Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Stellate cells are in utero markers of pancreatic disease in cystic fibrosis.

BACKGROUND: Pancreatic fibrosis is an early diagnostic feature of the common inherited disorder cystic fibrosis (CF). Many people with CF (pwCF) are pancreatic insufficient from birth and the replacement of acinar tissue with cystic lesions and fibrosis is a progressive phenotype that may later lead to diabetes. Little is known about the initiating events in the fibrotic process though it may be a sequela of inflammation in the pancreatic ducts resulting from loss of CFTR impairing normal fluid secretion. Here we use a sheep model of CF (CFTR-/-) to examine the evolution of pancreatic disease through gestation. METHODS: Fetal pancreas was collected at six time points from 50-days of gestation through to term, which is equivalent to ~ 13 weeks to term in human. RNA was extracted from tissue for bulk RNA-seq and single cells were prepared from 80-day, 120-day and term samples for scRNA-seq. Data were validated by immunochemistry. RESULTS: Transcriptomic evidence from bulk RNA-seq showed alterations in the CFTR-/- pancreas by 65-days of gestation, which are accompanied by marked pathological changes by 80-days of gestation. These include a fibrotic response, confirmed by immunostaining for COL1A1, αSMA and SPARC, together with acinar loss. Moreover, using scRNA-seq we identify a unique cell population that is significantly overrepresented in the CFTR-/- animals at 80- and 120-days gestation, as are stellate cells at term. CONCLUSION: The transcriptomic changes and cellular imbalance that we observe likely have pivotal roles in the evolution of CF pancreatic disease and may provide therapeutic opportunities to delay or prevent pancreatic destruction in CF.

Who Am I? Self-concept in Adults with Cystic Fibrosis: Association with Anxiety and Depression.

Cystic Fibrosis (CF) is a progressive condition resulting in reduced lung function and strongly associated with elevated anxiety and depression symptoms. Self-concept refers to an individual's overarching sense of identity, a positive level of which is widely associated with reduced anxiety and depression. There is a significant lack of self-concept research within CF. This study explores the association between self-concept and anxiety and depression in adults with CF. 64 adults living with CF in Western Australia completed validated online questionnaires (Generalised Anxiety Disorder-7, Patient Health Questionnaire-9, Tennessee Self-Concept Questionnaire 2: Short-Form) and consented to the collection of medical data. Descriptive, t-test, correlation and multiple regression analysis were undertaken. Higher levels of self-concept were associated with lower levels of anxiety and depression symptoms. Lower self-concept levels were a significant predictor of increased anxiety and depression symptoms after accounting for physical health status. Mean self-concept scores for those who required mental health intervention were significantly lower compared to those that did not. This study identifies a significant relationship between self-concept and anxiety and depression in adults with CF. Further research is required to establish causation and test the feasibility of self-concept interventions in reducing anxiety and depression symptoms.

Elexacaftor Mediates the Rescue of F508del CFTR Functional Expression Interacting with MSD2.

Cystic fibrosis (CF) is one of the most frequent lethal autosomal recessive diseases affecting the Caucasian population. It is caused by loss of function variants of the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein located on the apical side of epithelial cells. The most prevalent CF-causing mutation, the deletion of phenylalanine at position 508 (F508del), is characterized by folding and trafficking defects, resulting in the decreased functional expression of the protein on the plasma membrane. Two classes of small-molecule modulators, termed potentiators and correctors, respectively, have been developed to rescue either the gating or the cellular processing of defective F508del CFTR. Kaftrio, a next-generation triple-combination drug, consisting of the potentiator ivacaftor (VX770) and the two correctors tezacaftor (VX661) and elexacaftor (VX445), has been demonstrated to be a life-changing therapeutic modality for the majority of people with CF worldwide. While the mechanism of action of VX770 and VX661 is almost known, the precise mechanism of action and binding site of VX445 have not been conclusively determined. We investigated the activity of VX445 on mutant F508del to identify the protein domains whose expression is mostly affected by this corrector and to disclose its mechanisms of action. Our biochemical analyses revealed that VX445 specifically improves the expression and the maturation of MSD2, heterologously expressed in HEK 293 cells, and confirmed that its effect on the functional expression of defective F508del CFTR is additive either with type I or type II CFTR correctors. We are confident that our study will help to make a step forward in the comprehension of the etiopathology of the CF disease, as well as to give new information for the development and testing of combinations of even more effective correctors able to target mutation-specific defects of the CFTR protein.

