prothrombotic platelet phenotype with an unique platelet miRNA signature, increased platelet count and plateletcrit. This correlated with a unique circulating protein biomarker signature. Taken together, these findings highlight platelets as sensitive adaptive sentinels and functional biomarkers of epigenetic drift within the cardiovascular compartment.

Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Antibiotics are life-saving medicines, but many bacteria now have the ability to resist their effects. For some infections, all frontline antibiotics are now ineffective. To treat infections caused by these highly resistant bacteria, clinicians must use so-called 'antibiotics of last resort'. These antibiotics include a drug called colistin, which is moderately effective, but often fails to eradicate the infection. One of the challenges to making colistin more effective is that its mechanism is poorly understood. Bacteria have two layers of protection against the outside world: an outer cell membrane and an inner cell membrane. To kill them, colistin must punch holes in both. First, it disrupts the outer membrane by interacting with molecules called lipopolysaccharides. But how it disrupts the inner membrane was unclear. Bacteria have evolved several different mechanisms that make them resistant to the effects of colistin. Sabnis et al. reasoned that understanding how these mechanisms protected bacteria could reveal how the antibiotic works to damage the inner cell membrane. Sabnis et al. examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that the antibiotic damages both layers of the cell surface in the same way, targeting lipopolysaccharide in the inner membrane as well as the outer membrane. Next, Sabnis et al. used this new information to make colistin work better. They found that the effects of colistin were magnified when it was combined with the experimental antibiotic murepavadin, which caused lipopolysaccharide to build up at the inner membrane. This allowed colistin to punch more holes through the inner membrane, making colistin more effective at killing bacteria. To find out whether this combination of colistin and murepavadin could work as a clinical treatment, Sabnis et al. tested it on mice with Pseudomonas aeruginosa infections in their lungs. Colistin was much better at killing Pseudomonas aeruginosa and treating infections when combined with murepavadin than it was on its own. Pseudomonas aeruginosa bacteria can cause infections in the lungs of people with cystic fibrosis. At the moment, patients receive colistin in an inhaled form to treat these infections, but it is not always successful. The second drug used in this study, murepavadin, is about to enter clinical trials as an inhaled treatment for lung infections too. If the trial is successful, it may be possible to use both drugs in combination to treat lung infections in people with cystic fibrosis.

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.

Elucidating the Biological Activity of Fish-Derived Collagen and Gelatine Hydrolysates using Animal Cell Culture - A Review.

A large percentage of a fish's weight is generally discarded during fish processing. Reducing the waste products of marine origin is a subject of great interest within the scientific community. Pelagic byproducts, such as the structural protein collagen, which can be generated during the processing of fish, have been proposed as an alternative to terrestrial, mammalian sources due to advantages including high availability and low risk of zoonotic disease transmission. Gelatine has multiple possible applications, ranging from nutraceutical applications to cosmetics and has the advantage of being generally regarded as safe. In this multidisciplinary review, the chemistry of gelatine and its parent protein collagen, the chemical reactions to generate their hydrolysates, and studies on their biological activities using animal cell culture are discussed.

Vascular and Microvascular Dysfunction Induced by Microgravity and Its Analogs in Humans: Mechanisms and Countermeasures.

Weightlessness and physical inactivity have deleterious cardiovascular effects. The space environment and its ground-based models offer conditions to study the cardiovascular effects of physical inactivity in the absence of other vascular risk factors, particularly at the macro- and microcirculatory levels. However, the mechanisms involved in vascular dysfunction and remodeling are not sufficiently studied in the context of weightlessness and its analogs including models of physical inactivity. Here, we summarize vascular and microvascular changes induced by space flight and observed in models of microgravity and physical inactivity and review the effects of prophylactic strategies (i.e., countermeasures) on vascular and microvascular function. We discuss physical (e.g., exercise, vibration, lower body negative pressure, and artificial gravity) and nutritional/pharmacological (e.g., caloric restriction, resveratrol, and other vegetal extracts) countermeasures. Currently, exercise countermeasure appears to be the most effective to protect vascular function. Although pharmacological countermeasures are not currently considered to fight vascular changes due to microgravity, nutritional countermeasures are very promising. Dietary supplements/natural health products, especially plant extracts, should be extensively studied. The best prophylactic strategy is likely a combination of countermeasures that are effective not only at the cardiovascular level but also for the organism as a whole, but this strategy remains to be determined.

Brain structure changes over time in normal and mildly impaired aged persons.

Structural brain changes in aging are known to occur even in the absence of dementia, but the magnitudes and regions involved vary between studies. To further characterize these changes, we analyzed paired MRI images acquired with identical protocols and scanner over a median 5.8-year interval. The normal study group comprised 78 elders (25M 53F, baseline age range 70-78 years) who underwent an annual standardized expert assessment of cognition and health and who maintained normal cognition for the duration of the study. We found a longitudinal grey matter (GM) loss rate of 2.56 ± 0.07 ml/year (0.20 ± 0.04%/year) and a cerebrospinal fluid (CSF) expansion rate of 2.97 ± 0.07 ml/year (0.22 ± 0.04%/year). Hippocampal volume loss rate was higher than the GM and CSF global rates, 0.0114 ± 0.0004 ml/year (0.49 ± 0.04%/year). Regions of greatest GM loss were posterior inferior frontal lobe, medial parietal lobe and dorsal cerebellum. Rates of GM loss and CSF expansion were on the low end of the range of other published values, perhaps due to the relatively good health of the elder volunteers in this study. An additional smaller group of 6 subjects diagnosed with MCI at baseline were followed as well, and comparisons were made with the normal group in terms of both global and regional GM loss and CSF expansion rates. An increased rate of GM loss was found in the hippocampus bilaterally for the MCI group.

