Refining the accuracy of validated target identification through coding variant fine-mapping in type 2 diabetes.

We aggregated coding variant data for 81,412 type 2 diabetes cases and 370,832 controls of diverse ancestry, identifying 40 coding variant association signals (P < 2.2 × 10-7); of these, 16 map outside known risk-associated loci. We make two important observations. First, only five of these signals are driven by low-frequency variants: even for these, effect sizes are modest (odds ratio ≤1.29). Second, when we used large-scale genome-wide association data to fine-map the associated variants in their regional context, accounting for the global enrichment of complex trait associations in coding sequence, compelling evidence for coding variant causality was obtained for only 16 signals. At 13 others, the associated coding variants clearly represent 'false leads' with potential to generate erroneous mechanistic inference. Coding variant associations offer a direct route to biological insight for complex diseases and identification of validated therapeutic targets; however, appropriate mechanistic inference requires careful specification of their causal contribution to disease predisposition

Butyrate Permeation across the Isolated Ovine Reticulum Epithelium

We hypothesized that, due to the high pH of this compartment, the reticulum epithelium displays particular features in the transport of short-chain fatty acids (SCFA). Ovine reticulum epithelium was incubated in Ussing chambers using a bicarbonate-free buffer solution containing butyrate (20 mmol L−1). p-hydroxymercuribenzoic acid (pHMB), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), or ouabain were added to the buffer solution as inhibitors of monocarboxylate transporters, sodium-proton-exchangers, or the Na+/K+-ATPase, respectively. The short-circuit current (Isc) and transepithelial conductance (Gt) were monitored continuously while the flux rates of 14C-labelled butyrate were measured in the mucosal-to-serosal (Jmsbut) or serosal-to-mucosal direction (Jsmbut). Under control conditions, the mean values of Isc and Gt amounted to 2.54 ± 0.46 µEq cm−2 h−1 and 6.02 ± 3.3 mS cm−2, respectively. Jmsbut was 2.1 ± 1.01 µmol cm−2 h−1 on average and about twice as high as Jsmbut. Incubation with ouabain reduced Jmsbut, while Jsmbut was not affected. The serosal addition of EIPA did not affect Jmsbut but reduced Jsmbut by about 10%. The addition of pHMB to the mucosal or serosal solution reduced Jmsbut but had no effect on Jsmbut. Mucosally applied pHMB provoked a transient increase in the Isc. The serosal pHMB sharply reduced Isc. Our results demonstrate that butyrate can be effectively transported across the reticulum epithelium. The mechanisms involved in this absorption differ from those known from the rumen epithelium

Signaling Cascade Involved in Rapid Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Dexamethasone

Impairment of mucociliary clearance with reduced airway fluid secretion leads to chronically inflamed airways. Cystic fibrosis transmembrane conductance regulator (CFTR) is crucially involved in airway fluid secretion and dexamethasone (dexa) has previously been shown to elevate CFTR activity in airway epithelial cells. However, the pathway by which dexa increases CFTR activity is largely unknown. We aimed to determine whether the increase of CFTR activity by dexa is achieved by non-genomic signaling and hypothesized that the phosphoinositide 3-kinase (PI3K) pathway is involved in CFTR stimulation. Primary rat airway epithelial cells and human bronchial submucosal gland-derived Calu-3 cells were analyzed in Ussing chambers and kinase activation was determined byWestern blots. Results demonstrated a critical involvement of PI3K and protein kinase B (AKT) signaling in the dexa-induced increase of CFTR activity, while serum and glucocorticoid dependent kinase 1 (SGK1) activity was not essential. We further demonstrated a reduced neural precursor cell expressed, developmentally downregulated 4-like (NEDD4L) ubiquitin E3 ligase activity induced by dexa, possibly responsible for the elevated CFTR activity. Finally, increases of CFTR activity by dexa were demonstrated within 30 min accompanied by rapid activation of AKT. In conclusion, dexa induces a rapid stimulation of CFTR activity which depends on PI3K/AKT signaling in airway epithelial cells. Glucocorticoids might thus represent, in addition to their immunomodulatory actions, a therapeutic strategy to rapidly increase airway fluid secretion

Integrated Steering and Coordination of Local Public Services in Germany – Reasons and Restrictions of an Innovative Governance Approach

