Social cheating in a Pseudomonas aeruginosa quorum-sensing variant.

Proc Natl Acad Sci U S A. 2019 Mar 07;:

Authors: Chen R, Déziel E, Groleau MC, Schaefer AL, Greenberg EP

Abstract
The opportunistic bacterial pathogen Pseudomonas aeruginosa has a layered acyl-homoserine lactone (AHL) quorum-sensing (QS) system, which controls production of a variety of extracellular metabolites and enzymes. The LasRI system activates genes including those coding for the extracellular protease elastase and for the second AHL QS system, RhlRI. Growth of P. aeruginosa on casein requires elastase production and LasR-mutant social cheats emerge in populations growing on casein. P. aeruginosa colonizes the lungs of individuals with the genetic disease cystic fibrosis (CF), and LasR mutants can be isolated from the colonized lungs; however, unlike laboratory-generated LasR mutants, many of these CF isolates have functioning RhlR-RhlI systems. We show that one such mutant can use the RhlR-RhlI system to activate expression of elastase and grow on casein. We carried out social-evolution experiments by growing this isolate on caseinate and, as with wild-type P. aeruginosa, elastase-negative mutants emerge as cheats, but these are not RhlR mutants; rather, they are mutants that do not produce the non-AHL Pseudomonas quinolone signal (PQS). Furthermore, we generated a RhlRI mutant and showed it had a fitness defect when growing together with the parent. Apparently, RhlR QS and PQS collude to support growth on caseinate in the absence of a functional LasR. Our findings provide a plausible explanation as to why P. aeruginosa LasR mutants, but not RhlR mutants, are common in CF lungs.

PMID: 30846553 [PubMed - as supplied by publisher]

Effects of exercise and airway clearance (PEP) on mucus clearance in cystic fibrosis: a randomised cross-over trial.

Eur Respir J. 2019 Mar 07;:

Authors: Dwyer TJ, Daviskas E, Zainuldin R, Verschuer J, Eberl S, Bye PTP, Alison JA

Abstract
Exercise improves mucus clearance in people without lung disease and those with chronic bronchitis. No study has investigated exercise alone for mucus clearance in cystic fibrosis (CF). The aim was to compare treadmill exercise to resting breathing and airway clearance with Positive Expiratory Pressure (PEP) therapy, on mucus clearance in adults with CF.Fourteen adults with mild to severe CF lung disease (FEV1 31-113% predicted) completed a three-day randomised, controlled, cross-over trial. Interventions were 20 min of: resting breathing (control); treadmill exercise at 60% of the participant's peak oxygen consumption; and PEP therapy (including huffing and coughing). Mucus clearance was measured using the radioaerosol technique and gamma camera imaging.Treadmill exercise improved whole lung mucus clearance compared to resting breathing (mean difference 3%, 95% CI 2-4), however exercise alone was less effective than PEP therapy (mean difference -7%, 95% CI -6- -8). When comparing treadmill exercise to PEP therapy, there were no significant differences in mucus clearance from the intermediate and peripheral lung regions, but significantly less clearance from the central lung region (likely reflecting the huffing and coughing that was only in PEP therapy).It is recommended to include huffing and coughing to maximise mucus clearance with exercise.

PMID: 30846472 [PubMed - as supplied by publisher]

Liver Failure in a Chinese Cystic Fibrosis Child With Homozygous R553X Mutation.