Development of dynamic cell and organotypic skin models, for the investigation of a novel visco-elastic burns treatment using molecular and cellular approaches.

BACKGROUND: Burn injuries are a major cause of morbidity and mortality worldwide. Despite advances in therapeutic strategies for the management of patients with severe burns, the sequelae are pathophysiologically profound, up to the systemic and metabolic levels. Management of patients with a severe burn injury is a long-term, complex process, with treatment dependent on the degree and location of the burn and total body surface area (TBSA) affected. In adverse conditions with limited resources, efficient triage, stabilisation, and rapid transfer to a specialised intensive care burn centre is necessary to provide optimal outcomes. This initial lag time and the form of primary treatment initiated, from injury to specialist care, is crucial for the burn patient. This study aims to investigate the efficacy of a novel visco-elastic burn dressing with a proprietary bio-stimulatory marine mineral complex (MXC) as a primary care treatment to initiate a healthy healing process prior to specialist care. METHODS: A new versatile emergency burn dressing saturated in a >90% translucent water-based, sterile, oil-free gel and carrying a unique bio-stimulatory marine mineral complex (MXC) was developed. This dressing was tested using LabSkin as a burn model platform. LabSkin a novel cellular 3D-dermal organotypic full thickness human skin equivalent, incorporating fully-differentiated dermal and epidermal components that functionally models skin. Cell and molecular analysis was carried out by in vitro Real-Time Cellular Analysis (RTCA), thermal analysis, and focused transcriptomic array profiling for quantitative gene expression analysis, interrogating both wound healing and fibrosis/scarring molecular pathways. In vivo analysis was also performed to assess the bio-mechanical and physiological effects of this novel dressing on human skin. RESULTS: This hybrid emergency burn dressing (EBD) with MXC was hypoallergenic, and improved the barrier function of skin resulting in increased hydration up to 24 h. It was demonstrated to effectively initiate cooling upon application, limiting the continuous burn effect and preventing local tissue from damage and necrosis. xCELLigence RTCA® on primary human dermal cells (keratinocyte, fibroblast and micro-vascular endothelial) demonstrated improved cellular function with respect to tensegrity, migration, proliferation and cell-cell contact (barrier formation) [1]. Quantitative gene profiling supported the physiological and cellular function finding. A beneficial quid pro quo regulation of genes involved in wound healing and fibrosis formation was observed at 24 and 48 h time points. CONCLUSION: Utilisation of this EBD + MXC as a primary treatment is an effective and easily applicable treatment in cases of burn injury, proving both a cooling and hydrating environment for the wound. It regulates inflammation and promotes healing in preparation for specialised secondary burn wound management. Moreover, it promotes a healthy remodelling phenotype that may potentially mitigate scarring. Based on our findings, this EBD + MXC is ideal for use in all pre-hospital, pre-surgical and resource limited settings.

DI-5-CUFFS: Venoconstrictive Thigh Cuffs Limit Body Fluid Changes but Not Orthostatic Intolerance Induced by a 5-Day Dry Immersion.

Venoconstrictive thigh cuffs are used by cosmonauts to ameliorate symptoms associated with cephalad fluid shift. A ground simulation of microgravity, using the dry immersion (DI) model, was performed to assess the effects of thigh cuffs on body fluid changes and dynamics, as well as on cardiovascular deconditioning. Eighteen healthy men (25-43 years), randomly divided into two groups, (1) control group or (2) group with thigh cuffs worn 10 h/day, underwent 5-day DI. Cardiovascular responses to orthostatic challenge were evaluated using the lower body negative pressure (LBNP) test; body fluid changes were assessed by bio-impedance and hormonal assay; plasma volume evolution was estimated using hemoglobin-hematocrit; subjective tolerance was assessed by questionnaires. DI induced a decrease in plasma volume of 15-20%. Reduction in total body water of 3-6% stabilized toward the third day of DI. This reduction was derived mostly from the extracellular compartment. During the acute phase of DI, thigh cuffs limited the decrease in renin and the increase in N-terminal prohormone of brain natriuretic peptide (NT-proBNP), the loss in total body water, and tended to limit the loss in calf volume, extracellular volume and plasma volume. At the later stable phase of DI, a moderate protective effect of thigh cuffs remained evident on the body fluids. Orthostatic tolerance time dropped after DI without significant difference between groups. Thigh cuff countermeasure slowed down and limited the loss of body water and tended to limit plasma loss induced by DI. These observed physiological responses persisted during periods when thigh cuffs were removed. However, thigh cuffs did not counteract decreased tolerance to orthostatic challenge.

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.