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On April 5, 2019, a World Trade Organization (WTO) panel (Panel) issued its decision in the dispute Russia – Measures Concerning Traffic in Transit. This decision is notable because it is the first instance in which a WTO panel has been called on to interpret the national security provision of the General Agreement on Tariffs and Trade (GATT 1994), Article XXI.

Cleavage of amyloid precursor protein (APP) by β-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-β peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1−/−mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1−/−mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1−/−mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1−/−mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENTGiven its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.

Dieses Kapitel befasst sich mit der neueren und aktuellen Literatur aus den Fachgebieten Psychosomatische Medizin, Psychiatrie und Palliativmedizin fur die klinischen Situationen und Zusammenhange, in denen sie fur die psychotherapeutische Betreuung und Begleitung von Patienten relevant sind. Allen drei Fachgebieten gemein ist die Tatsache, dass sie vor allem dann konsiliarisch oder interprofessionell-integrativ hinzugezogen werden, wenn es sich um kritische, lebensbedrohliche oder lebensverkurzende Situationen handelt. Als Grundlage fur die weitergehende Zusammenfassung der aktuellen Literatur der einzelnen Fachgebiete wird eine theoretische Einfuhrung vorangestellt, die sich mit dieser alle drei Gebiete vereinenden Tatsache in der Arzt-Patient-Beziehung befasst. Die Diversitatserfahrung, die als spezielles Phanomen in der Palliativmedizin auftritt wurde bereits in Kap. 3 eingefuhrt (Kap. 3). In diesem Kapitel findet das philosophische Konzept seine konkrete Anwendung in der Praxis psychotherapeutischer Interventionen. Das Grundverstandnis wesentlicher existentiell-psychologischer Zusammenhange kann hilfreich sein, um die Ergebnisse relevanter Fachgebietsstudien besser einordnen zu konnen und ihren klinischen Wert in Einbettung eines theoretischen Kontexts zu bewerten.

The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is deemed a major culprit in Alzheimer's disease, but accumulating evidence indicates that there is more to the enzyme than driving the amyloidogenic processing of the amyloid precursor protein. For example, BACE1 has emerged as an important regulator of neuronal activity through proteolytic and, most unexpectedly, also through nonproteolytic interactions with several ion channels. Here, we identify and characterize the voltage-gated K+channel 3.4 (Kv3.4) as a new and functionally relevant interaction partner of BACE1. Kv3.4 gives rise to A-type current with fast activating and inactivating kinetics and serves to repolarize the presynaptic action potential. We found that BACE1 and Kv3.4 are highly enriched and remarkably colocalized in hippocampal mossy fibers (MFs). In BACE1−/−mice of either sex, Kv3.4 surface expression was significantly reduced in the hippocampus and, in synaptic fractions thereof, Kv3.4 was specifically diminished, whereas protein levels of other presynaptic K+channels such as KCa1.1 and KCa2.3 remained unchanged. The apparent loss of presynaptic Kv3.4 affected the strength of excitatory transmission at the MF–CA3 synapse in hippocampal slices of BACE1−/−mice when probed with the Kv3 channel blocker BDS-I. The effect of BACE1 on Kv3.4 expression and function should be bidirectional, as predicted from a heterologous expression system, in which BACE1 cotransfection produced a concomitant upregulation of Kv3.4 surface level and current based on a physical interaction between the two proteins. Our data show that, by targeting Kv3.4 to presynaptic sites, BACE1 endows the terminal with a powerful means to regulate the strength of transmitter release.SIGNIFICANCE STATEMENTThe β-secretase β-site APP-cleaving enzyme 1 (BACE1) is infamous for its crucial role in the pathogenesis of Alzheimer's disease, but its physiological functions in the intact nervous system are only gradually being unveiled. Here, we extend previous work implicating BACE1 in the expression and function of voltage-gated Na+and K+channels. Specifically, we characterize voltage-gated K+channel 3.4 (Kv3.4), a presynaptic K+channel required for action potential repolarization, as a novel interaction partner of BACE1 at the mossy fiber (MF)–CA3 synapse of the hippocampus. BACE1 promotes surface expression of Kv3.4 at MF terminals, most likely by physically associating with the channel protein in a nonenzymatic fashion. We advance the BACE1–Kv3.4 interaction as a mechanism to strengthen the temporal control over transmitter release from MF terminals.