Front Pediatr. 2019;7:36

Authors: Li H, Lin L, Hu X, Li C, Zhang H

Abstract
Cystic fibrosis (CF) is a relatively rare disease in Asians with various clinical characteristics, including CF-associated liver disease (CFLD), which is a common early non-pulmonary complication. This case report describes a Chinese CF patient harboring a homozygous nonsense mutation (c.1657C>T, p.R553X) who was failure to thrive and had intermittently diarrhea during the first year after birth. Liver function test of the patient showed the mildly and intermittently elevated alanine aminotransferase (ALT) levels ranging from 70 to 92 U/L and aspartate aminotransferase (AST) levels ranging from 80 to 90 U/L, which began at 8 months of age and lasted for 4 years without CF diagnosis. In addition, abdominal computed tomography (CT) revealed diffuse fatty infiltration of the liver at 4 years old and gradually developed hepatic cirrhosis. Subsequently, cirrhosis rapidly progressed with obvious splenomegaly and pancreatic insufficiency and the patient died of liver failure with coagulopathy by the age of 6 years old. Pediatricians should remain vigilant to avoid failure to diagnose CF, the occurrence of which may be underestimated, and pay greater attention to the patients with atypical clinical manifestations in Asian countries.

PMID: 30842938 [PubMed]

Antimicrobial Activity of α-Peptide/β-Peptoid Lysine-Based Peptidomimetics Against Colistin-Resistant Pseudomonas aeruginosa Isolated From Cystic Fibrosis Patients.

Front Microbiol. 2019;10:275

Authors: Molchanova N, Wang H, Hansen PR, Høiby N, Nielsen HM, Franzyk H

Abstract
Pseudomonas aeruginosa infection is a predominant cause of morbidity and mortality in patients with cystic fibrosis infection and with a compromised immune system. Emergence of bacterial resistance renders existing antibiotics inefficient, and therefore discovery of new antimicrobial agents is highly warranted. In recent years, numerous studies have demonstrated that antimicrobial peptides (AMPs) constitute potent agents against a range of pathogenic bacteria. However, AMPs possess a number of drawbacks such as susceptibility to proteolytic degradation with ensuing low bioavailability. To circumvent these undesired properties of AMPs unnatural amino acids or altered backbones have been incorporated to provide stable peptidomimetics with retained antibacterial activity. Here, we report on antimicrobial α-peptide/β-peptoid lysine-based peptidomimetics that exhibit high potency against clinical drug-resistant P. aeruginosa strains obtained from cystic fibrosis patients. These clinical strains possess phoQ and/or pmrB mutations that confer high resistance to colistin, the last-resort antibiotic for treatment of infections caused by P. aeruginosa. The lead peptidomimetic LBP-2 demonstrated a 12-fold improved anti-pseudomonal activity as compared to colistin sulfate as well as favorable killing kinetics, similar antibiofilm activity, and moderate cytotoxicity.

PMID: 30842761 [PubMed]

Evaluation of bone disease in patients with cystic fibrosis and end-stage lung disease.

J Bras Pneumol. 2019 Feb 28;45(1):e20170280

Authors: Robinson CA, Hofer M, Benden C, Schmid C

Abstract
OBJECTIVE: Bone disease is a common comorbidity in patients with cystic fibrosis (CF). We sought to determine risk factors and identify potential biochemical markers for CF-related bone disease (CFBD) in a unique cohort of CF patients with end-stage lung disease undergoing lung transplantation (LTx) evaluation.
METHODS: All of the CF patients who were evaluated for LTx at our center between November of 1992 and December of 2010 were included in the study. Clinical data and biochemical markers of bone turnover, as well as bone mineral density (BMD) at the lumbar spine and femoral neck, were evaluated. Spearman's rho and multivariate logistic regression analysis were used.
RESULTS: A total of 102 adult CF patients were evaluated. The mean age was 28.1 years (95% CI: 26.7-29.5), and the mean body mass index was 17.5 kg/m2 (95% CI: 17.2-18.2). Mean T-scores were -2.3 and -1.9 at the lumbar spine and femoral neck, respectively, being lower in males than in females (-2.7 vs. -2.0 at the lumbar spine and -2.2 vs. -1.7 at the femoral neck). Overall, 52% had a T-score of < -2.5 at either skeletal site. The homozygous Phe508del genotype was found in 57% of patients without osteoporosis and in 60% of those with low BMD. Mean T-scores were not particularly low in patients with severe CFTR mutations. Although the BMI correlated with T-scores at the femoral neck and lumbar spine, serum 25-hydroxyvitamin D and parathyroid hormone levels did not.
CONCLUSIONS: CFBD is common in CF patients with end-stage lung disease, particularly in males and patients with a low BMI. It appears that CF mutation status does not correlate with CFBD. In addition, it appears that low BMD does not correlate with other risk factors or biochemical parameters. The prevalence of CFBD appears to have recently decreased, most likely reflecting increased efforts at earlier diagnosis and treatment.

