Analysis of CF patient survival confirms STAT3 as a CF-modifying gene with changing impact over time.

INTRODUCTION AND AIM: The signal transducer and activator of transcription 3 (STAT3) has been identified as one of the cystic fibrosis (CF) modifying genes. In this study, we aimed to assess the association between STAT3 genotype and CF patient survival over several decades and to investigate the effect of STAT3 inhibition on epithelial CFTR expression. METHODS: We analyzed the informative genetic marker STAT3Sat for its association with survival in 174 p.Phe508del-CFTR homozygous CF patients treated at the CF center in Hannover spanning birth cohorts from >3 decades (1959-1994). Furthermore, we treated two epithelial cell lines with STAT3 inhibitors and monitored changes of CFTR protein expression by western blot. RESULTS: Only for p.Phe508del-CFTR homozygous patients born prior to 1975, survival was significantly influenced by STAT3sat genotype (P = 0.023). The expression levels of STAT3 and CFTR positively correlated in epithelial cell lines (P = 0.01). CONCLUSIONS: Our results in different birth cohorts identified a time-dependent impact of STAT3 genotype on CF patients' survival and found that improved symptomatic treatment of later-born CF patients obviates STAT3's modifying influence. Consistent with our previous results, STAT3-specific inhibition resulted in increased CFTR expression in the epithelial cell line 16HBE14o-. Thus, care should be taken when CF-modifying genes are studied in cross-sectional cohorts as the impact of modifying genes might not be invariant in the light of changing therapeutic regimens.

Impact of elexacaftor/tezacaftor/ivacaftor therapy on lung clearance index and magnetic resonance imaging in children with cystic fibrosis and one or two F508del alleles.

BACKGROUND: We recently demonstrated that elexacaftor/tezacaftor/ivacaftor (ETI) improves the lung clearance index (LCI) and abnormalities in lung morphology detected by magnetic resonance imaging (MRI) in adolescent and adult patients with cystic fibrosis (CF). However, real-world data on the effect of ETI on these sensitive outcomes of lung structure and function in school-age children with CF have not been reported. The aim of this study was therefore to examine the effect of ETI on the LCI and the lung MRI score in children aged 6-11 years with CF and one or two F508del alleles. METHODS: This prospective, observational, multicentre, post-approval study assessed the longitudinal LCI up to 12 months and the lung MRI score before and 3 months after initiation of ETI. RESULTS: A total of 107 children with CF including 40 heterozygous for F508del and a minimal function mutation (F/MF) and 67 homozygous for F508del (F/F) were enrolled in this study. Treatment with ETI improved the median (interquartile range (IQR)) LCI in F/MF (-1.0 (-2.0- -0.1); p<0.01) and F/F children (-0.8 (-1.9- -0.2); p<0.001) from 3 months onwards. Further, ETI improved the median (IQR) MRI global score in F/MF (-4.0 (-9.0-0.0); p<0.01) and F/F children (-3.5 (-7.3- -0.8); p<0.001). CONCLUSIONS: ETI improves early abnormalities in lung ventilation and morphology in school-age children with CF and at least one F508del allele in a real-world setting. Our results support early initiation of ETI to reduce or even prevent lung disease progression in school-age children with CF.

Effects of Elexacaftor/Tezacaftor/Ivacaftor Therapy on Lung Clearance Index and Magnetic Resonance Imaging in Patients with Cystic Fibrosis and One or Two F508del Alleles.

Rationale: We recently demonstrated that triple-combination CFTR (cystic fibrosis transmembrane conductance regulator) modulator therapy with elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) improves CFTR function in airway and intestinal epithelia to 40-50% of normal in patients with cystic fibrosis (CF) with one or two F508del alleles. In previous studies, this improvement of CFTR function was shown to improve clinical outcomes; however, effects on the lung clearance index (LCI) determined by multiple-breath washout and abnormalities in lung morphology and perfusion detected by magnetic resonance imaging (MRI) have not been studied. Objectives: To examine the effect of ELX/TEZ/IVA on LCI and lung MRI scores in patients with CF and one or two F508del alleles aged ⩾12 years. Methods: This prospective, observational, multicenter, postapproval study assessed LCI and lung MRI scores before and 8-16 weeks after initiation of ELX/TEZ/IVA. Measurements and Main Results: A total of 91 patients with CF, including 45 heterozygous for F508del and a minimal function mutation (MF) and 46 homozygous for F508del, were enrolled in this study. Treatment with ELX/TEZ/IVA improved LCI in F508del/MF (-2.4; interquartile range [IQR], -3.7 to -1.1; P < 0.001) and F508del homozygous (-1.4; IQR, -2.4 to -0.4; P < 0.001) patients. Furthermore, ELX/TEZ/IVA improved the MRI global score in F508del/MF (-6.0; IQR, -11.0 to -1.3; P < 0.001) and F508del homozygous (-6.5; IQR, -11.0 to -1.3; P < 0.001) patients. Conclusions: Our data demonstrate that improvement of CFTR function by ELX/TEZ/IVA improves lung ventilation and abnormalities in lung morphology, including airway mucus plugging and wall thickening, in adolescent and adult patients with CF and one or two F508del alleles in a real-world, postapproval setting. Clinical trial registered with www.clinicaltrials.gov (NCT04732910).

