Search

The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline BAP1 mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (w-) and a promoter trap (KO) into human mesothelial cells. BAP1w-/KO MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture–mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in BAP1w-/KO MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines. Implications: Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.

The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline BAP1 mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (w-) and a promoter trap (KO) into human mesothelial cells. BAP1w-/KO MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture–mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in BAP1w-/KO MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines.Implications:Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.

Metabolomics MeT5A isogenics

Supplementary Methods, References and List of Figures and Tables

Fig. S1. SNP6.0 analysis of parental MeT5A-BAP1+/+ cells.Fig. S2. Expression of catalytically active BAP1 in parental MeT5A.Fig. S3. Characterisation of BAP1 expression in gene-edited MeT5A.Fig. S4. Whole genome sequencing of gene-edited MeT5A.Fig. S5. Proliferative profiles for gene-edited MeT5A.Fig. S6. Enriched GO terms in gene-edited MeT5A SILAC-MS.Fig. S7. Enriched KEGG pathways in gene-edited MeT5A SILAC-MS include EMT.Fig. S8. Metabolite responses to BAP1 mutation in isogenic MeT5A.Fig. S9. Immunoblotting for differentially expressed metabolic enzymes identified by SILAC-MS in isogenic MeT5A cell lines.Fig. S10. Characterisation of BAP1-status for MPM cell panel.Fig. S11. Evaluating correlation between BAP1 and selected metabolic enzymes in a panel of MPM cell lines and the TCGA MESO pan-cancer dataset.Fig. S12. Validation of ASS1 response to BAP1 alteration.Fig. S13. Improved prognosis for epithelioid MPM patients with loss of nBAP1 and increased expression of ASS1.Fig. S14. Relationship between BAP1 and ASS1 transcripts in the TCGA Pan-Cancer datasets for other cancer types.Fig. S15. The influence of BAP1-status on response to inhibition of purine metabolism.Fig. S16. The influence of BAP1-status on response to ASS1 inhibition.

SUMMARYPhase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes.GRAPHICAL ABSTRACT

Societal biosecurity – measures built into everyday society to minimize risks from pests and diseases – is an important aspect of managing epidemics and pandemics. We aimed to identify societal options for reducing the transmission and spread of respiratory viruses. We used SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as a case study to meet the immediate need to manage the COVID-19 pandemic and eventually transition to more normal societal conditions, and to catalog options for managing similar pandemics in the future. We used a ‘solution scanning’ approach. We read the literature; consulted psychology, public health, medical, and solution scanning experts; crowd-sourced options using social media; and collated comments on a preprint. Here, we present a list of 519 possible measures to reduce SARS-CoV-2 transmission and spread. We provide a long list of options for policymakers and businesses to consider when designing biosecurity plans to combat SARS-CoV-2 and similar pathogens in the future. We also developed an online application to help with this process. We encourage testing of actions, documentation of outcomes, revisions to the current list, and the addition of further options.

ObjectivesSophisticated scores have been proposed for prognostication of mortality due to SARS-CoV-2 but perform inconsistently. We conducted these meta-analyses to uncover why and to pragmatically seek a single dependable biomarker for mortality.DesignWe searched the PubMed database for the keywords ‘SARS-CoV-2’ with ‘biomarker name’ and ‘mortality’. All studies published from 01stDecember 2019 to 30thJune 2021 were surveyed. To aggregate the data, themetalibrary in R was used to report overall mean values and 95% confidence intervals. We fitted a random effects model to obtain pooled AUCs and associated 95% confidence intervals for the European/North American, Asian, and overall datasets.Setting and ParticipantsData was collected from 131 studies on SARS-CoV-2 PCR-positive general hospital adult admissions (n=76,169 patients in total).Main Outcome MeasuresWe planned a comparison of pooled area under curves (AUCs) from Receiver Operator Characteristic curves plotted for admission D-dimer, CRP, urea, troponin and interleukin-6 levels.Main ResultsBiomarker effectiveness varies significantly in different regions of the world. Admission CRP levels are a good prognostic marker for mortality due to SARS-CoV-2 in Asian countries, with a pooled area under curve (AUC) of 0.83 (95% CI 0.80-0.85), but only an average predictor of mortality in Europe/North America, with a pooled AUC of 0.67 (95% CI 0.63-0.71,PConclusionsBiomarker effectiveness for prognosticating SARS-CoV-2 mortality varies significantly by geographical location. We propose that biomarkers and by extension prognostic scores need to be tailored for specific populations. This also implies that validation of commonly used prognostic scores for other conditions should occur before they are used in different populations.Summary boxSection 1: What is already known on this topicBiomarkers such as CRP, D-dimer, and interleukin-6 have been proven to have prognostic value in SARS-CoV-2. However prognostic scores using these as building blocks perform unevenly in different locations.Section 2: What this study addsCommonly used biomarkers for SARS-CoV-2 have different efficacy in different parts of the world. For example, admission CRP and interleukin-6 levels are good prognostic markers for mortality in Asian countries but only average in Europe and North America. Prognostic markers and scores cannot be ‘transplanted’ from one region to another. This has implications not just for SARS-CoV-2 but also for scores in other conditions.