Role of Nasal Nitric Oxide in Primary Ciliary Dyskinesia and Other Respiratory Conditions in Children.

Nitric oxide (NO) is produced within the airways and released with exhalation. Nasal NO (nNO) can be measured in a non-invasive way, with different devices and techniques according to the age and cooperation of the patients. Here, we conducted a narrative review of the literature to examine the relationship between nNO and some respiratory diseases with a particular focus on primary ciliary dyskinesia (PCD). A total of 115 papers were assessed, and 50 were eventually included in the review. nNO in PCD is low (below 77 nL/min), and its measurement has a clear diagnostic value when evaluated in a clinically suggestive phenotype. Many studies have evaluated the role of NO as a molecular mediator as well as the association between nNO values and genotype or ciliary function. As far as other respiratory diseases are concerned, nNO is low in chronic rhinosinusitis and cystic fibrosis, while increased values have been found in allergic rhinitis. Nonetheless, the role in the diagnosis and prognosis of these conditions has not been fully clarified.

Clinical and Functional Characteristics of the E92K CFTR Gene Variant in the Russian and Turkish Population of People with Cystic Fibrosis.

The pathogenic variant E92K (c.274G > A) of the CFTR gene is rare in America and Europe, but it is common for people with cystic fibrosis from Russia and Turkey. We studied the effect of the E92K genetic variant on the CFTR function. The function of the CFTR channel was studied using the intestinal current measurements (ICM) method. The effects of CFTR modulators on the restoration of the CFTR function were studied in the model of intestinal organoids. To assess the effect of E92K on pre-mRNA splicing, the RT-PCR products obtained from patients' intestinal organoid cultures were analyzed. Patients with the genetic variant E92K are characterized by an older age of diagnosis compared to homozygotes F508del and a high frequency of pancreatic sufficiency. The results of the sweat test and the ICM method showed partial preservation of the function of the CFTR channel. Functional analysis of CFTR gene expression revealed a weak effect of the E92K variant on mRNA-CFTR splicing. Lumacaftor (VX-809) has been shown to restore CFTR function in an intestinal organoid model, which allows us to consider the E92K variant as a promising target for therapy with CFTR correctors.

Personalized Selection of a CFTR Modulator for a Patient with a Complex Allele [L467F;F508del].

The presence of complex alleles in the CFTR gene can lead to difficulties in diagnosing cystic fibrosis and cause resistance to therapy with CFTR modulators. Tezacaftor/ivacaftor therapy for 8 months in a patient with the initially established F508del/F508del genotype did not lead to an improvement in her condition-there was no change in spirometry and an increase in the patient's weight, while there was only a slight decrease in NaCl values, measured by a sweat test. The intestinal current measurements of the patient's rectal biopsy showed no positive dynamics in the rescue of CFTR function while taking tezacaftor/ivacaftor. The assumption that the patient had an additional mutation in the cis position was confirmed by sequencing the CFTR gene, and the complex allele [L467F;F508del] was identified. Based on the rescue of CFTR function by elexacaftor/tezacaftor/ivacaftor obtained using forskolin-induced swelling on intestinal organoids, the patient was prescribed therapy with this targeted drug. The use of elexacaftor/tezacaftor/ivacaftor for 7 months resulted in a significant improvement in the patient's clinical condition.

Discovery and SAR of 4-aminopyrrolidine-2-carboxylic acid correctors of CFTR for the treatment of cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease resulting from mutations on both copies of the CFTR gene. Phenylalanine deletion at position 508 of the CFTR protein (F508del-CFTR) is the most frequent mutation in CF patients. Currently, the most effective treatments of CF use a dual or triple combination of CFTR correctors and potentiators. In triple therapy, two correctors (C1 and C2) and a potentiator are employed. Herein, we describe the identification and exploration of the SAR of a series of 4-aminopyrrolidine-2-carboxylic acid C2 correctors of CFTR to be used in conjunction with our existing C1 corrector series for the treatment of CF.

Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.

