A novel tool to measure extracellular glutamate in the Zebrafish nervous system in vivo

Glutamate is the major excitatory neurotransmitter in the brain. Its release and eventual recycling are key to rapid sustained neural activity. We have paired the gfap promoter region with the glutamate reporter molecule, iGluSnFR, to drive expression in glial cells throughout the nervous system. Tg(gfap:iGluSnFR) is expressed on the glial membrane of Müller glia cells in the retina, which rapidly respond to stimulation and the release of extracellular glutamate. As glial cells are associated with most, if not all, synapses, Tg(gfap:iGluSnFR) is a novel and exciting tool to measure neuronal activity and extracellular glutamate dynamics in many regions of the nervous system. Glutamate is the major excitatory neurotransmitter in the brain. Its release and eventual recycling are key to rapid sustained neural activity.1 Glial cells play a key role in the uptake and recycling of glutamate from the synaptic cleft. iGluSnFR has been used to study synaptic activity by measuring glutamate dynamics in the zebrafish nervous system.2,3 Previous work has also used iGluSnFR in glial cells; however, this was done transiently in the mouse using viral vectors.2,4 As such, we designed a transgene to stably express iGluSnFR in the glial cells of the zebrafish nervous system. We report a novel transgenic zebrafish, Tg(gfap:iGluSnFR), that displays the glutamate-sensitive fluorescent reporter iGluSnFR specifically on the membrane of glial cells (Figure 1A–C). This molecule is expressed on the glial membrane in many brain regions and rapidly responds to stimulation and the release of extracellular glutamate (Figure 1D–F, Supplementary Data; Supplementary Data are available online at www.liebertpub.com/zeb). Thus, pairing the sensitivity of iGluSnFR and optical transparency of the zebrafish provides a powerful tool for understanding glutamate dynamics in neural tissues in vivo

Meeting in the Middle: Towards Successful Multidisciplinary Bioimage Analysis Collaboration

With an increase in subject knowledge expertise required to solve specific biological questions, experts from different fields need to collaborate to address increasingly complex issues. To successfully collaborate, everyone involved in the collaboration must take steps to "meet in the middle". We thus present a guide on truly cross-disciplinary work using bioimage analysis as a showcase, where it is required that the expertise of biologists, microscopists, data analysts, clinicians, engineers, and physicists meet. We discuss considerations and best practices from the perspective of both users and technology developers, while offering suggestions for working together productively and how this can be supported by institutes and funders. Although this guide uses bioimage analysis as an example, the guiding principles of these perspectives are widely applicable to other cross-disciplinary work

GliaMorph: A modular image analysis toolkit to quantify Müller glial cell morphology

Cell morphology is critical for all cell functions. This is particularly true for glial cells as they rely on complex shape to contact and support neurons. However, methods to quantify complex glial cell shape accurately and reproducibly are lacking. To address this, we developed the image analysis pipeline "GliaMorph". GliaMorph is a modular analysis toolkit developed to perform (i) image pre-processing, (ii) semi-automatic region-of-interest (ROI) selection, (iii) apicobasal texture analysis, (iv) glia segmentation, and (v) cell feature quantification. Müller Glia (MG) have a stereotypic shape linked to their maturation and physiological status. We here characterized MG on three levels, including (a) global image-level, (b) apicobasal texture, and (c) regional apicobasal vertical-to-horizontal alignment. Using GliaMorph we quantified MG development on a global and single-cell level, showing increased feature elaboration and subcellular morphological rearrangement in the zebrafish retina. As proof-of-principle, we analysed expression changes in a mouse glaucoma model, identifying subcellular protein localization changes in MG. Together, GliaMorph enables an in-depth understanding of MG morphology in the developing and diseased retina

Unexpected phenotypic and molecular changes of combined glucocerebrosidase and acid sphingomyelinase deficiency

Heterozygous variants in GBA1 encoding glucocerebrosidase (GCase) are the most common genetic risk factor for Parkinson's disease (PD). Moreover, sporadic PD patients also have a substantial reduction of GCase activity. Genetic variants in SMPD1 are also overrepresented in PD cohorts, whilst a reduction of its encoded enzyme (ASM) activity is linked to an earlier age of PD onset. Despite both converging on the ceramide pathway, how combined deficiencies of both enzymes may interact to modulate PD has yet to be explored. Therefore, we created a double knock out (DKO) zebrafish line for both gba1 and smpd1 to test for an interaction in vivo, hypothesising an exacerbation of phenotypes in the DKO compared to single mutants. Unexpectedly, DKOs maintained conventional swimming behaviour and had normalised neuronal gene expression signatures when compared to single mutants. We further identified rescue of mitochondrial Complexes I and IV in DKOs. Despite having an unexpected rescue effect, our results confirm ASM as a modifier of GBA1 deficiency in vivo. Our study highlights the need for validating how genetic variants and enzymatic deficiencies may interact in vivo

Study Design and Cohort Comparability in a Study of Major Cardiovascular Events in New Users of Prucalopride Versus Polyethylene Glycol 3350