The pleural lining of the thorax regulates local immunity, inflammation and repair. A variety of conditions, both benign and malignant including pleural mesothelioma, can affect this tissue. A lack of knowledge concerning the mesothelial and stromal cells comprising the pleura has hampered the development of targeted therapies. Here we present the first comprehensive single cell transcriptomic atlas of the human parietal pleura and demonstrate its utility in elucidating pleural biology. We confirm the presence of known universal fibroblasts and describe novel, potentially pleural-specific, fibroblast subtypes. We also present transcriptomic characterisation of multiplein vitromodels of benign and malignant mesothelial cells, and characterise these through comparison within vivotranscriptomic data. While bulk pleural transcriptomes have been reported previously, this is the first study to provide resolution at single cell level. We expect our pleural cell atlas will prove invaluable to those studying pleural biology and disease. For example, it has already enabled us to shed light on the transdifferentiation of mesothelial cells allowing us to develop a simple method for prolonging mesothelial cell differentiation in vitro.

Funder: British Lung Foundation (BLF); doi: https://doi.org/10.13039/501100000351, Funder: Royal Society through a University Research Fellowship and the Engineering and Physical Sciences Research Council (EPRSC), Funder: China Scholarship Council (CSC); doi: https://doi.org/10.13039/501100004543, Mesothelioma is an aggressive cancer that is associated with exposure to asbestos. Although asbestos is banned in several countries, including the UK, an epidemic of mesothelioma is predicted to affect middle-income countries during this century owing to their heavy consumption of asbestos. The prognosis for patients with mesothelioma is poor, reflecting a failure of conventional chemotherapy that has ultimately resulted from an inadequate understanding of its biology. However, recent work has revolutionised the study of mesothelioma, identifying genetic and pathophysiological vulnerabilities, including the loss of tumour suppressors, epigenetic dysregulation and susceptibility to nutrient stress. We discuss how this knowledge, combined with advances in immunotherapy, is enabling the development of novel targeted therapies.

Heart failure and associated cachexia is an unresolved and important problem. We report a new model of severe heart failure that consistently results in cachexia. Mice lacking the integrated stress response (ISR) induced eIF2α phosphatase, PPP1R15A, exhibit a dilated cardiomyopathy and severe weight loss following irradiation, whilst wildtype mice are unaffected. This is associated with increased expression of Gdf15 in the heart and increased levels of GDF15 in the circulation. We provide evidence that blockade of GDF15 activity prevents cachexia and slows the progression of heart failure. Our data suggests that cardiac stress mediates a GDF15 dependent pathway that drives weight loss and worsens cardiac function. We show relevance of GDF15 to lean mass and protein intake with patients with heart failure. Blockade of GDF15 could constitute a novel therapeutic option to limit cardiac cachexia and improve clinical outcomes in patients with severe systolic heart failure.

Summary Objectives At least a third of patients go on to suffer a recurrence following a first spontaneous pneumothorax. Surgical intervention reduces the risk of recurrence and has been advocated as a primary treatment for pneumothorax. But surgery exposes patients to the risks of anaesthesia and in some cases can cause chronic pain. Risk stratification of patients to identify those most at risk of recurrence would help direct the most appropriate patients to early intervention. Many studies have addressed the role of thoracic computerized tomography (CT) in identifying those individuals at increased risk of recurrence, but a consensus is lacking. Aim Our objective was to clarify whether CT provides valuable prognostic information for recurrent pneumothorax. Design Meta-analysis. Methods We conducted an exhaustive search of the literature for thoracic CT imaging and pneumothorax, and then performed a meta-analysis using a random effects model to estimate the common odds ratio and standard error. Results Here, we show by meta-analysis of data from 2475 individuals that emphysematous change on CT scan is associated with a significant increased odds ratio for recurrent pneumothorax ipsilateral to the radiological abnormality (odds ratio 2.49, 95% confidence interval 1.51–4.13). Conclusions The association holds true for primary spontaneous pneumothorax when considering emphysematous changes including blebs and bullae. Features, such as bullae at the azygoesophageal recess or increased Goddard score similarly predicted recurrent secondary pneumothorax, as shown by subgroup analysis. Our meta-analysis suggests that CT scanning has value in risk stratifying patients considering surgery for pneumothorax.

