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BACKGROUND:Among the many consequences of loss of CFTR protein function, a significant reduction of the secretion of bicarbonate (HCO3-) in cystic fibrosis (CF) is a major pathogenic feature. Loss of HCO3- leads to abnormally low pH and impaired mucus clearance in airways and other exocrine organs, which suggests that NaHCO3 inhalation may be a low-cost, easily accessible therapy for CF. OBJECTIVE:To evaluate the safety, tolerability, and effects of inhaled aerosols of NaHCO3 solutions (4.2% and 8.4%). METHODS:An experimental, prospective, open-label, pilot, clinical study was conducted with 12 CF volunteer participants over 18 years of age with bronchiectasis and pulmonary functions classified as mildly to severely depressed. Sputum rheology, pH, and microbiology were examined as well as spirometry, exercise performance, quality-of-life assessments, dyspnea, blood count, and venous blood gas levels. RESULTS:Sputum pH increased immediately after inhalation of NaHCO3 at each clinical visit and was inversely correlated with rheology when all parameters were evaluated: [G' (elasticity of the mucus) = - 0.241; G″ (viscosity of the mucus) = - 0.287; G* (viscoelasticity of the mucus) = - 0.275]. G* and G' were slightly correlated with peak flow, forced expiratory volume in 1 s (FEV1), and quality of life; G″ was correlated with quality of life; sputum pH was correlated with oxygen consumption (VO2) and vitality score in quality of life. No changes were observed in blood count, venous blood gas, respiratory rate, heart rate, peripheral oxygen saturation of hemoglobin (SpO2), body temperature, or incidence of dyspnea. No adverse events associated with the study were observed. CONCLUSION:Nebulized NaHCO3 inhalation appears to be a safe and well tolerated potential therapeutic agent in the management of CF. Nebulized NaHCO3 inhalation temporarily elevates airway liquid pH and reduces sputum viscosity and viscoelasticity.

Cemeteries existing within cities are often omitted from the “green space” narrative despite their high levels of vegetation. Given the relatively small areas of green space in many cities, it is important to appropriately manage these landscapes to ensure that residents can access green spaces and enjoy the many benefits they offer. The purpose of our paper is to demonstrate that cemeteries should be managed and researched as urban green spaces that provide ecosystem services. We compared and contrasted cemeteries with urban parks and used their similarities and differences, as well as a review of existing cemetery and other green-space literature, to discuss how cemeteries can provide a wide variety of ecosystem services. We found that cemeteries and parks both have high levels of vegetation, similar perceived safety issues, and some common rules and regulations, while differing in their consideration as public spaces, effect on real-estate values, historical–cultural value, and funding and management goals. Despite the differences, we believe that the vegetation (particularly trees), monuments, other infrastructure, and atmosphere within cemeteries make them well-suited to providing ecosystem services such as recreation, human health and restoration, stormwater management, microclimate regulation, aesthetics, and so on. Cemeteries can also potentially provide ecosystem “disservices” such as allergens, invasive/dangerous/poisonous species, and the degradation of groundwater quality. However, we believe that the potential for ecosystem services far outweighs the potential for ecosystem disservices in urban cemeteries, and as such we believe they should be studied and managed as green spaces with functions beyond those of interment and mourning. Given the general superiority of trees over other vegetation in providing a diversity of ecosystem services, we urge cemetery managers to consider options for increases and improvements in cemetery tree populations.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a “capillary”-Ussing chamber (area ≈1 mm2) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ([Formula: see text]), which can be enhanced pharmacologically by cAMP as well as Ca2+-mediated agonists. Concurrent secretion and absorption of surface liquid along with [Formula: see text] secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

In dendritic river systems, gene flow is expected to occur primarily within watersheds. Yet, rare cross‐watershed transfers can also occur, whether mediated by (often historical) geological events or (often contemporary) human activities. We explored these events and their potential evolutionary consequences by analyzing patterns of neutral genetic variation (microsatellites) and adaptive phenotypic variation (male color) in wild guppies (Poecilia reticulata) distributed across two watersheds in northern Trinidad. We found the expected signatures of within‐watershed gene flow; yet we also inferred at least two instances of cross‐watershed gene flow—one in the upstream reaches and one further downstream. The upstream cross‐watershed event appears to be very recent (41 ± 13 years), suggesting dispersal via recent flooding or undocumented human‐mediated transport. The downstream cross‐watershed event appears to be considerably older (577 ± 265 years), suggesting a role for rare geological or climatological events. Alongside these strong signatures of both contemporary and historical gene flow, we found little evidence of impacts on presumably adaptive phenotypic differentiation, except perhaps in the one instance of very recent cross‐watershed gene flow. Selection in this system seems to overpower gene flow—at least on the spatiotemporal scales investigated here.

