Mechanisms of Coenzyme Q10 Blood-Brain Barrier Transport

Coenzyme Q10 (CoQ10) deficiencies are unique among mitochondrial respiratory chain (MRC) disorders in that they are potentially treatable. While there is clear evidence, both clinically and biochemically, for the improvement of peripheral abnormalities associated with CoQ10 deficiency following CoQ10 supplementation, neurological symptoms are only partially ameliorated. The reasons for the refractory nature of the neurological sequelae associated with a CoQ10 deficiency are as yet unknown and may be a consequence of irreversible damage prior to CoQ10 supplementation, the retention of CoQ10 in the blood-brain barrier (BBB) itself, or simply reflect poor transport of CoQ10 across the BBB. This thesis presents the first isolated investigations into the mechanisms that govern bi-directional BBB transport of CoQ10 and its synthetic analogue, idebenone, using normal and pathophysiological cell models relevant to disorders of CoQ10 biosynthesis. The mouse BBB endothelial cell line bEnd.3 and porcine primary brain endothelial cells (PBECs) co-cultured with primary astrocytes were used to assess transcytosis from ‘blood-to-brain’ or ‘brain-to-blood’, revealing that although CoQ10 can traverse the BBB, CoQ10 is being effluxed back to the blood, which could explain the refractory nature of CoQ10 therapy, whereas, idebenone appeared to cross the BBB passively. Using inhibitors of known transport systems for lipoproteins, the circulatory bio-carriers of CoQ10 in vivo, three systems mediating the BBB transport of lipoprotein-bound CoQ10 were identified. Inhibitors of the scavenger receptor class B type 1 (SR-B1), BLT-1, and the receptor for advanced glycation end products (RAGE), FPS-ZM1, reduced uptake of lipoprotein-bound CoQ10 towards the brain, implicating RAGE and SR-B1 as modes for CoQ10 brain uptake.. In the reverse direction, the low-density lipoprotein receptor-related protein-1 (LRP-1) inhibitor, RAP, reduced efflux of lipoprotein-bound CoQ10 towards the blood, implicating LRP-1 as a major impediment to brain entry of CoQ10. This study is the first to generate a BBB endothelial cell model of CoQ10 deficiency, using para-aminobenzoic acid (pABA) to pharmacologically induce a depletion of cellular CoQ10 status, resulting in a global reduction of MRC enzyme activities. The CoQ10 deficient BBB models were leakier to large permeability markers, with poor BBB tight-junction formation, and altered CoQ10 transport dynamics in favour of an increased net efflux towards the blood, suggesting BBB pathophysiology is key to the neurological presentation and refractory nature of CoQ10 supplementation in symptomatic patients. In addition, the effects of vitamin E, a common clinical co-therapy in the ‘mito-cocktail’, and simvastatin were assessed. Interestingly, vitamin E co-administration reduced net efflux of CoQ10 from the brain. It is unknown why this occurs, but oxidative effects on the BBB transporters and/or carrier-lipoproteins may be factors to consider. In-line with its deleterious effect on CoQ10 biosynthesis, simvastatin therapy appeared to disrupt BBB integrity, increasing the paracellular leak of the BBB. This would be detrimental to normal brain homeostasis, particularly given the BBBs major role in limiting brain entry of the small molecule plasma excitotoxins, calcium, and glutamate. Throughout this study CoQ10 was quantified using a novel and rapid mass spectrometric method (ESI+ LC-MS/MS), which could potentially enable detection of CoQ10 in the CSF of patients presenting with neurological symptoms, perhaps providing a new analytical tool for the diagnosis of CoQ10 deficiencies in clinical laboratories. In conclusion, this thesis has demonstrated for the first time the pathophysiological consequences of a CoQ10 deficiency on the BBB. It has highlighted the impact of a deficit in CoQ10 status on CoQ10 delivery to the brain parenchyma and has elucidated some of the mechanisms by which CoQ10 is transported across the BBB, which are ultimately dictated by lipoprotein interactions. Additionally, this thesis outlines the potential dangers of statin therapy in patients with an underlying or established MRC dysfunction. Overall, this thesis provides insights into the limitations of CoQ10 supplementation as a therapy for neurological disorders associated with MRC dysfunction and indicates that further work will be required to improve the delivery of exogenous CoQ10 across the BBB, alongside a need for further investigations into the composition of the widely administered ‘mito-cocktail’

Face Masks and Cough Etiquette Reduce the Cough Aerosol Concentration of Pseudomonas aeruginosa in People with Cystic Fibrosis

People with cystic fibrosis (CF) generate Pseudomonas aeruginosa in droplet nuclei during coughing. The use of surgical masks has been recommended in healthcare settings to minimize pathogen transmission between patients with CF.To determine if face masks and cough etiquette reduce viable P. aeruginosa aerosolized during coughing.Twenty-five adults with CF and chronic P. aeruginosa infection were recruited. Participants performed six talking and coughing maneuvers, with or without face masks (surgical and N95) and hand covering the mouth when coughing (cough etiquette) in an aerosol-sampling device. An Andersen Cascade Impactor was used to sample the aerosol at 2 meters from each participant. Quantitative sputum and aerosol bacterial cultures were performed, and participants rated the mask comfort levels during the cough maneuvers.During uncovered coughing (reference maneuver), 19 of 25 (76%) participants produced aerosols containing P. aeruginosa, with a positive correlation found between sputum P. aeruginosa concentration (measured as cfu/ml) and aerosol P. aeruginosa colony-forming units. There was a reduction in aerosol P. aeruginosa load during coughing with a surgical mask, coughing with an N95 mask, and cough etiquette compared with uncovered coughing (P