BackgroundThe COVID-19 pandemic has presented substantial new challenges to clinical and research teams. Our objective was to analyse the experience of investigators and research delivery staff regarding the research response to COVID-19 in order to identify these challenges as well as solutions for future pandemic planning.MethodsWe conducted a survey of diverse research staff involved in delivery of COVID-19 clinical trials across the UK. This was delivered online across centres linked to the NIHR Respiratory Translational Research Collaboration. Responses were analysed using a formal thematic analysis approach to identify common themes and recommendations.Results83 survey participants from ten teaching hospitals provided 922 individual question responses. Respondents were involved in a range of research delivery roles but the largest cohort (60%) was study investigators. A wide range of research experiences were captured, including early and late phase trials. Responses were coded into overarching themes. Among common observations, complex protocols without adaptation to a pandemic were noted to have hampered recruitment. Recommendations included the need to develop and test pandemic-specific protocols, and make use of innovations in information technology. Research competition needs to be avoided and drug selection processes should be explicitly transparent.ConclusionsDelivery of clinical trials, particularly earlier phase trials, in a pandemic clinical environment is highly challenging, and was reactive rather than anticipatory. Future pandemic studies should be designed and tested in advance, making use of pragmatic study designs as far as possible and planning for integration between early and later phase trials and regulatory frameworks.

Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets., Alpha-1 Foundation Grifols Alpha-1-Antitrypsin Laurell’s Training Award National Biofilms Innovation Centre MINECO MedImmune Infinitus NIH The American Cancer Society The Pershing Square Sohn Cancer Research Award The Chan Zuckerberg Initiative UK Dementia Research Institute grant Canadian Institutes of Health Research US Alzheimer Society Integrated Biological Imaging Network

Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α

The lungs are exposed to a range of environmental toxins (including cigarette smoke, air pollution, asbestos) and pathogens (bacterial, viral and fungal), and most respiratory diseases are associated with local or systemic hypoxia. All of these adverse factors can trigger endoplasmic reticulum (ER) stress. The ER is a key intracellular site for synthesis of secretory and membrane proteins, regulating their folding, assembly into complexes, transport and degradation. Accumulation of misfolded proteins within the lumen results in ER stress, which activates the unfolded protein response (UPR). Effectors of the UPR temporarily reduce protein synthesis, while enhancing degradation of misfolded proteins and increasing the folding capacity of the ER. If successful, homeostasis is restored and protein synthesis resumes, but if ER stress persists, cell death pathways are activated. ER stress and the resulting UPR occur in a range of pulmonary insults and the outcome plays an important role in many respiratory diseases. The UPR is triggered in the airway of patients with several respiratory diseases and in corresponding experimental models. ER stress has been implicated in the initiation and progression of pulmonary fibrosis, and evidence is accumulating suggesting that ER stress occurs in obstructive lung diseases (particularly in asthma), in pulmonary infections (some viral infections and in the setting of the cystic fibrosis airway) and in lung cancer. While a number of small molecule inhibitors have been used to interrogate the role of the UPR in disease models, many of these tools have complex and off-target effects, hence additional evidence (eg, from genetic manipulation) may be required to support conclusions based on the impact of such pharmacological agents. Aberrant activation of the UPR may be linked to disease pathogenesis and progression, but at present, our understanding of the context-specific and disease-specific mechanisms linking these processes is incomplete. Despite this, the ability of the UPR to defend against ER stress and influence a range of respiratory diseases is becoming increasingly evident, and the UPR is therefore attracting attention as a prospective target for therapeutic intervention strategies.

Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension., Responding to general demand from mechanobiology, user-friendly small-molecule fluorescent probes are introduced to image tension changes selectively in any membrane of interest within live cells

The 7th Birt-Hogg-Dubé (BHD) International Symposium convened virtually in October 2021. The meeting attracted more than 200 participants internationally and highlighted recent findings in a variety of areas, including genetic insight and molecular understanding of BHD syndrome, structure and function of the tumor suppressor Folliculin (FLCN), therapeutic and clinical advances as well as patients’ experiences living with this malady.

