Front Pharmacol. 2024 Sep 27;15:1495855. doi: 10.3389/fphar.2024.1495855. eCollection 2024.

ABSTRACT

[This corrects the article DOI: 10.3389/fphar.2024.1403649.].

PMID:39399470 | PMC:PMC11466882 | DOI:10.3389/fphar.2024.1495855

medRxiv [Preprint]. 2024 Sep 26:2024.09.25.24314390. doi: 10.1101/2024.09.25.24314390.

ABSTRACT

Arsenic is associated with lung disease and experimental models suggest that arsenic-induced degradation of the chloride channel CFTR (cystic fibrosis transmembrane conductance regulator) is a mechanism of arsenic toxicity. We examined associations between arsenic exposure, sweat chloride concentration (measure of CFTR function), and pulmonary function among 285 adults in Bangladesh. Participants with sweat chloride ≥ 60 mmol/L had higher arsenic exposures than those with sweat chloride < 60 mmol/L (water: median 77.5 µg/L versus 34.0 µg/L, p = 0.025; toenails: median 4.8 µg/g versus 3.7 µg/g, p = 0.024). In linear regression models, a one-unit µg/g increment in toenail arsenic was associated with a 0.59 mmol/L higher sweat chloride concentration, p < 0.001. We found that toenail arsenic concentration was associated with increased odds of airway obstruction (OR: 1.97, 95%: 1.06, 3.67, p = 0.03); however, sweat chloride concentration did not mediate this association. Our findings suggest that sweat chloride concentration may be a novel biomarker for arsenic exposure and also that arsenic likely acts on the lung through mechanisms other than CFTR dysfunction.

PMID:39399016 | PMC:PMC11469388 | DOI:10.1101/2024.09.25.24314390

Mol Ther Nucleic Acids. 2024 Sep 16;35(4):102339. doi: 10.1016/j.omtn.2024.102339. eCollection 2024 Dec 10.

ABSTRACT

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two-halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558, which inhibit non-homologous end-joining (NHEJ) and micro-homology mediated end-joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2- to 3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

PMID:39398224 | PMC:PMC11470261 | DOI:10.1016/j.omtn.2024.102339

Rev Med Liege. 2024 Oct;79(10):664-669.

ABSTRACT

Since January 2020, neonatal screening for cystic fibrosis (CF-NBS) has been implemented in the Wallonia-Brussels Federation. It's based on the immunoreactive trypsin (IRT1) assay between day 2 and day 4, associated with a 12 CFTR pathogenic variants analysis and with an IRT control on day 21. The aim of this study is to evaluate the performance of our CF-NBS in Liège according to the quality criteria defined by the European Cystic Fibrosis Society's working group on neonatal screening. After four years, 58.762 newborns have been screened. Nineteen children with cystic fibrosis were diagnosed : 14 by NBS, 3 following a meconium ileus, 1 by family history and 1 false negative diagnosed on clinical basis. Furthermore, 39 healthy carriers and 2 uncertain diagnosis (CFSPID) were identified. The sensitivity of CF NBS is 93,3 % (target ≥ 95 %), the positive predictive value (PPV) 17,7 % (target ≥ 30 %). Increasing the TIR1 threshold by 0,1 in 0,1 from P99 to P99,5, would be associated with a lower sensitivity and a non-significant improvement of PPV. A national assessment of CF NBS needs to be carried out.

PMID:39397555

Beijing Da Xue Xue Bao Yi Xue Ban. 2024 Oct 18;56(5):763-774.

ABSTRACT

OBJECTIVE: To detect the cystic fibrosis transmembrane transduction regulator (CFTR) gene mutations and congenital bilateral absence of vas deferens (CBAVD) susceptibility gene mutations in patients with CBAVD, and to explore their association with the risk of CBAVD.

METHODS: Whole-exome sequencing and Sanger sequencing validation were conducted on the pathogenic genes CFTR, adhesion G protein-coupled receptor G2 (ADGRG2), sodium channel epithelial 1 subunit beta (SCNN1B), carbonic anhydrase 12 (CA12), and solute carrier family 9 member A3 (SLC9A3) in thirteen cases of isolated CBAVD patients. The polymorphic loci, intron and flanking sequences of CFTR gene were amplified by polymerase chain reaction (PCR) followed by Sanger sequencing. Bioinformatics methods were employed for conservative analysis and deleterious prediction of novel susceptibility gene mutations in CBAVD. Genetic analysis was performed on the pedigree of one out of thirteen patients with CBAVD to evaluate the risk of inheritance in offspring.

