Biochemical and Structural Insights into FIH-Catalysed Hydroxylation of Transient Receptor Potential Ankyrin Repeat Domains.

Transient receptor potential (TRP) channels have important roles in environmental sensing in animals. Human TRP subfamily A member 1 (TRPA1) is responsible for sensing allyl isothiocyanate (AITC) and other electrophilic sensory irritants. TRP subfamily vanilloid member 3 (TRPV3) is involved in skin maintenance. TRPV3 is a reported substrate of the 2-oxoglutarate oxygenase factor inhibiting hypoxia-inducible factor (FIH). We report biochemical and structural studies concerning asparaginyl hydroxylation of the ankyrin repeat domains (ARDs) of TRPA1 and TRPV3 catalysed by FIH. The results with ARD peptides support a previous report on FIH-catalysed TRPV3 hydroxylation and show that, of the 12 potential TRPA1 sequences investigated, one sequence (TRPA1 residues 322-348) undergoes hydroxylation at Asn336. Structural studies reveal that the TRPA1 and TRPV3 ARDs bind to FIH with a similar overall geometry to most other reported FIH substrates. However, the binding mode of TRPV3 to FIH is distinct from that of other substrates.

Effects of patient room layout on viral accruement on healthcare professionals' hands.

Healthcare professionals (HCPs) are exposed to highly infectious viruses, such as norovirus, through multiple exposure routes. Understanding exposure mechanisms will inform exposure mitigation interventions. The study objective was to evaluate the influences of hospital patient room layout on differences in HCPs' predicted hand contamination from deposited norovirus particles. Computational fluid dynamic (CFD) simulations of a hospital patient room were investigated to find differences in spatial deposition patterns of bioaerosols for right-facing and left-facing bed layouts under different ventilation conditions. A microbial transfer model underpinned by observed mock care for three care types (intravenous therapy (IV) care, observational care, and doctors' rounds) was applied to estimate HCP hand contamination. Viral accruement was contrasted between room orientation, care type, and by assumptions about whether bioaerosol deposition was the same or variable by room orientation. Differences in sequences of surface contacts were observed for care type and room orientation. Simulated viral accruement differences between room types were influenced by mostly by differences in bioaerosol deposition and by behavior sequences when deposition patterns for the room orientations were similar. Differences between care types were likely driven by differences in hand-to-patient contact frequency, with doctors' rounds resulting in the greatest predicted viral accruement on hands.

Ribosomal oxygenases are structurally conserved from prokaryotes to humans.

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases.

Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed ß-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl ß-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl ß-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general.

13C-Carbamylation as a mechanistic probe for the inhibition of class D ß-lactamases by avibactam and halide ions.

The class D (OXA) serine ß-lactamases are a major cause of resistance to ß-lactam antibiotics. The class D enzymes are unique amongst ß-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that ß-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the 'hydrolytic water' molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D ß-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.

Anti-inflammatory effects of elexacaftor/tezacaftor/ivacaftor in adults with cystic fibrosis heterozygous for F508del.

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1ß (p<0.0013, p = 0.0476), IL-6 (p = 0.0109, p = 0.0216) and TNF (p = 0.0028, p = 0.0033) levels in CF serum and following PBMCs stimulation respectively. The corresponding mRNA levels were also found to be reduced in stimulated PBMCs, as well as reduced ASC specks and caspase-1 levels, indicative of NLRP3-mediated production of pro-inflammatory cytokines, IL-1ß and IL-18. While ETI therapy is highly effective at reducing sweat chloride and improving lung function, it also displays potent anti-inflammatory properties, which are likely to contribute to improved long-term clinical outcomes.

The interaction of isopenicillin N synthase with homologated substrate analogues δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-Xaa characterised by protein crystallography.