BackgroundAlveolar epithelial cell dysfunction plays an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), but remains incompletely understood. Some monogenic forms of pulmonary fibrosis are associated with expression of mutant surfactant protein C (SFTPC). The commonest pathogenic mutant, I73T, mislocalises to the alveolar epithelial cell plasma membrane and displays a toxic gain of function. Because the mechanisms explaining the link between this mutant and IPF are incompletely understood, we sought to interrogate SFTPC trafficking in health and disease to understand the functional significance of SFTPC-I73T relocalisation.MethodsWe performed mechanistic analysis of SFTPC trafficking in a cell model that reproduces the in vivo phenotype and validated findings in human primary alveolar organoids.ResultsWe show that wild-type SFTPC takes an unexpected indirect trafficking route via the plasma membrane and undergoes the first of multiple cleavage events before reaching the multivesicular body (MVB) for further processing. SFTPC-I73T takes this same route, but its progress is retarded both at the cell surface and due to failure of trafficking into the MVB. Unable to undergo onward trafficking, it is recycled to the plasma membrane as a partially cleaved intermediate.ConclusionThese data show for the first time that all SFTPC transits the cell surface during normal trafficking, and the I73T mutation accumulates at the cell surface through both retarded trafficking and active recycling. This understanding of normal SFTPC trafficking and how the I73T mutant disturbs it provides novel insight into SFTPC biology in health and disease, and in the contribution of the SFTPC mutant to IPF development.

Familial spontaneous pneumothorax (FSP) accounts for 10% of primary spontaneous pneumothoraces. Appropriate investigation of FSP enables early diagnosis of serious monogenic diseases and the practice of precision medicine. Here, we show that a pneumothorax genetics multidisciplinary team (MDT) can efficiently diagnose a range of syndromic causes of FSP. A sizeable group (73.6%) of clinically unclassifiable FSPs remains. Using whole genome sequencing we demonstrate that most of these cases are not known monogenic disorders. Therefore, clinico-radiological assessment by an MDT has high sensitivity for currently known clinically important monogenic causes of FSP, which has relevance for the design of efficient pneumothorax services.

Background and Objective: Malignant pleural effusion (MPE) often results in debilitating symptoms. Relief of dyspnoea and improvement in quality of life can be achieved with either talc pleurodesis or insertion of an indwelling tunneled pleural catheter (IPC). The former requires a lengthy hospital stay and the latter is associated with lower pleurodesis rates. In response to limited hospital bed capacity, we developed a pragmatic approach in managing MPE by combining thoracoscopic talc poudrage and insertion of IPC into a single day case procedure. We present data on the safety and efficacy of this approach.Methods: Patients who had undergone the abovementioned procedure between 2017 and 2020 were analyzed. Demographic data, hospital length of stay (LOS), histological diagnosis, rates of pleurodesis success and procedural related complications were collated. Patients were followed-up for 6 months.Results: Forty-five patients underwent the procedure. Mean age was 68.5 ± 10.4 years and 56% were male. Histological diagnosis was achieved in all cases. 86.7% of patients were discharged on the day of the procedure. Median LOS was 0 (IQR 0–0) days. Successful pleurodesis was attained in 77.8% at 6-month follow-up. No procedure related deaths or IPC related infections were recorded.Conclusion: Ambulatory thoracoscopic poudrage and IPC insertion is a safe and effective option in the management of MPE. All patients received a definitive pleural intervention with 77.8% pleurodesis success at 6-months and majority of them discharged on the same day. Future randomized trials are required to confirm these findings.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress, resulting in activation of the unfolded protein response (UPR) that aims to restore protein homeostasis. However, the UPR also plays an important pathological role in many diseases, including metabolic disorders, cancer and neurological disorders. Over the last decade, significant effort has been invested in targeting signalling proteins involved in the UPR and an array of drug-like molecules is now available. However, these molecules have limitations, the understanding of which is crucial for their development into therapies. Here, we critically review the existing ER stress and UPR-directed drug-like molecules, highlighting both their value and their limitations.

Pneumothorax is an important complication of COVID-19 [1, 2]. Based on a series of 60 individuals, we previously estimated that 0.91% of people admitted to hospital with COVID-19 develop pneumothorax [1]. Males accounted for three quarters of those affected, and patients requiring non-invasive or invasive ventilatory support appeared at elevated risk. In a separate series of ventilated patients with COVID-19, barotrauma, defined as pneumothorax or pneumomediastinum, was found to be an independent risk for death [2]. During the pandemic, treatment strategies have evolved, influenced by large randomised controlled trials and clinical experience. Following the landmark results from the RECOVERY trial [3], dexamethasone became standard of care for patients requiring supplemental oxygen. Following the first UK wave between March to June 2020, use of non-invasive respiratory support became more common [4, 5]. Such changes could plausibly alter the incidence of pneumothorax caused by COVID-19. Indeed, a recent small study reported an increase in pneumothoraces in the second wave of COVID-19 in Italy, speculating that dexamethasone use might have been causal [6]., Population level data from 131 679 patients show that COVID-19 pneumothorax occurs in 0.97% of admitted patients, especially males and smokers, and is associated with increased mortality.