Due to the institutional differentiation of the public sector, which lead to a hardly comprehensible network of organisations from local administration, public economy and the third sector, the critical question of the importance of the integrated steering and coordination (ISC) of public tasks in municipalities arises in local government research and practice. Following an interdisciplinary approach, the aim of this contribution is (1) to examine the idea of the ISC of local public services from the perspective of policy science, New Public Management and public governance and (2) to identify – based on a qualitative empirical study – reasons and restrictions of its implementation. It is discussed why ISC is at the same time a feasible and guiding approach, but in part also a hardly practicable idea. With regard to this crucial controversy, five perspectives for further local public sector reforms are presented. From the perspective of innovative governance, the paper might help us to stimulate critical thinking about alternative approaches to problem solving and decision making in the public sector

Butyrate Protects Porcine Colon Epithelium from Hypoxia-Induced Damage on a Functional Level

The large intestinal epithelium is confronted with the necessity to adapt quickly to varying levels of oxygenation. In contrast to other tissues, it meets this requirement successfully and remains unharmed during (limited) hypoxic periods. The large intestine is also the site of bacterial fermentation producing short-chain fatty acids (SCFA). Amongst these SCFA, butyrate has been reported to ameliorate many pathological conditions. Thus, we hypothesized that butyrate protects the colonocytes from hypoxic damage. We used isolated porcine colon epithelium mounted in Ussing chambers, incubated it with or without butyrate and simulated hypoxia by changing the gassing regime to test this hypothesis. We found an increase in transepithelial conductance and a decrease in short-circuit current across the epithelia when simulating hypoxia for more than 30 min. Incubation with 50 mM butyrate significantly ameliorated these changes to the epithelial integrity. In order to characterize the protective mechanism, we compared the effects of butyrate to those of iso-butyrate and propionate. These two SCFAs exerted similar effects to butyrate. Therefore, we propose that the protective effect of butyrate on colon epithelium under hypoxia is not (only) based on its nutritive function, but rather on the intracellular signaling effects of SCFA

Epigenetic Drifts during Long-Term Intestinal Organoid Culture

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. We found that, besides the expected changes in short-term culture, the organoids showed profound changes in their epigenomes also during the long-term culture. The most prominent were epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation is disturbed

Glucocorticoids Equally Stimulate Epithelial Na+ Transport in Male and Female Fetal Alveolar Cells

Preterm infants frequently suffer from respiratory distress syndrome (RDS), possibly due to lower expression of epithelial Na+ channels (ENaC). RDS incidence is sex-specific, affecting males almost twice as often. Despite the use of antenatal glucocorticoids (GCs), the sex difference persists. It is still controversial whether both sexes benefit equally from GCs. We previously showed that Na+ transport is higher in female compared with male fetal distal lung epithelial (FDLE) cells. Since GCs increase Na+ transport, we hypothesized that their stimulating effect might be sex-specific. We analyzed FDLE cells with Ussing chambers and RT-qPCR in the presence or absence of fetal serum. In serum-free medium, GCs increased the ENaC activity and mRNA expression, independent of sex. In contrast, GCs did not increase the Na+ transport in serum-supplemented media and abolished the otherwise observed sex difference. Inhibition of the GC receptor in the presence of serum did not equalize Na+ transport between male and female cells. The GC-induced surfactant protein mRNA expression was concentration and sex-specific. In conclusion, female and male FDLE cells exhibit no sex difference in response to GCs with regard to Na+ transport, and GR activity does not contribute to the higher Na+ transport in females

Involvement of the putative anion transporter 1 (SLC26A6) in permeation of short chain fatty acids and their metabolites across the basolateral membrane of ovine ruminal epithelium: Involvement of the putative anion transporter 1 (SLC26A6) inpermeation of short chain fatty acids and their metabolites across thebasolateral membrane of ovine ruminal epithelium