PurposeCystic fibrosis (CF) is a multisystem genetic disease caused by dysfunction of the epithelial anionic channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Decreased mucociliary clearance because of thickened mucus is part of the pulmonary disease pathophysiology. It is controversial if the thickened airway surface liquid (ASL) is caused by the deficient chloride secretion and excessive sodium (through ENaC) and water hyperabsorption from the periciliar fluid or by the lack of bicarbonate secretion with relative acidification of the ASL. Correlations between the magnitude of in vivo chloride conductance with phenotypic characteristics and CF genotype can help to elucidate these mechanisms and direct to new treatments.MethodsNasal potential difference was measured in 28 CF patients (age from 0.3 to 28 year) and correlated with pulmonary function, pancreatic phenotype, pulmonary colonization and genotype severity.ResultsThe CFTR-chloride conductance was better in older patients (r = 0.40; P = 0.03), in patients with better pulmonary function (r = 0.48; P = 0.01), and was associated with genotype severity. Higher chloride diffusion in the presence of a favorable chemical gradient was associated with Pseudomonas aeruginosa negativity (P

Research Highlights: A large body of research highlighted the important contributions that urban forests make to cities and their inhabitants. However, our urban forests face threats from issues such as rapid urbanization, climate change, and the spread of pests and diseases. As such, proactive and effective management is necessary to ensure their long-term sustainability. Given the multiple spatial and temporal scales on which threats can arise, effective management needs to account for these scales and adjust accordingly. The degree to which this currently happens is unclear. Background and Objectives: The aim of this study was to determine the role of multi-scale management in urban forestry, using cemeteries in Malm&ouml, Sweden as a case study. Cemeteries can provide extensive tree canopy but are not readily considered in urban forest management. We sought to determine (1) the threats to the current cemetery tree populations, (2) the extent of multi-scale cemetery tree management, (3) whether tree management plans promote multi-scale management, and (4) how cemetery tree management can be improved. Materials and Methods: Malm&ouml, cemetery tree inventories were analyzed with respect to size class and species diversity. Existing cemetery tree management plans were examined to determine the spatial and temporal scales of their recommendations. Interviews were conducted with cemetery managers to determine management priorities and actions. Results: We found that cemetery tree populations in Malm&ouml, suffer from a lack of age class and species diversity. Management tends to occur on short time scales and efforts focus mainly on addressing individual trees, although some consideration is given to large-scale species diversification. The management plans previously created for these cemetery trees make recommendations for age class and species diversification but are yet to be used extensively by cemetery managers. Conclusions: The long-term stability of Malm&ouml, &rsquo, s cemetery tree populations is threatened by a lack of species and age diversity. Current management efforts emphasize addressing small-scale issues. Although there is a desire to improve species diversity, this can cause conflict with existing cultural values.

Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). With the discovery of Ivacaftor and Orkambi, it has been shown that CFTR function can be partially restored by administering one or more small molecules. These molecules aim at either enhancing the amount of CFTR on the cell surface (correctors) or at improving the gating function of the CFTR channel (potentiators). Here we describe the discovery of a novel potentiator GLPG1837, which shows enhanced efficacy on CFTR mutants harboring class III mutations compared to Ivacaftor, the first marketed potentiator. The optimization of potency, efficacy, and pharmacokinetic profile will be described.

The small airways of the lungs are under constant assault from the pathogens and debris in the air that they must conduct to alveoli. Although hygiene is of paramount importance for respiratory health, the underlying principles of airway clearance have not been well integrated or established. Newly emerging concepts of simultaneous absorption and secretion of airway surface liquid (ASL) and the role of [Formula: see text] in the maturation of mucins have advanced from experimental evidence as well as observations from the congenital disease cystic fibrosis (CF) to present a novel model that integrates microanatomy with organ physiology to meet the constant challenge of cleaning small airways.

β-adrenergically stimulated sweat secretion depends on the function of the cystic fibrosis transmembrane conductance regulator (CFTR) and discriminates between cystic fibrosis (CF) patients and healthy controls. Therefore, we sought to determine the feasibility, safety, and efficacy of assaying β-adrenergic sweating in children identified by CF newborn screening to help determine prognoses for individuals with CFTR-related metabolic syndrome (CRMS). Preschool age children with a positive newborn screening test for CF participated in this cross-sectional study. Sweat rates were measured by evaporimetery (cyberDERM, inc.) as transepidermal water losses (g H2O/m2/h) before and after selectively stimulating sweat glands either cholinergically or β-adrenergically. Net peak sweat responses assayed as evaporation rates were compared between CF and CRMS cohorts. After a pilot test in adults, children between 4 and 6 years of age were evaluated (CF, n = 16; CRMS, n = 10). The test protocol was well tolerated; electrocardiograms and vital signs were within normal range for all subjects. The mean evaporative sweat rates in both groups in response to cholinergic stimulation were similar (CF, 60.3 ± 23.8; CRMS, 57.7 ± 13.9; p = 0.72) as well as to β-adrenergic stimulation (CF, 1.1 ± 1.7; CRMS, 2.0 ± 2.0; p = 0.14). The β-adrenergic sweat test is safe and well tolerated by young children. However, the β-adrenergic sweat secretion rates as measured by evaporimetery did not discriminate between CF and CRMS cohorts.