β-site APP-cleaving enzyme 1 (BACE1)is a major player in the pathogenesis of Alzheimer’sdisease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques either require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduceSiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photo- physical and pharmacologic properties, and evaluate its behavior in solution, in overexpression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion (STED), and dynamic single molecule microscopy. First functional studies withSiR-BACE1on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.

The increasing problem of antibiotic resistance has been related, among other factors, to the extensive use of antibiotics in pig farming. To be able to reduce antibiotic usage in this type of livestock breeding, it may be important to know which internal characteristics of farmers are related to their antibiotic treatment practices, next to the external characteristics, such as husbandry conditions. We conducted a survey among Swiss farmers of fattening pigs to investigate their attitudes and habits related to antibiotic usage, as well as their perception of antibiotic resistance and of policy measures intended to reduce antibiotic usage in pig farming. In addition, we related farmers׳ personal variables, attitudes and habits, and their farm characteristics to their actual antibiotic usage at three time points during the fattening period. The farmers in our study appeared to be little aware of the risks of antibiotic usage in pig husbandry. Results seemed to imply that different attitudes and habits were related to antibiotic usage at the three time points during fattening. At the beginning of the fattening period, the farmers׳ habits were mainly related to their antibiotic usage. Perceived risks and cautious behaviour regarding antibiotics were important predictors of farmers׳ perceived impact of policy measures to reduce antibiotic usage. We suggest that promoting habits that reduce antibiotic use among farmers and increasing their risk awareness related to antibiotics in pig husbandry will most likely reduce the use of these substances in pig farming.

β-site APP-cleaving enzyme 1 (BACE1) has become infamous for its pivotal role in the pathogenesis of Alzheimer's disease (AD). Consequently, BACE1 represents a prime target in drug development. Despite its detrimental involvement in AD, it should be quite obvious that BACE1 is not primarily present in the brain to drive mental decline. In fact, additional functions have been identified. In this review, we focus on the regulation of ion channels, specifically voltage-gated sodium and KCNQ potassium channels, by BACE1. These studies provide evidence for a highly unexpected feature in the functional repertoire of BACE1. Although capable of cleaving accessory channel subunits, BACE1 exerts many of its physiologically significant effects through direct, non-enzymatic interactions with main channel subunits. We discuss how the underlying mechanisms can be conceived and develop scenarios how the regulation of ion conductances by BACE1 might shape electric activity in the intact and diseased brain and heart.

A previously described method for the non-aqueous fractionation of cereal flours by sedimentation in non-aqueous solvents was carried out using flours of three wheat cultivars differing in baking performance, as well as one rye and one barley flour. The method was based on differences in the densities of starch (higher) and protein (lower). Thus, suspending finely milled flour in an inert solvent mixture with a density in between the densities of starch and protein yielded a sedimented starch fraction and a protein-rich fraction at the surface of the solvent. Further purification of this upper fraction provided a protein fraction, a middle fraction, and a lipid fraction. The protein fractions were examined by means of rheological methods such as micro-extension tests and creep-recovery tests. They also were reconstituted to standard flour with a protein content of 13.5%, which was used for micro-scale baking tests. Compared to aqueous isolated gluten, the hydrated protein fractions from wheat were much more extensible and had a lower resistance to extension. The baking performance of the wheat protein fractions was superior to gluten and comparable to the native wheat flours. The protein fraction from rye gave a wheat-like bread crumb, whereas the barley protein was not suited for bread making.