Objectives: The impulse oscillometry system (IOS) provides an alternative method of lung function testing for patients in whom forced expiratory manoeuvres are contraindicated, such as those with inherited vascular connective tissue disorders. Here we examine the role of IOS in the diagnosis and monitoring of asthma in such patients through a clinical case series and literature review.Methods: The clinical case series comprised of data from 12 patients with inherited connective tissue disorders representing 32 clinical encounters. Of these, 11 encounters were for asthma diagnosis and 21 were for asthma monitoring. Symptoms, exhaled nitric oxide (FeNO) and IOS were assessed at each encounter.Results: In the clinical case series, 5 of 6 patients with likely asthma (as determined by physician review and exhaled nitric oxide testing) had abnormal IOS parameters compared with 0 of 5 of those with unlikely asthma. In the monitoring group, 11 encounters resulted in treatment escalation (demonstrating suboptimal control), and 8 resulted in no change to treatment (good control). Six of 11 of those with suboptimal control had abnormalities in ≥3 IOS parameters, with R5 and R5-20 most frequently affected. Only 1 of 8 of those with good control had abnormalities in ≥3 IOS parameters.Conclusions: IOS can be used as an alternative to conventional lung function testing to support the diagnosis and monitoring of asthma when forced expiratory manoeuvres are contraindicated. Larger studies are required to establish severity and treatment escalation thresholds and provide clearer comparisons with spirometry values.

BackgroundPulmonary arterial hypertension (PAH) covers a range of life-limiting illnesses characterized by increased pulmonary arterial pressures leading to right heart failure and death, if untreated. 15-25% of patients have genetic mutations, the most common affecting bone morphogenetic protein receptor type 2 (BMPR2). The aim was to define an inflammatory cytokine profile in BMPR2-mutation positive patients and analyze their influence on survival.MethodsLevels of cytokines were measured in plasma samples from BMPR2-mutation positive patients (BMPR2mut, n=54), patients without any driving mutations (n=54), and healthy controls (n=56) recruited from the United Kingdom cohort.FindingsBMPR2-mutation positive patients and patients without mutations had high levels of interleukin-6, interleukin-8, tumor necrosis factor-α, and vascular endothelial growth factor-A compared to controls. Only BMPR2-mutation carrying patients had higher G-CSF levels compared to controls. VEGF-A levels were substantially higher in patients without mutations compared to the BMPR2mut group. Interleukin-6 was a significant discriminator for mortality in the BMPR2mut cohort (cumulative survival with interleukin-6≥1.6pg/ml at 3 years was 65% compared to 96% with interleukin-6P=0·0013). N-Terminal pro-B-Type natriuretic peptide levels did not discriminate for survival in our BMPR2mut cohort (cumulative survival for patients with an NT-proBNP>130ng/ml at 3 years was 76% compared to 84% for patients with an NT-proBNP≤130ng/ml, P=0·37). NT-proBNP outperformed interleukin-6 in PAH without mutations.InterpretationBMPR2-mutation positivity has a direct impact not only on inflammatory profiles but also on effectiveness of prognostic biomarkers. In our BMPR2-mutation positive cohort IL-6 was the strongest prognostic biomarker and NT-proBNP failed to discriminate for survival.Key messagesWhat is the key question?Do pulmonary arterial hypertension patients who are BMPR2-mutation positive have a different cytokine signature than PAH patients without mutations?What is the bottom line?BMPR2-mutation positive and PAH patients without mutations display different patterns of cytokine elevation and these cytokines differ in the way they influence transplant-free survival.Why read on?In our cohort of BMPR2-mutation positive patients, IL-6 is the best prognostic biomarker while NT-proBNP failed to discriminate for survival – this implies that prognostic biomarkers and by inference treatments could be genotype-specific.Graphical AbstractTAKE HOME MESSAGEBMPR2-mutation positive patients have different inflammatory and growth factor profiles compared to PAH patients without mutations. Interleukin-6 is an effective biomarker for transplant-free survival in our cohort of BMPR2-mutation positive patients while NT-proBNP is ineffective. Conversely, NT-proBNP appears to be a more effective biomarker for pulmonary arterial patients without any mutations.