RESULTS: Exome sequencing revealed CFTR gene exon mutations in only six of the thirteen CBAVD patients, with six missense mutations c.2684G>A(p.Ser895Asn), c.4056G>C(p.Gln1352His), c.2812G>(p.Val938Leu), c.3068T>G(p.Ile1023Arg), c.374T>C(p.Ile125Thr), c.1666A>G(p.Ile556Val)), and one nonsense mutation (c.1657C>T(p.Arg553Ter). Among these six patients, two also had the CFTR homozygous p.V470 site, additionally, mutations in CFTR gene exon regions were not detected in the remaining seven patients. Within the thirteen CBAVD patients, three carried the homozygous p.V470 polymorphic site, four carried the 5T allele, two carried the TG13 allele, and ten carried the c.-966T>G site. Four CBAVD patients simultaneously carried 2-3 of the aforementioned CFTR gene mutation sites. Susceptibility gene mutations in CBAVD among the thirteen patients included one ADGRG2 missense mutation c.2312A>G(p.Asn771Ser), two SLC9A3 missense mutations c.2395T>C(p.Cys799Arg), c.493G>A(p.Val165Ile), one SCNN1B missense mutation c.1514G>A(p.Arg505His), and one CA12 missense mutation c.1061C>T (p.Ala354Val). Notably, the SLC9A3 gene c.493G>A (p.Val165Ile) mutation site was first identified in CBAVD patients. The five mutations exhibited an extremely low population mutation frequency in the gnomAD database, classifying them as rare mutations. Predictions from Mutation Taster and Polyphen-2 software indicated that the harmfulness level of the SLC9A3 gene c.493G>A (p.Val165Ile) site and the SCNN1B gene c.1514G>A (p.Arg505His) site were disease causing and probably damaging. The genetic analysis of one pedigree revealed that the c.1657C>T (p.Arg553Ter) mutation in the proband was a de novo mutation, as neither the proband's father nor mother carried this mutation. The proband and his spouse conceived a daughter through assisted reproductive technology, and the daughter inherited the proband's pathogenic mutation c.1657C>T (p.Arg553Ter).

CONCLUSION: CFTR gene mutations remain the leading cause of CBAVD in Chinese patients; however, the distribution and frequency of mutations differ from data reported in other domestic and international studies, highlighting the need to expand the CFTR mutation spectrum in Chinese CBAVD patients. The susceptibility genes ADGRG2, SLC9A3, SCNN1B, and CA12 may explain some cases of CBAVD without CFTR mutations. Given the lack of specific clinical manifestations in CBAVD patients, it is recommended that clinicians conduct further physical examinations and consider scrotal or transrectal ultrasound before making a defi-nitive diagnosis. It is advisable to employ CFTR gene mutation testing in preconception genetic screening to reduce the risk of CBAVD and cystic fibrosis in offspring.

PMID:39397452

Lancet Respir Med. 2024 Oct 9:S2213-2600(24)00305-9. doi: 10.1016/S2213-2600(24)00305-9. Online ahead of print.

NO ABSTRACT

PMID:39395437 | DOI:10.1016/S2213-2600(24)00305-9

Expert Opin Investig Drugs. 2024 Oct 15:1-8. doi: 10.1080/13543784.2024.2416982. Online ahead of print.

ABSTRACT

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is characterized by progressive inflammation during therapy. Cystic fibrosis (CF), alpha-one antitrypsin deficiency (AATD), and non-CF bronchiectasis are also chronic respiratory disorders with inflammation and progression that share many similarities with COPD. Therefore, various anti-inflammatory approaches are currently being investigated, and protein phosphatase 2A (PP2A) activators may represent one such approach.

AREAS COVERED: Systematic review of papers published from 2000-to date on the anti-inflammatory role of endogenous PP2A, the consequences of its inhibition by smoking, and the beneficial effects of its activation in COPD.

EXPERT OPINION: PP2A activation is a plausible therapeutic approach in COPD and related disorders, such as CF, AATD, and non-CF bronchiectasis, although the available evidence is still mostly experimental. Metformin repurposing and consideration of inhalation for some of the molecules discussed in this study are promising approaches.