OBJECTIVES: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. METHODS: After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. RESULTS: Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. CONCLUSIONS: We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

Xylitol as a Hydrophilization Moiety for a Biocatalytically Synthesized Ibuprofen Prodrug.

Biocatalyzed synthesis can be exploited to produce high-value products, such as prodrugs. The replacement of chemical approaches with biocatalytic processes is advantageous in terms of environmental prevention, embracing the principles of green chemistry. In this work, we propose the covalent attachment of xylitol to ibuprofen to produce an IBU-xylitol ester prodrug. Xylitol was chosen as a hydrophilizer for the final prodrug, enhancing the water solubility of ibuprofen. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) extensively used as an analgesic, anti-inflammatory, and antipyretic. Despite being the third-most-prescribed medicine in the world, the aqueous solubility of ibuprofen is just 21 mg/L. This poor water solubility greatly limits the bioavailability of ibuprofen. We aimed to functionalize ibuprofen with xylitol using the reusable immobilized N435 biocatalyst. Instead of a biphasic media, we proposed a monophasic reaction environment. The characterization of the IBU-xylitol ester was performed by 1H, 13C-NMR, DEPT, COSY, HMQC, HMBC, FTIR, and MS spectroscopy. Preliminary in vitro tests showed that this enzymatically synthesized prodrug of ibuprofen reduced the expression of the interleukin 8 genes in human bronchial epithelial cells (IB3-1) from cystic fibrosis (CF) patients.

Mucins and CFTR: Their Close Relationship.

Mucociliary clearance is a critical defense mechanism for the lungs governed by regionally coordinated epithelial cellular activities, including mucin secretion, cilia beating, and transepithelial ion transport. Cystic fibrosis (CF), an autosomal genetic disorder caused by the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, is characterized by failed mucociliary clearance due to abnormal mucus biophysical properties. In recent years, with the development of highly effective modulator therapies, the quality of life of a significant number of people living with CF has greatly improved; however, further understanding the cellular biology relevant to CFTR and airway mucus biochemical interactions are necessary to develop novel therapies aimed at restoring CFTR gene expression in the lungs. In this article, we discuss recent advances of transcriptome analysis at single-cell levels that revealed a heretofore unanticipated close relationship between secretory MUC5AC and MUC5B mucins and CFTR in the lungs. In addition, we review recent findings on airway mucus biochemical and biophysical properties, focusing on how mucin secretion and CFTR-mediated ion transport are integrated to maintain airway mucus homeostasis in health and how CFTR dysfunction and restoration of function affect mucus properties.

A Splice Switch in SIGIRR Causes a Defect of IL-37-Dependent Anti-Inflammatory Activity in Cystic Fibrosis Airway Epithelial Cells.

Cystic fibrosis (CF) is a hereditary disease typically characterized by infection-associated chronic lung inflammation. The persistent activation of toll-like receptor (TLR) signals is considered one of the mechanisms for the CF hyperinflammatory phenotype; however, how negative regulatory signals of TLRs associate with CF inflammation is still elusive. Here, we showed that the cell surface expression of a single immunoglobulin interleukin-1 receptor (IL-1R)-related molecule (SIGIRR), a membrane protein essential for suppressing TLRs- and IL-1R-dependent signals, was remarkably decreased in CF airway epithelial cells compared to non-CF cells. Notably, CF airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the SIGIRR that lacks exon 8 (Δ8-SIGIRR), which results in the production of a C-terminal truncated form of the SIGIRR. Δ8-SIGIRR was expressed intracellularly, and its over-expression abolished the cell surface expression and function of the full-length SIGIRR (WT-SIGIRR), indicating its dominant-negative effect leading to the deficiency of anti-inflammatory activity in CF cells. Consistently, IL-37, a ligand for the SIGIRR, failed to suppress viral dsRNA analogue poly(I:C)-dependent JNK activation and IL-8 production, confirming the reduction in the functional WT-SIGIRR expression in the CF cells. Together, our studies reveal that SIGIRR-dependent anti-inflammatory activity is defective in CF airway epithelial cells due to the unique splicing switch of the SIGIRR gene and provides the first evidence of IL-37-SIGIRR signaling as a target of CF airway inflammation.

Evaluation of the Complex p.[Leu467Phe;Phe508del] CFTR Allele in the Intestinal Organoids Model: Implications for Therapy.