A method for the fractionation of wheat, rye, and barley flours without using aqueous solvents was developed. The separation of protein and starch was based on differences in their densities. Therefore, ball-milled flour was suspended in a mixture of inert solvents (toluene/tetrachoroethene) with a density of 1.47 g/cm 3 and centrifuged. Owing to its higher density, the starch fraction was obtained as sediment whereas the protein fraction (PF) formed a layer on the surface of the solvent because of its lower density. The PF was enriched in a solvent mixture with a density of 1.355 g/cm 3 yielding a middle fraction (sediment) and the enriched PF (upper layer). The latter was then defatted with toluene (0.87 g/cm) providing a lipid fraction in addition. The influence of ball milling under air or in the sedimentation solvent on the yield and the purity of the fractions was studied. Three varieties of wheat, and one rye and barley variety were fractionated by the optimised method and the obtained fractions were characterised by chemical methods e.g. gel permeation chromatography, SDS electrophoresis, and a combined extraction/HPLC method.

KCNQ1 (Kv7.1) proteins form a homotetrameric channel, which produces a voltage-dependent K+ current. Co-assembly of KCNQ1 with the auxiliary beta-subunit KCNE1 strongly up-regulates this current In cardiac myocytes, KCNQ1/E1 complexes are thought to give rise to the delayed rectifier current I-Ks, which contributes to cardiac action potential repolarization. We report here that the type I membrane protein BACE1 (beta-site APP-cleaving enzyme 1), which is best known for its detrimental role in Alzheimer's disease, but is also, as reported here, present in cardiac myocytes, serves as a novel interaction partner of KCNQ1. Using HEK293T cells as heterologous expression system to study the electrophysiological effects of BACE1 and KCNE1 on KCNQ1 in different combinations, our main findings were the following: (1) BACE1 slowed the inactivation of KCNQ1 current producing an increased initial response to depolarizing voltage steps. (2) Activation kinetics of KCNQ1/E1 currents were significantly slowed in the presence of co-expressed BACE1. (3) BACE1 impaired reconstituted cardiac I-Ks when cardiac action potentials were used as voltage commands, but interestingly augmented the I-Ks of ATP-deprived cells, suggesting that the effect of BACE1 depends on the metabolic state of the cell. (4) The electrophysiological effects of BACE1 on KCNQ1 reported here were independent of its enzymatic activity, as they were preserved when the proteolytically inactive variant BACE1 D289N was co-transfected in lieu of BACE1 or when BACE1-expressing cells were treated with the BACE1-inhibiting compound C3. (5) Co-immunoprecipitation and fluorescence recovery after photobleaching (FRAP) supported our hypothesis that BACE1 modifies the biophysical properties of I-Ks by physically interacting with KCNQ1 in a beta-subunit-like fashion. Strongly underscoring the functional significance of this interaction, we detected BACE1 in human iPSC-derived cardiomyocytes and murine cardiac tissue and observed decreased I-Ks in atrial cardiomyocytes of BACE1-deficient mice. (C) 2015 Elsevier Ltd. All rights reserved.

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment andcis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2–3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2–3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.

The β-secretase BACE1 is widely known for its pivotal role in the amyloidogenic pathway leading to Alzheimer's disease, but how its action on transmembrane proteins other than the amyloid precursor protein affects the nervous system is only beginning to be understood. We report here that BACE1 regulates neuronal excitability through an unorthodox, nonenzymatic interaction with members of the KCNQ (Kv7) family that give rise to the M-current, a noninactivating potassium current with slow kinetics. In hippocampal neurons from BACE1−/−mice, loss of M-current enhanced neuronal excitability. We relate the diminished M-current to the previously reported epileptic phenotype of BACE1-deficient mice. In HEK293T cells, BACE1 amplified reconstituted M-currents, altered their voltage dependence, accelerated activation, and slowed deactivation. Biochemical evidence strongly suggested that BACE1 physically associates with channel proteins in a β-subunit-like fashion. Our results establish BACE1 as a physiologically essential constituent of regular M-current function and elucidate a striking new feature of how BACE1 impacts on neuronal activity in the intact and diseased brain.