PMID:39394816 | DOI:10.1080/13543784.2024.2416982

J Dent Res. 2024 Oct 12:220345241271943. doi: 10.1177/00220345241271943. Online ahead of print.

ABSTRACT

Tissue-specific immune responses are critical determinants of health-maintaining homeostasis and disease-related dysbiosis. In the context of COVID-19, oral immune responses reflect local host-pathogen dynamics near the site of infection and serve as important "windows to the body," reflecting systemic responses to the invading SARS-CoV-2 virus. This study leveraged multiplex technology to characterize the salivary SARS-CoV-2-specific immunological landscape (37 cytokines/chemokines and 11 antibodies) during early infection. Cytokine/immune profiling was performed on unstimulated cleared whole saliva collected from 227 adult SARS-CoV-2+ participants and 37 controls. Statistical analysis and modeling revealed significant differential abundance of 25 cytokines (16 downregulated, 9 upregulated). Pathway analysis demonstrated early SARS-CoV-2 infection is associated with local suppression of oral type I/III interferon and blunted natural killer-/T-cell responses, reflecting a potential novel immune-evasion strategy enabling infection. This virus-associated immune suppression occurred concomitantly with significant upregulation of proinflammatory pathways including marked increases in the acute phase proteins pentraxin-3 and chitinase-3-like-1. Irrespective of SARS-CoV-2 infection, prior vaccination was associated with increased total α-SARS-CoV-2-spike (trimer), -S1 protein, -RBD, and -nucleocapsid salivary antibodies, highlighting the importance of COVID-19 vaccination in eliciting mucosal responses. Altogether, our findings highlight saliva as a stable and accessible biofluid for monitoring host responses to SARS-CoV-2 over time and suggest that oral-mucosal immune dysregulation is a hallmark of early SARS-CoV-2 infection, with possible implications for viral evasion mechanisms.

PMID:39394771 | DOI:10.1177/00220345241271943

Methods Cell Biol. 2024;189:153-168. doi: 10.1016/bs.mcb.2024.05.008. Epub 2024 Jun 12.

ABSTRACT

Dendritic cells (DCs), and especially so conventional type I DCs (cDC1s), are fundamental regulators of anticancer immunity, largely reflecting their superior ability to engulf tumor-derived material and process it for cross-presentation on MHC Class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). Thus, investigating key DC functions including (but not limited to) phagocytic capacity, expression of CTL-activating ligands on the cell surface, and cross-presentation efficacy is an important component of multiple immuno-oncology studies. Unfortunately, DCs are terminally differentiated cells, implying that they cannot be propagated indefinitely in vitro and hence must be generated ad hoc from circulating or bone marrow-derived precursors, which presents several limitations. Here, we propose a simple, cytofluorometric method to quantify phenotypic activation markers including CD80, CD86 and MHC class II molecules on the surface of a conditionally immortalized immature DC line that can be indefinitely propagated in vitro but also driven into maturation at will with a simple change in culture conditions. Upon appropriate scaling and automatization, this approach is compatible with high-throughput screening programs for the discovery of novel DC activators that do not suffer from batch variability and other limitations associated with the generation of fresh DCs.

PMID:39393881 | DOI:10.1016/bs.mcb.2024.05.008

J Biol Chem. 2024 Oct 9:107873. doi: 10.1016/j.jbc.2024.107873. Online ahead of print.

ABSTRACT

A-kinase anchoring proteins (AKAPs) are key orchestrators of cyclic AMP (cAMP) signaling that act by recruiting protein kinase A (PKA) in proximity of its substrates and regulators to specific subcellular compartments. Modulation of AKAPs function offers the opportunity to achieve compartment-restricted modulation of the cAMP/PKA axis, paving the way to new targeted treatments. For instance, blocking the AKAP activity of PI3Kγ improves lung function by inducing cAMP-mediated bronchorelaxation, ion transport and anti-inflammatory responses. Here, we report the generation of a non-natural peptide, DRI-Pep #20, optimized to disrupt the AKAP function of PI3Kγ. DRI-Pep #20 mimicked the native interaction between the N-terminal domain of PI3Kγ and PKA, demonstrating nanomolar affinity for PKA, high resistance to protease degradation and high permeability to the pulmonary mucus barrier. DRI-Pep #20 triggered cAMP elevation both in vivo in the airway tract of mice upon intratracheal administration, and in vitro in bronchial epithelial cells of cystic fibrosis (CF) patients. In CF cells, DRI-Pep #20 rescued the defective function of the cAMP-operated channel cystic fibrosis conductance regulator (CFTR), by boosting the efficacy of approved CFTR modulators. Overall, this study unveils DRI-Pep #20 as a potent PI3Kγ/PKA disruptor for achieving therapeutic cAMP elevation in chronic respiratory disorders.

