Function of Reelin Signaling in the Tangential Migration of Dopaminergic Neurons

Midbrain dopaminergic neurons (mDA neurons) are involved in the regulation of voluntary movement, reward behavior and cognitive processes. mDA neurons are born in the floor plate of the ventral midbrain, from where they migrate to form three anatomically distinct structures: the laterally positioned substantia nigra (SN), the medially positioned ventral tegmental area (VTA), and the posterior retrorubral field (RRF). Previous studies of the developing mDA system have shown that both SN- and VTA-mDA neurons undergo radial migration between embryonic (E) day 9.5-13.5. Between E12.5 and E15.5, SN-mDA neurons migrate tangentially to separate from the VTA-mDA neurons and take up a more lateral position. However, while the tangential migration of SN-mDA neurons is important for the correct anatomical positioning of the dopaminergic system, little is known about the extracellular signaling cues and dynamic cell morphologies that underlie this process. The first step in studying migration is visualizing cell movements at high resolution. To provide easy visual access to the neuronal population under investigation, migration is often studied in organotypic slice culture preparations. Migrating mDA neurons form dense clusters in the developing ventral midbrain. In order to study mDA migration in detail, it is therefore essential to mosaically label mDA neurons. Furthermore, to characterize mDA migration, data-analysis protocols need to be developed for tracking a large number of migrating mDA neurons and tracing their cell morphologies in 3D. This study establishes an imaging pipeline for high-resolution 2-photon microscopy of migrating mDA neurons in organotypic slices. Using time-lapse 2-photon images, this study tracks the trajectories, neuronal speeds and cell morphologies of migrating mDA neurons in 3D. Reelin, an extracellular matrix protein and a well-established regulator of neuronal migration, is known to be important for SN-mDA tangential migration. However, the exact role of Reelin signaling in this process is not completely understood. Disabled 1 (DAB1) is the intracellular mediator of the Reelin signal. The binding of Reelin to its receptors apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR) activates DAB1 (via phosphorylation). By activating the downstream effectors of Reelin, phosphorylated DAB1 brings about changes.in cell adhesion properties and cytoskeletal stability thereby regulating cell migration. In this study, Dab1 was specifically inactivated in differentiated mDA neurons. This resulted in an ectopic medial localization of parts of the SN and an intermingling of SN and VTA-mDA neurons. These data indicate that Reelin signaling is directly required in mDA neurons for their correct localization. Applying the image acquisition and data-analysis pipeline developed in this study to mDA neurons in embryonic organotypic slices from control and Dab1-/- (Dab1 knockout) mice, the speeds, trajectories and underlying cell morphologies of mDA neurons were examined in the presence and absence of Reelin signaling. Using these techniques, this study characterizes the migratory modes and morphological changes underlying SN-mDA tangential migration and demonstrates that Reelin promotes laterally-biased movements in mDA neurons during their slow migration mode. Furthermore, Reelin stabilizes leading process morphology and increases the probability of fast, laterally-directed migration of mDA neurons. Overall, this study characterizes in detail the migratory and cell morphological characteristics of developing mDA neurons and elucidates the complex role of Reelin in modulating mDA neuronal speed, migratory trajectory and dynamic cell morphology.Dopaminerge Neuronen des Mittelhirns (mDA-Neurone) sind an der Regulation von extrapyramidaler Motorik, Belohnungsverhalten und kognitiven Prozessen beteiligt. mDA-Neurone entstehen in der Bodenplatte des ventralen