Expression and localization of epithelial aquaporins in the adult human lung

Aquaporins (AQPs) facilitate water transport across epithelia and play an important role in normal physiology and disease in the human airways. We used in situ hybridization and immunofluorescence to determine the expression and cellular localization of AQPs 5, 4, and 3 in human airway sections. In nose and bronchial epithelia, AQP5 is expressed at the apical membrane of columnar cells of the superficial epithelium and submucosal gland acinar cells. AQP4 was detected in basolateral membranes in ciliated ducts and by in situ in gland acinar cells. AQP3 is present on basal cells of both superficial epithelium and gland acinus. In these regions AQPs 5, 4, and 3 are appropriately situated to permit transepithelial water permeability. In the small airways (proximal and terminal bronchioles) AQP3 distribution shifts from basal cell to surface expression (i.e., localized to the apical membrane of proximal and terminal bronchioles) and is the only AQP identified in this region of the human lung. The alveolar epithelium has all three AQPs represented, with AQP5 and AQP4 localized to type I pneumocytes and AQP3 to type II cells. This study describes an intricate network of AQP expression that mediates water transport across the human airway epithelium

Permeabilization via the P2X7 purinoreceptor reveals the presence of a Ca2+ -activated Cl- conductance in the apical membrane of murine tracheal epithelial cells

Calcium-activated Cl- secretion is an important modulator of regulated ion transport in murine airway epithelium and is mediated by an unidentified Ca2+-stimulated Cl- channel. We have transfected immortalized murine tracheal epithelial cells with the cDNA encoding the permeabilizing P2X7 purinoreceptor (P2X7-R) to selectively permeabilize the basolateral membrane and thereby isolate the apical membrane Ca2+-activated Cl- current. In P2X7-R-permeabilized cells, we have demonstrated that UTP stimulates a Cl- current across the apical membrane of CF and normal murine tracheal epithelial cells. The magnitude of the UTP-stimulated current was significantly greater in CF than in normal cells. Ion substitution studies demonstrated that the current exhibited a permselectivity sequence of Cl- > I- > Br- > gluconate-. We have also determined a rank order of potency for putative Cl- channel blockers: niflumic acid ≥ 5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanostilbene-2,2'-disulfonate > glybenclamide >> diphenlyamine-2-carboxylate, tamoxifen, and p-tetra-sulfonato-tetra-methoxy-calix[4]arene. Complete characterization of this current and the corresponding single channel properties could lead to the development of a new therapy to correct the defective airway surface liquid in cystic fibrosis patients

Probing Rock Type, Fe Redox State, and Transition Metal Contents with Six-Window VNIR Spectroscopy Under Venus Conditions

VEM-window data are shown to distinguish among key rock types on Venus, and evaluate redox state and transition metal contents of Venus surface rocks

Transmembrane protein 16A (TMEM16A) is a Ca2+ -regulated Cl- secretory channel in mouse airways

For almost two decades, it has been postulated that calcium-activated Cl- channels (CaCCs) play a role in airway epithelial Cl- secretion, but until recently, the molecular identity of the airway CaCC(s) was unknown. Recent studies have unequivocally identified TMEM16A as a glandular epithelial CaCC. We have studied the airway bioelectrics of neonatal mice homozygous for a null allele of Tmem16a (Tmem16a-/ -) to investigate the role of this channel in Cl- secretion in airway surface epithelium. When compared with wild-type tracheas, the Tmem16a-/- tracheas exhibited a >60% reduction in purinoceptor (UTP)-regulated CaCC activity. Other members of the Tmem16 gene family,including Tmem16f and Tmem16k, were also detected by reverse transcription-PCR in neonatal tracheal epithelium, suggesting that other family members could be considered as contributing to the small residual UTP response. TMEM16A, however, appeared to contribute little to unstimulated Cl- secretion, whereas studies with cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice and wild-type littermates revealed that unstimulated Cl- secretion reflected ∼50% CFTR activity and ∼50% non-Tmem16a activity. Interestingly, the tracheas of both the Tmem16a-/- and the CFTR-/- mice exhibited similar congenital cartilaginous defects that may reflect a common Cl- secretory defect mediated by the molecularly distinct Cl- channels. Importantly, the residual CaCC activity in Tmem16a-/- mice appeared inadequate for normal airway hydration because Tmem16a-/- tracheas exhibited significant, neonatal, lumenal mucus accumulation. Our data suggest that TMEM16A CaCC-mediated Cl- secretion appears to be necessary for normal airway surface liquid homeostasis

Quantitation and localization of ENaC subunit expression in fetal, newborn, and adult mouse lung

The newborn lung is cleared of fetal liquid by active Na+ transport. The heterotrimeric (α, β, γ) epithelial Na+ channel, ENaC, mediates this process. To understand the role of individual ENaC subunits in Na+ transport during development, we quantified murine ENaC (mENaC) subunit messenger RNA (mRNA) expression levels of fetal, neonatal, and adult mouse lung by Northern blot analysis and studied regional expression by in situ hybridization. αmENaC and γmENaC mRNA expression increased sharply in late fetal gestation and reached near-adult levels by Day 1 of postnatal life. βmENaC expression increased more gradually through late fetal and early postnatal life and increased progressively until adulthood. In situ hybridization studies showed similar localization patterns of αmENaC and γmENaC subunit expression in fetal and postnatal lung. γmENaC and αmENaC subunits were initially localized to fetal lung bud tubules and by late gestation both subunits were expressed in all regions (acinar and bronchiolar) of the distal lung epithelium. βmENaC was detected from 16 d gestation onward and was expressed most intensely in small airways. There was little expression of βmENaC in the alveolar region. In postnatal lung all three subunits were expressed intensely in small airways. In adult lung, αmENaC and γmENaC were expressed in a pattern consistent with an alveolar type II (ATII) cell distribution. The timing of quantitative changes in mENaC subunit expression is consistent with a role of Na+ transport in liquid clearance of the perinatal lung. Intense expression of mENaC subunits in medium and small airway epithelium and in ATII cells suggests that these regions are a primary location for liquid absorption in the perinatal and post-natal murine lung