The secretion and management of readily transportable airway surface liquid (ASL) along the respiratory tract is crucial for the clearance of debris and pathogens from the lungs. In proximal large airways, submucosal glands (SMGs) can produce ASL. However, in distal small airways, SMGs are absent, although the lumens of these airways are, uniquely, highly plicated. Little is known about the production and maintenance of ASL in small airways, but using electrophysiology, we recently found that native porcine small airways simultaneously secrete and absorb. How these airways can concurrently transport ASL in opposite directions is puzzling. Using high expression of the Na-K-2Cl cotransport (NKCC) 1 protein (SLC12a2) as a phenotypic marker for fluid secretory cells, immunofluorescence microscopy of porcine small airways revealed two morphologically separated sets of luminal epithelial cells. NKCC1 was abundantly expressed by most cells in the contraluminal regions of the pleats but highly expressed very infrequently by cells in the luminal folds of the epithelial plications. In larger proximal airways, the acini of SMGs expressed NKCC1 prominently, but cells expressing NKCC1 in the surface epithelium were sparse. Our findings indicate that, in the small airway, cells in the pleats of the epithelium secrete ASL, whereas, in the larger proximal airways, SMGs mainly secrete ASL. We propose a mechanism in which the locations of secretory cells in the base of pleats and of absorptive cells in luminal folds physically help maintain a constant volume of ASL in small airways.

Progress in cystic fibrosis (CF) over the past few decades has been nothing short of miraculous on several fronts: median survival in the United States has increased from 15 years in 1975 to 41 years in 2013, with predictions into the 50s for children born this century.1 Our understanding of the genetic and cellular underpinnings of CF have similarly grown exponentially, beginning in the 1980s with the identification of the cellular ion transport defect2 and the discovery of the gene, dubbed CFTR (cystic fibrosis transmembrane conductance regulator).3 Now in the past 3 years, with the advent of mutation-specific therapies, we have entered a new era that promises even greater improvement in the quality and the length of lives. The first of these small molecule therapies to come to fruition (and to market) was Vertex Pharmaceuticals' (Boston, Massachusetts) ivacaftor (sold as Kalydeco), which received FDA approval in 2012. In a clinical trial, ivacaftor brought about dramatic improvements in pulmonary funct...

Introduction/Objective Proliferative activity is an essential prognostic and treatment indicator for neuroendocrine tumors (NET). Ki-67 proliferation index, if reported by unaided microscopic estimation on hot-spot locations could lead to variability and inconsistencies. This study aims to compare the Ki-67 assessment of NETs by visual estimation versus automated digital image analysis (Roche iCoreo/Virtuoso). Methods 212 patients with Ki-67-graded GI NETs (117 G1; 61 G2; 34 G3) from 2010 to 2019 were reassessed using digital image analysis quantification of hot spot areas of at least 500 cells (average 800 cells). Revised tumor grades were assigned according to the European Neuroendocrine Tumor Society guidelines and the 2010 World Health Organization classification and compared to initially reported grade. Results We found 75% concordance for G1, with 22% of cases upgraded to G2 and 3% of cases upgraded to G3. For G2, there was 70.5% agreement, with 13.1% of cases downgraded to G1 and 16.4% upgraded to G3. For G3, there was 100% agreement, (kappa=0.64, overall). Retrospective review of discordant G3 cases revealed cases with known metastasis, small fragments of tissue, or polyps. Scanning and scoring required approximately 10 minutes per case. Conclusion Our data shows the time/effort difference of visually estimating versus automated digital analysis may lead to significant classification errors in these tumors. Although digital analysis has limitations, including tumor heterogeneity, misidentification of tumor cells, and poor immunostaining which could require manual counting by a pathologist, this rigor should be reinforced and explicitly stated to increase accuracy and reproducibility of grading.

