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Background: The rehabilitation process following cochlear implant (CI) surgery is carried out in a multimodal therapy according to German national guidelines and includes technical and medical aftercare. In times of the corona pandemic surgery and rehabilitation appointments were cancelled or delayed leading to a more difficult access to auditory rehabilitation. Newly implemented hygiene modalities due to the SARS-CoV‑2 pandemic have changed medical aftercare and the rehabilitation process. The aim of this study was to evaluate the quality of rehabilitation under corona conditions., Material and Methods: An anonymous survey of adult cochlear implant patients was carried out by a non-standardized questionnaire. Demographics were analyzed and the quality of medical aftercare, speech therapy, technical aftercare, psychological support and the hygiene modalities were compared to previous rehabilitation stays., Results: In total 109 patients completed the questionnaire. The quality of rehabilitation and individual therapy were rated as qualitatively similar or improved. The threat of the pandemic and fear of corona were rated unexpectedly high with 68% and 50%, respectively. The hygiene measures during the rehabilitation stay eased subjective fears at the same time. The majority of patients were annoyed by wearing face masks but visors, protection shields and social distancing were more tolerated., Conclusion: The implementation of the new hygiene modalities within the therapeutic rehabilitation setting was well-accepted by patients allowing access to auditory rehabilitation. A successful rehabilitation should ensure a fear-free environment by adhering to the necessary hygiene modalities.

IntroductionImproving the surveillance of tuberculosis (TB) is especially important for multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. The large amount of publicly available whole genome sequencing (WGS) data for TB gives us the chance to re-use data and to perform additional analyses at a large scale.AimWe assessed the usefulness of raw WGS data of global MDR/XDR Mycobacterium tuberculosis isolates available from public repositories to improve TB surveillance.MethodsWe extracted raw WGS data and the related metadata of M. tuberculosis isolates available from the Sequence Read Archive. We compared this public dataset with WGS data and metadata of 131 MDR- and XDR M. tuberculosis isolates from Germany in 2012 and 2013.ResultsWe aggregated a dataset that included 1,081 MDR and 250 XDR isolates among which we identified 133 molecular clusters. In 16 clusters, the isolates were from at least two different countries. For example, Cluster 2 included 56 MDR/XDR isolates from Moldova, Georgia and Germany. When comparing the WGS data from Germany with the public dataset, we found that 11 clusters contained at least one isolate from Germany and at least one isolate from another country. We could, therefore, connect TB cases despite missing epidemiological information.ConclusionWe demonstrated the added value of using WGS raw data from public repositories to contribute to TB surveillance. Comparing the German with the public dataset, we identified potential international transmission events. Thus, using this approach might support the interpretation of national surveillance results in an international context.

Background: The muscle relaxant methocarbamol is widely used for the treatment of muscle spasms and pain syndromes. To elucidate molecular mechanisms of its action, we studied its influence on neuromuscular transmission, on isometric muscle force, and on voltage-gated Na + channels., Methods: Neuromuscular transmission was investigated in murine diaphragm-phrenic nerve preparations and muscle force studied on mouse soleus muscles. Na v 1.4 channels and Na v 1.7 channels were functionally expressed in eukaryotic cell lines., Results: Methocarbamol, at 2 mM, decreased the decay of endplate currents, slowed the decay of endplate potentials and reduced tetanic force of soleus muscles. The drug reversibly inhibited current flow through muscular Na v 1.4 channels, while neuronal Na v 1.7 channels were unaffected., Conclusions: The study provides evidence for peripheral actions of methocarbamol on skeletal muscle. Muscular Na + channels are a molecular target of methocarbamol. Since Na v 1.7 currents were unaffected, methocarbamol is unlikely to exert its analgesic effect by directly blocking Na v 1.7 channels., (© 2020 The Authors. Muscle & Nerve published by Wiley Periodicals LLC.)

Background: The rehabilitation process following cochlear implant (CI) surgery is carried out in a multimodal therapy according to German national guidelines and includes technical and medical aftercare. In times of the corona pandemic surgery and rehabilitation appointments were cancelled or delayed leading to a more difficult access to auditory rehabilitation. Newly implemented hygiene modalities due to the SARS-CoV‑2 pandemic have changed medical aftercare and the rehabilitation process. The aim of this study was to evaluate the quality of rehabilitation under corona conditions., Material and Methods: An anonymous survey of adult cochlear implant patients was carried out by a non-standardized questionnaire. Demographics were analyzed and the quality of medical aftercare, speech therapy, technical aftercare, psychological support and the hygiene modalities were compared to previous rehabilitation stays., Results: In total 109 patients completed the questionnaire. The quality of rehabilitation and individual therapy were rated as qualitatively similar or improved. The threat of the pandemic and fear of corona were rated unexpectedly high with 68% and 50%, respectively. The hygiene measures during the rehabilitation stay eased subjective fears at the same time. The majority of patients were annoyed by wearing face masks but visors, protection shields and social distancing were more tolerated., Conclusion: The implementation of the new hygiene modalities within the therapeutic rehabilitation setting was well-accepted by patients allowing access to auditory rehabilitation. A successful rehabilitation should ensure a fear-free environment by adhering to the necessary hygiene modalities.

Accounting guidelines exist for the recording of carbon flows in terrestrial and coastal ecosystems. Shelf sea sediments, while considered an important carbon store, have yet to receive comparable scrutiny. Here, we explore whether effective management of carbon stocks accumulating in shelf seas could contribute towards a nation's greenhouse gas emissions reduction targets. We review the complexities of carbon transport and fate in shelf seas, and the geopolitical challenges of carbon accounting in climate governance because of the transboundary nature of carbon flows in the marine environment. New international accounting guidance and governance frameworks are needed to prompt climate action.

Objectives: Current pediatric guidelines allow noninvasive diagnosis of celiac disease in selected children. We investigated in a large cohort study whether the severity of villous atrophy at diagnosis is associated with clinical characteristics or long-term health outcomes, thus having a prognostic significance., Methods: Comprehensive medical data on 906 children with celiac disease were analyzed. Long-term health outcomes of 503 adult patients diagnosed in childhood were moreover assessed with a specific study questionnaire and validated Gastrointestinal Symptom Rating Scale (GSRS) and Psychological General Well-Being (PGWB) questionnaires. Patients were classified into 3 groups according to the severity of villous atrophy at diagnosis, and all variables were compared., Results: Altogether 34% of the patients had partial, 40% subtotal, and 26% total villous atrophy. Children with milder lesions were diagnosed more recently (median year 2007 vs 2006 vs 2001, respectively, P < 0.001), more often by screening (30% vs 25% vs 17%, P < 0.001) and they suffered less often from anemia (16% vs 21% vs 32%, P < 0.001) and growth disturbances (22% vs 36% vs 54%, P < 0.001) and had lower transglutaminase-2 antibody levels (median 64 U/L vs 120 U/L vs 120 U/L, P < 0.001). There was no difference in other disease features.Altogether 212 adults diagnosed in childhood completed the questionnaires. Severity of villous atrophy at childhood diagnosis did not predict presence of complications or comorbidities, persistent symptoms, and self-perceived health, quality of life or adherence to a gluten-free diet in adulthood., Conclusion: Presence of advanced villous atrophy at diagnosis is associated with more severe clinical characteristics but not with poorer long-term health and treatment outcomes.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1 + stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.

