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In vitro models of the blood–brain barrier (BBB) are critical tools for the study of BBB transport and the development of drugs that can reach the CNS. Brain endothelial cells grown in culture are often used to model the BBB; however, it is challenging to maintain reproducible BBB properties and function. ‘BBB organoids’ are obtained following coculture of endothelial cells, pericytes and astrocytes under low-adhesion conditions. These organoids reproduce many features of the BBB, including the expression of tight junctions, molecular transporters and drug efflux pumps, and hence can be used to model drug transport across the BBB. This protocol provides a comprehensive description of the techniques required to culture and maintain BBB organoids. We also describe two separate detection approaches that can be used to analyze drug penetration into the organoids: confocal fluorescence microscopy and mass spectrometry imaging. Using our protocol, BBB organoids can be established within 2–3 d. An additional day is required to analyze drug permeability. The BBB organoid platform represents an accurate, versatile and cost-effective in vitro tool. It can easily be scaled to a high-throughput format, offering a tool for BBB modeling that could accelerate therapeutic discovery for the treatment of various neuropathologies.

\"Acceptance of new technology and systems by drivers is an important area of concern to governments, automotive manufacturers and equipment suppliers, especially technology that has significant potential to enhance safety. To be acceptable, new technology must be useful and satisfying to use. If not, drivers will not want to have it, in which case it will never achieve the intended safety benefit. Even if they have the technology, drivers may not use it if it is deemed unacceptable, or may not use it in the manner intended by the designer. At worst, they may seek to disable it\"-Provided by publisher.

Purpose As tourism destinations grapple with declines in tourist arrivals due to COVID-19 measures, scholarly debate on overtourism remains active, with discussions on solutions that could be enacted to contain the excessive regrowth of tourism and the return of “overtourism”. As social science holds an important role and responsibility to inform the debate on overtourism, this paper aims to understand overtourism by examining it as a discursive formation. Design/methodology/approach The paper explores recurring thematic threads in scholarly overtourism texts, given the phrases coherence as a nodal-point is partially held in place by a collective body of texts authored by a network of scholars who have invested in it. The paper uses interdiscursivity as an interpretative framework to identify overlapping thematic trajectories found in existing discourses. Findings Overtourism, as a discursive formation, determines what can and should be said about the self-evident “truths” of excessive tourist arrivals, the changes tourists bring to destinations and the range of discursive solutions available to manage or end overtourism. As the interpellation of these thematic threads into scholarly texts is based on a sense of crisis and urgency, the authors find that the themes contain rhetoric, arguments and metaphors that problematise tourists and construct them as objects in need of control and correction. Originality/value While the persistence of the discursive formation will be determined by the degree to which scholarly and other actors recognise themselves in it, this paper may enable overtourism scholars to become aware of the limits of their discursive domain and help them to expand the discourse or weave a new one. 题目: 通过互斥性将过度旅游解构为一种话语形式 设计/方法论/方法 本文探讨了过度旅游研究文本中反复出现的主题线索, 鉴于这些短语连贯性作为一个节点, 由学者们组成的网络所研究的一组文本所组成。本文将互辩作为一个解释框架来识别现有语篇中重叠的主题轨迹。 目的 随着旅游目的地应对因新冠病毒−19措施而导致的游客人数下降, 关于过度旅游的学术辩论仍然活跃, 通过讨论可以制定的解决方案, 以遏制旅游业的过度再生和“过度旅游”的再现。由于社会科学在有关过度旅游的辩论中扮演着重要的角色和责任, 本文试图通过将其作为一种话语形式进行考察来理解过度旅游。 结果 作为一种话语形式, 过度旅游展现了过量游客所带来的不言而喻的“真相”、游客给目的地带来的变化以及可用于管理或结束过度旅游的一系列解决方案。由于这些主题线索在学术文本中的质询是基于危机感和紧迫感, 我们发现这些主题包含修辞、论据和隐喻, 使游客感到困惑, 并将其构建为需要控制和纠正的对象。 创新/价值 尽管话语形成的持续性将取决于学者和其他参与者在其中认识到自己的程度, 但本文可能会使过度旅游学者意识到其话语领域的局限性, 并帮助他们扩展话语或构建新的话语。 Descubriendo el sobreturismo como formación discursiva a través de la inter-discursividad Propósito A medida que los destinos turísticos lidian con la disminución de las llegadas de turistas debido a las medidas del COVID-19, el debate académico sobre el “sobreturismo” permanece activo, con discusiones sobre soluciones que podrían promulgarse para contener el crecimiento excesivo del turismo y el regreso del “sobreturismo”. La ciencia tiene un papel importante y una responsabilidad en informar sobre el debate del “sobreturismo”, este artículo busca entender el constructo de “sobreturismo”, examinándolo como una formación discursiva. Diseño/metodología/enfoque El artículo explora los hilos temáticos recurrentes en los textos académicos sobre el “sobreturismo”, dada la coherencia de las frases como un punto nodal, mantenida parcialmente en su lugar, por un cuerpo colectivo de textos escritos por una red de académicos, los cuales han invertido tiempo en ellos. El artículo utiliza la inter-discursividad como marco interpretativo a la hora de identificar trayectorias temáticas superpuestas que se encuentran en las disertaciones existentes. Hallazgos El sobreturismo, como formación discursiva, determina lo que puede y debe decirse sobre las “verdades” evidentes de la llegada excesiva de turistas, los cambios que los turistas traen a los destinos y la gama de soluciones discursivas disponibles para gestionar o acabar con el “sobreturismo”. Como la interpelación de estos hilos temáticos en textos académicos se basa en un sentido de crisis y urgencia, encontramos que los temas contienen retórica, argumentos y metáforas que problematizan a los turistas y los construyen como objetos que necesitan control y corrección. Originalidad/valor Si bien la persistencia de la formación discursiva estará determinada por el grado en que los académicos y otros actores se reconozcan en ella, este artículo puede permitir a los estudiosos del “sobreturismo” tomar conciencia de los límites de su dominio discursivo y ayudarlos a expandir el discurso o tejer un discurso nuevo.