Urban cemeteries represent an unusual type of greenspace that can provide a wide range of ecosystem benefits and services if they have mature tree populations. However, the extent to which tree management is prioritized in cemeteries alongside tasks associated with the burial and commemoration of the deceased is unknown. Given the large differences that exist amongst burial traditions and cemetery management around the world, it seems likely that cemetery tree management will not be uniform across countries. To investigate this, we compared cemetery governance and management in Halifax (Canada) and Malmo (Sweden) to determine which factors influence their cemetery tree populations, with a specific focus on tree planting. An inventory of places in which trees could be planted in cemeteries indicated that a large number of trees could theoretically be planted in both cities’ cemeteries. However, manager interviews and a review of cemetery legislation indicated multiple dimensions of governance that result in the very real prospect that precious few of these plantable spots will actually become occupied by a tree. These dimensions differed between the two cities, with financial constraints and limited knowledge about trees playing a large role in Halifax, while in Malmo, legislation and public opinion influence the extent to which tree planting occurs. As such, the cities require different solutions to promote tree planting in their cemeteries to avoid future canopy loss and ensure the long-term continued provision of current ecosystem benefits and services.

We investigated the effects of bicarbonate on the growth of several different bacteria as well as its effects on biofilm formation and intracellular cAMP concentration in Pseudomonas aeruginosa. Biofilm formation was examined in 96-well plates, with or without bicarbonate. The cAMP production of bacteria was measured by a commercial assay kit. We found that NaHCO3 (100 mmol l-1) significantly inhibited, whereas NaCl (100 mmol l-1) did not influence the growth of planktonic bacteria. MIC and MBC measurements indicated that the effect of HCO 3 - is bacteriostatic rather than bactericidal. Moreover, NaHCO3 prevented biofilm formation as a function of concentration. Bicarbonate and alkalinization of external pH induced a significant increase in intracellular cAMP levels. In conclusion, HCO 3 - impedes the planktonic growth of different bacteria and impedes biofilm formation by P. aeruginosa that is associated with increased intracellular cAMP production. These findings suggest that aerosol inhalation therapy with HCO 3 - solutions may help improve respiratory hygiene in patients with cystic fibrosis and possibly other chronically infected lung diseases.

SUMMARY Injurious pecking is considered a major economic concern and affects all sectors of commercial poultry production. Though extensive research has been performed examining feather pecking in layer chickens, little information exists regarding feather or head pecking behavior in domestic turkey flocks, and less still in commercial settings. The objective of the present field study was to examine the development of injurious feather and head pecking in tom turkeys raised in 2 different commercial environments. At a commercial facility, 49,332 beak-conditioned tom turkeys were placed in 8 barns (5,000–7,500 turkeys/barn) and studied through 16 wk of age. Approximately half were housed in control barns in March 2010; these barns were environmentally controlled (artificial light and tunnel ventilation) in both rearing and growing, with a density of 0.16 m 2 /bird for rearing through 4.5 wk and 0.39 m 2 /bird for growing through 16 wk. The remaining turkeys were housed in curtain-sided barns in April 2010; the 4 rearing barns were environmentally controlled with a density of 0.10 m 2 /bird, whereas the 4 growing barns had natural light and ventilation and a density of 0.36 to 0.38 m 2 /bird through 16 wk. Two barns from each growing environment were provided with multicolored plastic balls as enrichment. Each housing and enrichment combination had 2 replicate barns. Behavior, weights, and feather condition were assessed every 3 wk. Mortalities and culls were recorded as they occurred. Turkeys in curtain-sided barns had worse feather condition and more culls and mortalities with severe pecking injuries compared with those in control barns. In both environments, severe and gentle pecking was observed throughout production, and the majority of culls and mortalities had severe pecking injuries. More controlled studies are needed to parse the causal factors of injurious pecking in toms.

Since the discovery of Cl(-) impermeability in cystic fibrosis (CF) and the cloning of the responsible channel, CF pathology has been widely attributed to a defect in epithelial Cl(-) transport. However, loss of bicarbonate (HCO3(-)) transport also plays a major, possibly more critical role in CF pathogenesis. Even though HCO3(-) transport is severely affected in the native pancreas, liver, and intestines in CF, we know very little about HCO3(-) secretion in small airways, the principle site of morbidity in CF. We used a novel, mini-Ussing chamber system to investigate the properties of HCO3(-) transport in native porcine small airways (∼ 1 mm φ). We assayed HCO3(-) transport across small airway epithelia as reflected by the transepithelial voltage, conductance, and equivalent short-circuit current with bilateral 25-mM HCO3(-) plus 125-mM NaGlu Ringer's solution in the presence of luminal amiloride (10 μM). Under these conditions, because no major transportable anions other than HCO3(-) were present, we took the equivalent short-circuit current to be a direct measure of active HCO3(-) secretion. Applying selective agonists and inhibitors, we show constitutive HCO3(-) secretion in small airways, which can be stimulated significantly by β-adrenergic- (cAMP) and purinergic (Ca(2+)) -mediated agonists, independently. These results indicate that two separate components for HCO3(-) secretion, likely via CFTR- and calcium-activated chloride channel-dependent processes, are physiologically regulated for likely roles in mucus clearance and antimicrobial innate defenses of small airways.