Summary Circulating polymers of α1-antitrypsin (α1AT) are neutrophil chemo-attractants and contribute to inflammation, yet cellular factors affecting their secretion remain obscure. We report on a genome-wide CRISPR-Cas9 screen for genes affecting trafficking of polymerogenic α1ATH334D. A CRISPR enrichment approach based on recovery of single guide RNA (sgRNA) sequences from phenotypically selected fixed cells reveals that cells with high-polymer content are enriched in sgRNAs targeting genes involved in “cargo loading into COPII-coated vesicles,” where “COPII” is coat protein II, including the cargo receptors lectin mannose binding1 (LMAN1) and surfeit protein locus 4 (SURF4). LMAN1- and SURF4-disrupted cells display a secretion defect extending beyond α1AT monomers to polymers. Polymer secretion is especially dependent on SURF4 and correlates with a SURF4-α1ATH334D physical interaction and with their co-localization at the endoplasmic reticulum (ER). These findings indicate that ER cargo receptors co-ordinate progression of α1AT out of the ER and modulate the accumulation of polymeric α1AT not only by controlling the concentration of precursor monomers but also by promoting secretion of polymers., Graphical abstract, Highlights • Genome-wide CRISPR screen for genes affecting trafficking of polymerogenic α1AT • CRISPR enrichment based on recovery of DNA from phenotypic selected fixed cells • Early secretory pathway as the strongest modifier of α1AT intracellular polymers • LMAN1 and SURF4 regulate the export of monomeric and polymeric α1AT out of the ER, Ordóñez et al. report that ER cargo receptors regulate α1AT trafficking and modulate polymeric α1AT accumulation by controlling the concentration of monomers and promoting polymers ER exit. The latter implicates conventional intracellular trafficking in the early steps of polymer secretion and sheds light on the biogenesis of pro-inflammatory circulating polymers.

Malignant pleural mesothelioma (MPM) is an aggressive cancer most commonly caused by prior exposure to asbestos. Median survival is 12–18 months, since surgery is ineffective and chemotherapy offers minimal benefit. Preclinical models that faithfully recapitulate the genomic and histopathological features of cancer are critical for the development of new treatments. The most commonly used models of MPM are two-dimensional cell lines established from primary tumours or pleural fluid. While these have provided some important insights into MPM biology, these cell models have significant limitations. In order to address some of these limitations, spheroids and microfluidic chips have more recently been used to investigate the role of the three-dimensional environment in MPM. Efforts have also been made to develop animal models of MPM, including asbestos-induced murine tumour models, MPM-prone genetically modified mice and patient-derived xenografts. Here, we discuss the available in vitro and in vivo models of MPM and highlight their strengths and limitations. We discuss how newer technologies, such as the tumour-derived organoids, might allow us to address the limitations of existing models and aid in the identification of effective treatments for this challenging-to-treat disease.

Severe α1‐antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1‐antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA‐encoded chemical library to undertake a high‐throughput screen to identify small molecules that bind to, and stabilise Z α1‐antitrypsin. The lead compound blocks Z α1‐antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1‐antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1‐antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that “mutation ameliorating” small molecules can block the aberrant polymerisation that underlies Z α1‐antitrypsin deficiency., A chemistry campaign has developed a small molecule that stabilises the severe Z deficiency mutant of α1‐antitrypsin. The lead compound binds to a cryptic pocket and blocks the conformational change and pathological polymerisation that underlie α1‐antitrypsin deficiency.

Background Pulmonary arterial hypertension (PAH) covers a range of life-limiting conditions characterized by increased mean pulmonary arterial pressures leading to right heart failure and eventually death, if left untreated. In approximately 25% of cases of idiopathic PAH genetic analysis reveals a mutation in one of several genes, the most common being bone morphogenetic type II receptor (BMPR2). There is substantial evidence implicating inflammation in the pathogenesis of PAH. We questioned whether potential inflammatory drivers were different in BMPR2-mutation positive PAH and in PAH without any mutations. Methods Patients with confirmed mutations in BMPR2 (BMPR2mut, n=54) and patients without any driving mutations (n=56) were recruited from the national UK- PAH cohort. Levels of IL-6, IL-8, IL-10, TNF-α, VEGF-A and G-CSF were measured in plasma samples from these and healthy controls (n=56). Results Both BMPR2­-mutation positive and PAH patients without mutations had high levels of IL-6, IL-8, TNF-α and VEGF-A compared to controls. Only PAH patients without mutations had higher levels of IL-10 while only BMPR2­-mutation carrying patients had higher levels of G-CSF compared to controls. VEGF-A levels were substantially higher in PAH without mutation compared to the BMPR2mut group. Only IL-6 was a significant discriminator for mortality in the BMPR2mut cohort (cumulative survival for patients with an IL-6 level of ≥1.6 pg/ml at 3 years is 65% compared to 96% for patients with an IL-6 level of 130 ng/ml at 3 years was 76% compared to 84% for patients with an NT-proBNP level of ≤130 ng/ml, P=0.37, figure 1B). The converse was true in PAH without mutation (cumulative survival for patients with an IL-6 level of >1.3 pg/ml at 3 years is 81% compared to 92% for patients with an IL-6 level of ≤1.3 pg/ml, P=0.048, figure 1C). Cumulative survival for patients with a NT-proBNP level of >239 ng/ml at 3 years was 80% compared to 96% for patients with NT-proBNP level of Conclusions BMPR2-mutation positive patients have a different inflammatory profile compared to PAH patients without mutations. The selection of biomarkers of inflammation to predict clinical outcomes may therefore differ between these groups.