Isopenicillin N synthase (IPNS) converts the linear tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into bicyclic isopenicillin N (IPN) in the central step in the biosynthesis of penicillin and cephalosporin antibiotics. Solution-phase incubation experiments have shown that IPNS turns over analogues with a diverse range of side chains in the third (valinyl) position of the substrate, but copes less well with changes in the second (cysteinyl) residue. IPNS thus converts the homologated tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-valine (AhCV) and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-allylglycine (AhCaG) into monocyclic hydroxy-lactam products; this suggests that the additional methylene unit in these substrates induces conformational changes that preclude second ring closure after initial lactam formation. To investigate this and solution-phase results with other tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-Xaa, we have crystallised AhCV and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-S-methylcysteine (AhCmC) with IPNS and solved crystal structures for the resulting complexes. The IPNS:Fe(II):AhCV complex shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the IPNS:Fe(II):AhCmC complex, by virtue of thioether coordination to iron. AhCmC occupies two distinct conformations, both distorted relative to the natural substrate ACV, in order to accommodate the extra methylene group in the second residue. Attempts to turn these substrates over within crystalline IPNS using hyperbaric oxygenation give rise to product mixtures.

The crystal structure of isopenicillin N synthase with a dipeptide substrate analogue.

Isopenicillin N synthase (IPNS) converts its linear tripeptide substrate δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to bicyclic isopenicillin N (IPN), the key step in penicillin biosynthesis. Solution-phase incubation experiments have shown that IPNS will accept and oxidise a diverse array of substrate analogues, including tripeptides that incorporate L-homocysteine as their second residue, and tripeptides with truncated side-chains at the third amino acid such as δ-L-α-aminoadipoyl-L-cysteinyl-D-α-aminobutyrate (ACAb), δ-L-α-aminoadipoyl-L-cysteinyl-D-alanine (ACA) and δ-L-α-aminoadipoyl-L-cysteinyl-glycine (ACG). However IPNS does not react with dipeptide substrates. To probe this selectivity we have crystallised the enzyme with the dipeptide δ-L-α-aminoadipoyl-L-homocysteine (AhC) and solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 Å resolution. This structure reveals an unexpected mode of peptide binding at the IPNS active site, in which the homocysteinyl thiolate does not bind to iron. Instead the primary mode of binding sees the homocysteinyl carboxylate coordinated to the metal, while its side-chain is oriented into the region of the active site normally occupied by the benzyl group of protein residue Phe211.

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases.

Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(-)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC(50)) values for the R-form of 2HG varied from approximately 25 µM for the histone N(ɛ)-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Physical activity promotion interventions in chronic airways disease: a systematic review and meta-analysis.

Physical inactivity is common in people with chronic airways disease (pwCAD) and associated with worse clinical outcomes and impaired quality of life. We conducted a systematic review and meta-analysis to characterise and evaluate the effectiveness of interventions promoting step-based physical activity (PA) in pwCAD. We searched for studies that included a form of PA promotion and step-count outcome measure. A random-effects model was used to determine the overall effect size using post-intervention values. 38 studies (n=32 COPD; n=5 asthma; n=1 bronchiectasis; study population: n=3777) were included. Overall, implementing a form of PA promotion resulted in a significant increase in step-count: median (IQR) 705 (183-1210) when compared with usual standard care: -64 (-597-229), standardised mean difference (SMD) 0.24 (95% CI: 0.12-0.36), p<0.01. To explore the impact of specific interventions, studies were stratified into subgroups: PA promotion+wearable activity monitor-based interventions (n=17) (SMD 0.37, p<0.01); PA promotion+step-count as an outcome measure (n=9) (SMD 0.18, p=0.09); technology-based interventions (n=12) (SMD 0.16, p=0.01). Interventions promoting PA, particularly those that incorporate wearable activity monitors, result in a significant and clinically meaningful improvement in daily step-count in pwCAD.

Anti-IL-5 biologics and rheumatoid arthritis: a single-centre 500 patient year exposure analysis.