The global population is becoming more urban, which has increased demand for built infrastructure. This has had negative impacts on the extent and quality of urban greenspace, including tree canopy cover. Tree-planting efforts have mainly focused on street rights-of-way, but consideration should also be given to alternative locations such as cemeteries, which are free of many of the challenges facing street trees. However, the primary function of cemeteries for the interment of human remains indicates that the social dimensions need to be considered before planting trees in these environments. We employed interception surveys and manager interviews to determine how cemetery trees are valued and what concerns people have about increasing tree cover in cemeteries in Halifax, Nova Scotia (Canada). Our results indicate that cemetery users highly value cemetery trees, particularly for shade provision, aesthetics, improved personal wellbeing, and their contribution to a sense of place. These values differ somewhat from previous values obtained using street surveys, suggesting that urban-forest values depend on the environment in which surveys take place. While cemetery managers valued trees for the creation of a park-like atmosphere, they focus their efforts primarily on interring remains, preserving history, and maintaining a neat lawn. Tree maintenance is mainly reactive, and concerns such as a lack of space and the potential for damage to monuments have limited tree-planting efforts. Unlike management personnel, cemetery users have few concerns about trees in cemeteries, and we believe they would not object to more trees being planted. Given the old age of most of the trees in Halifax cemeteries, it is likely that cemetery tree cover will decline in the future due to a lack of planting and natural regeneration. Based on the high value placed on trees by cemetery users, this may have negative implications for the future role of cemeteries as urban greenspace.

Estimates of seaweed nutrient sequestration ability in open-water, Integrated Multi-Trophic Aquaculture (IMTA) required to ‘balance’ nutrients from fish cages have typically assigned a specific nutrient load for a specific fish biomass. The resultant culture area and densities of seaweeds required for full equivalent nutrient sequestration may have meaning only to experienced aquaculturists. Consequently, a novel ratio model is proposed which determines the weight ratio of harvested seaweeds required to sequester an equivalent weight of soluble inorganic nutrients loaded per unit growth of fish. Soluble inorganic nutrient excretion from Atlantic salmon, Salmo salar, was estimated using a semi-stochastic nutritional mass balance approach. Oxygen demand was estimated using respiratory quotients. Nutrient contents of the IMTA kelps, Alaria esculenta and Saccharina latissima, were measured at harvest times, and net oxygen production was estimated using the photosynthetic equation. To quantify uncertainty, input parameters were assigned theoretical distributions (based on empirical or literature data) and the model was run using a stratified sampling approach (Latin Hypercube) over multiple iterations, to generate distributions of weight ratios for the various nutrients. A mathematical simulation of nutrient loading from a salmon farm over a full production cycle was estimated using mean loading values per unit growth, with monthly growth estimated by a thermal growth coefficient (TGC). Results showed that one kilogramme of Atlantic salmon growth (large fish fed a typical commercial feed, ± standard deviation) resulted in the excretion of 29.49 ± 4.20 g nitrogen, 2.26 ± 2.25 g phosphorus and the respiration of 243.38 ± 48.28 g carbon. Dissolved oxygen requirements for 1 kg of growth were 455.29 ± 86.24 g. Salmon smolts placed in cages in spring and harvested 21 months later, load approximately 4 times more nutrients in the second grow-out season. The mean ratios of A. esculenta weight required to sequester nutrients excreted per unit weight of S. salar production were 6.7(± 1.5):1 for nitrogen, 4.8(± 3.0):1 for phosphorus, and 5.8(± 1.4):1 for carbon. Oxygen could be supplied at a weight ratio of 4.1(± 1.0):1. The mean ratios of S. latissima were 12.9(± 2.7):1 for nitrogen, 10.5(± 6.2):1 for phosphorus, and 10.2(± 2.2):1 for carbon. Oxygen could be supplied at a weight ratio of 7.2(± 1.5):1. A. esculenta appears to have almost twice the nutrient sequestration capacity per wet weight than S. latissima. However, culture densities of S. latissima are 1.5 times greater than those for A. esculenta and when spatially weighted this difference is reduced to 1–1.5 times. Numbers of rafts for both kelp species required for full nutrient sequestration from a commercial scale salmon farm exceed the number of rafts which can be practically deployed within a typical site lease area. However, not all inorganic nutrients from cultured fish will be available to IMTA seaweeds, nor should 100% nutrient sequestration need be the only successful endpoint in such systems. These aspects should be considered when assessing the net value of kelps in open-water IMTA systems.