PMID: 30843951 [PubMed - in process]

Pyruvate-depleting conditions induce biofilm dispersion and enhance the efficacy of antibiotics in killing biofilms in vitro and in vivo.

Sci Rep. 2019 Mar 06;9(1):3763

Authors: Goodwine J, Gil J, Doiron A, Valdes J, Solis M, Higa A, Davis S, Sauer K

Abstract
The formation of biofilms is a developmental process initiated by planktonic cells transitioning to the surface, which comes full circle when cells disperse from the biofilm and transition to the planktonic mode of growth. Considering that pyruvate has been previously demonstrated to be required for the formation of P. aeruginosa biofilms, we asked whether pyruvate likewise contributes to the maintenance of the biofilm structure, with depletion of pyruvate resulting in dispersion. Here, we demonstrate that the enzymatic depletion of pyruvate coincided with the dispersion of established biofilms by S. aureus and laboratory and clinical P. aeruginosa isolates. The dispersion response was dependent on pyruvate fermentation pathway components but independent of proteins previously described to contribute to P. aeruginosa biofilm dispersion. Using porcine second-degree burn wounds infected with P. aeruginosa biofilm cells, we furthermore demonstrated that pyruvate depletion resulted in a reduction of biofilm biomass in vivo. Pyruvate-depleting conditions enhanced the efficacy of tobramycin killing of the resident wound biofilms by up to 5-logs. Our findings strongly suggest the management of pyruvate availability to be a promising strategy to combat biofilm-related infections by two principal pathogens associated with wound and cystic fibrosis lung infections.

PMID: 30842579 [PubMed - in process]

Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

Biomed Pharmacother. 2019 Mar;111:1249-1259

Authors: Kittayaruksakul S, Sawasvirojwong S, Noitem R, Pongkorpsakol P, Muanprasat C, Chatsudthipong V

Abstract
Constitutive androstane receptor (CAR) belonging to the nuclear receptor superfamily plays an important role in the xenobiotic metabolism and disposition. It has been reported that CAR regulates the expression of the ATP-binding cassette (ABC) transporters in the intestine, such as multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 2/3 (MRP2 and MRP3). In this study, we investigated the role of CAR in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport in T84 human colonic epithelial cells and mouse intestinal tissues. Treatments of T84 cell monolayers with specific CAR agonists (CITCO and phenytoin at concentrations of 1 μM and 5 μM, respectively) for 24 h decreased transepithelial Cl- secretion in response to cAMP-dependent agonist. This inhibition was abolished by coincubation of CITCO with a CAR antagonist, CINPA1. We confirmed that an inhibitory effect of CAR agonists was not due to their cytotoxicity. Basolateral membrane permeabilization experiments also revealed that activation of CAR decreased apical Cl- current stimulated by both CPT-cAMP and genistein (a direct CFTR activator). Such activation also reduced both mRNA and protein expression of CFTR. Furthermore, CITCO decreased cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers. In ICR mice, administration of TCPOBOP (3 mg/kgBW), a murine-specific CAR agonist, for 7 days produced significant decreases in CFTR mRNA and protein expressions in intestinal tissues. Interestingly, TCPOBOP also inhibited CT-induced intestinal fluid accumulation in mice. This is the first evidence showing that CFTR was downregulated by CAR activation in the intestine. Our findings suggest that CAR has potential as a new drug target for treatment of condition with hyperactivity/ hyperfunction of CFTR especially secretory diarrheas.