OBJECTIVE: The increasing use of biological therapies has led to the paradoxical finding that monoclonal antibody therapy for one inflammatory disease can sometimes induce another inflammatory disease. Recently, the use of anti-IL-5 (IL, interleukin) antibody therapies for severe asthma has been associated with the onset of rheumatoid arthritis (RA) and other inflammatory rheumatological disease. We undertook this audit to identify the prevalence of this finding across a large clinical cohort of patients receiving anti-IL-5 therapy. METHODS: All patients currently receiving mepolizumab or benralizumab for severe asthma across the Leeds Teaching Hospitals NHS Trust's (LTHT) Respiratory Service were included. Electronic records for each patient were searched to identify clinical and biochemical manifestations of inflammatory rheumatological disease following the initiation of anti-IL-5 therapy. RESULTS: 142 patients, with a mean duration of 3.5 years on therapy, were included (89 mepolizumab, 53 benralizumab). 17 patients developed new arthralgias (nine mepolizumab, eight benralizumab), however only one of these patients (on mepolizumab) had raised acute phase reactants and newly positive anti-CCP antibody (ACPA) and rheumatoid factor and was the only patient to receive a formal diagnosis of RA. CONCLUSION: Although ACPA positive RA has now been reported in a handful of case reports, we noted a very low rate of evolution into RA or inflammatory arthritis, at least in the short-medium term under anti-IL-5 therapy. This challenges the emerging suggestion that anti-IL-5 biologics may be triggering RA.

Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors.

Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.

Isopenicillin N synthase binds δ-(L-α-aminoadipoyl)-L-cysteinyl-D-thia-allo-isoleucine through both sulfur atoms.

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution-phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ-L-α-aminoadipoyl-L-cysteinyl-D-isoleucine (ACI) and AC-D-allo-isoleucine (ACaI) to penam products, the isosteric sulfur-containing peptides AC-D-thiaisoleucine (ACtI) and AC-D-thia-allo-isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe(II) :ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.

The crystal structure of isopenicillin N synthase with δ-((L)-α-aminoadipoyl)-(L)-cysteinyl-(D)-methionine reveals thioether coordination to iron.

Isopenicillin N synthase (IPNS) catalyses cyclization of δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N (IPN), the central step in penicillin biosynthesis. Previous studies have shown that IPNS turns over a wide range of substrate analogues in which the valine residue of its natural substrate is replaced with other amino acids. IPNS accepts and oxidizes numerous substrates that bear hydrocarbon sidechains in this position, however the enzyme is less tolerant of analogues presenting polar functionality in place of the valinyl isopropyl group. We report a new ACV analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-d-methionine (ACM), which incorporates a thioether in place of the valinyl sidechain. ACM has been synthesized using solution phase methods and crystallized with IPNS. A crystal structure has been elucidated for the IPNS:Fe(II):ACM complex at 1.40Å resolution. This structure reveals that ACM binds in the IPNS active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57Å. The sulfur of the cysteinyl thiolate sits 2.36Å from the metal.

Structural and biochemical analyses reveal how ornithine acetyl transferase binds acidic and basic amino acid substrates.

Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes.

Ward based inhaler technique service reduces exacerbations of asthma and COPD.

BACKGROUND: The management of asthma and COPD is largely dependent on patients being able to use their inhaled medication correctly, but poor inhaler technique continues to be a recurring theme in studies and clinical practice. This is associated with poor disease control, increased risk of exacerbations and hospital admissions, and so there is a need to redesign services for patients to optimise their medicines use. METHODS: A novel ward-based dedicated inhaler technique service was developed, and pharmacy support workers trained to provide this, focusing on optimising inhaler technique using a checklist and recommending protocol-guided inhaler device switches. Inpatients on adult respiratory wards with a diagnosis of exacerbation of asthma or COPD consented to receive this service, and the impact on exacerbations and hospital admissions were compared in the 6-months before and after the intervention. RESULTS: 266 adults (74 asthma, 188 COPD, and four asthma-COPD overlap) received the inhaler technique service. Six-month exacerbation and hospital admission data were available for 184 subjects. Optimising inhaler technique achieved a significant reduction in the combined asthma and COPD annualised rate of moderate-to-severe exacerbations (Rate Ratio [RR] 0.75, p < 0.05) and annualised rate of hospital admissions (RR 0.57, p < 0.0005). Improvements were also observed in future length of stay (- 1.6 days) and the average cost of admission (-£748). CONCLUSIONS: This novel inhaler technique service produced a significant reduction in the rate of moderate-to-severe exacerbations of asthma and COPD, and a reduction in the rate hospital admissions, length of stay and average cost of admission.