PMID:39393573 | DOI:10.1016/j.jbc.2024.107873

Pediatr Pulmonol. 2024 Oct 11. doi: 10.1002/ppul.27296. Online ahead of print.

ABSTRACT

BACKGROUND: Bronchopulmonary dysplasia, a sequela of preterm birth, is the most common chronic respiratory disorder in infancy, and the second most common in children. Despite this, clinical care remains highly variable with guidelines supported by limited evidence, and do not provide specific guidance for timing of clinical follow-up, echocardiography, modalities of pulmonary function testing, etc. OBJECTIVE/METHODS: To further our understanding of care delivery for BPD, we sought to describe outpatient care patterns at tertiary care centers through survey data from 27 well-established BPD programs.

RESULTS: We observed variability in referral patterns to outpatient BPD clinics, ancillary services provided, indications for follow-up echocardiograms, availability of lung function testing, and criteria for discharge from care.

CONCLUSION: More comprehensive and detailed clinical guidelines similar to other pulmonary diseases such as asthma and cystic fibrosis should be developed to help standardize care and may improve long term outcomes.

PMID:39392254 | DOI:10.1002/ppul.27296

Cell Insight. 2024 Sep 18;3(6):100201. doi: 10.1016/j.cellin.2024.100201. eCollection 2024 Dec.

ABSTRACT

Preclinical models serve as indispensable tools in translational medicine. Specifically, patient-derived models such as patient-derived xenografts (PDX), induced pluripotent stem cells (iPSC), organoids, and recently developed technique of conditional reprogramming (CR) have been employed to reflect the host characteristics of diseases. CR technology involves co-culturing epithelial cells with irradiated Swiss-3T3-J2 mouse fibroblasts (feeder cells) in the presence of a Rho kinase (ROCK) inhibitor, Y-27632. CR technique facilitates the rapid conversion of both normal and malignant cells into a "reprogrammed stem-like" state, marked by robust in vitro proliferation. This is achieved without reliance on exogenous gene expression or viral transfection, while maintaining the genetic profile of the parental cells. So far, CR technology has been used to study biology of diseases, targeted therapies (precision medicine), regenerative medicine, and noninvasive diagnosis and surveillance. Respiratory diseases, ranking as the third leading cause of global mortality, pose a significant burden to healthcare systems worldwide. Given the substantial mortality and morbidity rates of respiratory diseases, efficient and rapid preclinical models are imperative to accurately recapitulate the diverse spectrum of respiratory conditions. In this article, we discuss the applications and future potential of CR technology in modeling various respiratory tract diseases, including lung cancer, respiratory viral infections (such as influenza and Covid-19 and etc.), asthma, cystic fibrosis, respiratory papillomatosis, and upper aerodigestive track tumors. Furthermore, we discuss the potential utility of CR in personalized medicine, regenerative medicine, and clinical translation.

PMID:39391007 | PMC:PMC11462205 | DOI:10.1016/j.cellin.2024.100201

Pulm Ther. 2024 Oct 10. doi: 10.1007/s41030-024-00275-x. Online ahead of print.

ABSTRACT

INTRODUCTION: There are limited real-world data on outcomes in patients with non-cystic fibrosis bronchiectasis (NCFBE). This study assessed clinical characteristics and survival in patients with NCFBE by baseline exacerbation rate.

METHODS: Patients with bronchiectasis (≥ 1 inpatient or ≥ 2 outpatient claims with a bronchiectasis diagnosis code, or one outpatient claim with bronchiectasis code and a chest computed tomography scan) were from the 100% Medicare Fee-for-Service database (Jan 2014-Dec 2020). Patients had continuous enrollment ≥ 12 months pre-index (baseline) and post-index (follow-up), with index a random bronchiectasis claim preceded by ≥ 12 months bronchiectasis history. Patients with cystic fibrosis were excluded. Patients were stratified by exacerbations during baseline (0, 1, or ≥ 2). Follow-up exacerbation rate and all-cause mortality were assessed. Controls were identified using a multistep direct matching approach. Time to death from index was estimated by Kaplan-Meier analyses.