Background: International contact-tracing (CT) following exposure during long-distance air travel is resource-intensive, whereas evidence for risk of tuberculosis (TB) transmission during international travel is weak. In this study, we systematically analyzed the information from international requests for CT received at the national level in Germany in order to evaluate the continued utility of the current approach and to identify areas for improvement., Methods: An anonymized archive of international CT notifications received by the Robert Koch Institute between 2010 and 2018 was searched for key parameters for data collection. A total of 31 parameters, such as characteristics of TB patients and their identified contacts, were extracted from each CT notification and collated into a dataset. Descriptive data analysis and trend analyses were performed to identify key characteristics of CT notifications, patients, and contacts over the years., Results: 192 CT notifications, each corresponding to a single TB index case, were included in the study, increasing from 12 in 2010 to 41 in 2018. The majority of notifications (N = 130, 67.7%) concerned international air travel, followed by private contact (N = 39, 20.3%) and work exposure (N = 16, 8.3%). 159 (82.8%) patients had sputum smear results available, of which 147 (92.5%) were positive. Of 119 (62.0%) patients with drug susceptibility testing results, most (N = 92, 77.3%) had pan-sensitive TB, followed by 15 (12.6%) with multi-drug resistant TB. 115 (59.9%) patients had information on infectiousness, of whom 99 (86.1%) were considered infectious during the exposure period. 7 (5.3%) patients travelled on long-distance flights despite a prior diagnosis of active TB. Of the 771 contact persons, 34 (4.4%) could not be reached for CT measures due to lack of contact information., Conclusion: The high variability in completeness of information contained within the international CT requests emphasizes the need for international standards for reporting of CT information. With the large proportion of TB patients reported to have travelled while being infectious in our study, we feel that raising awareness among patients and health professionals to detect TB early and prevent international long-distance travel during the infectious disease phase should be a cornerstone strategy to safeguard against possible transmission during international travel.

Key Points: Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin- and dysferlin-deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin-deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy., Abstract: Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin- and dysferlin-deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin-deficient mice. Single-unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp-and-hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy., (© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet's intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets ( p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring.

Next-generation sequencing based base-by-base distance measures have become an integral complement to epidemiological investigation of infectious disease outbreaks. This study introduces PANPASCO, a computational pan-genome mapping based, pairwise distance method that is highly sensitive to differences between cases, even when located in regions of lineage specific reference genomes. We show that our approach is superior to previously published methods in several datasets and across different Mycobacterium tuberculosis lineages, as its characteristics allow the comparison of a high number of diverse samples in one analysis-a scenario that becomes more and more likely with the increased usage of whole-genome sequencing in transmission surveillance., Competing Interests: The authors have declared that no competing interests exist.

Agrin is a multi-domain protein best known for its essential function during formation of the neuromuscular junction. Alternative mRNA splicing at sites named y and z in the C-terminal part of agrin regulates its interaction with a receptor complex consisting of the agrin-binding low-density lipoprotein receptor-related protein 4 (Lrp4) and the muscle-specific kinase (MuSK). Isoforms with inserts at both splice sites bind to Lrp4, activate MuSK and are synaptogenic at the neuromuscular junction. Agrin is also expressed as a transmembrane protein in the central nervous system (CNS) but its function during interneuronal synapse formation is unclear. Recently we demonstrated that transfection of a full-length cDNA coding for transmembrane agrin (TM-agrin) in cultured embryonic cortical neurons induced an Lrp4-dependent but MuSK-independent increase in dendritic glutamatergic synapses and an Lrp4- and MuSK-independent reduction of inhibitory synapses. Here we show that presynaptic specializations were similarly affected by TM-agrin overexpression. In addition, we mapped the regions within TM-agrin responsible for TM-agrin's effects on dendritic aggregates of synapse-associated proteins. We show that the presence of a four amino acid insert at splice site y is essential for the increase in the density of puncta containing the postsynaptic density protein 95 kDa. This effect was independent of splice site z. The reduction of the gephyrin puncta density was independent of the entire extracellular part of TM-agrin but required a highly conserved serine residue in the intracellular domain of TM-agrin. These results provide further evidence for a function of TM-agrin during CNS synaptogenesis and demonstrate that different domains and alternative splicing of TM-agrin differentially affect excitatory and inhibitory synapse formation in cultured embryonic CNS neurons., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Around 10 million people worldwide contract tuberculosis every year. According to the World Health Organization (WHO), approximately one-quarter of the world's population is latently infected with Mycobacterium tuberculosis. In Ger- many, the incidence of tuberculosis was in decline over several decades but rose in 2015 to 7.3 new cases per 100 000 persons. In 2018, a total of 5429 new cases were documented, corresponding to 6.5 new cases per 100 000 persons., Methods: This article is based on literature retrieved by a selective search in PubMed and on the authors' clinical experience., Results: Tuberculosis involves the lungs in almost 75% of patients but can generally involve any organ. In Germany, the majority of patients come from high-incidence countries. If a patient's differential diagnosis includes tuberculosis, the main tests for the detection of the pathogen in sputum and tissue samples are culture (the gold standard), microscopy, and nucleic acid amplification tests. Imaging studies are also used for diagnosis and follow-up. The standard treatment consists of a combination of isoniazid, rifampicin, ethambutol, and pyrazinamide, followed by a combination of isoniazid and rifampicin only. Liver damage is one of the more common adverse effects of this treatment, arising in 2.4% of patients. Multidrug-resistant tuberculosis, which is rare in Germany (around 100 cases per year), should be treated in special- ized centers., Conclusion: Rapid diagnosis and targeted treatment are essential to prevent an unfavorable course of the disease as well as its transmission to other individuals. In patients presenting with unclear symptoms, tuberculosis should always be considered as a differential diagnosis. The diagnosis of latent tuberculosis and decision-making regarding its treatment are difficult because of the lack of specific biomarkers and of relevant data from clinical trials.

Key Points: Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d-tubocurarine and α-bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium-3 also enhanced stretch-evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine-tune proprioceptive afferent sensitivity., Abstract: Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ-motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp-and-hold stretches and sinusoidal vibrations before and after the addition of the competitive and non-competitive AChR blockers d-tubocurarine and α-bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp-and-hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high-affinity choline transporter antagonist hemicholinium-3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine-tuning muscle spindle sensitivity., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