At least a third of patients go on to suffer a recurrence following a first spontaneous pneumothorax. Surgical intervention reduces the risk of recurrence and has been advocated as a primary treatment for pneumothorax. But surgery exposes patients to the risks of anaesthesia and in some cases can cause chronic pain. Risk stratification of patients to identify those most of risk of recurrence would help direct the most appropriate patients to early intervention. Many studies have addressed the role of thoracic CT in identifying those individuals at increased risk of recurrence, but a consensus as to whether CT provides valuable prognostic information is lacking. Here we show by meta-analysis of data from 2475 individuals that emphysematous change on CT scan is associated with a significant increased odds ratio for recurrent pneumothorax ipsilateral to the radiological abnormality (OR 2.49, 95% CI 1.51 to 4.13). The association holds true for primary spontaneous pneumothorax when considering emphysematous changes including blebs and bullae. Features such as bullae at the azygoesophageal recess or increased Goddard score similarly predicted recurrent secondary pneumothorax. Our meta-analysis suggests that CT scanning has value in risk stratifying patients considering surgery for pneumothorax.

Introduction and Objectives Birt–Hogg–Dube (BHD) syndrome is an autosomal dominant condition characterised by skin fibrofolliculoma, lung cysts, spontaneous pneumothorax and increased risk of renal cell carcinoma (RCC). The thoracic radiological features of BHD vary from solitary to multiple lung cysts with a lower zone and peripheral predominance. The purpose of our study was to evaluate the presence of pulmonary cysts on computed tomography (CT) in patients who underwent nephrectomy for RCC and integrate these findings with the established European BHD Consortium criteria to identify those patients with a potential diagnosis of BHD. Methods We retrospectively reviewed chest CT imaging and hospital records from the last 6 years of patients who had been diagnosed with RCC and treated with nephrectomy. We investigated if the patients fulfilled the BHD consortium criteria (1 major criteria: genetically confirmed Folliculin germline mutation, or two minor criteria: presence of multiple/lower zone predominant lung cysts on CT, and either age Results In our cohort of 1288 patients with RCC, 120 (9.3%, age at time of diagnosis 61±11 years, M:F 88:32) were found to have at least one pulmonary cyst. Of these, 48 (3.7%) had multiple/lower zone predominant cysts. Twenty-seven patients (2.1%) fulfilled the BHD consortium criteria: three cases had genetically confirmed BHD, while 24 patients (1.9%) met at least two minor criteria. Genetic evaluation in patients with suspicious imaging appearance of BHD is in progress. Twenty-eight patients had died at the time of data collection, and of these 15 had cysts in a lower zone distribution. Conclusion Although BHD remains a rare condition, understanding the association of varying radiological and clinical features can provide an opportunity for improving its detection. Our findings suggest the potential need for a dedicated pathway for RCC patients with incidental lung cysts, where they may benefit from referral for genetic workup and/or multidisciplinary teammeeting review. The implications of a diagnosis of BHD are numerous, including genetic counselling for the patient and their family, regular imaging follow-up and potential lifestyle modifications due to the increased risk of pneumothorax.

Face masks and personal respirators are used to curb the transmission of SARS-CoV-2 in respiratory droplets; filters embedded in some personal protective equipment could be used as a non-invasive sample source for applications, including at-home testing, but information is needed about whether filters are suited to capture viral particles for SARS-CoV-2 detection. In this study, we generated inactivated virus-laden aerosols of 0.3–2 microns in diameter (0.9 µm mean diameter by mass) and dispersed the aerosolized viral particles onto electrostatic face mask filters. The limit of detection for inactivated coronaviruses SARS-CoV-2 and HCoV-NL63 extracted from filters was between 10 to 100 copies/filter for both viruses. Testing for SARS-CoV-2, using face mask filters and nasopharyngeal swabs collected from hospitalized COVID-19-patients, showed that filter samples offered reduced sensitivity (8.5% compared to nasopharyngeal swabs). The low concordance of SARS-CoV-2 detection between filters and nasopharyngeal swabs indicated that number of viral particles collected on the face mask filter was below the limit of detection for all patients but those with the highest viral loads. This indicated face masks are unsuitable to replace diagnostic nasopharyngeal swabs in COVID-19 diagnosis. The ability to detect nucleic acids on face mask filters may, however, find other uses worth future investigation.