RESULTS: Exacerbation analysis included 92,529 patients with NCFBE and 92,529 matched controls. Exacerbations were common (43% had ≥ 1 exacerbation), with patients with more baseline exacerbations more likely to have ≥ 2 exacerbations during follow-up (11.4%, 24.2%, and 46.8% of patients with 0, 1, and ≥ 2 baseline exacerbations, respectively). Survival analysis included 110,298 patients with NCFBE and 110,298 controls. Time to death was shorter in patients with more baseline exacerbations (P < 0.0001). Five-year survival was 55.3%, 62.6%, and 65.4% for patients with ≥ 2, 1, and 0 baseline exacerbations, respectively, compared with 64.1% for controls.

CONCLUSIONS: In these patients with NCFBE, exacerbations were common. History of exacerbations was associated with future exacerbations and increased all-cause mortality.

PMID:39390311 | DOI:10.1007/s41030-024-00275-x

J Cyst Fibros. 2024 Oct 9:S1569-1993(24)01785-5. doi: 10.1016/j.jcf.2024.09.017. Online ahead of print.

ABSTRACT

RATIONALE: CF care guidelines recommend chronic inhaled antibiotics for chronic Pseudomonas aeruginosa (Pa) lung infection. These medications are costly, time consuming and prescription needs may change with improved outcomes.

OBJECTIVES: We determined the proportion of pwCF with chronic, intermittent or negative Pa infection categories, their clinical and demographic characteristics, factors associated with inhaled antibiotic prescription and changes between 2011 and 2019.

METHODS: This cohort study using the U.S. CF Foundation patient registry for pwCF >2 years, no prior lung transplant, and with ≥3 respiratory cultures/year determined chronic inhaled antibiotics (≥3 months per calendar year) and Pa infection status from encounter level data. Outcomes and odds of prescription for relevant clinical factors were evaluated using generalized estimating equation models with additional interaction between the predictor and the calendar year to examine changes of predictors over time.

RESULTS: Proportion of pwCF with chronic and intermittent Pa decreased and antibiotic prescription rates increased for these groups and decreased for Pa negative pwCF. Hispanic ethnicity, female sex, pancreatic insufficiency, CF diabetes, and ivacaftor/lumacaftor were associated with higher antibiotic prescriptions for each Pa status. Among Pa-negative pwCF prescriptions were higher with Burkholderia spp. (1.17, (CI95 1.03,1.34)) or MRSA (OR 1.45, (1.26,1.68)) but decreased between 2011 and 2019. For Aspergillus OR increased to 1.6,(1.3,1.8) in 2019. Prescriptions for pwCF on ivacaftor decreased, becoming lower in 2019 for chronic (OR 0.7, (0.5,0.8)) and Pa-negative pwCF (OR 0.7, (0.5,0.8)).

CONCLUSIONS: Factors predicting inhaled antibiotic prescription differed between 2011 and 2019 indicating changes in health and care for pwCF even prior to triple-modulators.

PMID:39389810 | DOI:10.1016/j.jcf.2024.09.017

Eur Respir J. 2024 Oct 10;64(4):2401573. doi: 10.1183/13993003.01573-2024. Print 2024 Oct.

NO ABSTRACT

PMID:39389615 | DOI:10.1183/13993003.01573-2024

Ann Am Thorac Soc. 2024 Oct 10. doi: 10.1513/AnnalsATS.202312-1070OC. Online ahead of print.

ABSTRACT

RATIONALE: The pathogenicity of Aspergillus in the cystic fibrosis (CF) airway is debated, leading to unclear clinical benefit of antifungal therapy for Aspergillus infection.

OBJECTIVE: To determine the real-world effectiveness of antifungal use in people with CF (PwCF) with Aspergillus species in the United States.

METHODS: We conducted a retrospective cohort study evaluating the association of antifungal use and respiratory outcomes in PwCF and Aspergillus positive cultures using the Cystic Fibrosis Foundation patient registry. Marginal structural models using inverse-probability treatment weighted estimators were used to test if antifungal exposure was associated with forced expiratory volume in one second percent predicted (FEV1pp) and pulmonary exacerbation rate, while controlling for fixed and time-varying confounders. We conducted sensitivity analyses on individuals with persistent Aspergillus and without concomitant allergic bronchopulmonary aspergillosis (ABPA).