The role of agrin, Lrp4 and MuSK, key organizers of neuromuscular synaptogenesis, in the developing CNS is only poorly understood. We investigated the role of these proteins in cultured mouse embryonic cortical neurons from wildtype and from Lrp4- and MuSK-deficient mice. Neurons from Lrp4-deficient mice had fewer but longer primary dendrites and a decreased density of puncta containing excitatory and inhibitory synapse-associated proteins. Neurons from MuSK-deficient mice had an altered dendritic branching pattern but no change in the density of puncta stained by antibodies against synapse-associated proteins. Transfection of TM-agrin compensated the dendritic branching deficits in Lrp4-deficient but not in MuSK-deficient neurons. TM-agrin transfection increased the density of excitatory synaptic puncta in MuSK-deficient but not in Lrp4-deficient mice and reduced the number of inhibitory synaptic puncta irrespective of MuSK and Lrp4 expression. Addition of purified soluble agrin to microisland cultures of cortical neurons revealed an Lrp4-dependent increase in the size and density of glutamatergic synaptic puncta and in mEPSC but not in mIPSC frequency and amplitude. Thus, agrin induced an Lrp4-independent increase in dendritic branch complexity, an Lrp4-dependent increase of excitatory synaptic puncta and an Lrp4- and MuSK-independent decrease in the density of puncta containing inhibitory synapse-associated proteins. These results establish selective roles for agrin, Lrp4 and MuSK during dendritogenesis and synaptogenesis in cultured CNS neurons., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A quantitative dried blood spot (DBS) method based on direct sampling by means of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is presented. Gadolinium-based contrast agents were used as model metallodrugs with a significant relevance for pharmaceutical applications. Challenges regarding the ablation of the complex blood-filter matrix were characterized and successfully addressed by a thorough adaption of the laser ablation conditions. Especially the laser fluence was optimized with respect to the particle size distribution of the generated aerosol as monitored by an optical particle counter. Thus, generation of micrometer-sized particles could be minimized in favor of smaller particles increasing the transport efficiency of the DBS ablation aerosol to the plasma and the recorded signal stability. Inhomogeneous blood drying on the porous filter paper could be compensated by the addition of an internal standard prior to blood spotting. To preserve the advantages of DBS sampling, such as small blood volumes and minimal invasiveness, the combined use of DBS and a capillary blood sampling system is demonstrated. By placing the internal standard into the capillary prior to blood sampling, a simple workflow usable for clinical application was implemented. The applicability of the developed method, achieving limits of detection and quantification in the low μg L -1 range and covering a linear range of over four orders of magnitude, was demonstrated for blood samples containing different concentrations of the gadolinium contrast agents gadopentetate and gadoterate., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Background: Key determinants for lesion formation in catheter ablation are contact force, radiofrequency (RF) power and time. The aim of this study was to evaluate the clinical applicability of ablation index (AI), a novel non-linear formula based on these components, and to compare AI with the conventional linear force-time interval (FTI) in pulmonary vein isolation (PVI). Methods and Results: Target AI ranges were defined for anatomical segments of the ipsilateral pulmonary veins. The operator was blinded to AI during PVI for the initial 11 patients (group A), and was unblinded for the remaining 23 patients (group B). We assessed (1) the clinical value of AI to avoid excessively high and low values with an operator blinded vs. non-blinded to AI; and (2) the relation of AI and FTI in predefined ranges. In group A, 235/564 lesions (41.7%) were in the predefined target range as compared with 1,171/1,412 lesions (82.9%) in group B (P<0.001). A given AI may correspond to a wide range of FTI, as reflected by a quartile coefficient of dispersion for AI of 0.11 vs. a quartile coefficient of dispersion for FTI of 0.36., Conclusions: Incorporating RF current power, the non-linear AI provides more comprehensive information during PVI compared with FTI. Given that the FTI for a given AI varies widely, the value of FTI in clinical practice is questionable.

Fluorescence-guided surgery (FGS) has been established as a powerful technique for glioblastoma resection. After oral application of the prodrug 5-aminolevulinic acid (5-ALA), protoporphyrin IX (PpIX) is formed as an intermediate of the heme-biosynthesis cascade and accumulates within the tumor. By intraoperative fluorescence microscopy, the specific PpIX fluorescence can be used to differentiate the tumor from healthy brain tissue. To investigate possible limitations of fluorescence diagnosis, the complementary use of molecular and elemental mass-spectrometry imaging (MSI) is presented. Matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) is used to examine the distribution of PpIX and heme b in human brain tumors. MALDI-MS/MS imaging is performed to validate MS data and improve the signal-to-noise ratio (S/N). Comparing the imaging results with histological evaluation, increased PpIX accumulation in areas of high tumor-cell density is observed. Heme b accumulation are only found in areas of blood vessels and hemorrhage, confirming the hampered transformation from PpIX to heme b in glioblastoma tissue. Investigation of non-neoplastic brain tissue and glioblastoma resected without external 5-ALA administration as control samples with true-negative fluorescence verified the absence of PpIX accumulation. Analysis of necrotic tumor tissue and gliosarcoma, one rare type of glioma appearing nonfluorescent during FGS, as case examples with false-negative-fluorescence diagnosis, revealed the absence of significant amounts of PpIX, indicating an impairment of PpIX formation. Molecular analysis is complemented by quantitative laser ablation-inductively coupled plasma (LA-ICP) MSI correlating heme b and Fe distribution. Mathematical pixel-by-pixel correlation of molecular and elemental data revealed a positive correlation with heteroscedasticity for the spatially resolved heme b signal intensities and Fe concentrations.

Purpose of Review: Muscle spindles are encapsulated mechanosensory receptors within skeletal muscle tissue that inform the central nervous system about the contractile status of each muscle. This information is required for any coordinated movement and for stable posture. This review summarizes recent findings regarding novel functions for proprioceptive feedback information, muscle spindle disease and the molecular basis of mechanoreception., Recent Findings: Muscle spindle function is not limited to regulating motor control but is also required for appropriate realignment of fractured bones, successful regeneration of spinal cord axons after injury and spinal alignment. Several proteins responsible for or modulating mechanotransduction in proprioceptive sensory neurons have been identified, including the Piezo2 channel as a candidate for the principal mechanotransduction channel. Many neuromuscular diseases are known to be accompanied by an impaired function of muscle spindles, resulting in a decline of motor performance and coordination in the patients., Summary: Our knowledge regarding the molecular basis of muscle spindle function is still incomplete. However, increasing our understanding of mechanotransduction in muscle spindles is a prerequisite for finding appropriate strategies to prevent injuries due to unstable gait and frequent falls.

Background: The advantages of 5-aminolevulinacid (5-ALA)-induced fluorescence-guided surgery in meningiomas are increasingly discussed. In this context, despite detectable tumor tissue in histopathologial analyses, no fluorescence was shown at the dura tail using the standard operating microscope. Thus, 5-ALA metabolism in this surgically important site remains unknown but needs to be elucidated when further evaluating indications of fluorescence-guided surgery in meningiomas., Method: We here present the spatially resolved identification of protoporphyrin IX (PpIX) in sphenoid ridge meningioma cryosections from a patient who underwent fluorescence-guided microsurgery using molecular imaging analysis by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS)., Results: Despite a strong fluorescence of the main tumor, no fluorescence could be detected at the dura tail using the standard operating microscope (blue-light, 405 nm). However, histopathological analyses clearly showed meningioma tissue. Remarkably, MALDI-MS/MS analysis revealed PpIX formation also at the non-fluorescing dura tail. However, no PpIX was detected in the tumor free dura mater., Conclusion: MALDI-MS/MS visualized a selective accumulation of PpIX within the tumor tissue including the dura tail. Thus, absence of fluorescence in the dura tail as visualized by the operating microscope is not caused by the lack of PpIX formation.