Efficient, non‐toxic anion transport by synthetic carriers in cells and epithelia

Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.info:eu-repo/semantics/publishe

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

CoQ10 Deficient Endothelial Cell Culture Model for the Investigation of CoQ10 Blood–Brain Barrier Transport

Primary coenzyme Q10 (CoQ10) deficiency is unique among mitochondrial respiratory chain disorders in that it is potentially treatable if high-dose CoQ10 supplements are given in the early stages of the disease. While supplements improve peripheral abnormalities, neurological symptoms are only partially or temporarily ameliorated. The reasons for this refractory response to CoQ10 supplementation are unclear, however, a contributory factor may be the poor transfer of CoQ10 across the blood–brain barrier (BBB). The aim of this study was to investigate mechanisms of CoQ10 transport across the BBB, using normal and pathophysiological (CoQ10 deficient) cell culture models. The study identifies lipoprotein-associated CoQ10 transcytosis in both directions across the in vitro BBB. Uptake via SR-B1 (Scavenger Receptor) and RAGE (Receptor for Advanced Glycation Endproducts), is matched by efflux via LDLR (Low Density Lipoprotein Receptor) transporters, resulting in no “net” transport across the BBB. In the CoQ10 deficient model, BBB tight junctions were disrupted and CoQ10 “net” transport to the brain side increased. The addition of anti-oxidants did not improve CoQ10 uptake to the brain side. This study is the first to generate in vitro BBB endothelial cell models of CoQ10 deficiency, and the first to identify lipoprotein-associated uptake and efflux mechanisms regulating CoQ10 distribution across the BBB. The results imply that the uptake of exogenous CoQ10 into the brain might be improved by the administration of LDLR inhibitors, or by interventions to stimulate luminal activity of SR-B1 transporters

A multi‐method approach to analyse changes in gully characteristics between 2009 and 2018 in southeast Nigeria

Gully erosion is the dominant environmental problem in southeast Nigeria and has led to loss of human and material resources. In this study, we evaluated changes in gully characteristics in southeast Nigeria and their potential drivers between 2009 and 2018 using a multi-method approach: analysis of high-resolution satellite imagery (2–5 m) and focus group discussions. Gully numbers increased from 26 to 39 and mean gully length increased from 0.39 to 0.43 km. We found that land adjacent to rivers had the highest concentration of gullies which is associated with an increase in slope angle from 10 to 58% up to 500 m from rivers. Regarding potential gully-drivers, land-use changes were observed. Non-vegetated lands increased from 58.6 to 144.7 km2 between 2009 and 2018, while reductions in fallow lands from 281.2 to 57.8 km2 were observed. Results from focus group meetings indicate there was no gullying in the area before the Nigerian civil war (1967–1970). War-time activities such as digging trenches and increased population density were said to have led to the formation of the oldest gullies in the area. Although war-time activities have ceased, meeting attendees believed that present land-use changes have increased the volume of surface runoff and thus enhancing gully erosion. Incorporating local knowledge in this study has therefore provided a valuable understanding on the key drivers of gullying that pre-dates the availability of freely available high-resolution satellite data

Alternating regimes of shallow and deep-sea diversification explain a species-richness paradox in marine fishes

R scripts and R packages,The deep sea contains a surprising diversity of life, including iconic fish groups such as anglerfishes and lanternfishes. Still, \u3e 65% of marine teleost fish species are restricted to the photic zone \u3c 200 m, which comprises less than 10% of the ocean’s total volume. From a macroevolutionary perspective, this paradox may be explained by three hypotheses: 1) shallow-water lineages have had more time to diversify than deep-sea lineages, 2) shallow-water lineages have faster rates of speciation than deep-sea lineages, or 3) shallow-to-deep sea transition rates limit deep-sea richness. Here we use phylogenetic comparative methods to test among these three non-mutually exclusive hypotheses. While we found support for all hypotheses, the disparity in species richness is better described as the uneven outcome of alternating phases that favored shallow or deep diversification over the past 200 million y. Shallow marine teleosts became incredibly diverse 100 million years ago during a period of warm temperatures and high sea level, suggesting the importance of reefs and epicontinental settings. Conversely, deep-sea colonization and speciation were favored during brief episodes when cooling temperatures increased the efficiency of the ocean’s carbon pump. Finally, time-variable ecological filters limited shallow-to-deep colonization for much of teleost history, which helped maintain higher shallow richness. A pelagic lifestyle and large jaws were associated with early deep-sea colonists, while a demersal lifestyle and a tapered body plan were typical of later colonists. Therefore, we also suggest that some hallmark characteristics of deep-sea fishes evolved prior to colonizing the deep sea