In the cohort of Russian patients with cystic fibrosis, the p.[Leu467Phe;Phe508del] complex allele (legacy name [L467F;F508del]) of the CFTR gene is understudied. In this research, we present the results of frequency evaluation of the [L467F;F508del] complex allele in the Russian Federation among patients with a F508del/F508del genotype, its effect on the clinical course of cystic fibrosis, the intestinal epithelium ionic channel function, and the effectiveness of target therapy. The frequency of the [L467F;F508del] complex allele among patients with homozygous F508del was determined with multiplex ligase-dependent probe amplification followed by polymerase chain reaction and fragment analysis. The function of ionic channels, including the residual CFTR function, and the effectiveness of CFTR modulators was analyzed using intestinal current measurements on rectal biopsy samples and the forskolin-induced swelling assay on organoids. The results showed that the F508del/[L467F;F508del] genotype is present in 8.2% of all Russian patients with F508del in a homozygous state. The clinical course of the disease in patients with the F508del/[L467F;F508del] genotype is severe and does not vary from the course in the cohort with homozygous F508del, although the CFTR channel function is significantly lower. For patients with the F508del/[L467F;F508del] genotype, we can recommend targeted therapy using a combined ivacaftor + tezacaftor + elexacaftor medication.

Two Siblings Homozygous for F508del-CFTR Have Varied Disease Phenotypes and Protein Biomarkers.

Two siblings with CF are homozygous for F508del (referred to as Subject A and Subject B). Despite having the same CFTR genotype and similar environment, these two subjects exhibited different disease phenotypes. We analyzed their medical records and CF Foundation Registry data and measured inflammatory protein mediators in their sputum samples. Then, we examined the longitudinal relationships between inflammatory markers and disease severity for each subject and compared between them. Subject A presented a more severe disease than Subject B. During the study period, Subject A had two pulmonary exacerbations (PEs) whereas Subject B had one mild PE. The forced expiratory volume in 1 s (FEV1, % predicted) values for Subject A were between 34-45% whereas for Subject B varied between 48-90%. Inflammatory protein mediators associated with neutrophils, Th1, Th2, and Th17 responses were elevated in sputum of Subject A compared with Subject B, and also in samples collected prior to and during PEs for both subjects. Neutrophilic elastase (NE) seemed to be the most informative biomarkers. The infectious burden between these two subjects was different.

Two Years of Newborn Screening for Cystic Fibrosis in North Macedonia: First Experience.

There is a widely accepted consensus on the benefits of newborn screening (NBS) for cystic fibrosis (CF) in terms of reduced disease severity, improved quality of life, lower treatment burden, and reduced costs. More and more countries in the world are introducing NBS for CF as a national preventive health program. Newborn screening for CF was introduced in the Republic of North Macedonia (RNM) in April, 2019, after a pilot study of 6 months in 2018. A two-step immunoreactive trysinogen (IRT-IRT) algorithm is performed, and then a sweat test for confirmation/exclusion of the CF diagnosis when the IRT values were both over the cutoff (70.0 and 45.0 ng/mL, respectively). In cases with confirmed diagnosis of CF (a sweat chloride concentration >60.0 mmol/L) or with intermediate sweat test results (a sweat chloride concentration of between 30.0 and 59.0 mmol/L), CF transmembrane conductance regulator (CFTR) mutation analysis is performed. By the end of 2020, over a period of 27 months, including the pilot study period, a total number of 43,139 newborns were screened for CF. Seventeen (0.039%) newborns were diagnosed with CF. In all newly discovered CF cases by screening, the diagnosis was confirmed by determination of the CFTR mutations. The most common CFTR mutation, F508del, was found with an overall incidence of 70.6%. Other more frequent mutations were G542X (11.8%) and N1303K (5.9%). Four mutations were found in one CFTR allele each: G1349D, G126D, 457TAT>G and CFTRdupexon22, with the last one being newly discovered with unknown consequences. An incredibly large difference was found in the incidence of the disease between the Macedonian and Albanian neonatal population, with almost four time higher prevalence among Albanians (1:4530 vs. 1:1284).

Determination of Cystic Fibrosis Mutation Frequency in Preterm and Term Neonates with Respiratory Tract Problems.