Nasal high-flow therapy as an adjunct to exercise in patients with cystic fibrosis: A pilot feasibility trial.

BACKGROUND: Exercise tolerance in people with CF and advanced lung disease is often reduced. While supplemental oxygen can improve oxygenation, it does not affect dyspnoea, fatigue or comfort. Nasal high-flow therapy (NHFT), thanks to its pathophysiological mechanisms, could improve exercise tolerance, saturation and dyspnoea. This study explores the feasibility of conducting a clinical trial of using NHFT in patients with CF during exercise. METHODS: A pilot, open-label, randomized crossover trial was performed, enroling 23 participants with CF and severe lung disease. Participants completed two treadmill walking test (TWT) with and without NHFT at 24-48 h interval. Primary outcome was trial feasibility, and exploratory outcomes were TWT distance (TWTD), SpO2, transcutaneous CO2, dyspnoea and comfort. RESULTS: Recruitment rate was 2.4 subjects/month with 1.3:1 screening-to-randomization ratio. No adverse events caused by NHFT were observed. Tolerability was good and data completion rate was 100%. Twenty subjects (91%) were included in the exploratory study. Mean difference in TWTD on NHFT was 19 m (95% CI [4.8 - 33.1]). SpO2 was similar, but respiratory rate and mean tcCO2 were lower on NHFT (mean difference = -3.9 breaths/min 95% CI [-5.9 - -1.9] and -0.22 kPa 95% CI [-0.4 - 0.04]). NHFT reduced exercise-induced dyspnoea and discomfort. CONCLUSION: Trials using NHFT in patients with CF during exercise are feasible. NHFT appears to improve walking distance, control respiratory rate, CO2, dyspnoea and improve comfort. A larger trial with a longer intervention is feasible and warranted to confirm the impact of NHFT in training programmes for patients with CF.

Noninvasive Ventilation in Cystic Fibrosis: Clinical Indications and Outcomes in a Large UK Adult Cystic Fibrosis Center.

BACKGROUND: Noninvasive ventilation (NIV) is routinely used to treat patients with cystic fibrosis and respiratory failure. However, evidence on its use is limited, with no data on its role in disease progression and outcomes. The aim of this study was to assess the indications of NIV use and to describe the outcomes associated with NIV in adults with cystic fibrosis in a large adult tertiary center. METHODS: A retrospective analysis of data captured prospectively on the unit electronic patient records was performed. All patients with cystic fibrosis who received NIV over a 10-y period were included in the study. A priori, 2 groups were identified based on length of follow-up, with 2 subgroups identified based on duration of NIV treatment. RESULTS: NIV was initiated on 64 occasions. The duration of follow-up was categorized as > 6 months or < 6 months in 31 (48.4%) and 33 (51.6%) occasions, respectively. The most common indications for starting NIV were chronic (48.5%) and acute (32.8%) hypercapnic respiratory failure. Among those with a follow-up > 6 months, subjects who stopped using NIV early showed a steady median (interquartile range) decline in FEV1 (pre-NIV: -0.04 [-0.35 to 0.03] L/y vs post-NIV: -0.07 [-0.35 to 0.01] L/y, P = .51), while among those who continued using it had an improvement in the rate of decline (pre-NIV: -0.25 [-0.52 to -0.02] L/y vs post-NIV: -0.07 [-0.13 to 0.16] L/y, P = .006). No differences in intravenous antibiotic requirement or pulmonary exacerbations were noted with the use of NIV. Pneumothorax and massive hemoptysis occurred independently in 4 cases. CONCLUSIONS: NIV is being used in cystic fibrosis as adjunct therapy for the management of advanced lung disease in a similar fashion to other chronic respiratory conditions. Adherence to NIV treatment can stabilize lung function but does not reduce pulmonary exacerbations or intravenous antibiotic requirement.