Background: The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan., Methods: On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis. In this molecular epidemiological study, we prospectively defined outbreak cases with 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) profiles; phenotypic resistance to isoniazid, rifampicin, ethambutol, pyrazinamide, and capreomycin; and corresponding drug resistance mutations. We whole-genome sequenced all Mycobacterium tuberculosis isolates and clustered them using a threshold of five single nucleotide polymorphisms (SNPs). We collated epidemiological data from host countries from the European Centre for Disease Prevention and Control., Findings: Between Feb 12, 2016, and April 19, 2017, 29 patients were diagnosed with multidrug-resistant tuberculosis in seven European countries. All originated from the Horn of Africa or Sudan, with all isolates two SNPs or fewer apart. 22 (76%) patients reported their travel routes, with clear spatiotemporal overlap between routes. We identified a further 29 MIRU-VNTR-linked cases from the Horn of Africa that predated the outbreak, but all were more than five SNPs from the outbreak. However all 58 isolates shared a capreomycin resistance-associated tlyA mutation., Interpretation: Our data suggest that source cases are linked to an M tuberculosis clone circulating in northern Somalia or Djibouti and that transmission probably occurred en route before arrival in Europe. We hypothesise that the shared mutation of tlyA is a drug resistance mutation and phylogenetic marker, the first of its kind in M tuberculosis sensu stricto., Funding: The Swiss Federal Office of Public Health, the University of Zurich, the Wellcome Trust, National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), the Medical Research Council, BELTA-TBnet, the European Union, the German Center for Infection Research, and Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG)., (Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Two feeding trials were conducted to investigate the effects of hydrolyzed (HY) or non-hydrolyzed (NHY) yeast (Kluyveromyces fragilis) in isoenergetic and isonitrogeneous diets in the postweaning period. In experiment 1, a total of 550 unsexed pigs (6.5 ± 0.5 kg BW), weaned at 24 ± 2 d of age, were allocated to five treatment groups, receiving either a control diet (CON) or diets with 1%, 3%, and 5% HY (groups HY1, HY3, and HY5, respectively), or a diet with 3% NHY (group NHY3). In experiment 2, a total of 48 male and female pigs (6.2 ± 0.3 kg BW, weaned at d 25) were allocated to three dietary groups (n = 8 replicates with two pigs) receiving a control diet (CON) or diets with 1% NHY or 1% HY. Eight animals were sacrificed 2 wk after weaning for histological investigations in the jejunum and colon, determination of apparent ileal digestibility (AID) of CP and ether extract (EE), and electrophysiological measurements in the jejunal tissue after addition of carbachol or l-glutamine using Ussing chambers. In experiment 1, different treatments had no significant effect on pig performance, but diet HY1 tended to increase ADG and G:F in wk 2 after weaning (P < 0.1). In experiment 2, diet HY1 increased feed intake in wk 2 (P < 0.05), whereas NHY yeast had no effect on feed intake. Villus height, villus/crypt ratio in jejunum (P < 0.05), and crypt depth in colon (P < 0.01) were increased in group HY1. Crypt depth in jejunum and small intestinal length were not affected by different treatments. The AID of CP and EE tended to increase in group HY1 (P < 0.1) compared with groups CON and NHY. In the Ussing chamber experiments, no changes in basal electrophysiological parameters were observed, and the reactions of the treatment groups to carbachol and l-glutamine were comparable. ADFI was positively correlated with different parameters of intestinal morphology (villus height, villus/crypt ratio, crypt depth in colon, length of small intestine), AID of CP, EE, and performance. The results suggest that a supplementation of 1% HY based on K. fragilis to pig diets may positively influence ADFI and intestinal morphology in pig in the early postweaning period (d 1 to 14).

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Objectives: Cochlear implant is regarded as a treatment option for hearing rehabilitation of adults with unilateral sensorineural hearing loss. A clear benefit has been experienced in regard to speech comprehension in noise, localization, and quality of life. The aim of this study was to investigate the benefit of cochlear implantation for children with congenital unilateral hearing loss., Study Design: Retrospective case series., Setting: Tertiary referral center; cochlear implant program., Patients: Ten children with congenital unilateral hearing loss., Intervention: After extensive consultation with the families and intensive counseling, the children received a cochlear implant., Main Outcome Measures: Categories of auditory performance, speech discrimination in open set, subjective assessment by Speech, Spatial and Qualities scale questionnaire., Conclusion: Eight of ten children use their cochlear implant consistently on a daily basis. Two children who were equipped with an implanted device at a later age tend to nonuse of the device. The evaluation of binaural hearing in small children is still difficult and methods have to be developed to allow objective assessment.

Lignocellulose is an alternative fiber source for dogs; however, it has not yet been studied as a feed ingredient for the nutrition of dogs. Eight adult Beagles were involved in the study, which consisted of 3 feeding periods of 8 to 12 wk each. All dogs received 3 different diets, which either had the same concentration of fiber sources (2.7% sugar beet pulp or lignocellulose) or were formulated for a similar concentration of approximately 3% crude fiber: 12% sugar beet pulp (highSBP; 3.1% crude fiber), 2.7% sugar beet pulp (lowSBP; 0.96% crude fiber), or 2.7% lignocellulose (LC; 2.4% crude fiber). Feces samples were collected at the end of each feeding period, and the apparent nutrient digestibility, daily amount, and DM content of feces and fecal cell numbers of relevant bacteria were analyzed. The daily feces amount was lower and the feces DM was higher when dogs were fed the LC diet and the lowSBP diet compared with the highSBP diet ( < 0.001). Apparent digestibility of CP, Na, and K was highest with the lowSBP diet followed by the LC and highSBP diets ( < 0.001). After feeding LC, the bacterial cell counts of spp., spp., and the cluster were reduced compared with feeding highSBP and even more reduced after feeding lowSBP ( < 0.001). The bacterial cell count of the cluster was lower in LC and lowSBP compared with highSBP ( < 0.001). The feces of dogs fed LC and lowSBP had lower concentrations of acetate ( < 0.001), propionate ( < 0.001), -butyrate ( = 0.015), total fatty acids ( < 0.001), and lactate ( < 0.001) compared with dogs fed highSBP. The concentration of -butyrate was higher in the feces of dogs fed with LC compared with dogs fed high and low sugar beet pulp (SBP; < 0.001). The pH of the feces of the LC-fed dogs was highest followed by lowSBP- and highSBP-fed dogs ( < 0.001). Depending on the concentration, the use of LC and SBP as fiber sources in dog feed has different impacts on the fecal microbiota and the apparent digestibility of nutrients. Therefore, different areas of application should be considered.