Cystic fibrosis (CF) is an autosomal recessive disease. The genetic transition occurs with CF transmembrane conductance regulator (CFTR) gene mutation. We aimed to determine the frequency of CF mutations and also new mutations in the CFTR gene in neonates with respiratory distress. Newborn babies hospitalized due to respiratory distress were included in the patient group. The control group consisted of infants who had no respiratory distress. The CFTR genes of both groups were analyzed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods. A total of 40 patients (20 in the patient group and 20 in the control group) were evaluated. The CFTR gene analysis was normal in 16 neonates in the patient group, whereas in others: A46D (c.137C>A) (n = 1), D1312G (c.3935A>G) (n = 1), R117H (c.350G>A) (n = 1), S1426P (c.4276T>C) (n = 1) heterozygotes were detected; CFTR gene analysis was normal at 14 neonates in the control group, whereas in others: E1228G (c.3683A>G) (n = 1), E217G (c.650A>G) (n = 1), E632TfsX9 (c1894_1895delAG) (n = 1), I807M (c.2421 A>G) (n = 2), S573F (c.1718C>T) (n = 1) heterozygotes were detected. There was no significant difference in the patient and control groups' CFTR gene analysis (p = 0.340). This study demonstrates the importance of CFTR gene analysis in asymptomatic newborn infants for follow-up and early diagnosis of CFTR-related disorders. In this study, a c.1894_1895delAG (E632TfsX9) heterozygous mutation detected in the CFTR gene in an asymptomatic newborn infant, was first encountered in the literature.

Prevalence of allergic bronchopulmonary aspergillosis in cystic fibrosis patients using two different diagnostic criteria.

Summary: Objective.There are different diagnostic criteria for the diagnosis of Allergic bronchopulmonary aspergillosis (ABPA) in CF patients. In this present study we evaluated the prevalence of ABPA in Iranian CF patients by two more usual diagnostic criteria as ISHAM working criteria (A) and CF Foundation Consensus Conference criteria (B). Methods.Eighty-six CF patients were included in the study. All CF patients underwent for Aspergillus skin prick test (AST), Aspergillus-specific IgE (sIgEAf) and Aspergillus-specific IgG (sIgGAf), total IgE. The ABPA prevalence was estimated by two diagnostic criteria, (A) and (B) and compared. Results. The frequency of positive AST, total IgE, sIgEAf and sIgGAf were 47 (54.6%), 9 (10.5%), 42 (48.8%) and 67 (77.9%), respectively. The obtained rate of ABPA prevalence (10.5%) was identical in two diagnostic criteria A and B (kappa value of 1.000). Conclusions.The applied diagnostic criteria had no significant effect on the reported rate of ABPA prevalence.

Case Report of a Successful Pregnancy in a Cystic Fibrosis Patient with The c.1521_1523delCTT/c.3718-2477C>t Genotypes.

The aim of this case report was to show the consequences of pregnancy in a cystic fibrosis (CF) patient with a rare mutation. We present a case of a patient with CF, pregnant for the second time, who gave birth to a healthy child. Her mutation status revealed the presence of relatively rare mutation c.3718-2477C>T that is associated with a milder phenotype of the disease. During pregnancy, her vital signs were within normal limits. She had no exacerbations after the third gestational month. Cystic fibrosis is the most common genetic disorder among Caucasians. Over the last few decades, the survival rate and the lifespan of patients with CF have increased progressively. This is why more affected women are choosing to become pregnant. Predictive factors for the pregnancy outcome are basal pulmonary function [measured by forced expiratory volume/1 second (FEV1)], nutritional status [measured by body mass index (BMI)], diabetes and bacterial colonization. The report of our case emphasizes the need for establishing the exact mutations in CF patients who plan to become pregnant in order to predict the possible outcomes of this specific period of life. Moreover, genetic counseling is strongly recommended for the right understanding of the pregnancy risks in such cases.

Pyrimidine Biosynthesis Regulates the Small-Colony Variant and Mucoidy in Pseudomonas aeruginosa through Sigma Factor Competition.