PMID: 30841439 [PubMed - in process]

Oral Azithromycin Use and the Recovery of Lung Function from Pulmonary Exacerbations Treated with Intravenous Tobramycin or Colistimethate in Adults with Cystic Fibrosis.

Ann Am Thorac Soc. 2019 Mar 06;:

Authors: Somayaji R, Russell R, Cogen JD, Goss CH, Nick SE, Saavedra MT, Taylor-Cousar JL, Nick JA, Nichols DP

Abstract
RATIONALE: The potential of azithromycin to alter the antimicrobial and clinical benefits of inhaled tobramycin in CF patients has been previously reported. The potential interaction between azithromycin and intravenous antibiotics in the treatment of pulmonary exacerbations is unknown.
OBJECTIVES: To determine if chronic azithromycin use as a concomitant therapy associated with change in lung function after receiving IV antibiotic regimens including tobramycin or colistimethate.
METHODS: Retrospective cohort study evaluating the effect of azithromycin with IV tobramycin or colistimethate in adult CF patients treated for a pulmonary exacerbation. The primary outcome was relative lung function recovery (FEV1) following exacerbation treatment. Generalized estimating equations were applied to account for repeated events with independent correlation structures and robust standard errors, incorporating several confounders.
RESULTS: 220 exacerbation events occurred in 121 patients in the tobramycin group (47% using azithromycin), and 207 exacerbation events occurred in 86 patients in the colistimethate group (59% using azithromycin). Azithromycin use was associated with less FEV1% recovery in patients treated with tobramycin (-3% relative FEV1% recovery [95% CI -7, 0.2] and -2.64% absolute FEV1% change [95% CI -4.52, -0.76]). Azithromycin use was associated with greater recovery of FEV1% when treated with colistimethate (+3% relative FEV1% recovery [95% CI -0.1, 7] and 2.00% absolute improvement in FEV1% [95% CI 0.13, 3.87]). The odds of 90% or 100% recovery to baseline FEV1% were lower with azithromycin use in the tobramycin cohort and higher with azithromycin use in the colistimethate cohort but were not statistically significant.
CONCLUSION: azithromycin use associated with a more favorable response in patients treated with IV colistimethate but a less favorable response in patients treated with IV tobramycin.

PMID: 30840835 [PubMed - as supplied by publisher]

Life in the cystic fibrosis upper respiratory tract influences competitive ability of the opportunistic pathogen Pseudomonas aeruginosa.

R Soc Open Sci. 2018 Sep;5(9):180623

Authors: Bara JJ, Matson Z, Remold SK

Abstract
Understanding characteristic differences between host-associated and free-living opportunistic pathogens can provide insight into the fundamental requirements for success after dispersal to the host environment, and more generally into the ecological and evolutionary processes by which populations respond to simultaneous selection on complex interacting traits. We examined how cystic fibrosis (CF)-associated and environmental isolates of the opportunistic pathogen Pseudomonas aeruginosa differ in the production of an ecologically important class of proteinaceous toxins known as bacteriocins, and how overall competitive ability depends on the production of and resistance to these bacteriocins. We determined bacteriocin gene content in a diverse collection of environmental and CF isolates and measured bacteriocin-mediated inhibition, resistance and the outcome of competition in a shared environment between all possible pairs of these isolates at 25°C and 37°C. Although CF isolates encoded significantly more bacteriocin genes, our phenotypic assays suggest that they have diminished bacteriocin-mediated killing and resistance capabilities relative to environmental isolates, regardless of incubation temperature. Notably, however, although bacteriocin killing and resistance profiles significantly predicted head-to-head competitive outcomes, CF and environmental isolates did not differ significantly in their competitive ability. This suggests that the contribution of bacteriocins to competitive ability involves selection on other traits that may be pleiotropically linked to interference competition mediated by bacteriocins.