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Two Cl- conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca2+-activated Cl- conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl-/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca2+ signal in response to mucosal nucleotides that may contribute to the increased Cl-/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca2+ signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height

Early phonological and sociocognitive skills as predictors of later language and social communication outcomes

Background:  Previous studies of outcome for children with early language delay have focused on measures of early language as predictors of language outcome. This study investigates whether very early processing skills (VEPS) known to underpin language development will be better predictors of specific language and social communication outcomes than measures of language itself. Method:  Participants were 163 children referred to clinical services with concerns about language at 2;6–3;6 years and followed up at 4–5 years. Novel assessments of phonological and sociocognitive processing were administered at Time 1 (T1), together with a standardised test of receptive and expressive language, and parental report of expressive vocabulary. The language test was re-administered at Time 2 (T2), together with assessments of morphosyntax and parental reports of social communication. Results:  Intercorrelations at and between T1 and T2 were high, and dissociations were rare. Ordinal regressions were run, entering predictors singly and simultaneously. With the exception of the phonological task, every early measure on its own was significantly predictive of most outcomes, and receptive language was the strongest all-round predictor. Results of simultaneous entry, controlling for the effect of other predictors, showed that early language was the strongest predictor of general language outcome, but early phonology was the strongest predictor of a measure of morphosyntax, and early sociocognition the strongest predictor of social communication. Conclusions:  Language measures which draw on a wide range of skills were the strongest overall predictors of general language outcomes. However, our VEPS measures were stronger predictors of specific outcomes. The clinical and theoretical implications of these findings are discussed

An Adeno-Associated Viral Vector Capable of Penetrating the Mucus Barrier to Inhaled Gene Therapy

Diffusion of the viral vectors evaluated in inhaled gene therapy clinical trials to date are largely hindered within airway mucus, which limits their access to, and transduction of, the underlying airway epithelium prior to clearance from the lung. Here, we discovered that adeno-associated virus (AAV) serotype 6 was able to rapidly diffuse through mucus collected from cystic fibrosis (CF) patients, unlike previously tested AAV serotypes. A point mutation of the AAV6 capsid suggests a potential mechanism by which AAV6 avoids adhesion to the mucus mesh. Significantly greater transgene expression was achieved with AAV6 compared to a mucoadhesive serotype, AAV1, in air-liquid interface cultures of human CF bronchial epithelium with naturally secreted mucus or induced mucus hypersecretion. In addition, AAV6 achieved superior distribution and overall level of transgene expression compared to AAV1 in the airways and whole lungs, respectively, of transgenic mice with airway mucus obstruction. Our findings motivate further evaluation and clinical development of AAV6 for inhaled gene therapy

Dimensionless cosmology

Although it is well known that any consideration of the variations of fundamental constants should be restricted to their dimensionless combinations, the literature on variations of the gravitational constant $G$ is entirely dimensionful. To illustrate applications of this to cosmology, we explicitly give a dimensionless version of the parameters of the standard cosmological model, and describe the physics of Big Bang Neucleosynthesis and recombination in a dimensionless manner. The issue that appears to have been missed in many studies is that in cosmology the strength of gravity is bound up in the cosmological equations, and the epoch at which we live is a crucial part of the model. We argue that it is useful to consider the hypothetical situation of communicating with another civilization (with entirely different units), comparing only dimensionless constants, in order to decide if we live in a Universe governed by precisely the same physical laws. In this thought experiment, we would also have to compare epochs, which can be defined by giving the value of any {\it one} of the evolving cosmological parameters. By setting things up carefully in this way one can avoid inconsistent results when considering variable constants, caused by effectively fixing more than one parameter today. We show examples of this effect by considering microwave background anisotropies, being careful to maintain dimensionlessness throughout. We present Fisher matrix calculations to estimate how well the fine structure constants for electromagnetism and gravity can be determined with future microwave background experiments. We highlight how one can be misled by simply adding $G$ to the usual cosmological parameter set

Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized low-density lipoproteins and in secretion of H2O2

Oxidized low-density lipoprotein (oxLDL) is considered one of the principal effectors of atherogenesis. To explore mechanisms by which oxLDL affects human mononuclear phagocytes, we incubated these cells in medium containing oxLDL, acetylated LDL (acLDL), or native LDL, or on surfaces coated with these native and modified lipoproteins. The presence of soluble oxLDL, acLDL, or native LDL in the medium did not stimulate H2O2 secretion by macrophages. In contrast, macrophages adherent to surfaces coated with oxLDL secreted three- to fourfold more H2O2 than macrophages adherent to surfaces coated with acLDL or native LDL. Freshly isolated blood monocytes secreted little H2O2 regardless of the substrate on which they were plated. H2O2 secretion was maximal in cells maintained for 4–6 d in culture before plating on oxLDL-coated surfaces. Fucoidan, a known ligand of class A macrophage scavenger receptors (MSR-A), significantly reduced macrophage adhesion to surfaces coated with oxLDL or acLDL. Monoclonal antibody SMO, which blocks oxLDL binding to CD36, did not inhibit adhesion of macrophages to oxLDL-coated surfaces but markedly reduced H2O2 secretion by these cells. These studies show that MSR-A is primarily responsible for adhesion of macrophages to oxLDL-coated surfaces, that CD36 signals H2O2 secretion by macrophages adherent to these surfaces, and that substrate-bound, but not soluble, oxLDL stimulates H2O2 secretion by macrophages