Mucoidy due to alginate overproduction by the Gram-negative bacterium Pseudomonas aeruginosa facilitates chronic lung infections in patients with cystic fibrosis (CF). We previously reported that disruption in de novo synthesis of pyrimidines resulted in conversion to a nonmucoid small-colony variant (SCV) in the mucoid P. aeruginosa strain (PAO581), which has a truncated anti-sigma factor, MucA25, that cannot sequester sigma factor AlgU (AlgT). Here, we showed that supplementation with the nitrogenous bases uracil or cytosine in growth medium complemented the SCV to normal growth, and nonmucoidy to mucoidy, in these mucA25 mutants. This conversion was associated with an increase in intracellular levels of UMP and UTP suggesting that nucleotide restoration occurred via a salvage pathway. In addition, supplemented pyrimidines caused an increase in activity of the alginate biosynthesis promoter (P algD ), but had no effect on P algU , which controls transcription of algU Cytosolic levels of AlgU were not influenced by uracil supplementation, yet levels of RpoN, a sigma factor that regulates nitrogen metabolism, increased with disruption of pyrimidine synthesis and decreased after supplementation of uracil. This suggested that an elevated level of RpoN in SCV may block alginate biosynthesis. To support this, we observed that overexpressing rpoN resulted in a phenotypic switch to nonmucoidy in PAO581 and in mucoid clinical isolates. Furthermore, transcription of an RpoN-regulated promoter increased in the mutants and decreased after uracil supplementation. These results suggest that the balance of RpoN and AlgU levels may regulate growth from SCV to mucoidy through sigma factor competition for P algDIMPORTANCE Chronic lung infections with P. aeruginosa are the main cause of morbidity and mortality in patients with cystic fibrosis. This bacterium overproduces a capsular polysaccharide called alginate (also known as mucoidy), which aids in bacterial persistence in the lungs and in resistance to therapeutic regimens and host immune responses. The current study explores a previously unknown link between pyrimidine biosynthesis and mucoidy at the level of transcriptional regulation. Identifying/characterizing this link could provide novel targets for the control of bacterial growth and mucoidy. Inhibiting mucoidy may improve antimicrobial efficacy and facilitate host defenses to clear the noncapsulated P. aeruginosa bacteria, leading to improved prognosis for patients with cystic fibrosis.

Case report of synchronous post-lung transplant colon cancers in the era of colorectal cancer screening recommendations in cystic fibrosis: screening "too early" before it's too late.

BACKGROUND: The increasing life expectancy of individuals with Cystic Fibrosis (CF) is likely to be associated with new age-related challenges, colorectal cancer (CRC) most notably; recent consensus recommendations for CRC screening published in 2018 represent an important early step in addressing the emerging awareness of CF as a gastrointestinal cancer syndrome. These recommendations, however, need to be further refined based on more systematic data. We discuss an illustrative first-ever case of synchronous CRC arising in a post-lung transplant individual with CF within the recommended surveillance interval after a well-documented prior normal colonoscopy. CASE PRESENTATION: A 51-year-old female individual with homozygous F508del CF, presents to clinic with abdominal discomfort and intermittent blood in stools. She had previously undergone bilateral lung transplantation 18 years earlier, as well as two kidney transplants related to immunosuppression-related nephrotoxicity. A diagnostic colonoscopy was performed which revealed the presence of two separate synchronous colon cancers in the cecum and transverse colon; she had undergone a colonoscopy three years prior to this exam which was structurally normal. Endoscopic quality indicators, including a good quality bowel preparation, colonoscopic withdrawal time > 12 min, and quarterly Adenoma Detection Rate (ADR) ranging from 50 to 70% for both male and female patients for the endoscopist from both colonoscopic exams, as well as secondary retrospective comparative review of the pertinent case images, diminish the risk for a "missed" cancer or advanced lesion on the index exam. These cancers did not demonstrate any immunohistochemical features suggestive of Lynch Syndrome, though the rapid progression to cancer within the surveillance interval (possibly non-polypoid in nature) is similar. This cancer presentation within the newly-established recommended colon cancer screening interval warrants concern. CONCLUSIONS: This case prompts serious discussion regarding the length of surveillance intervals in the post-transplant CF population (a population at 20-30 times greater risk for CRC compared to the general non-CF population), as well as the importance of documenting endoscopic quality benchmarks, particularly if a narrative of interval CRC development continues to develop with further prospective monitoring and multi-center experience.