A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits.

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian Gα subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A2B receptor (hA2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: Gαi, Gαs, Gαq, and Gα12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the Gα subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103(3.50) and I107(3.54) are vital too in G protein-coupling and the activation of the hA2BR, whereas L213(IL3) is more important in G protein inactivation. Substitution of S235(6.36) to alanine provided the most divergent G protein-coupling profile. Finally, L236(6.37) substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.

ECFS standards of care on CFTR-related disorders: Identification and care of the disorders.

This is the third paper in the series providing updated information and recommendations for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (CFTR-RD). This paper covers the individual disorders, including the established conditions - congenital absence of the vas deferens (CAVD), diffuse bronchiectasis and chronic or acute recurrent pancreatitis - and also other conditions which might be considered a CFTR-RD, including allergic bronchopulmonary aspergillosis, chronic rhinosinusitis, primary sclerosing cholangitis and aquagenic wrinkling. The CFTR functional and genetic evidence in support of the condition being a CFTR-RD are discussed and guidance for reaching the diagnosis, including alternative conditions to consider and management recommendations, is provided. Gaps in our knowledge, particularly of the emerging conditions, and future areas of research, including the role of CFTR modulators, are highlighted.

Blood mRNA biomarkers for detection of treatment response in acute pulmonary exacerbations of cystic fibrosis.

BACKGROUND: Acute pulmonary exacerbations accelerate pulmonary decline in cystic fibrosis (CF). There is a critical need for better predictors of treatment response. OBJECTIVE: To test whether expression of a panel of leucocyte genes directly measured from whole blood predicts reductions in sputum bacterial density. METHODS: A previously validated 10-gene peripheral blood mononuclear cell (PBMC) signature was prospectively tested in PBMC and whole blood leucocyte RNA isolated from adult subjects with CF at the beginning and end of treatment for an acute pulmonary exacerbation. Gene expression was simultaneously quantified from PBMCs and whole blood RNA using real-time PCR amplification. Test characteristics including sensitivity, specificity, positive and negative predictive values were calculated and receiver operating characteristic curves determined the best cut-off to diagnose a microbiological response. The findings were then validated in a smaller independent sample. RESULTS: Whole blood transcript measurements are more accurate than forced expiratory volume in 1 s (FEV(1)) or C reactive protein (CRP) alone in identifying reduction of airway infection. When added to FEV(1), the whole blood gene panel improved diagnostic accuracy from 64% to 82%. The specificity of the test to detect reduced infection was 88% and the positive predictive value for the presence of persistent infection was 86%. The area under the curve for detecting treatment response was 0.81. Six genes were the most significant predictors for identifying reduction in airway bacterial load beyond FEV(1) or CRP alone. The high specificity of the test was replicated in the validation cohort. CONCLUSIONS: The addition of blood leucocyte gene expression to FEV(1) and CRP enhances specificity in predicting reduced pulmonary infection and may bolster the assessment of CF treatment outcomes.

Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP.

Stimulation of human neutrophils with chemoattractants FMLP or platelet activating factor (PAF) results in different but overlapping functional responses. We questioned whether these differences might reflect patterns of intracellular signal transduction. Stimulation with either PAF or FMLP resulted in equivalent phosphorylation and activation of the mitogen-activated protein kinase (MAPk) homologue 38-kD murine MAP kinase homologous to HOG-1 (p38) MAPk. Neither FMLP nor PAF activated c-jun NH2-terminal MAPk (JNKs). Under identical conditions, FMLP but not PAF, resulted in significant p42/44 (ERK) MAPk activation. Both FMLP and PAF activated MAP kinase kinase-3 (MKK3), a known activator of p38 MAPk. Both MAP ERK kinase kinase-1 (MEKK1) and Raf are activated strongly by FMLP, but minimally by PAF. Pertussis toxin blocked FMLP-induced activation of the p42/44 (ERK) MAPk cascade, but not that of p38 MAPk. A specific p38 MAPk inhibitor (SK&F 86002) blocked superoxide anion production in response to FMLP and reduced adhesion and chemotaxis in response to PAF or FMLP. These results demonstrate distinct patterns of intracellular signaling for two chemoattractants and suggest that selective activation of intracellular signaling cascades may underlie different patterns of functional responses.

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

Glucose >200 mg/dL during Continuous Glucose Monitoring Identifies Adult Patients at Risk for Development of Cystic Fibrosis Related Diabetes.