PMID: 30839703 [PubMed]

Loss of miR-17~92 results in dysregulation of Cftr in nephron progenitors.

Am J Physiol Renal Physiol. 2019 Mar 06;:

Authors: Phua YL, Chen KH, Hemker SL, Marrone AK, Bodnar AJ, Liu X, Clugston A, Kostka D, Butterworth MB, Ho J

Abstract
We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse modelresults in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment due to renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrate increased Cystic fibrosis transmembrane conductance regulator( Cftr) expression. We show that one member of the cluster, miR-19b, is sufficient to repress Cftrexpression in vitro, and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19bregulates Cftrexpression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.

PMID: 30838872 [PubMed - as supplied by publisher]

Change in lung clearance index with microbiological status in children with cystic fibrosis.

Pediatr Pulmonol. 2019 Mar 05;:

Authors: Walicka-Serzysko K, Postek M, Milczewska J, Sands D

Abstract
The impact of infections caused by bacteria, especially Gram-negative, on the progression of lung disease in cystic fibrosis is well established. Decline in pulmonary function commence already at early age. In this group of patients, the lung clearance index seems to be a better marker than FEV1 allowing non-invasive monitoring of changes in small airways. The aim of this study was to investigate the association between the microbiological status and LCI derived from multiple breath washout (MBW) technique as well as FEV1 and FVC in children suffering from cystic fibrosis. Over the 1-year recruitment period, 136 CF patients aged 5-18 with: Staphylococcus aureus (n-27), Pseudomonas aeruginosa (first time (n-27), intermittent (n-9), and chronic (34) infection), Aspergillus fumigatus (n-6) and without pathogenic flora (n-33) were included in the study. Patients had performed a spirometry and MBW test during the visit at outpatient clinic. The study showed that the lung clearance index in patients infected with Aspergillus fumigatus was significantly higher (P < 0.05) than in those with normal throat flora. There was also statistically significant differences in the lung clearance index obtained in subjects with chronic Pseudomonas aeruginosa infection and those with first Pseudomonas aeruginosa infection (P < 0.05). Furthermore, significant statistical differences (P < 0.05) were observed between the groups of patients with chronic Pseudomonas aeruginosa infection FEV1  > 70% and FEV1  < 70%. In conclusion, LCI was associated with microbiological status of CF patients. Chronic lung infections, especially Aspergillus fumigatus and Pseudomonas aeruginosa, were associated with increased LCI. Early eradication of pathological flora positively affects the maintenance of lower LCI.

PMID: 30838817 [PubMed - as supplied by publisher]

Assessing arthritis in the context of cystic fibrosis.

Pediatr Pulmonol. 2019 Mar 05;:

Authors: Clarke EA, Watson P, Freeston JE, Peckham DG, Jones AM, Horsley A

Abstract
Inflammatory arthritis in the context of cystic fibrosis (CF) can represent a diagnostic and therapeutic challenge. Poor recognition and under-treatment of musculoskeletal conditions increases symptom burden, affects quality of life, and may lead to changes to an individual's ability to carry out activities of daily living and to exercise. A careful assessment and multidisciplinary approach is essential when considering a diagnosis of CF-associated arthritis (CFA), both in terms of identifying other treatable conditions, such as rheumatoid arthritis, and effectively addressing symptoms. In this collaboration between CF specialists and Rheumatologists, we consider joint symptoms in patients with CF, with a focus on CFA. We offer a differential diagnosis list and consider steps to assess and manage CF patients presenting with arthralgia including appropriate up-to-date rheumatological assessment.

PMID: 30838784 [PubMed - as supplied by publisher]

TNF receptor signalling in autoinflammatory diseases.