This study used microelectrodes to record pH profiles in fresh shelf sea sediment cores collected across a range of different sediment types within the Celtic Sea. Spatial and temporal variability was captured during repeated measurements in 2014 and 2015. Concurrently recorded oxygen microelectrode profiles and other sedimentary parameters provide a detailed context for interpretation of the pH data. Clear differences in profiles were observed between sediment type, location and season. Notably, very steep pH gradients exist within the surface sediments (10-20 mm), where decreases greater than 0.5 pH units were observed. Steep gradients were particularly apparent in fine cohesive sediments, less so in permeable sandier matrices. We hypothesise that the gradients are likely caused by aerobic organic matter respiration close to the sediment-water interface or oxidation of reduced species at the base of the oxic zone (NH 4 + , Mn 2+ , S 2+ , S - ). Statistical analysis suggests the variability in the depth of the pH minima is controlled spatially by the oxygen penetration depth, and seasonally by the input and remineralisation of deposited organic phytodetritus. Below the pH minima the observed pH remained consistently low to maximum electrode penetration (ca. 60 mm), indicating an absence of sub-oxic processes generating H + or balanced removal processes within this layer. Thus, a climatology of sediment surface porewater pH is provided against which to examine biogeochemical processes. This enhances our understanding of benthic pH processes, particularly in the context of human impacts, seabed integrity, and future climate changes, providing vital information for modelling benthic response under future climate scenarios., (© The Author(s) 2017.)

Continental shelf sediments are globally important for biogeochemical activity. Quantification of shelf-scale stocks and fluxes of carbon and nutrients requires the extrapolation of observations made at limited points in space and time. The procedure for selecting exemplar sites to form the basis of this up-scaling is discussed in relation to a UK-funded research programme investigating biogeochemistry in shelf seas. A three-step selection process is proposed in which (1) a target area representative of UK shelf sediment heterogeneity is selected, (2) the target area is assessed for spatial heterogeneity in sediment and habitat type, bed and water column structure and hydrodynamic forcing, and (3) study sites are selected within this target area encompassing the range of spatial heterogeneity required to address key scientific questions regarding shelf scale biogeochemistry, and minimise confounding variables. This led to the selection of four sites within the Celtic Sea that are significantly different in terms of their sediment, bed structure, and macrofaunal, meiofaunal and microbial community structures and diversity, but have minimal variations in water depth, tidal and wave magnitudes and directions, temperature and salinity. They form the basis of a research cruise programme of observation, sampling and experimentation encompassing the spring bloom cycle. Typical variation in key biogeochemical, sediment, biological and hydrodynamic parameters over a pre to post bloom period are presented, with a discussion of anthropogenic influences in the region. This methodology ensures the best likelihood of site-specific work being useful for up-scaling activities, increasing our understanding of benthic biogeochemistry at the UK-shelf scale., (© The Author(s) 2017.)

Results from a 1D setup of the European Regional Seas Ecosystem Model (ERSEM) biogeochemical model were compared with new observations collected under the UK Shelf Seas Biogeochemistry (SSB) programme to assess model performance and clarify elements of shelf-sea benthic biogeochemistry and carbon cycling. Observations from two contrasting sites (muddy and sandy) in the Celtic Sea in otherwise comparable hydrographic conditions were considered, with the focus on the benthic system. A standard model parameterisation with site-specific light and nutrient adjustments was used, along with modifications to the within-seabed diffusivity to accommodate the modelling of permeable (sandy) sediments. Differences between modelled and observed quantities of organic carbon in the bed were interpreted to suggest that a large part (>90%) of the observed benthic organic carbon is biologically relatively inactive. Evidence on the rate at which this inactive fraction is produced will constitute important information to quantify offshore carbon sequestration. Total oxygen uptake and oxic layer depths were within the range of the measured values. Modelled depth average pore water concentrations of ammonium, phosphate and silicate were typically 5-20% of observed values at the muddy site due to an underestimate of concentrations associated with the deeper sediment layers. Model agreement for these nutrients was better at the sandy site, which had lower pore water concentrations, especially deeper in the sediment. Comparison of pore water nitrate with observations had added uncertainty, as the results from process studies at the sites indicated the dominance of the anammox pathway for nitrogen removal; a pathway that is not included in the model. Macrofaunal biomasses were overestimated, although a model run with increased macrofaunal background mortality rates decreased macrofaunal biomass and improved agreement with observations. The decrease in macrofaunal biomass was compensated by an increase in meiofaunal biomass such that total oxygen demand remained within the observed range. The permeable sediment modification reproduced some of the observed behaviour of oxygen penetration depth at the sandy site. It is suggested that future development in ERSEM benthic modelling should focus on: (1) mixing and degradation rates of benthic organic matter, (2) validation of benthic faunal biomass against large scale spatial datasets, (3) incorporation of anammox in the benthic nitrogen cycle, and (4) further developments to represent permeable sediment processes., (© Springer International Publishing AG 2017.)

Shelf sediments play a vital role in global biogeochemical cycling and are particularly important areas of oxygen consumption and carbon mineralisation. Total benthic oxygen uptake, the sum of diffusive and faunal mediated uptake, is a robust proxy to quantify carbon mineralisation. However, oxygen uptake rates are dynamic, due to the diagenetic processes within the sediment, and can be spatially and temporally variable. Four benthic sites in the Celtic Sea, encompassing gradients of cohesive to permeable sediments, were sampled over four cruises to capture seasonal and spatial changes in oxygen dynamics. Total oxygen uptake (TOU) rates were measured through a suite of incubation experiments and oxygen microelectrode profiles were taken across all four benthic sites to provide the oxygen penetration depth and diffusive oxygen uptake (DOU) rates. The difference between TOU and DOU allowed for quantification of the fauna mediated oxygen uptake and diffusive uptake. High resolution measurements showed clear seasonal and spatial trends, with higher oxygen uptake rates measured in cohesive sediments compared to the permeable sediment. The significant differences in oxygen dynamics between the sediment types were consistent between seasons, with increasing oxygen consumption during and after the phytoplankton bloom. Carbon mineralisation in shelf sediments is strongly influenced by sediment type and seasonality., (© The Author(s) 2017.)

Shelf seas and their associated benthic habitats represent key systems in the global carbon cycle. However, the quantification of the related stocks and flows of carbon are often poorly constrained. To address benthic carbon storage in the North-West European continental shelf, we have spatially predicted the mass of particulate organic carbon (POC) stored in the top 10 cm of shelf sediments in parts of the North Sea, English Channel and Celtic Sea using a Random Forest model, POC measurements on surface sediments from those seas and relevant predictor variables. The presented model explains 78% of the variance in the data and we estimate that approximately 250 Mt of POC are stored in surficial sediments of the study area (633,000 km 2 ). Upscaling to the North-West European continental shelf area (1,111,812 km 2 ) yielded a range of 230-882 Mt of POC with the most likely estimate being on the order of 476 Mt. We demonstrate that the largest POC stocks are associated with coarse-grained sediments due to their wide-spread occurrence and high dry bulk densities. Our results also highlight the importance of coastal sediments for carbon storage and sequestration. Important predictors for POC include mud content in surficial sediments, annual average bottom temperature and distance to shoreline, with the latter possibly a proxy for terrestrial inputs. Now that key variables in determining the spatial distribution of POC have been identified, it is possible to predict future changes to the POC stock, with the presented maps providing an accurate baseline against which to assess predicted changes., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2017.)

