The Laminin-Induced Phosphorylation of PKCδ Regulates AQP4 Distribution and Water Permeability in Rat Astrocytes.

In astrocytes, the water-permeable channel aquaporin-4 (AQP4) is concentrated at the endfeet that abut the blood vessels of the brain. The asymmetric distribution of this channel is dependent on the function of dystroglycan (DG), a co-expressed laminin receptor, and its associated protein complex. We have demonstrated that the addition of laminin to astrocytes in culture causes the clustering of AQP4, DG, and lipid rafts. The last, in particular, have been associated with the initiation of cell signaling. As laminin binding to DG in muscle cells can induce the tyrosine phosphorylation of syntrophin and laminin requires tyrosine kinases for acetylcholine receptor clustering in myotubes, we asked if signal transduction might also be involved in AQP4 clustering in astrocytes. We analyzed the timecourse of AQP4, DG, and monosialotetrahexosylganglioside (GM1) clustering in primary cultures of rat astrocytes following the addition of laminin, and determined that the clustering of DG precedes that of AQP4 and GM1. We also showed that laminin induces the formation of phosphotyrosine-rich clusters and that the tyrosine kinase inhibitor, genistein, disrupts the laminin-induced clustering of both ß-DG and AQP4. Using the Kinexus antibody microarray chip, we then identified protein-serine kinase C delta (PKCδ) as one of the main proteins exhibiting high levels of tyrosine phosphorylation upon laminin treatment. Selective inhibitors of PKC and siRNA against PKCδ disrupted ß-DG and AQP4 clustering, and also caused water transport to increase in astrocytes treated with laminin. Our results demonstrate that the effects of laminin on AQP4 localization and function are relayed, at least in part, through PKC signaling.

Impact of a simulation-based ergonomics training curriculum on work-related musculoskeletal injury risk in colonoscopy.

BACKGROUND AND AIMS: Endoscopists are at risk of developing musculoskeletal injuries (MSIs), and few receive training on ergonomics. The aim of this study was to determine the impact of a simulation-based ergonomics training curriculum (ETC) on work-related MSI risk during clinical colonoscopy. METHODS: Novice endoscopists underwent a simulation-based ETC and were compared with an historical control group who received simulation-based training without ergonomics training. The ETC included a didactic lecture and video on ergonomics in colonoscopy, feedback from supervisors on ergonomics, and an ergonomics checklist to augment feedback and promote self-reflection. Participants were assessed using the rapid entire body assessment (REBA) and rapid upper limb assessment (RULA). The primary outcome was participants' REBA scores during 2 clinical colonoscopies 4 to 6 weeks after training. RESULTS: In clinical colonoscopy, the ETC group had superior REBA scores (clinical procedure 1: median score, 6 vs 11; P < .001; clinical procedure 2: median score, 6 vs 10; P < .001). In a simulated colonoscopy, the ETC group did not have significantly different REBA or RULA scores between baseline, immediately after training, and 4 to 6 weeks after (REBA: median scores of 5, 5, and 5, respectively; P > .05; RULA: median scores of 6, 6, and 6, respectively; P > .05). The control group had worsening REBA and RULA scores during the study timeline (REBA: median scores of 5 at baseline, 9 immediately after training, and 9 at 4-6 weeks after training; P < .001; RULA: median scores of 6, 7, and 7, respectively; P = .04) during simulated procedures. CONCLUSIONS: A simulation-based ETC is associated with reduced risk of MSI during endoscopy. Although the REBA score was improved, the intervention group was still within the medium-risk range.

Prevalence and predictors of antibiotic use among children visiting the Emergency Department in a Tertiary Hospital in Malaysia.

Inappropriate use of antibiotics in human and animal is one of the causes of antimicrobial resistance. This study evaluates the prevalence and predictors of antibiotic use among pediatric patients visiting the Emergency Department (ED) in Malaysia. A retrospective cross-sectional study was conducted in the ED of a tertiary hospital. Data of children aged 2 to 11 years who visited the ED from January-May 2015 were extracted from the patient's assessment forms. A total of 549 children were included in the analysis (median age 5 years) of which 54.3% were boys. Upper respiratory tract infections (URTI) were the most common diagnosis. Antibiotic was prescribed in 43.5% of the children. Children who visited the ED during the weekend (OR, 1.65; 95% confidence interval (CI) 1.13-2.40, P = 0.009), those diagnosed with URTI (OR 3.81; 95% CI, 2.45-5.93, P < 0.001) and those with a longer duration of fever (OR, 1.31; 95% CI, 1.15-1.48, P < 0.001) were more likely to have an antibiotic prescription.Conclusions: Antibiotic was prescribed in more than one-third of children who visited the ED and antibiotic use was associated with visits during the weekend, URTI, and duration of fever. Antimicrobial stewardship program is recommended in the pediatric ED to improve appropriate use of antibiotics.What is Known:•Use of antibiotic among children increases the risk of antibiotic resistance and adverse drug reactions.•Patient-, clinical-, and prescriber-related factors are three important domain associated with antibiotic use in children.What is New:•Fever and upper respiratory tract infections account for majority of antibiotic prescriptions.•The "weekend effect" influences the use of antibiotics in the ED while upper respiratory tract infection is the strongest predictor of antibiotic use among children visiting the ED.