Int Immunol. 2019 Mar 06;:

Authors: Jarosz-Griffiths HH, Holbrook J, Lara-Reyna S, McDermott MF

Abstract
Autoinflammatory syndromes are a group of disorders characterised by recurring episodes of inflammation as a result of specific defects in the innate immune system. Patients with autoinflammatory disease present with recurrent outbreaks of chronic systemic inflammation that are mediated by innate immune cells, for the most part. A number of these diseases arise from defects in the tumour necrosis factor (TNF) receptor signalling pathway leading to elevated levels of inflammatory cytokines. Elucidation of the molecular mechanisms of these recently defined autoinflammatory diseases has led to a greater understanding of the mechanisms of action of key molecules involved in TNFR signalling, particularly those involved in ubiquitination, as found in haploinsufficiency of A20 (HA20), otulipenia/otulin-related autoinflammatory syndrome (ORAS) and linear ubiquitin chain assembly complex (LUBAC) deficiency. In this review we also address other TNFR signalling disorders such as (TNF) receptor-associated periodic syndrome (TRAPS), RELA haploinsufficiency, RIPK1-associated immunodeficiency and autoinflammation, X-linked ectodermal dysplasia and immunodeficiency (X-EDA-ID) and we review the most recent advances surrounding these diseases and therapeutic approaches currently used to target these diseases. Finally, we explore therapeutic advances in TNF-related immune based therapies and explore new approaches to target disease-specific modulation of autoinflammatory diseases.

PMID: 30838383 [PubMed - as supplied by publisher]

The Role of Autophagy in Eosinophilic Airway Inflammation.

Immune Netw. 2019 Feb;19(1):e5

Authors: Lee J, Kim HS

Abstract
Autophagy is a homeostatic mechanism that discards not only invading pathogens but also damaged organelles and denatured proteins via lysosomal degradation. Increasing evidence suggests a role for autophagy in inflammatory diseases, including infectious diseases, Crohn's disease, cystic fibrosis, and pulmonary hypertension. These studies suggest that modulating autophagy could be a novel therapeutic option for inflammatory diseases. Eosinophils are a major type of inflammatory cell that aggravates airway inflammatory diseases, particularly corticosteroid-resistant inflammation. The eosinophil count is a useful tool for assessing which patients may benefit from inhaled corticosteroid therapy. Recent studies demonstrate that autophagy plays a role in eosinophilic airway inflammatory diseases by promoting airway remodeling and loss of function. Genetic variant in the autophagy gene ATG5 is associated with asthma pathogenesis, and autophagy regulates apoptotic pathways in epithelial cells in individuals with chronic obstructive pulmonary disease. Moreover, autophagy dysfunction leads to severe inflammation, especially eosinophilic inflammation, in chronic rhinosinusitis. However, the mechanism underlying autophagy-mediated regulation of eosinophilic airway inflammation remains unclear. The aim of this review is to provide a general overview of the role of autophagy in eosinophilic airway inflammation. We also suggest that autophagy may be a new therapeutic target for airway inflammation, including that mediated by eosinophils.

PMID: 30838160 [PubMed]

Profiling the susceptibility of Pseudomonas aeruginosa strains from acute and chronic infections to cell-wall-targeting immune proteins.

Sci Rep. 2019 Mar 05;9(1):3575

Authors: Torrens G, Barceló IM, Pérez-Gallego M, Escobar-Salom M, Tur-Gracia S, Munar-Bestard M, González-Nicolau MDM, Cabrera-Venegas YJ, Rigo-Rumbos EN, Cabot G, López-Causapé C, Rojo-Molinero E, Oliver A, Juan C