Rationale. Cystic fibrosis related diabetes (CFRD) is the most common comorbidity in patients with CF. In spite of increased screening, diagnosis, and treatment of CFRD, the mortality rate in patients with CFRD still far exceeds the mortality rate in those without CFRD. Guidelines suggest that screening for CFRD be performed annually using the 2-hour 75-gram oral glucose tolerance test (OGTT). Adherence to recommended screening has been poor, with only approximately one-quarter of adults with CF undergoing OGTT in 2014. Use of continuous glucose monitoring (CGM) for diagnosis may become an alternative. Objectives. Our objective was to determine whether abnormal CGM predicts subsequent development of CFRD, lung function, and body mass index (BMI) decline and increased rate of CF pulmonary exacerbations in adults with CF. Methods. In a prospective single center pilot trial from September 2009 to September 2010, 21 adult patients due for routine OGTT were recruited to complete simultaneous 3-day CGM and 2-hour 75 gram OGTT. Subsequently, clinical information was reviewed from 2008 to 2015. Conclusions. There was a moderate correlation between interpreted results of 2-hour OGTT and CGM (p = 0.03); CGM indicated a greater level of glucose impairment than OGTT. Glucose >200 mg/dL by CGM predicted development of CFRD (p = 0.0002).

The metabolic effects of pregnancy in cystic fibrosis.

OBJECTIVE: Our purpose was to determine glucose tolerance in pregnant women with cystic fibrosis (CF) and to relate glucose tolerance to insulin sensitivity, hepatic glucose production, and protein turnover. METHODS: We studied 8 CF women during pregnancy (CFPreg). Results were compared with those from 9 pregnant controls (PregCont) and 8 nonpregnant CF women (CFCont). The following metabolic studies were conducted: oral glucose tolerance test (OGTT), hyperinsulinemic euglycemic clamp, stable isotope infusion of [1-13C]leucine and [6,6-2H2]glucose for measurement of whole body protein turnover and hepatic glucose production (HGP), respectively. Indirect calorimetry was used to measure resting energy expenditure (REE), and food intake was measured by 3-day food journals. Fat-free mass was measured by total body potassium 40K scan. RESULTS: All but one CFPreg developed diabetes by the end of the second trimester and had significantly lower insulin secretion and more insulin resistance than PregCont. Hepatic glucose production was significantly higher and suppression by insulin was less in CF subjects, and protein breakdown was significantly higher. Insulin resistance and HGP increased during pregnancy similarly in CFPreg and PregCont groups. CONCLUSION: Pregnancy in CF is associated with decreased insulin sensitivity and high HGP, in addition to inherent decreased insulin secretion. Pregnancy in CF is also associated with increased protein turnover and less response to insulin's anticatabolic effect. These changes appear to predispose the pregnant CF women to early development of diabetes and poor weight gain.

Pharmacokinetics and tolerability of oral sildenafil in adults with cystic fibrosis lung disease.

RATIONALE: Airway inflammation is central to cystic fibrosis (CF) pathophysiology. Pre-clinical models have shown that phosphodiesterase inhibitors (PDEi) like sildenafil have anti-inflammatory activity. PDEi have not been studied in CF subjects. OBJECTIVES: We evaluated the pharmacokinetics, tolerability, and safety of sildenafil in subjects with CF. Sputum biomarkers were used to explore efficacy. METHODS: An open-label pilot study of oral sildenafil administration was conducted in adults with mild to moderate CF lung disease. Subjects received oral sildenafil 20 or 40 mg p.o. t.i.d. for 6 weeks. MEASUREMENTS AND MAIN RESULTS: Twenty subjects completed the study. Estimated elimination rate constants were statistically different in subjects with CF compared to previously published non-CF subjects. Side effects were generally mild. There were no drug-related serious adverse events. Sputum neutrophil elastase activity decreased. CONCLUSIONS: Subjects with CF may eliminate sildenafil at a faster rate than non-CF subjects. Sildenafil administration was safe in subjects with CF and decreased sputum elastase activity. Sildenafil warrants further study as an anti-inflammatory in CF.

An investigation of aspects of design of resin-bonded bridges in general dental practice and hospital services.

PURPOSE OF STUDY: To assess the design of resin-bonded bridges (RBBs) by dentists, the quality of information provided to a dental laboratory, and aid identification of best practice. DESIGN: A retrospective case series study of slips for RBB construction, sent by hospital and general dental practitioners to a dental laboratory based in a postgraduate dental institute in Scotland, UK. MAIN FINDINGS: 384 forms were reviewed. For single-tooth replacements, 40% of designs for upper anterior bridges and 46% for lower posterior bridges were fixed-fixed (F-F) when a cantilever design was a better option. Thirty-six (9.3%) of RBB designs involved double abutting. On the laboratory forms, fewer than five (1%) cases included instructions about the thickness of the metal framework and in 48% there was no reference to the extension of the metal framework. PRINCIPAL CONCLUSIONS: For single-tooth replacements, a relatively high percentage of dentists prescribed a fixed-fixed design for RBBs, despite the evidence advocating the use of a cantilever design. A significant number of dentists used double abutments. The information provided to the laboratory for the construction of resin-bonded bridges was often insufficient.

A clinical overview of removable prostheses: 4. Technological considerations when designing removable partial dentures.