Towards a better understanding of AQP4's role in astrocytic process extension: An Editorial for 'Involvement of aquaporin-4 in laminin-enhanced process formation of mouse astrocytes in 2D culture: Roles of dystroglycan and a-syntrophin in aquaporin-4 expression' on page 495.

At the blood-brain-barrier (BBB), blood vessels are surrounded by the endfoot structures formed by astroglial cells. The latter are themselves part of a larger syncytial network of astrocytes connected to each other via a complex web of processes. The water-permeable channel aquaporin-4 (AQP4) is expressed at high concentrations at these endfeet, held in place by the dystrophin glycoprotein complex (DGC), a collection of proteins that act as a bridge linking AQP4 to the laminin-containing basal lamina found in between the blood vessels and the astrocytic endfeet. Although AQP4, supported by the DGC, has well-established roles in facilitating certain neurological processes, and in mediating the removal of excess water from the brain in certain disease states, relatively few studies have looked at the importance of these components in the regulation of the extension of the processes that are so characteristic of astrocytes. In this Editorial Highlight, we discuss an article by Sato et al., published in this issue of the Journal of Neurochemistry, which attempts to address this question.

Financial Conflicts of Interest in Propensity Score-Matched Studies Evaluating Biologics and Biosimilars for Inflammatory Bowel Disease.

Background: Propensity score matching (PSM), a statistical technique that estimates a treatment effect by accounting for predictor covariates, has been used to evaluate biologics for inflammatory bowel disease (IBD). Financial conflicts of interest are prevalent in the marketing of biologic medications. It is unclear whether this burden of conflicts is present among authors of PSM studies comparing IBD biologics and biosimilars. Objective: This study was aimed to determine the prevalence of financial conflicts of interest among authors of PSM studies evaluating IBD biologics and biosimilars. Methods: We conducted a systematic search for PSM studies comparing biologics and biosimilars in IBD treatment. We identified 21 eligible studies. Two independent authors extracted self-declared conflicts from the disclosures section. Each participating author was searched on the Centers for Medicare & Medicaid Services Open Payments to identify payment amounts and undisclosed conflicts. Primary outcome was the prevalence of author conflicts. Secondary analyses assessed for an association between conflict prevalence and reporting of positive outcomes. Results: Among 283 authors, conflicts were present among 41.0% (116 of 283). Twenty-three per cent (27 of 116) of author conflicts involved undisclosed payments. Studies with positive outcomes were significantly more likely to include conflicted authors than neutral studies (relative risk = 2.34, 95% confidence interval: 1.71 to 3.21, P < 0.001). Conclusions: Overall, we found a high burden of undisclosed conflicts among authors of PSM studies comparing IBD biologics and biosimilars. Given the importance of PSM studies as a means for biologic comparison and the potential for undue industry influence from these payments, authors should ensure greater transparency with reporting of industry relationships.

Regulation of Kir4.1 and AQP4 expression and stability at the basolateral domain of epithelial MDCK cells by the extracellular matrix.

The proper targeting of ion channels to specialized domains is crucial for cell function. Kir4.1, the inwardly rectifying potassium channel, and aquaporin-4 (AQP4), the type 4 water-permeable channel, are localized at the basolateral domain of polarized epithelial cells; however, the mechanisms involved in their localization have yet to be determined. In this study, we investigated the role of the extracellular matrix in the localization of these channels in polarized Madin-Darby canine kidney (MDCK) cells. MDCK cells expressing green fluorescent protein-Kir4.1 or -AQP4 were cultured on laminin-1 or fibronectin and examined by confocal microscopy and cell surface biotinylation to assess plasma membrane expression of Kir4.1 and AQP4. Our data show that laminin-1 and fibronectin induce a significant increase in cell surface expression of both channels at the basolateral domain. Using fluorescence recovery after photobleaching, we demonstrate that laminin-1 and fibronectin reduce the diffusion rates of these channels. Finally, we show that the laminin receptor dystroglycan is important for cell surface expression of Kir4.1 but not AQP4. However, laminin-1 increases cell surface expression of both channels in cells deficient for dystroglycan, indicating that other receptors are involved. Indeed, RGD-containing peptides, which inhibit fibronectin binding to certain integrins, prevent the fibronectin-induced increase in Kir4.1 and AQP4 cell surface expression and reverse the laminin- and fibronectin-induced reduction in both channels' diffusion rates. Similarly, the αvß3-integrin function-blocking antibody alters the reduction of AQP4 diffusion rates induced by both laminin and fibronectin, suggesting that αvß3-integrin plays a role in the stabilization of APQ4 at the basolateral domain of epithelial cells.