Abstract
In the current scenario of high antibiotic resistance, the search for therapeutic options against Pseudomonas aeruginosa must be approached from different perspectives: cell-wall biology as source of bacterial weak points and our immune system as source of weapons. Our recent study suggests that once the permeability barrier has been overcome, the activity of our cell-wall-targeting immune proteins is notably enhanced, more in mutants with impaired peptidoglycan recycling. The present work aims at analyzing the activity of these proteins [lysozyme and Peptidoglycan-Recognition-Proteins (PGLYRPs)], alone or with a permeabilizer (subinhibitory colistin) in clinical strains, along with other features related to the cell-wall. We compared the most relevant and complementary scenarios: acute (bacteremia) and chronic infections [early/late isolates from lungs of cystic fibrosis (CF) patients]. Although a low activity of lysozyme/PGLYRPs per se (except punctual highly susceptible strains) was found, the colistin addition significantly increased their activity regardless of the strains' colistin resistance levels. Our results show increased susceptibility in late CF isolates, suggesting that CF adaptation renders P. aeruginosa more vulnerable to proteins targeting the cell-wall. Thus, our work suggests that attacking some P. aeruginosa cell-wall biology-related elements to increase the activity of our innate weapons could be a promising therapeutic strategy.

PMID: 30837659 [PubMed - in process]

Trimethylation of Elongation Factor-Tu by the Dual Thermoregulated Methyltransferase EftM Does Not Impact Its Canonical Function in Translation.

Sci Rep. 2019 Mar 05;9(1):3553

Authors: Prezioso SM, Duong DM, Kuiper EG, Deng Q, Albertí S, Conn GL, Goldberg JB

Abstract
The Pseudomonas aeruginosa methyltransferase EftM trimethylates elongation factor-Tu (EF-Tu) on lysine 5 to form a post-translational modification important for initial bacterial adherence to host epithelial cells. EftM methyltransferase activity is directly temperature regulated. The protein stability of EftM is tuned with a melting temperature (Tm) around 37 °C such that the enzyme is stable and active at 25 °C, but is completely inactivated by protein unfolding at higher temperatures. This leads to higher observable levels of EF-Tu trimethylation at the lower temperature. Here we report an additional layer of thermoregulation resulting in lower eftM mRNA transcript level at 37 °C compared to 25 °C and show that this regulation occurs at the level of transcription initiation. To begin to define the impact of this system on P. aeruginosa physiology, we demonstrate that EF-Tu is the only observable substrate for EftM. Further, we interrogated the proteome of three different wild-type P. aeruginosa strains, their eftM mutants, and these mutants complemented with eftM and conclude that trimethylation of EF-Tu by EftM does not impact EF-Tu's canonical function in translation. In addition to furthering our knowledge of this Pseudomonas virulence factor, this study provides an intriguing example of a protein with multiple layers of thermoregulation.

PMID: 30837495 [PubMed - in process]

Readiness for transition and healthcare satisfaction in adolescents with complex medical conditions.

Child Care Health Dev. 2019 Mar 05;:

Authors: Haarbauer-Krupa J, Alexander NM, Mee L, Johnson A, Wise J, Gupta NA, Schechter MS, Wasilewski-Masker K, Marchak JG

Abstract
BACKGROUND: The purpose of this study is to examine contributions to patient perceptions of transition readiness and satisfaction with care among adolescents and young adults (AYA) with complex health conditions engaging in pediatric care.
METHODS: Participants included 94 patients aged 14-20 years (M= 16.41, SD = 1.56) with cystic fibrosis (n=31), sickle cell disease (n=27), and solid organ transplants (n=36). Participants completed self-report questionnaires and medical providers completed measures of their medication regimen complexity. One-way analysis of variance (ANOVA) compared differences between disease groups on study variables. Pearson product-moment correlation coefficients and linear regression models evaluated factors associated with AYA reported transition readiness and satisfaction with healthcare.
RESULTS: There were no significant differences between disease groups on patient reported transition readiness, barriers to medication adherence, healthcare self-management or satisfaction. Patient age, self-reported healthcare responsibility, medication barriers, and academic performance predicted a large portion of the variance in AYA perceptions of transition readiness (R2 = 0.27, F (4, 83)= 7.74, p< 0.001, Cohen's f2= 0.37). Patient gender, self-reported healthcare responsibility, and medication barriers predicted a medium portion of the variance in AYA satisfaction with healthcare (R2 = 0.23, F (3, 88) = 8.56, p< 0.001, Cohen's f2= 0.30).
CONCLUSIONS: Patient perceptions of healthcare self-management and barriers to medication adherence are important predictors of readiness for transition and satisfaction with care. Considering a holistic approach that includes these factors allows for improved understanding of individual needs for transition interventions that can improve adult outcomes for individuals with complex health conditions.