Assessment of the effects of sediment metal contamination on biological assemblages and function remains a key question in marine management, especially in relation to disposal activities. However, the appropriate description of bioavailable metal concentrations within pore-waters has rarely been reported. Here, metal behaviour and availability at contaminated dredged material disposal sites within UK waters were investigated using Diffusive Gradient in Thin films (DGT). Three stations, representing contrasting history and presence of dredge disposal were studied. Depth profiles of five metals were derived using DGT probes as well as discrete analysis of total metal concentrations from sliced cores. The metals analysed were: iron and manganese, both relevant to sediment biogeochemistry; cadmium, nickel and lead, classified as priority pollutants. DGT time-integrated labile flux profiles of the metals display behaviour consistent with increasingly reduced conditions at depth and availability to DGT (iron and manganese), subsurface peaks and a potential sedimentary source to the water column related to the disposal activity (lead and nickel) and release to pore-water linked to decomposition of enriched phytodetritus (cadmium). DGT data has the potential to improve our current understanding of metal behaviour at impacted sites and is suitable as a monitoring tool. DGT data can provide information on metal availability and fluxes within the sediment at high depth-resolution (5mm steps). Differences observed in the resulting profiles between DGT and conventional total metal analysis illustrates the significance of considering both total metals and a potentially labile fraction. The study outcomes can help to inform and improve future disposal site impact assessment, and could be complemented with techniques such as Sediment Profile Imagery for improved biologically relevance, spatial coverage and cost-effective monitoring and sampling of dredge material disposal sites. Additionally, the application of this technology could help improve correlative work on biological impacts under national and international auspices when linking biological effects to more biologically relevant metal concentrations., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

For decades the neuromuscular junction (NMJ) has been a favorite preparation to investigate basic mechanisms of synaptic function and development. As its function is to transmit action potentials in a 1:1 ratio from motor neurons to muscle fibers, the NMJ shows little or no functional plasticity, a property that makes it poorly suited to investigate mechanisms of use-dependent adaptations of synaptic function, which are thought to underlie learning and memory formation in the brain. On the other hand, the NMJ is unique in that the differentiation of the subsynaptic membrane is regulated by one major factor secreted from motor neurons, agrin. As a consequence, myotubes grown on a laminin substrate that is focally impregnated with recombinant neural agrin closely resemble the situation in vivo, where agrin secreted from motor neurons binds to the basal lamina of the NMJ's synaptic cleft to induce and maintain the subsynaptic muscle membrane. We provide here a detailed protocol through which acetylcholine receptor clusters are induced in cultured myotubes contacting laminin-attached agrin, enabling molecular, biochemical and cell biological analyses including high resolution microscopy in 4D. This preparation is ideally suited to investigate the mechanisms involved in the assembly of the postsynaptic muscle membrane, providing distinct advantages over inducing AChR clusters using soluble agrin.

NG2-glia in the adult brain are known to proliferate and differentiate into mature and myelinating oligodendrocytes throughout lifetime. However, the role of these newly generated oligodendrocytes in the adult brain still remains little understood. Here we took advantage of the Sox10-iCreER T2 x CAG-eGFP x Esco2 fl/fl mouse line in which we can specifically ablate proliferating NG2-glia in adult animals. Surprisingly, we observed that the generation of new oligodendrocytes in the adult brain was severely affected, although the number of NG2-glia remained stable due to the enhanced proliferation of non-recombined cells. This lack of oligodendrogenesis led to the elongation of the nodes of Ranvier as well as the associated paranodes, which could be locally rescued by myelinating oligodendrocytes differentiated from transplanted NG2-glia deriving from wildtype mice. Repetitive measurements of conduction velocity in the corpus callosum of awake animals revealed a progressive deceleration specifically in the mice lacking adult oligodendrogenesis that resulted in progressive motor deficits. In summary, here we demonstrated for the first time that axon function is not only controlled by the reliable organization of myelin, but also requires a dynamic and continuous generation of new oligodendrocytes in the adult brain. GLIA 2016;64:2201-2218., (© 2016 Wiley Periodicals, Inc.)

Limited drug penetration into tumor tissue is a significant factor to the effectiveness of cancer therapy. Tumor spheroids, a 3D cell culture model system, can be used to study drug penetration for pharmaceutical development. In this study, a method for quantitative bioimaging of platinum group elements by laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS) is presented. Different matrix-matched standards were used to develop a quantitative LA-ICP-MS method with high spatial resolution. To investigate drug penetration, tumor spheroids were incubated with platinum complexes (Pt(II)acetylacetonate, cisplatin) and the palladium tagged photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). Distribution and accumulation of the pharmaceuticals were determined with the developed method., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The uptake of mercury species in the model organism Drosophila melanogaster was investigated by elemental bioimaging using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS). The mercury distribution in Drosophila melanogaster was analyzed for the three species mercury(II) chloride, methylmercury chloride, and thimerosal after intoxication. A respective analytical method was developed and applied to the analysis of the entire Drosophila melanogaster first, before a particular focus was directed to the cerebral areas of larvae and adult flies. For quantification of mercury, matrix-matched standards based on gelatin were prepared. Challenges of spatially dissolved mercury determination, namely, strong evaporation issues of the analytes and an inhomogeneous distribution of mercury in the standards due to interactions with cysteine containing proteins of the gelatin were successfully addressed by complexation with meso-2,3-dimercaptosuccinic acid (DMSA). No mercury was detected in the cerebral region for mercury(II) chloride, whereas both organic species showed the ability to cross the blood-brain barrier. Quantitatively, the mercury level in the brain exceeded the fed concentration indicating mercury enrichment, which was approximately 3 times higher for methylmercury chloride than for thimerosal.

The reversible molecular interconversion behaviour of a synthesised oxime (2-phenylpropanaldehyde oxime; (C6H5)CH(CH3)CHN(OH)) was investigated by both, single dimensional gas chromatography (1D GC) and comprehensive two-dimensional gas chromatography (GC×GC). Previous studies on small molecular weight oximes were extended to this larger aromatic oxime (molar mass 149.19gmol(-1)) with interest in the extent of interconversion, enantioselective resolution, and retention time. On a polyethylene glycol (PEG; wax-type) column, a characteristic interconversion zone between two antipodes of E and Z isomers was formed by molecules which have undergone isomerisation on the column (E⇌Z). The extent of interconversion was investigated by varying chromatographic conditions (oven temperature and carrier flow rate) to understand the nature of the behaviour observed. The extent of interconversion was negligible in both enantioselective and methyl-phenylpolysiloxane phase-columns, correlating with the low polarity of the stationary phase. In order to obtain isomerisation along with enantio-resolution, a wax-type and an enantioselective column were coupled in either enantioselective-wax or wax-enantioselective order. The most appropriate column arrangement was selected for study by using a GC×GC experiment with either a wax-phase or phenyl-methylpolysiloxane phase as (2)D column. In addition to evaluation of these fast elution columns, a long narrow-bore enantioselective column (10m) was introduced as (2)D, providing an enantioselective-PEG (coupled-column ensemble: (1)D1+(1)D2)×enantioselective ((2)D) column combination. In this instance, the (1)D1 enantioselective column provides enantiomeric separation of the corresponding enantiomers ((R) and (S)) of (E)- and (Z)-2-phenylpropanaldehyde oxime, followed by E/Z isomerisation in the coupled (1)D2 PEG (reactor) column. The resulting chromatographic interconversion region was modulated and separated into either E/Z isomers (achiral (2)D column) or into the respective (R) and (S) enantiomers of the E/Z isomers when using a (2)D enantioselective column. With this arrangement, the isomers underneath the broad interconversion plateau in 1D elution profiles, including the enantiomers, could be resolved, illuminating salient features and understanding of the molecular reversible process of the interconverting molecules during the chromatographic elution. The two-dimensional patterns (contour plots), resulting from the combination of interconversion process and chiral separation, are discussed phenomenologically., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aims of the present multi-center study were to investigate the extent of mental health problems in adolescents with a hearing loss and cochlear implants (CIs) in comparison to normal hearing (NH) peers and to investigate possible relations between the extent of mental health problems of young CI users and hearing variables, such as age at implantation, or functional gain of CI. The survey included 140 adolescents with CI (mean age = 14.7, SD = 1.5 years) and 140 NH adolescents (mean age = 14.8, SD = 1.4 years), their parents and teachers. Participants were matched by age, gender and social background. Within the CI group, 35 adolescents were identified as "risk cases" due to possible and manifest additional handicaps, and 11 adolescents were non-classifiable. Mental health problems were assessed with the Strengths and Difficulties Questionnaire (SDQ) in the versions "Self," "Parent," and "Teacher." The CI group showed significantly more "Peer Problems" than the NH group. When the CI group was split into a "risk-group" (35 "risk cases" and 11 non-classifiable persons) and a "non-risk group" (n = 94), increased peer problems were perceived in both CI subgroups by adolescents themselves. However, no further differences between the CI non-risk group and the NH group were observed in any rater. The CI risk-group showed significantly more hyperactivity compared to the NH group and more hyperactivity and conduct problems compared to the CI non-risk group. Cluster analyses confirmed that there were significantly more adolescents with high problems in the CI risk-group compared to the CI non-risk group and the NH group. Adolescents with CI, who were able to understand speech in noise had significantly less difficulties compared to constricted CI users. Parents, teachers, and clinicians should be aware that CI users with additionally special needs may have mental health problems. However, peer problems were also experienced by CI adolescents without additional handicaps.

Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.

In a feeding experiment, the probiotic Bacillus cereus var. Toyoi was fed to sows and piglets in order to test whether it influences the stress response of enterocytes, thereby causing intestinal immune activation, possibly accompanied by an impairment of the epithelial integrity. The impact of B. cereus on the piglets' intestinal enterocytes (EC) and on the communicating intraepithelial lymphocytes (IEL) was investigated during the weaning phase where significant immunological changes might be expected. The expression of the stress-induced MHC class I-related molecule 2 (MIC2) and the UL16 binding protein (ULBP) was measured in jejunal EC and the frequencies of the main present IEL populations in the jejunum were monitored. To find out which of the IEL populations can be activated by the stress-induced molecules the sorted IEL were tested for the expression of the activating natural killer receptor 2D (NKG2D). The piglets fed with B. cereus showed an impaired intestinal barrier function shortly after weaning. However, a significant impact on the expression of stress-induced molecules was not observed. The mRNA expression of NKG2D was confirmed in intraepithelial CD5+ γδ T cells. The ratio of IEL (CD45+) to EC was lower in the B. cereus treated group, which could be explained by lower frequencies of CD8αβ+ T cells in the jejunal epithelium (p ≤ 0.005 for ages 32 and 34). Although a consistently increased expression of stress-induced MHC class I-related molecules was not found, this study suggests a negative impact of B. cereus on the intestinal barrier function and supports immune-modulating properties of the probiotic feed supplement., (Copyright © 2014. Published by Elsevier B.V.)

Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibers, called intrafusal fibers, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. We show that AChRs are concentrated at the γ-motoneuron endplate as well as in the equatorial region where they colocalize with the sensory nerve ending. In addition to the AChRs, the contact site between sensory nerve ending and intrafusal muscle fiber contains a high concentration of choline acetyltransferase, vesicular acetylcholine transporter and the AChR-associated protein rapsyn. Moreover, bassoon, a component of the presynaptic cytomatrix involved in synaptic vesicle exocytosis, is present in γ-motoneuron endplates but also in the sensory nerve terminal. Finally, we demonstrate that during postnatal development of the γ-motoneuron endplate, the AChR subunit stoichiometry changes from the γ-subunit-containing fetal AChRs to the ε-subunit-containing adult AChRs, similar and approximately in parallel to the postnatal subunit maturation at the neuromuscular junction. In contrast, despite the onset of ε-subunit expression during postnatal development the γ-subunit remains detectable in the equatorial region by subunit-specific antibodies as well as by analysis of muscle spindles from mice with genetically-labeled AChR γ-subunits. These results demonstrate an unusual maturation of the AChR subunit composition at the annulospiral endings and suggest that in addition to the recently described glutamatergic secretory system, the sensory nerve terminals are also specialized for cholinergic synaptic transmission, synaptic vesicle storage and exocytosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Because the extracellular matrix protein agrin is essential for neuromuscular junction formation and maintenance, we tested the hypothesis that autoantibodies against agrin are present in sera from patients with myasthenia gravis (MG)., Methods: We determined the presence of anti-agrin antibodies in 54 sera from patients with generalized MG using a solid-phase ELISA with purified mini-agrin protein. Thirty of the 54 sera were seronegative for antibodies against the acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK), 15 had elevated levels of anti-MuSK, and 9 had elevated levels of anti-AChR autoantibodies. Sixteen sera from healthy volunteers served as control., Results: Five sera with elevated levels of anti-agrin antibodies were identified. The concentration of the antibodies ranged between 0.04 and 0.12 nM. Four of the 5 agrin-positive sera were also positive for anti-MuSK, one was positive for anti-AChR, and 2 had elevated levels of anti-low-density lipoprotein receptor-related protein 4 (LRP4) autoantibodies. Some of the sera stained adult mouse neuromuscular junctions and reacted with native mini-agrin expressed in 293HEK cells., Conclusions: The results provide evidence for agrin as a novel target protein for autoantibodies in patients with MG. Anti-agrin antibodies were always detected in combination with autoantibodies against MuSK, LRP4, or AChRs, indicating a high incidence of autoantibodies against several neuromuscular proteins in the agrin-positive MG cases., (© 2014 American Academy of Neurology.)

We examined the influence of dietary fermentable protein (fCP) and fermentable carbohydrates (fCHO) on the colonic epithelial response to histamine in pigs. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO, low fCP/high fCHO, high fCP/low fCHO and high fCP/high fCHO. After 21-23 days, the pigs were killed and tissue from the proximal colon was stimulated with carbachol, histamine, PGE2 or sodium hydrogen sulphide in Ussing chambers. Changes in short-circuit current and tissue conductance were measured. Diamine oxidase, histamine N-methyltransferase, stem cell growth factor receptor, Fc-epsilon receptor I and cystic fibrosis transmembrane conductance regulator gene expression was determined. Activities of diamine oxidase and histamine N-methyltransferase and numbers of colonic mast cells were measured. The change in the short-circuit current in response to histamine was lower (P = 0.002) and tended to be lower for PGE2 (P = 0.053) in high fCP groups compared to low fCP groups, irrespective of fCHO. Additionally, the change in tissue conductance after the application of histamine was lower (P = 0.005) in the high fCP groups. The expression of histamine N-methyltransferase mRNA (P = 0.033) and the activities of diamine oxidase (P = 0.001) and histamine N-methyltransferase (P = 0.006) were higher with high fCP in comparison with low fCP. The expression of mast cell markers, stem cell growth factor receptor (P = 0.005) and Fc-epsilon receptor I (P = 0.049) was higher with high fCP diets compared to diets low in fCP, whereas the mast cell count did not differ between groups. The expression of the cystic fibrosis transmembrane conductance regulator was reduced (P = 0.001) with high fCP diets compared to low fCP diets. The lower epithelial response to histamine and PGE2 and elevated epithelial histamine inactivation suggests an adaptation to high fCP diets.