RESULTS: A total of 14,754 individuals with Aspergillus positive cultures between 2006 and 2019 were identified. Antifungals were prescribed to 3,575 (24.2%) unique PwCF during the study period. Antifungal use was not associated with FEV1pp (adjusted estimate -0.96 percentage points, 95% CI -2.21,0.29). Antifungal use was associated with 29% increased rate of pulmonary exacerbations requiring intravenous (IV) antibiotics (adjusted IRR=1.29, 95% CI 1.22, 1.37). In sensitivity analyses limited to individuals without ABPA< antifungals were associated with 1.88 lower FEV1pp (95% CI -3.35, -0.41) and an increased rate of pulmonary exacerbations (adjusted IRR= 1.30, 95% CI 1.21,1.40). Whereas, in patients with persistent Aspergillus and persistent Aspergillus without concomitant ABPA, antifungals were not associated with FEV1pp.

CONCLUSIONS: Antifungal therapy in PwCF and Aspergillus positive cultures was not associated with improvements in FEV>1pp. While antifungal therapy was associated with increased risk for pulmonary exacerbations, this could reflect confounding by severity of disease. Randomized clinical trials examining the clinical efficacy of antifungals in Aspergillus infections are warranted.

PMID:39388639 | DOI:10.1513/AnnalsATS.202312-1070OC

Proc Natl Acad Sci U S A. 2024 Oct 15;121(42):e2414768121. doi: 10.1073/pnas.2414768121. Epub 2024 Oct 10.

ABSTRACT

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of CF. The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex, which was previously found to modulate ribosome collisions during "preemptive quality control" of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest that interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

PMID:39388263 | DOI:10.1073/pnas.2414768121

bioRxiv [Preprint]. 2024 Sep 25:2024.09.25.615034. doi: 10.1101/2024.09.25.615034.

ABSTRACT

The electrophile methylglyoxal (MG) is produced by microorganisms and host cells through central metabolic pathways. MG is a highly reactive electrophile, so it must be rapidly detoxified to prevent damaging modifications to macromolecules. Pseudomonas aeruginosa , a pathogen of concern due to its ability develop multidrug resistance, causes many types of infections that have been associated with elevated MG levels, including cystic fibrosis (CF). P. aeruginosa isolates commonly have mutations that lead to LasR loss-of-function (LasR-) and we found that lasR mutations confer sensitivity to MG in multiple strain backgrounds. LasR-strains have increased activity of the CbrAB two-component system which represses Crc regulation of metabolism. Here, we show that higher CbrAB activity and low Crc activity renders cells sensitive to MG. We found that P. aeruginosa LasR-strains are more sensitive to MG and have lower intracellular reduced glutathione (GSH) compared to their LasR+ comparators. Consistent with published reports, mutants lacking gloA3 , which encodes a MG-glyoxalase, and mutants lacking GSH biosynthesis enzymes ( gshA or gshB ) were sensitive to MG. Exogenous GSH rescued MG sensitivity in LasR-strains and gshA or gshB mutants, but not in a gloA3 mutant strain. We propose that low GSH levels in LasR-strains contribute to increased sensitivity to MG and H 2 O 2 .

SIGNIFICANCE: Methylglyoxal is a highly reactive metabolite that is detected in various disease states, including those where Pseudomonas aeruginosa is present and MG resistance requires the glutathione-dependent glyoxalase enzyme GloA3 enzyme. This study reveals that P . aeruginosa strains with LasR mutations, which are commonly found in clinical isolates, are more sensitive to methylglyoxal (MG) and hydrogen peroxide due to lower intracellular glutathione levels and high activity of the CbrAB-Crc regulatory pathway. This could be significant for understanding the selective pressures that drive P. aeruginosa evolution in infection sites, as well as a better understanding of LasR-strain metabolism in infections such as those associated with cystic fibrosis.

PMID:39386711 | PMC:PMC11463435 | DOI:10.1101/2024.09.25.615034

bioRxiv [Preprint]. 2024 Sep 25:2024.09.24.614743. doi: 10.1101/2024.09.24.614743.