Introduction: Microbial fermentation of carbohydrates in forestomach of ruminants produces large amounts of short-chain fatty acids (SCFA, mainly acetic acid, propionic acid, and n-butyric acid). The majority of these substrates is taken up directly across the ruminal wall. After luminal uptake into the epithelial cells, SCFA mainly occur in the dissociated form due to the intracellular pH of ~7.4. Moreover, a big portion of SCFA is metabolised within the cytosol. Main end products of epithelial SCFA metabolism are ketone bodies (D-3-hydroxybutyric acid and acetoacetic acid) and lactic acid. Both intact SCFA and ketone bodies and lactate need to be efficiently extruded from the ruminal epithelial cells to prevent a lethal drop of intracellular pH and counteract osmotic load of the cytosol. All these substances are less lipophilic in comparison to the undissociated form of SCFA. Thus, dissociated SCFA (SCFA-) and their metabolites need Protein mediated mechanisms for the extrusion across the basolateral side of ruminal epithelium. One mechanism suggested to be involved in the extrusion of SCFA- across basolateral membrane of the ruminal epithelium is the monocarboxylate transporter 1 (MCT1). Functionally, MCT1 was first assumed to operate as proton-coupled transporter for monocarboxylates including SCFA. Nonetheless, a recent study found a bicarbonate dependent anion exchange mechanism which turned out to be sensitive to MCT1 Inhibitors at the basolateral side of the ruminal epithelium pointing to the ability of MCT1 to act as an anion exchanger. However, in these experiments the inhibition of MCT1 abolished bicarbonate dependent transport only by half. This suggests the involvement of further anion exchanger(s) in the transport of SCFA across the basolateral membrane of ruminal epithelium. Promising candidates to underlie this exchange are the putative Anion exchanger 1 (PAT1) and a transport protein designated „down-regulated in adenoma“ (DRA). Materials and Methods: Sheep rumen epithelium was mounted in Ussing Chambers under short-circuit conditions. Radioactively labelled acetate (ac) was added to the serosal side. Serosal to mucosal flux of ac (Jsm ac) was measured with or without anion Exchange inhibitors (50 mM NO3- or 1 mM DIDS) or the MCT1 inhibitor p-hydroxy mercuribenzoic acid (pHMB; 1.5 mM) in the serosal buffer solution. The inhibitors were added alone or in combination with each other. Furthermore, mucosal to serosal flux of radioactivelly labelled ac or butyrate (bu) (Jms ac, bu) was measured in the presence or absence of SO42-, Cl- or NO3- (50 mM respectively) as exchange substrate in the serosal buffer solution. Immunohistochemical staining was conducted to locate PAT1 and DRA by use of commercially available antibodies. Results: NO3- and pHMB significantly reduced Jsm ac by 57 % and 51 %, respectively. When pHMB was applied after pre-incubation with NO3- an additional inhibition of Jsm ac was observed. Vice versa, NO3- further inhibited Jsm ac when epithelia were pre-incubated with pHMB before. DIDS had no inhibitory effect on SCFA flux. Serosal presence of SO42- or Cl- enhanced Jms ac significantly. Regarding bu, Cl- or SO4 2- also enhanced Jms bu significantly. The different anions available in the serosal buffer solution numerically enhanced Jms in the order of SO4 2- > Cl- for both ac and bu, which corresponds to the known affinity sequence of PAT1 and DRA. Immunohistochemistry revealed localization of PAT 1 in the stratum basale, whereas DRA was not detectable using this method. Conclusions: Basically, this study supports the suggestion that MCT1 works as an Anion exchanger in ruminal epithelium. In addition, it clearly shows that there is at least one further anion exchanger involved in the basolateral extrusion of SCFA and their metabolites. The functional and immunohistochemical findings suggest that PAT1 holds a significant role in this respect.:1 Introduction 1 2 Literature Review 3 2.1 Importance of short-chain fatty acid production of ruminants 3 2.2 Apical uptake of short-chain fatty acids from the rumen 5 2.2.1 Apical uptake of undissociated SCFA from the rumen 6 2.2.2 Apical uptake of dissociated fatty acids from the rumen 8 2.3 Intraepithelial metabolism of short-chain fatty acids 9 2.4 Mechanisms for the basolateral discharge of the short-chain fatty acids 11 2.4.1 Basolateral extrusion of short-chain fatty acids in other gastrointestinal tract epithelia 12 2.4.2 Basolateral extrusion of short-chain fatty acids in ruminal epithelium 14 2.4.3 Further candidate proteins for extrusion of SCFA- in exchange for HCO3 - 19 2.4.3.1 Putative Anion transporter 1 (PAT1 = SLC26A6) 19 2.4.3.2 Down-regulated in adenoma (DRA = SLC26A3) 21 2.4.3.3 Anion exchanger 2 (AE2 = SLC4A2) 22 2.5 Literature implications for this study 23 3 Materials and Methods 24 3.1 Animals 24 3.2 Ussing chamber studies 24 3.2.1 Buffer solutions 24 3.2.2 Preparation of ruminal epithelium 25 3.2.3 Incubation 25 3.2.4 Electrophysiological parameters 26 3.3 Experimental procedure 27 3.3.1 Determination of the unidirectional SCFA flux rate 29 3.4 Experimental Setups 30 3.4.1 Sensitivity of Jsm ac to inhibitors 30 3.4.1.1 Effect of nitrate and pHMB on Jsm ac 30 3.4.1.2 Effect of DIDS, NO3 - and pHMB on Jsm ac 31 3.4.2 Effect of the basolateral replacement of the anions on the extrusion of SCFA 32 3.4.2.1 Effect of Cl- and NO3 - on Jms of acetate and butyrate 32 3.4.2.2 Effect of SO4 2- on Jms of acetate and butyrate 32 3.4.3 Effect of different anions available in the serosal solution on Jms of acetate and butyrate 33 3.5 Immunohistochemistry 34 3.5.1 Preparation of the samples. 34 3.5.2 Fixation and staining of the samples. 34 3.5.3 Evaluation 35 3.6 Statistical analysis 36 4 Results 37 4.1 Inhibitors sensitivity 37 4.1.1 Effect of nitrate and pHMB on Jsm ac 37 4.1.2 Effect of DIDS, pHMB and NO3 - on Jsm ac 41 4.2 Effect of Cl- and NO3 - on Jms of acetate and butyrate 43 4.2.1 Effect of SO4 2- on Jms of acetate and butyrate 44 4.3 Effect of Cl-, NO3 - or SO4 2- when present in the serosal solution for 150 min 49 4.4 Immunohistochemistry 52 5 Discussion 54 5.1Ussing chamber experiments 56 5.1.1 Effect of Cl- and NO3 - on Jms of acetate 56 5.1.2 Effect of nitrate and pHMB on Jsm of acetate 57 5.1.3 Effect of DIDS, pHMB or NO3 - on Jsm of acetat 58 5.1.4 Effect of SO4 2- on Jms of acetate 59 5.1.5 Comparison between different anions as exchange substrate for the basolateral extrusion of acetate 60 5.2 Immunohistochemistry 62 5.3 Comparison between basolateral extrusion of butyrate and acetate 62 5.4 Conclusions 64 6 Summary 66 7 Zusammenfassung 68 8 References 70 Ac Aknowledgement