Small ruminant lentiviruses (SLRV) are major production limiting diseases of sheep and goats and are major source of economic loss to producers. There is currently no treatment or vaccine, and the prevalence of SRLV infection across the Ontario is unknown. A preliminary study of 45 Ontario goat herds was used to estimate the prevalence of SRLV infection. Blood was sampled and analyzed with an ELISA test for the presence of SRLV-specific antibodies as an indirect indicator of infection. Additionally, a telephone survey was conducted to identify management factors that are associated with prevalence of infection. The prevalence of SRLV in the 482 goats from 30 dairy goat farms was estimated to be 80.4% and 17.0% from the 225 goats on 15 meat goat farms. Significant differences were found between both prevalence and antibody levels risk in young (≤12 months) and older (>12 months) goats in the dairy sector with older goats slowing a greater prevalence and higher antibody levels as farm risk increased. In the meat sector, there was also a significant difference between antibody levels and risk between young and older goats, however, older meat goats tended to have lower antibody levels than younger goats.

Evidence from the pathology in cystic fibrosis (CF) and recent results in vitro indicate that HCO3- is required for gel-forming mucins to form the mucus that protects epithelial surfaces. Mucus formation and release is a complex process that begins with an initial intracellular phase of synthesis, packaging and apical granule exocytosis that is followed by an extracellular phase of mucin swelling, transport and discharge into a lumen. Exactly where HCO3- becomes crucial in these processes is unknown, but we observed that in the presence of HCO3-, stimulating dissected segments of native mouse intestine with 5-hydroxytryptamine (5-HT) and prostaglandin E2 (PGE2) induced goblet cell exocytosis followed by normal mucin discharge in wild-type (WT) intestines. CF intestines that inherently lack cystic fibrosis transmembrane conductance regulator (CFTR)-dependent HCO3- secretion also demonstrated apparently normal goblet cell exocytosis, but in contrast, this was not followed by similar mucin discharge. Moreover, we found that even in the presence of HCO3-, when WT intestines were stimulated only with a Ca2+-mediated agonist (carbachol), exocytosis was followed by poor discharge as with CF intestines. However, when the Ca2+-mediated agonist was combined with a cAMP-mediated agonist (isoproterenol (isoprenaline) or vasoactive intestinal peptide) in the presence of HCO3- both normal exocytosis and normal discharge was observed. These results indicate that normal mucus formation requires concurrent activation of a Ca2+-mediated exocytosis of mucin granules and an independent cAMP-mediated, CFTR-dependent, HCO3- secretion that appears to mainly enhance the extracellular phases of mucus excretion.

The design of swine breeding programs for the tropics requires knowledge of genetic parameters for economically important traits determined under tropical conditions. A literature review found a total of 468 heritability (h 2 ) estimates, 368 genetic correlations and 254 phenotypic correlations for 35 traits across 117 peer-reviewed articles published from 1974 to 2009 and covering tropical Africa, Southeast Asia, the Caribbean and Latin America. A model that incorporated between and within study variance component was used to obtain weighted means and variances for all parameter estimates. Weighted means and standard errors of direct and maternal heritability, common litter effects and the correlation between direct and maternal effects are given for various reproduction, growth and carcass traits. The weighted means and confidence intervals for the genetic and phenotypic correlations between the trait groups are also given. Weighted least-squares analyses of the h 2 estimates were performed by fitting a number of fixed effects and covariates for each trait as appropriate for the data. Breed, data origin, estimation method, data age and location of study were found to be significant (Po 0.05) for majority of the traits analysed. These results indicate the relevance of having local, population-specific genetic parameter estimates for the tropics. The weighted mean estimates of genetic parameters presented here are recommended for use when reliable estimates are not available for a specific tropical pig population. This is quite appropriate because it uses the vast resource of published genetic parameter estimates effectively.

Two new single-step reactions allowing the construction of two heterocyclic motifs are reported. Both processes involve 5-substituted 1,3,4-thiadiazol-2- amines as starting materials. Reaction with 1-fluoro-2-nitrobenzenes in presence of Hünig's base permitted the convenient synthesis of various 3-substituted 1-aryl-5-amino-1,2,4-triazoles. Reaction with 2-chloro-3- nitropyridines and 2-chloro-5-nitropyridines in similar conditions readily gave access to a number of [1,2,4]triazolo[1,5-a]pyridines. In absence of base, adducts composed by one aromatic unit and two thiadiazole units were formed. This observation and previous work by Anders and co-workers led to the suggestion of plausible mechanisms for these reactions.