Mittelhirns, von wo aus sie zu drei anatomisch unterschiedlichen Strukturen wandern: der seitlich positionierten Substantia Nigra (SN), dem medial positionierten ventralen Tegmentalbereich (VTA) und dem posterioren Retrorubralfeld (RRF). Frühere Studien des sich entwickelnden mDA-Systems haben gezeigt, dass sowohl SN- wie auch VTA-mDA-Neurone eine radiale Migration zwischen dem embryonalen Tag (E) 9,5-13,5 durchlaufen. Zwischen E12,5 und E15,5 wandern SN-mDA-Neurone tangential, um sich von den VTA-mDA-Neuronen zu trennen und eine lateralere Position einzunehmen. Über die extrazellulären Signalstoffe und dynamischen Zellmorphologien, die der mDA-Migration zugrunde liegen, ist jedoch wenig bekannt. Der erste Schritt zur Untersuchung der Migration ist die Visualisierung von Zellbewegungen mit hoher Auflösung. Um einen einfache Visualisierung der zu untersuchenden neuronalen Population zu ermöglichen, wird die Zellmigration oft in organotypischen Schnittkulturpräparaten untersucht. Migrierende mDA-Neurone sind im sich entwickelnden ventralen Mittelhirn dicht gedrängt. Um die mDA-Migration im Detail zu untersuchen, ist es daher unerlässlich, mDA-Neuronen mosaikartig zu markieren. Darüber hinaus müssen zur Charakterisierung der mDA-Migration Protokolle zur Datenanalyse entwickelt werden, um eine große Anzahl von migrierenden mDA-Neuronen verfolgen und deren Zellmorphologien dreidimensional charakterisieren zu können. Die vorliegende Studie nutzt die hochauflösende 2-Photonen Mikroskopie zur dreidimensionalen Bildgebung migrierender mDA-Neurone in organotypischen Schnittkulturen. Anhand der gewonnenen Zeitraffer-2-Photonen-Bildern werden die Bewegungsbahnen, neuronalen Geschwindigkeiten und Zellmorphologien migrierender mDA-Neurone in 3D analysiert. Reelin ist ein extrazelluläres Matrixprotein, das neuronale Migration in verschiedenen Gehirnbereichen regelt. Für die tangentiale SN-mDA-Migration ist Reelin ebenfalls wichtig, aber die genaue Rolle des Reelin-Signalwegs in diesem Prozess ist nicht vollständig verstanden. Disabled 1 (DAB1) ist der intrazelluläre Effektor des Reelin-Signals. Die Bindung von Reelin an seine Rezeptoren Apolipoprotein E Receptor 2 (ApoER2) und Very Low Density Lipoprotein Receptor (VLDLR) aktiviert DAB1 durch Phosphorylierung. Durch die Aktivierung von nachgeschalteten Effektoren bewirkt phosphoryliertes DAB1 Veränderungen der Adhäsionseigenschaften und der zytoskelettalen Stabilität der Zelle und reguliert so die Zellmigration. In dieser Dissertation wurde Dab1 in differenzierten mDA-Neuronen spezifisch inaktiviert. Dies führte zu einer ektopischen medialen Lokalisation von Teilen der SN und einer teilweisen Vermischung von SN- und VTA-mDA-Neuronen. Diese Daten deuten darauf hin, dass die Aktivierung des Reelin-Signalwegs in SN-mDA-Neuronen für ihre korrekte Lokalisierung erforderlich ist. Unter Anwendung der in dieser Studie entwickelten Protokolle für die Bildgebung und Datenanalyse von mDA-Neuronen in embryonalen organotypischen Schnittkulturen von Kontroll- und Dab1-/- (Dab1 Knockout)-Mäusen wurden die Geschwindigkeiten, Bewegungsbahnen sowie die zugrunde liegenden Zellmorphologien von mDA-Neuronen in Gegenwart und Abwesenheit von Reelin-Signalen untersucht. Mit diesen Techniken charakterisiert diese Studie die Migrationsmodi und morphologischen Veränderungen, die der SN-mDA tangentialen Migration zugrunde liegen, und zeigt, dass Reelin lateral orientierte Bewegungen in mDA-Neuronen während ihres langsamen Migrationsmodus fördert. Darüber hinaus stabilisiert Reelin die Morphologie des Leitsaums und erhöht die Wahrscheinlichkeit einer schnellen, lateral gerichteten Migration von mDA-Neuronen. Insgesamt charakterisiert diese Studie im Detail die migratorischen und zellmorphologischen Eigenschaften der sich entwicklenden mDA-Neuronen und verdeutlicht die komplexe Rolle von Reelin bei der Modulation der neuronalen Geschwindigkeit, den migratorischen Bewegungsbahnen und der dynamischen Zellmorphologie dieser Neurone