INTRODUCTION: Given prior safety experience with other 5-HT4 agonists for chronic constipation, an observational, population-based cohort study in five data sources from Germany, Sweden, and the UK was conducted to evaluate the cardiovascular safety of prucalopride. OBJECTIVES: Our objective is to describe the methods and resulting comparability of cohorts in a multi-database, multinational study of prucalopride initiators and polyethylene glycol 3350 (PEG) initiators following a harmonized protocol. METHODS: Prucalopride initiators were matched on age, sex, and index date to PEG initiators (1:5 ratio). Study exposures, cardiovascular risk factors, and other covariates were identified from healthcare utilization codes harmonized across databases. Cardiovascular outcomes were identified using database-specific algorithms based on diagnosis codes. The propensity score (PS) in each database was estimated using logistic regression, with prucalopride versus PEG as the outcome and including clinically relevant variables associated with major adverse cardiovascular events. RESULTS: In total, 12,030 prucalopride initiators and 59,985 PEG initiators were identified. After matching and trimming, cohorts from the UK and Sweden were well-balanced for cardiovascular risk factors and cancer. However, in Germany, PEG initiators remained older and sicker than prucalopride initiators. The prevalence of these characteristics also differed from those in the UK and Sweden. The pooled analyses included only data from the UK and Sweden. CONCLUSIONS: Matching, trimming, and PS stratification yielded comparable cohorts in four of five data sources. Use of these methods could not achieve balance for key covariates within the German cohort, likely due to reimbursement differences in Germany

Transmission Spectroscopy of the Habitable Zone Exoplanet LHS 1140 b with JWST/NIRISS

LHS 1140 b is the second-closest temperate transiting planet to Earth with an equilibrium temperature low enough to support surface liquid water. At 1.730 ± 0.025 R ⊕, LHS 1140 b falls within the radius valley separating H2-rich mini-Neptunes from rocky super-Earths. Recent mass and radius revisions indicate a bulk density significantly lower than expected for an Earth-like rocky interior, suggesting that LHS 1140 b could be either a mini-Neptune with a small envelope of hydrogen (∼0.1% by mass) or a water world (9%–19% water by mass). Atmospheric characterization through transmission spectroscopy can readily discern between these two scenarios. Here we present two JWST/NIRISS transit observations of LHS 1140 b, one of which captures a serendipitous transit of LHS 1140 c. The combined transmission spectrum of LHS 1140 b shows a telltale spectral signature of unocculted faculae (5.8σ), covering ∼20% of the visible stellar surface. Besides faculae, our spectral retrieval analysis reveals tentative evidence of residual spectral features, best fit by Rayleigh scattering from a N2-dominated atmosphere (2.3σ), irrespective of the consideration of atmospheric hazes. We also show through Global Climate Models (GCMs) that H2-rich atmospheres of various compositions (100×, 300×, 1000× solar metallicity) are ruled out to >10σ. The GCM calculations predict that water clouds form below the transit photosphere, limiting their impact on transmission data. Our observations suggest that LHS 1140 b is either airless or, more likely, surrounded by an atmosphere with a high mean molecular weight. Our tentative evidence of a N2-rich atmosphere provides strong motivation for future transmission spectroscopy observations of LHS 1140 b

Cardiovascular Safety of Prucalopride in Patients with Chronic Constipation:A Multinational Population-Based Cohort Study

INTRODUCTION: The serotonin 5-HT4 receptor agonist prucalopride is approved in the European Union for the treatment of chronic constipation. This offered the unique opportunity to include real-world observational data on cardiovascular safety in the new drug application for approval of prucalopride in the USA. METHODS: This observational population-based cohort study (EUPAS9200) conducted in five data sources (three in the UK, one in Sweden, and one in Germany [which was subsequently excluded from the pooled analyses]) aimed to estimate the pooled adjusted incidence rate ratio for major adverse cardiovascular events (defined as hospitalization for non-fatal acute myocardial infarction or stroke, and in-hospital cardiovascular death) in adult initiators of prucalopride compared with initiators of polyethylene glycol 3350 (PEG) following a common protocol. Standardized incidence rates and incidence rate ratios of major adverse cardiovascular events were derived using propensity score stratification. Sensitivity analyses explored the impact of exposure definition, outcome categories, interim cancer, and unmeasured confounding. RESULTS: The pooled analyses included 5715 initiators of prucalopride and 29,372 initiators of PEG. Average duration of use was 175 days for prucalopride and 82 days for PEG. The pooled standardized incidence rate per 1000 person-years (95% confidence interval) of major adverse cardiovascular events was 6.57 (3.90–10.39) for patients initiating prucalopride and 10.24 (6.97–14.13) for PEG. The pooled adjusted incidence rate ratio for major adverse cardiovascular events was 0.64 (95% confidence interval 0.36–1.14). Results remained consistent in various sensitivity analyses. CONCLUSIONS: The pooled incidence rate ratio estimate was consistent with no indication of an increased risk above the pre-specified safety threshold of 3.00 for major adverse cardiovascular events in patients with chronic constipation using prucalopride as compared with PEG

Picturing the nation : The Celtic periphery as discursive other in the archaeological displays of the museum of Scotland

Using the archaeological displays at the Museum of Scotland in Edinburgh, this paper examines the exhibition as a site of identity creation through the negotiations between categories of same and Other. Through an analysis of the poetics of display, the paper argues that the exhibition constructs a particular relationship between the Celtic Fringe and Scottish National identity that draws upon the historical discourses of the Highlands and Islands of Scotland as a place and a time \u27apart\u27. This will be shown to have implications for the display of archaeological material in museums but also for contemporary understandings of Scottish National identity. <br /