Agrin plays a major role in the coalescence of the aquaporin-4 clusters induced by gamma-1-containing laminin.

The basement membrane that seperates the endothelial cells and astrocytic endfeet that comprise the blood-brain barrier is rich in collagen, laminin, agrin, and perlecan. Previous studies have demonstrated that the proper recruitment of the water-permeable channel aquaporin-4 (AQP4) to astrocytic endfeet is dependent on interactions between laminin and the receptor dystroglycan. In this study, we conducted a deeper investigation into how the basement membrane might further regulate the expression, localization, and function of AQP4, using primary astrocytes as a model system. We found that treating these cells with laminin causes endogenous agrin to localize to the cell surface, where it co-clusters with ß-dystroglycan (ß-DG). Conversely, agrin sliencing profoundly disrupts ß-DG clustering. As in the case of laminin111, Matrigel™, a complete basement membrane analog, also causes the clustering of AQP4 and ß-DG. This clustering, whether induced by laminin111 or Matrigel™ is inhibited when the astrocytes are first incubated with an antibody against the γ1 subunit of laminin, suggesting that the latter is crucial to the process. Finally, we showed that laminin111 appears to negatively regulate AQP4-mediated water transport in astrocytes, suppressing the cell swelling that occurs following a hypoosmotic challenge. This suppression is abolished if DG expression is silenced, again demonstrating the central role of this receptor in relaying the effects of laminin.

Mitochondrial lipid droplet formation as a detoxification mechanism to sequester and degrade excessive urothelial membranes.

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due to its coverage by urothelial plaques consisting of 2D crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Because mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism.

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation.

The reperfusion of ischemic brain tissue following a cerebral stroke causes oxidative stress, and leads to the generation of reactive oxygen species (ROS). Apart from inflicting oxidative damage, the latter may also trigger the upregulation of aquaporin 4 (AQP4), a water-permeable channel expressed by astroglial cells of the blood-brain barrier (BBB), and contribute to edema formation, the severity of which is known to be the primary determinant of mortality and morbidity. The mechanism through which this occurs remains unknown. In the present study, we have attempted to address this question using primary astrocyte cultures treated with hydrogen peroxide (H2O2) as a model system. First, we showed that H2O2 induces a significant increase in AQP4 protein levels and that this is inhibited by the antioxidant N-acetylcysteine (NAC). Second, we demonstrated using cell surface biotinylation that H2O2 increases AQP4 cell-surface expression independently of it's increased synthesis. In parallel, we found that caveolin-1 (Cav1) is phosphorylated in response to H2O2 and that this is reversed by the Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). PP2 also abrogated the H2O2-induced increase in AQP4 surface levels, suggesting that  the phosphorylation of tyrosine-14 of Cav1 regulates  this  process. We  further  showed  that dominant-negative Y14F and phosphomimetic Y14D mutants caused a decrease and increase in AQP4 membrane expression respectively, and that the knockdown of Cav1 inhibits the increase in AQP4 cell-surface, expression following H2O2 treatment. Together, these findings suggest that oxidative stress-induced Cav1 phosphorylation modulates AQP4 subcellular distribution and therefore may indirectly regulate AQP4-mediated water transport.

Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes.

The water-permeable channel aquaporin-4 (AQP4) is highly expressed in perivascular astrocytes of the mammalian brain and represents the major conduit for water across the blood-brain barrier. Within these cells, AQP4 is found in great quantities at perivascular endfoot sites but is detected in lesser amounts at the membrane domains within the brain parenchyma. We had previously established that this polarization was regulated by the interaction between dystroglycan (DG), an extracellular matrix receptor that is co-expressed with AQP4, and the laminin that is contained within the perivascular basal lamina. In the present study, we have attempted to describe the mechanisms that underlie this regulation, using primary astrocyte cultures. Via biotinylation, we found that the cell-surface expression of AQP4 is DG-dependent and is potentiated by laminin. We also determined that this laminin-dependent increase occurs not through an upregulation of total AQP4 levels, but rather from a redirection of AQP4 from an intracellular, EEA-1-associated pool to the cell surface. We then demonstrated an association between DG and dynamin and showed that dynamin functioned in conjunction with clathrin to regulate surface AQP4 amounts. Furthermore, we observed that DG preferentially binds to the inactive forms of dynamin, suggesting that this interaction was inhibitory for AQP4 endocytosis. Finally, we showed that laminin selectively upregulates the cell-surface expression of the M23 isoform of AQP4. Our data therefore indicate that the dual interation of DG with laminin and dynamin is involved in the regulation of AQP4 internalization, leading to its asymmetric enrichment at perivascular astrocyte endfeet.

Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.

Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes.

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.