Section 121 of the 1997 FDA Modernization Act (FDAMA) required that PET drugs be prepared according to U.S. Pharmacopeia (USP) compounding standards and PET drug monographs until the Food and Drug Administration (FDA) established appropriate approval processes and good manufacturing practice (GMP)

β-Adrenergically induced sweat secretion offers an expedient method to assess native cystic fibrosis transmembrane conductance regulator (CFTR) secretory function in vivo.To evaluate the sensitivity, specificity, and reliability of a test based on the activity and secretory function of CFTR in the sweat gland.Primary and validation trials with prospectively ascertained healthy control subjects, obligate heterozygotes, and patients with a CFTR-related disorder and CF (pancreatic sufficient and insufficient).Diagnostic accuracy and reliability of β-adrenergic sweat secretory rates using an evaporimeter was assessed and compared with sweat chloride concentrations. The cholinergically stimulated mean sweat rate did not differ among groups. The mean maximal β-adrenergically stimulated sweat rate in heterozygotes was about half the rate of healthy control subjects, and completely absent in pancreatic-insufficient patients with CF and pancreatic-sufficient patients with CF (P0.0001). Subjects with a CFTR-related disorder showed reduced or absent β-adrenergic sweat secretion. The β-adrenergic secretory response demonstrated high diagnostic accuracy (area under a characteristic receiver-operator curve = 0.99; 95% confidence interval, 0.97-1.00) and reliability (intraclass correlation, 0.90; 95% confidence interval, 0.81-0.95). The diagnostic cutoff level for CF, derived from the primary trial, correctly identified all control subjects, heterozygotes, and patients with CF in the validation cohort, whereas concurrent sweat chloride measurements misclassified one heterozygote and five subjects with CF. The cholinergic and β-adrenergic sweat secretion rates were lower in women compared with men (P0.001).β-Adrenergic sweat secretion rate determined by evaporimetry is an accurate and reliable technique to assess different levels of CFTR function and to identify patients with CF.

Native small airways must remain wet enough to be pliable and support ciliary clearance, but dry enough to remain patent for gas flow. The airway epithelial lining must both absorb and secrete ions to maintain a critical level of fluid on its surface. Despite frequent involvement in lung diseases, the minuscule size has limited studies of peripheral airways. To meet this challenge, we used a capillary to construct an Ussing chamber (area

The invitation to present the 2010 Hans Ussing lecture for the Epithelial Transport Group of the American Physiological Society offered me a unique, special, and very surprising opportunity to join in saluting a man whom I met only once, but whose work was the basis, not only for my career, but also for finding the molecular defect in the inherited disease cystic fibrosis (CF). In this context, I will venture to make the tribute with a new explanation of why a mutation in a single gene that codes for an anion channel can cause devastation of multiple epithelial systems with pathogenic mucus. In so doing, I hope to raise awareness of a new role for that peculiar anion around which so much physiology revolves, HCO3−. I begin by introducing CF pathology as I question the name of the disease as well as the prevalent view of the basis of its pathology by considering: 1) mucus, 2) salt, and 3) HCO3−. I then present recent data showing that HCO3− is required for normal mucus discharge, and I will close with conjecture as to how HCO3− may support mucus discharge and why the failure to transport this electrolyte is pathogenic in CF.

The impact of small anions on the physical properties of gel-forming mucin has been almost overlooked relative to that of cations. Recently, based on the coincident abnormalities in HCO3−secretion and abnormal mucus formed in the hereditary disease cystic fibrosis (CF), HCO3−was hypothesized to be critical in the formation of normal mucus by virtue of its ability to sequester Ca2+from condensed mucins being discharged from cells. However, direct evidence of the impact of HCO3−on mucus properties is lacking. Herein, we demonstrate for the first time that mucin diffusivity (∼1/viscosity) increases as a function of [HCO3−]. Direct measurements of exocytosed mucin-swelling kinetics from airway cells showed that mucin diffusivity increases by ∼300% with 20 mM extracellular HCO3−concentration. Supporting data indicate that HCO3−reduces free Ca2+concentration and decreases the amount of Ca2+that remains associated with mucins. The results demonstrate that HCO3−enhances mucin swelling and hydration by reducing Ca2+cross-linking in mucins, thereby decreasing its viscosity and likely increasing its transportability. In addition, HCO3−can function as a Ca2+chelator like EGTA to disperse mucin aggregates. This study indicates that poor HCO3−availability in CF may explain why secreted mucus remains aggregated and more viscous in affected organs. These insights bear on not only the fundamental pathogenesis in CF, but also on the process of gel mucus formation and release in general.

Cervical mucus thinning and release during the female reproductive cycle is thought to rely mainly on fluid secretion. However, we now find that mucus released from the murine reproductive tract critically depends upon concurrent bicarbonate (HCO(3)(-)) secretion. Prostaglandin E(2) (PGE(2))- and carbachol-stimulated mucus release was severely inhibited in the absence of serosal HCO(3)(-), HCO(3)(-) transport, or functional cystic fibrosis transmembrane conductance regulator (CFTR). In contrast to mucus release, PGE(2)- and carbachol-stimulated fluid secretion was not dependent on bicarbonate or on CFTR, but was completely blocked by niflumic acid. We found stimulated mucus release was severely impaired in the cystic fibrosis F508 reproductive tract, even though stimulated fluid secretion was preserved. Thus, CFTR mutations and/or poor bicarbonate secretion may be associated with reduced female fertility associated with abnormal mucus and specifically, may account for the increased viscosity and lack of cyclical changes in cervical mucus long noted in women with cystic fibrosis.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl(-) conductance (gCFTR) in the apical membrane is completely deactivated following alpha-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through alpha-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10(-8) M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.