This fourth article in a series concerning the prescription of removable partial dentures is a précis of the technical aspects of RPD construction, commencing with the definitive or 'working' impressions, although all impressions ought to be considered as 'working' impressions.

A clinical overview of removable prostheses: 5. Diagnosis and treatment of RPD problems.

This, the fifth and final article in the series, addresses the diagnoses and treatment of problems which may arise following provision of removable partial dentures (RPDs). These include difficulties seating the denture, pain and discomfort, looseness and functional problems.

A clinical overview of removable prostheses: 2. Impression making for partial dentures.

This, the second article in a series on the prescription of removable partial dentures, will deal with the issue of primary impression and primary casts for partial dentures. The principles of definitive impressions and master cast planning will be described.

A clinical overview of removable prostheses: 3. Principles of design for removable partial dentures.

Removable partial dentures (RPDs) should not be made for patients unless they are necessary. Most partial dentures have the potential to cause some damage to the teeth and supporting tissues, however well they are designed and constructed; the criteria for selecting such devices were described in a previous article. In general there is merit in, wherever possible, reducing tissue coverage as much as possible when RPDs are being planned. This article, the third in a series on the prescription of RPDs, discusses the design principles involved.

A clinical overview of removable prostheses: introduction.

This series of articles has been written with the intention of simplifying the processes involved in the prescription of removable partial dentures. The scene is set in this introduction, and the first article addresses basic clinical and patient-related factors involved in decision-making before commencing active prosthodontic treatment. The second paper will outline a variety of impression techniques for primary and definitive impressions, while the third discusses designing principles. The fourth article is a brief overview of some technological aspects of removable partial denture-making and the fifth attempts to provide a useful guide showing how to diagnose and manage common clinical problems associated with removable partial dentures.

A clinical overview of removable prostheses: 1. Factors to consider in planning a removable partial denture.

This is the first article in a series on the prescription of removable partial dentures. It addresses basic clinical and patient-related factors involved in decision-making before commencing active prosthodontic treatment. Further papers will outline a variety of impression techniques for primary and definitive impression, discuss designing principles, give an overview of some technological aspects of removable partial denture-making and provide guidelines on how to diagnose and manage common clinical problems associated with removable partial dentures.

d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves.

d-[6-(14)C]Glucosone that had been prepared enzymically from d-[6-(14)C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, (14)C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-(14)C]glucose underwent considerable redistribution during AA formation, whereas (14)C from [6-(14)C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-(14)C]sorbosone was found to be equivalent to [6-(14)C]glucose as a source of (14)C for AA. In the presence of 0.1% d-glucosone, conversion of [6-(14)C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.

Activation of a p38 mitogen-activated protein kinase in human neutrophils by lipopolysaccharide.

Stimulation of human neutrophils by LPS is central to the pathogenesis of sepsis and the adult respiratory distress syndrome. The intracellular signaling pathway that results in cellular responses following LPS stimulation in neutrophils is unknown. We report that exposure of neutrophils to LPS results in the phosphorylation and activation of a p38 mitogen-activated protein (MAP) kinase, occurring in a concentration-dependent manner, with maximum response at 20 to 25 min. Partial purification of a p38 MAP kinase by ion exchange chromatography established it as distinct from the p42/p44 (extracellular signal-regulated kinases (ERK-1 and ERK-2) MAP kinases). Activation of the p38 MAP kinase by LPS in human neutrophils occurs via CD14, a proposed LPS receptor, and requires the presence of plasma containing the LPS-binding protein. This intracellular signaling pathway is independent of protein kinase C and does not involve Raf, MAP/ERK kinase kinase-1, MAP/ERK kinase-1, or MAP/ERK kinase-2 and does not result in the activation of the p42/p44 ERK MAP kinases or the c-jun N-terminal kinases.

Pulmonary capillaritis and diffuse alveolar hemorrhage. A primary manifestation of polymyositis.

Polymyositis is often complicated by either usual interstitial pneumonitis, diffuse alveolar damage, cellular interstitial pneumonitis, or bronchiolitis obliterans-organizing pneumonia. Pulmonary capillaritis, a distinct interstitial reaction associated with diffuse alveolar hemorrhage, occurs with the systemic vasculitides and some collagen vascular diseases. It has not been described in patients with polymyositis. We describe two patients who developed a relatively acute onset of polymyositis based on the appearance of a severe proximal myopathy, elevated serum creatine phosphokinase levels, compatible electromyographic abnormalities, inflammatory muscle biopsies, and in one case, the presence of serum anti-Jo-1 antibodies. Concomitant with their muscle disease, they developed respiratory failure that proved to be pulmonary capillaritis with varying degrees of diffuse alveolar hemorrhage as well as bronchiolitis obliterans-organizing pneumonia. Although those reactions are reported to occur with other collagen vascular diseases, these two cases are the first reports of pulmonary capillaritis and diffuse alveolar hemorrhage complicating polymyositis.