Effects of Elexacaftor/Tezacaftor/Ivacaftor Therapy on CFTR Function in Patients with Cystic Fibrosis and One or Two F508del Alleles.

Rationale: The CFTR (cystic fibrosis transmembrane conductance regulator) modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) was shown to improve clinical outcomes and sweat chloride concentration in patients with cystic fibrosis (CF) and one or two F508del alleles. However, the effect of ELX/TEZ/IVA on CFTR function in the airways and intestine has not been studied. Objectives: To assess the effect of ELX/TEZ/IVA on CFTR function in airway and intestinal epithelia in patients with CF and one or two F508del alleles aged 12 years and older. Methods: This prospective, observational, multicenter study assessed clinical outcomes including FEV1% predicted and body mass index and the CFTR biomarkers sweat chloride concentration, nasal potential difference, and intestinal current measurement before and 8-16 weeks after initiation of ELX/TEZ/IVA. Measurements and Main Results: A total of 107 patients with CF including 55 patients with one F508del and a minimal function mutation and 52 F508del homozygous patients were enrolled in this study. In patients with one F508del allele, nasal potential difference and intestinal current measurement showed that ELX/TEZ/IVA improved CFTR function in nasal epithelia to a level of 46.5% (interquartile range [IQR], 27.5-72.4; P < 0.001) and in intestinal epithelia to 41.8% of normal (IQR, 25.1-57.6; P < 0.001). In F508del homozygous patients, ELX/TEZ/IVA exceeded improvement of CFTR function observed with TEZ/IVA and increased CFTR-mediated Cl- secretion to a level of 47.4% of normal (IQR, 19.3-69.2; P < 0.001) in nasal and 45.9% (IQR, 19.7-66.6; P < 0.001) in intestinal epithelia. Conclusions: Treatment with ELX/TEZ/IVA results in effective improvement of CFTR function in airway and intestinal epithelia in patients with CF and one or two F508del alleles. Clinical trial registered with www.clinicaltrials.gov (NCT04732910).

The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

Direct RNA Nanopore Sequencing of Pseudomonas aeruginosa Clone C Transcriptomes.

The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNAArg, and the tRNAArg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs. IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that-if replicated in other species-will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.

Wochenende - modular and flexible alignment-based shotgun metagenome analysis.

BACKGROUND: Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. RESULTS: Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. Results are completely transparent and filters can be adjusted by the user. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization and the calculation of growth rates, as well as integration and subsequent plotting of pipeline results as heatmaps or heat trees. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. CONCLUSION: Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende .

Bacterial tRNA landscape revisited.

The updated Wobble Hypothesis reasonably explains why some 40 tRNA species are sufficient to decode the 61 amino acid codons of the Universal Genetic Code. However, we still have no clue why eubacteria lack tRNA isoacceptors with ANN anticodons, whereas eukaryotes universally lack eight GNN anticodons, only one of which is also absent in bacteria. Direct tRNA sequencing could resolve the patterns of nucleoside modification that had been driving the divergent evolution in prokaryotes and eukaryotes, but this task will require the development of AI-supported base-callers that can recognize modified nucleosides without any subsequent analytical verification. Our knowledge of the bacterial tRNA landscape is moreover broadened by the recent discovery of antisense tRNAs and tRNA-derived fragments that should be examined in their roles for gene expression, translation, bacterial physiology or metabolism.

Rescue from Pseudomonas aeruginosa Airway Infection via Stem Cell Transplantation.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

What Makes Pseudomonas aeruginosa a Pathogen?

Pseudomonas aeruginosa is classified as an opportunistic pathogen that causes a wide range of infections in humans, involving nearly all body systems, that vary from local to systemic and from self-limiting to life-threatening. This chapter outlines the features that have made P. aeruginosa a human pathogen. Each section starts with the argument in the heading followed by the epidemiological and/or experimental supportive evidence.