Data from the Indian drug regulator and from Clinical Trials Registry-India does not always match

IntroductionIn India, regulatory trials, which require the drug regulator’s permission, must be registered with the Clinical Trials Registry-India (CTRI) as of 19 March 2019. In this study, for about 300 trials, we aimed to identify the CTRI record that matched the trial for which the regulator had given permission. After identifying ‘true pairs’, our goal was to determine whether the sites and Principal Investigators mentioned in the permission letter were the same as those mentioned in the CTRI record.MethodsWe developed a methodology to compare the regulator’s permission letters with CTRI records. We manually validated 151 true pairs by comparing the titles, the drug interventions, and the indications. We then examined discrepancies in their trial sites and Principal Investigators.ResultsOur findings revealed substantial variations in the number and identity of sites and Principal Investigators between the permission letters and the CTRI records.DiscussionThese discrepancies raise concerns about the accuracy and transparency of regulatory trials in India. We recommend easier data extraction from regulatory documents, cross-referencing regulatory documents and CTRI records, making public the changes to approval letters, and enforcing oversight by Institutional Ethics Committees for site additions or deletions. These steps will increase transparency around regulatory trials running in India

Improving cold chain technologies through the use of phase change material

Gemstone Team FRESHVaccine-preventable diseases are responsible for about 25% of the 10 million deaths occurring annually for children under five years of age. The World Health Organization's Expanded Programmes on Immunization succeed in providing standardized guidelines for vaccine storage and distribution, but often fail to accommodate the unique infrastructure between and within countries. In order to better regulate the temperature of vaccines as they travel through countries, we have selected and characterized an appropriate phase change material (PCM) that will resist temperature fluctuations outside of a range of 2-8 °C, based on appropriate thermophysical properties. Additionally, we have integrated the selected PCM within a geometrically and thermally optimized cold box, maintaining long-term stabilization of temperatures within a range of 2-8 °C. In meeting these objectives, we have demonstrated the feasibility of a technological solution that may be readily implemented in the existing vaccine distribution supply chain, or that holds potential to be the centerpiece for new, more efficient vaccine distribution strategies

Zero-Shot Multi-View Indoor Localization via Graph Location Networks

Indoor localization is a fundamental problem in location-based applications. Current approaches to this problem typically rely on Radio Frequency technology, which requires not only supporting infrastructures but human efforts to measure and calibrate the signal. Moreover, data collection for all locations is indispensable in existing methods, which in turn hinders their large-scale deployment. In this paper, we propose a novel neural network based architecture Graph Location Networks (GLN) to perform infrastructure-free, multi-view image based indoor localization. GLN makes location predictions based on robust location representations extracted from images through message-passing networks. Furthermore, we introduce a novel zero-shot indoor localization setting and tackle it by extending the proposed GLN to a dedicated zero-shot version, which exploits a novel mechanism Map2Vec to train location-aware embeddings and make predictions on novel unseen locations. Our extensive experiments show that the proposed approach outperforms state-of-the-art methods in the standard setting, and achieves promising accuracy even in the zero-shot setting where data for half of the locations are not available. The source code and datasets are publicly available at https://github.com/coldmanck/zero-shot-indoor-localization-release.Comment: Accepted at ACM MM 2020. 10 pages, 7 figures. Code and datasets available at https://github.com/coldmanck/zero-shot-indoor-localization-releas

Knowledge and awareness of informed consent among orthodontists and patients: A pilot study

Aim: Despite fixed professional opinion of what might constitute optimal treatment, patients must be informed of the various treatment options available in orthodontics to manage their clinical problem. The purpose of this study was to compare and evaluate the knowledge and awareness among practicing orthodontists and patients with regard to informed consent in clinical practice and research. Materials and Methods: Twenty-five orthodontists and 25 patients were enrolled in a questionnaire study which was descriptive and cross-sectional in the nature. The questionnaire focused on the following aspects; contents of informed consent, at what age and who can give consent. Results: The study showed a majority of orthodontists (79.14%) were aware of knowledge regarding informed consent when compared to patients(35.14%). Conclusion: The overall result showed the huge gap that exists between orthodontists and patients and thus making it categorical for patients to be more involved in the decision-making process

Representation from India in multinational, interventional, phase 2 or 3 trials registered in Clinical Trials Registry-India: A cross-sectional study.

In multinational trials that have run in India, we wished to determine whether there was too much (60% or higher) recruitment from India. We downloaded all trial records from Clinical Trials Registry-India, CTRI, and stored them in a local SQLite database. We queried records registered in a recent 8-year period, ie 2013-2020 and evaluated the fraction of local participants in interventional Phase 2 or Phase 3 studies. 62 trials were completed, with completion dates available. Five trials (8%) had 60% or more planned recruitment from India. Four of the five (7% of 62) had a foreign sponsor, and therefore there was an unfair burden-benefit ratio on the Indian population. Seven trials (11%), of which six (10% of 62) had foreign sponsors, had 60% or more (of the total) actual recruitment from India, and for two trials (both with foreign sponsors), the data were meaningless. There were 362 studies that were listed as not completed, although, given their start date and estimated duration, some of them ought to have been. Twenty five cases (7% of 362) had 60% or more planned recruitment from India. Of these, 18 (5% of 362) had foreign sponsors and were potentially problematic. Even allowing for some delays in completion, 128 (35% of 362) studies ought to have been completed by the time of our study. As such, we identified several problematic trials for which the planned recruitment from India in multinational studies was 60% or more. We also identified trials in which the actual recruitment was significantly higher than the planned recruitment. Further, the records of several studies that were probably completed were not updated in CTRI in a timely manner. The Indian drug regulator needs to be particularly alert to the planned, or actual, over-recruitment of participants from India. Further, CTRI, alone or in collaboration with the regulator, needs to ensure that multinational trial records for the enrollment fields in particular are updated, in a timely manner