ABSTRACT

Staphylococcus aureus is one of the most common pathogens isolated from the lungs of people with cystic fibrosis (CF), but little is known about its ability to colonize this niche. We performed a Tn-seq screen to identify genes necessary for S. aureus growth in media prepared from ex vivo CF sputum. We identified 19 genes that were required for growth in all sputum media tested and dozens more that were required for growth in at least one sputum medium. Depleted mutants of interest included insertions in many genes important for surviving metal starvation as well as the primary regulator of cysteine metabolism cymR . To investigate the mechanisms by which these genes contribute to S. aureus growth in sputum, we quantified low-molecular-weight thiols, nutrient transition metals, and the host metal-sequestration protein calprotectin in sputum from 11 individuals with CF. In all samples, the abundance of calprotectin exceeded nutrient metal concentration, explaining the S. aureus requirement for metal-starvation genes. Further, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements of S. aureus . Deletion of the cysteine importer genes tcyA and tcyP in the Δ cymR background restored growth to wild-type levels in CF sputum, suggesting that the mechanism by which cymR is required for growth in sputum is to prevent uncontrolled import of cysteine or cystine from this environment. Overall, this work demonstrates that calprotectin and cysteine limit S. aureus growth in CF sputum.

IMPORTANCE: Staphylococcus aureus is a major cause of lung infections in people with cystic fibrosis (CF). This work identifies genes required for S. aureus growth in this niche, which represent potential targets for anti-Staphylococcal treatments. We show that genes involved in surviving metal starvation are required for growth in CF sputum. We also found that the primary regulator of cysteine metabolism, CymR, plays a critical role in preventing cysteine intoxication during growth in CF sputum. To support these models, we analyzed sputum from 11 individuals with CF to determine concentrations of calprotectin, nutrient metals, and low-molecular-weight thiols, which have not previously been quantified together in the same samples.

PMID:39386554 | PMC:PMC11463553 | DOI:10.1101/2024.09.24.614743

Front Pharmacol. 2024 Sep 25;15:1460692. doi: 10.3389/fphar.2024.1460692. eCollection 2024.

ABSTRACT

Exosomes, membrane-bound extracellular vesicles, ranging from approximately 30-200 nm in diameter, are released by almost all cell types and play critical roles in intercellular communication. In response to the prevailing stress, the exosome-bound protein signatures vary in abundance and composition. To identify the bronchoalveolar lavage fluid (BALF) exosome-bound proteins associated with mucoinflammatory lung disease and to gain insights into their functional implications, we compared BALF exosomes-derived proteins from adult Scnn1b transgenic (Scnn1b-Tg+) and wild type (WT) mice. A total of 3,144 and 3,119 proteins were identified in BALF exosomes from Scnn1b-Tg+ and WT mice, respectively. Using cutoff criteria (Log2 fold-change > 1 and adjusted p-value < 0.05), the comparison of identified proteins revealed 127 and 30 proteins that were significantly upregulated and downregulated, respectively, in Scnn1b-Tg+ versus WT mice. In addition, 52 and 27 proteins were exclusively enriched in Scnn1b-Tg+ and WT mice, respectively. The identified exosome-bound proteins from the homeostatic airspaces of WT mice were mostly relevant to the normal physiological processes. The protein signatures enriched in the BALF exosomes of Scnn1b-Tg+ mice were relevant to macrophage activation and mucoinflammatory processes. Ingenuity pathway analyses revealed that the enriched proteins in Scnn1b-Tg+ mice contributed to the inflammatory responses and antimicrobial defense pathways. Selective proteins including, RETNLA/FIZZ1, LGALS3/Galectin-3, S100A8/MRP8, and CHIL3/YM1 were immunolocalized to specific cell types. The comparative analysis between enriched BALF exosome proteins and previously identified differentially upregulated genes in Scnn1b-Tg+ versus WT mice suggested that the compartment-/cell-specific upregulation in gene expression dictates the enrichment of their respective proteins in the lung airspaces. Taken together, this study demonstrates that the BALF exosome-bound protein signatures reflect disease-relevant disturbances. Our findings suggest that the exosomes carry disease-relevant protein signatures that can be used as a diagnostic as well as predictive biomarkers for mucoinflammatory lung disease.

PMID:39386033 | PMC:PMC11461968 | DOI:10.3389/fphar.2024.1460692