A novel ERP paradigm was employed to investigate conceptual expansion, a central component of creative thinking. Participants were presented with word pairs, consisting of everyday objects and uses for these objects, which had to be judged based on the two defining criteria of creative products: unusualness and appropriateness. Three subject-determined trial types resulted from this judgement: high unusual and low appropriate (nonsensical uses), low unusual and high appropriate (common uses), and high unusual and high appropriate (creative uses). Word pairs of the creative uses type are held to passively induce conceptual expansion. The N400 component was not specifically modulated by conceptual expansion but was, instead, generally responsive as a function of unusualness or novelty of the stimuli (nonsense=creative>common). Explorative analyses in a later time window (500-900 ms) revealed that ERP activity in this phase indexes appropriateness (nonsense>creative=common). In the discussion of these findings with reference to the literature on semantic cognition, both components are proposed as indexing processes relevant to conceptual expansion as they are selectively involved in the encoding and integration of a newly established semantic connection between two previously unrelated concepts., (Copyright © 2013 Elsevier B.V. All rights reserved.)

ApoE, ApoE receptors and APP cooperate in the pathogenesis of Alzheimer's disease. Intriguingly, the ApoE receptor LRP4 and APP are also required for normal formation and function of the neuromuscular junction (NMJ). In this study, we show that APP interacts with LRP4, an obligate co-receptor for muscle-specific tyrosine kinase (MuSK). Agrin, a ligand for LRP4, also binds to APP and co-operatively enhances the interaction of APP with LRP4. In cultured myotubes, APP synergistically increases agrin-induced acetylcholine receptor (AChR) clustering. Deletion of the transmembrane domain of LRP4 (LRP4 ECD) results in growth retardation of the NMJ, and these defects are markedly enhanced in APP(-/-);LRP4(ECD/ECD) mice. Double mutant NMJs are significantly reduced in size and number, resulting in perinatal lethality. Our findings reveal novel roles for APP in regulating neuromuscular synapse formation through hetero-oligomeric interaction with LRP4 and agrin and thereby provide new insights into the molecular mechanisms that govern NMJ formation and maintenance. DOI:http://dx.doi.org/10.7554/eLife.00220.001.

Ussing chambers are frequently used for in vitro evaluation of intestinal transport physiology. The current study describes investigating the jejunal tissue from laying hens using a specific preparation method and evaluates the effect of glutamine in the maintenance buffer. Tunica mucosa was stripped from 104 jejunal samples from 10 hens and stabilised by a net device. Fifty samples were maintained with modified Krebs-Henseleit buffer (Control), 54 samples with additional 5 mM glutamine (Group Gln). The percentage of responding samples varied between 87 and 100%. Mean short circuit current (ΔI sc,) [µA/cm(2)] of samples exposed to 10 mM glucose in the Control group and Group Gln was 17.0 and 14.6 (p = 0.836), respectively, of samples exposed to 100 µM phloridzin -13.3 and -11.8 (p = 0.712), respectively, and of samples exposed to 100 µM carbachol 4.7 and 3.7 (p = 0.450), respectively. In conclusion, the net-supported method enabled a reliable investigation of jejunum from laying hens. Glutamine in the maintenance buffer was of no significant benefit.

The present investigations were conducted to test the effects of T-2 toxin on electrophysiological variables of jejunal epithelium of chicken. Jejunal segments of broilers were monitored in Ussing chambers in the presence of T-2 toxin at the levels of 0 (negative control), 0 (methanol/vehicle control), 0.1, 1, 5, and 10 μg/ml of buffer. T-2 toxin did not affect basal values of short circuit current (I(sc)), transmural potential difference, or tissue conductivity in the jejunal epithelium. T-2 toxin also did not statistically affect glucose-induced electrophysiological variables during the first 3 min of glucose induction. Compared to the vehicle control, the ouabain-sensitive I(sc) was negatively affected (P = 0.008) only under 5 μg of T-2 toxin/ml. Increasing levels of T-2 toxin negatively affected the ouabain-sensitive I(sc) in a cubic (P = 0.007) fashion. These data indicate that acute exposure to moderate levels of T-2 toxin may progressively impair the cation gradient across the jejunal epithelium.

Objectives: Recently, several studies have shown that a cochlear implant is a suitable treatment for hearing rehabilitation of adults with unilateral sensorineural hearing loss (UHL), and benefits for speech comprehension in noise and localization have been demonstrated. The aim of this clinical study was to evaluate the benefit of cochlear implantation in children with UHL in comparison to their unaided listening situation., Study Design: Prospective repeated measures single subject design., Setting: Tertiary referral center; cochlear implant (CI) program, Patients: Three children (4, 10, and 11 yr) with noncongenital UHL resulting from different causes were enrolled in the study., Intervention: After extensive consultation with each family and confirming CI-candidacy, each child received a cochlear implant., Main Outcome Measures: Open set speech recognition measures in competing background noise and tests of sound localization were performed for unaided preoperative and CI-aided postimplant intervals after 6 and 12 months. The parent and child versions of the Speech, Spatial and Qualities scale (SSQ) were used for subjective evaluation of hearing at preimplant and 12-month postimplant intervals., Conclusion: All children consistently use their CI 1 year postimplant. Our preliminary results suggest binaural hearing benefits for speech understanding in noise, localization ability and subjectively perceived hearing ability for school-aged children with UHL. Trends for CI-benefits in younger preschoolaged children are confirmed through subjective assessment reported by the parents. The benefits in hearing performance suggest binaural integration and processing of electric and acoustic stimulation arriving at 2 different ears is possible for children with UHL.

Conceptual expansion, one of the core operations in creative cognition, was investigated in the present ERP study. An experimental paradigm using novel metaphoric, nonsensical and literal phrases was employed where individual differences in conceptual knowledge organization were accounted for by using participants' responses to categorize the stimuli to each condition. The categorization was determined by their judgment of the stimuli on the two defining criteria of creativity: unusualness and appropriateness. Phrases judged as unusual and appropriate were of special interest as they are novel and unfamiliar phrases thought to passively induce conceptual expansion. The results showed a graded N400 modulation for phrases judged to be unusual and inappropriate (nonsense) or unusual and appropriate (conceptual expansion, novel metaphorical) relative to usual and appropriate (literal) phrases. The N400 is interpreted as indexing greater effort to retrieve semantic information and integrate the novel concepts presented through the phrases. Analyses of the later time-window showed an ongoing negativity that was graded in the same manner as the N400. The findings attest to the usefulness of investigating creative cognition using event-related electrophysiology., (Copyright © 2012 Elsevier Inc. All rights reserved.)