PMID: 30836446 [PubMed - as supplied by publisher]

Nonsense Mediated RNA Decay Pathway Inhibition Restores Expression and Function of W1282X CFTR.

Am J Respir Cell Mol Biol. 2019 Mar 05;:

Authors: Keenan MM, Huang L, Jordan NJ, Wong E, Cheng Y, Valley HC, Mahiou J, Liang F, Bihler H, Mense M, Guo S, Monia BP

Abstract
RATIONALE: The recessive genetic disease cystic fibrosis is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Approximately 10% of cystic fibrosis patients have at least one allele with a nonsense mutation in CFTR. Nonsense mutations generate premature termination codons that can subject mRNA transcripts to rapid degradation through the nonsense mediated mRNA decay (NMD) pathway. Currently, there are no approved therapies specifically targeting nonsense mutations in CFTR.
OBJECTIVES: Here, we identify antisense oligonucleotides (ASOs) targeting the nonsense mediated decay factor SMG1 to inhibit the NMD pathway and determine their effects on the W1282X CFTR mutation.
METHODS: First, we develop and validate two in vitro models of the W1282X CFTR mutation. Next, we treat these cells with antisense oligonucleotides to inhibit nonsense mediated decay and measure the effects of these treatments on W1282X expression and function.
MEASUREMENTS AND MAIN RESULTS: SMG1-ASO mediated NMD inhibition upregulates the RNA, protein and surface-localized protein expression of the truncated W1282X gene product. Additionally, these ASOs increase the CFTR chloride channel function in cells homozygous for the W1282X mutation.
CONCLUSIONS: Our approach suggests a new therapeutic strategy for patients harboring nonsense mutations and may be beneficial as a single agent in CF patients with the W1282X mutation.

PMID: 30836009 [PubMed - as supplied by publisher]

Tubuloids derived from human adult kidney and urine for personalized disease modeling.

Nat Biotechnol. 2019 Mar;37(3):303-313

Authors: Schutgens F, Rookmaaker MB, Margaritis T, Rios A, Ammerlaan C, Jansen J, Gijzen L, Vormann M, Vonk A, Viveen M, Yengej FY, Derakhshan S, de Winter-de Groot KM, Artegiani B, van Boxtel R, Cuppen E, Hendrickx APA, van den Heuvel-Eibrink MM, Heitzer E, Lanz H, Beekman J, Murk JL, Masereeuw R, Holstege F, Drost J, Verhaar MC, Clevers H

Abstract
Adult stem cell-derived organoids are three-dimensional epithelial structures that recapitulate fundamental aspects of their organ of origin. We describe conditions for the long-term growth of primary kidney tubular epithelial organoids, or 'tubuloids'. The cultures are established from human and mouse kidney tissue and can be expanded for at least 20 passages (>6 months) while retaining a normal number of chromosomes. In addition, cultures can be established from human urine. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. We apply tubuloids to model infectious, malignant and hereditary kidney diseases in a personalized fashion. BK virus infection of tubuloids recapitulates in vivo phenomena. Tubuloids are established from Wilms tumors. Kidney tubuloids derived from the urine of a subject with cystic fibrosis allow ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function.

PMID: 30833775 [PubMed - in process]

Comment on Comparison of lung clearance index determined by washout of N2 and SF6 in infants and preschool children with cystic fibrosis.

J Cyst Fibros. 2019 Mar 01;:

Authors: Horsley A, Nissenbaum C, Guglani L, Weiner D

PMID: 30833123 [PubMed - as supplied by publisher]