Axo-glial interdependance in peripheral nerve development

During the development of the peripheral nervous system, axons and myelinating Schwann cells form a unique symbiotic unit, which is realized by a finely tuned network of molecular signals and reciprocal interactions. The importance of this complex interplay becomes evident after injury or in diseases in which aspects of axo-glial interaction are perturbed. This Review focuses on the specific interdependence of axons and Schwann cells in peripheral nerve development that enables axonal outgrowth, Schwann cell lineage progression, radial sorting and, finally, formation and maintenance of the myelin sheath

Maximum Relative Entropy of Coherence for Quantum Channels

Based on the resource theory for quantifying the coherence of quantum channels, we introduce a new coherence quantifier for quantum channels via maximum relative entropy. We prove that the maximum relative entropy for coherence of quantum channels is directly related to the maximally coherent channels under a particular class of superoperations, which results in an operational interpretation of the maximum relative entropy for coherence of quantum channels. We also introduce the conception of sub-superchannels and sub-superchannel discrimination. For any quantum channels, we show that the advantage of quantum channels in sub-superchannel discrimination can be exactly characterized by the maximum relative entropy of coherence for quantum channels. Similar to the maximum relative entropy of coherence for channels, the robustness of coherence for quantum channels has also been investigated. We show that the maximum relative entropy of coherence for channels provides new operational interpretations of robustness of coherence for quantum channels and illustrates the equivalence of the dephasing-covariant superchannels, incoherent superchannels, and strictly incoherent superchannels in these two operational tasks