The generation of effective and safe nanoagents for biological applications requires their physicochemical characteristics to be tunable, and their cellular interactions to be well characterized. Here, the controlled synthesis is developed for preparing high‐aspect ratio gold nanotubes (AuNTs) with tailorable wall thickness, microstructure, composition, and optical characteristics. The modulation of optical properties generates AuNTs with strong near infrared absorption. Surface modification enhances dispersibility of AuNTs in aqueous media and results in low cytotoxicity. The uptake and trafficking of these AuNTs by primary mesothelioma cells demonstrate their accumulation in a perinuclear distribution where they are confined initially in membrane‐bound vesicles from which they ultimately escape to the cytosol. This represents the first study of the cellular interactions of high‐aspect ratio 1D metal nanomaterials and will facilitate the rational design of plasmonic nanoconstructs as cytosolic nanoagents for potential diagnosis and therapeutic applications.

Regulated phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by the endoplasmic reticulum (ER) stress-activated protein kinase PERK modulates protein synthesis and couples the production of ER client proteins with the organelle's capacity to fold and process them. PERK activation by ER stress is known to involve transautophosphorylation, which decorates its unusually long kinase insert loop with multiple phosphoserine and phosphothreonine residues. We report that PERK activation and phosphorylation selectively enhance its affinity for the nonphosphorylated eIF2 complex. This switch correlates with a marked change to the protease sensitivity pattern, which is indicative of a major conformational change in the PERK kinase domain upon activation. Although it is dispensable for catalytic activity, PERK's kinase insert loop is required for substrate binding and for eIF2α phosphorylation in vivo. Our findings suggest a novel mechanism for eIF2 recruitment by activated PERK and for unidirectional substrate flow in the phosphorylation reaction.

The respiratory tract and its resident immune cells face daily exposure to stress, both from without and from within. Inhaled pathogens, including severe acute respiratory syndrome coronavirus 2, and toxins from pollution trigger a cellular defence system that reduces protein synthesis to minimise viral replication or the accumulation of misfolded proteins. Simultaneously, a gene expression programme enhances antioxidant and protein folding machineries in the lung. Four kinases (PERK, PKR, GCN2 and HRI) sense a diverse range of stresses to trigger this “integrated stress response”. Here we review recent advances identifying the integrated stress response as a critical pathway in the pathogenesis of pulmonary diseases, including pneumonias, thoracic malignancy, pulmonary fibrosis and pulmonary hypertension. Understanding the integrated stress response provides novel targets for the development of therapies.

Introduction Pneumothorax and pneumomediastinum have both been noted to complicate cases of COVID-19 requiring hospital admission. We report the largest case series yet described of patients with both these pathologies that includes non-ventilated patients. Methods Cases were collected retrospectively from UK hospitals with inclusion criteria limited to a diagnosis of COVID-19 and the presence of either pneumothorax or pneumomediastinum. Patients included in the study presented between March and June 2020. Details obtained from the medical record included demographics, radiology, laboratory investigations, clinical management and survival. Results Seventy-one patients from 16 centres were included in the study, of whom 60 patients had pneumothoraces (six also with pneumomediastinum), whilst 11 patients had pneumomediastinum alone. Two of these patients had two distinct episodes of pneumothorax, occurring bilaterally in sequential fashion, bringing the total number of pneumothoraces included to 62. Clinical scenarios included patients who had presented to hospital with pneumothorax, patients who had developed pneumothorax or pneumomediastinum during their inpatient admission with COVID-19 and patients who developed their complication whilst intubated and ventilated, either with or without concurrent extracorporeal membrane oxygenation. Survival at 28 days was not significantly different following pneumothorax (63.1%±6.5%) or isolated pneumomediastinum (53.0%±18.7%; p=0.854). The incidence of pneumothorax was higher in males. The 28-day survival was not different between the sexes (males 62.5%±7.7% versus females 68.4%±10.7%; p=0.619). Patients above the age of 70 had a significantly lower 28-day survival than younger individuals (≥70 years 41.7%±13.5% survival versus, Roughly 1% of patients admitted with COVID-19 develop pneumothorax. This can occur without pre-existing lung disease or mechanical ventilation. Two thirds of patients survive, but age >70 years and acidosis are associated with poor prognosis.