The mechanisms underlying mucus-associated pathologies in cystic fibrosis (CF) remain obscure. However, recent studies indicate that CF transmembrane conductance regulator (CFTR) is required for bicarbonate (HCO3–) transport and that HCO3– is critical for normal mucus formation. We therefore investigated the role of HCO3– in mucus secretion using mouse small intestine segments ex vivo. Basal rates of mucus release in the presence or absence of HCO3– were similar. However, in the absence of HCO3–, mucus release stimulated by either PGE2 or 5-hydroxytryptamine (5-HT) was approximately half that stimulated by these molecules in the presence of HCO3–. Inhibition of HCO3– and fluid transport markedly reduced stimulated mucus release. However, neither absence of HCO3– nor inhibition of HCO3– transport affected fluid secretion rates, indicating that the effect of HCO3– removal on mucus release was not due to decreased fluid secretion. In a mouse model of CF (mice homozygous for the most common human CFTR mutation), intestinal mucus release was minimal when stimulated with either PGE2 or 5-HT in the presence or absence of HCO3–. These data suggest that normal mucus release requires concurrent HCO3– secretion and that the characteristically aggregated mucus observed in mucin-secreting organs in individuals with CF may be a consequence of defective HCO3– transport.

The activities of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and the amiloride-sensitive epithelial Na(+) channel (ENaC) are acutely coordinated in the sweat duct. However, the mechanisms responsible for cross-talk between these ion channels are unknown. Previous studies indicated that luminal pH of sweat ducts varies over 3 pH units and that the cytoplasmic pH affects both CFTR and ENaC. Therefore, using basolaterally alpha-toxin-permeabilized apical membrane preparations of sweat ducts as an experimental system, we tested the hypothesis that the cytosolic pH may mediate the cross-talk between CFTR and ENaC. We showed that while luminal pH had no effect, cytosolic pH acutely affected ENaC activity. That is, acidic pH inhibited, while basic pH activated, ENaC. pH regulation of ENaC appears to be independent of CFTR or endogenous kinase activities because basic pH independently stimulated ENaC (1) in normal ducts even when CFTR was deactivated, (2) in CF ducts that lack CFTR in the plasma membranes and (3) after blocking endogenous kinase activity with staurosporine. Considering the evidence of Na(+)/H(+) exchange (NHE) activity as shown by the expression of mRNA and function of NHE in the basolateral membrane of the sweat duct, we postulate that changes in cytosolic Na(+) ([Na(+)]( i )) may alter cytosolic pH (pH( i )) as salt loads into the cell during electrolyte absorption. These changes may play a role in coordinating the activities of ENaC and CFTR during transepithelial salt transport.

With the advent of numerous candidate drugs for therapy in cystic fibrosis (CF), there is an urgent need for easily interpretable assays for testing their therapeutic value. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) abolished beta-adrenergic but not cholinergic sweating in CF. Therefore, the beta-adrenergic response of the sweat gland may serve both as an in vivo diagnostic tool for CF and as a quantitative assay for testing the efficacy of new drugs designed to restore CFTR function in CF. Hence, with the objective of defining optimal conditions for stimulating beta-adrenergic sweating, we have investigated the components and pharmacology of sweat secretion using cell cultures and intact sweat glands. We studied the electrical responses and ionic mechanisms involved in beta-adrenergic and cholinergic sweating. We also tested the efficacy of different beta-adrenergic agonists. Our results indicated that in normal subjects the cholinergic secretory response is mediated by activation of Ca(2+)-dependent Cl(-) conductance as well as K(+) conductances. In contrast, the beta-adrenergic secretory response is mediated exclusively by activation of a cAMP-dependent CFTR Cl(-) conductance without a concurrent activation of a K(+) conductance. Thus, the electrochemical driving forces generated by beta-adrenergic agonists are significantly smaller compared with those generated by cholinergic agonists, which in turn reflects in smaller beta-adrenergic secretory responses compared with cholinergic secretory responses. Furthermore, the beta-adrenergic agonists, isoproprenaline and salbutamol, induced sweat secretion only when applied in combination with an adenylyl cyclase activator (forskolin) or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, aminophylline or theophylline). We surmise that to obtain consistent beta-adrenergic sweat responses, levels of intracellular cAMP above that achievable with a beta-adrenergic agonist alone are essential. beta-Adrenergic secretion can be stimulated in vivo by concurrent iontophoresis of these drugs in normal, but not in CF, subjects.