Plant shoot systems derive from the shoot apical meristems (SAMs), pools of stems cells that are regulated by a feedback between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. The maize heterotrimeric G protein α subunit COMPACT PLANT2 (CT2) functions with CLV receptors to regulate meristem development. In addition to the sole canonical Gα CT2, maize also contains three eXtra Large GTP-binding proteins (XLGs), which have a domain with homology to Gα as well as additional domains. By either forcing CT2 to be constitutively active, or by depleting XLGs using CRISPR-Cas9, here we show that both CT2 and XLGs play important roles in maize meristem regulation, and their manipulation improved agronomic traits. For example, we show that expression of a constitutively active CT2 resulted in higher spikelet density and kernel row number, larger ear inflorescence meristems (IMs) and more upright leaves, all beneficial traits selected during maize improvement. Our findings suggest that both the canonical Gα, CT2 and the non-canonical XLGs play important roles in maize meristem regulation and further demonstrate that weak alleles of plant stem cell regulatory genes have the capacity to improve agronomic traits.

N -methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives. Selective targeting individual subtypes of N-methyl-D-aspartate receptors (NMDARs) is a desirable therapeutic strategy for neurological disorders. Here, the authors report identification of a functional antibody that specifically targets and allosterically down-regulates ion channel activity of the GluN1—GluN2B NMDAR subtype.

Topotecan is cytotoxic to glioma cells but is clinically ineffective because of drug delivery limitations. Systemic delivery is limited by toxicity and insufficient brain penetrance, and, to date, convection-enhanced delivery (CED) has been restricted to a single treatment of restricted duration. To address this problem, we engineered a subcutaneously implanted catheter-pump system capable of repeated, chronic (prolonged, pulsatile) CED of topotecan into the brain and tested its safety and biological effects in patients with recurrent glioblastoma. We did a single-centre, open-label, single-arm, phase 1b clinical trial at Columbia University Irving Medical Center (New York, NY, USA). Eligible patients were at least 18 years of age with solitary, histologically confirmed recurrent glioblastoma showing radiographic progression after surgery, radiotherapy, and chemotherapy, and a Karnofsky Performance Status of at least 70. Five patients had catheters stereotactically implanted into the glioma-infiltrated peritumoural brain and connected to subcutaneously implanted pumps that infused 146 μM topotecan 200 μL/h for 48 h, followed by a 5–7-day washout period before the next infusion, with four total infusions. After the fourth infusion, the pump was removed and the tumour was resected. The primary endpoint of the study was safety of the treatment regimen as defined by presence of serious adverse events. Analyses were done in all treated patients. The trial is closed, and is registered with ClinicalTrials.gov, NCT03154996. Between Jan 22, 2018, and July 8, 2019, chronic CED of topotecan was successfully completed safely in all five patients, and was well tolerated without substantial complications. The only grade 3 adverse event related to treatment was intraoperative supplemental motor area syndrome (one [20%] of five patients in the treatment group), and there were no grade 4 adverse events. Other serious adverse events were related to surgical resection and not the study treatment. Median follow-up was 12 months (IQR 10–17) from pump explant. Post-treatment tissue analysis showed that topotecan significantly reduced proliferating tumour cells in all five patients. In this small patient cohort, we showed that chronic CED of topotecan is a potentially safe and active therapy for recurrent glioblastoma. Our analysis provided a unique tissue-based assessment of treatment response without the need for large patient numbers. This novel delivery of topotecan overcomes limitations in delivery and treatment response assessment for patients with glioblastoma and could be applicable for other anti-glioma drugs or other CNS diseases. Further studies are warranted to determine the effect of this drug delivery approach on clinical outcomes. US National Institutes of Health, The William Rhodes and Louise Tilzer Rhodes Center for Glioblastoma, the Michael Weiner Glioblastoma Research Into Treatment Fund, the Gary and Yael Fegel Foundation, and The Khatib Foundation.

Context-dependent inhibition of N -methyl-D-aspartate (NMDA) receptors has important therapeutic implications for the treatment of neurological diseases that are associated with altered neuronal firing and signaling. This is especially true in stroke, where the proton concentration in the afflicted area can increase by an order of magnitude. A class of allosteric inhibitors, the 93-series, shows greater potency against GluN1-GluN2B NMDA receptors in such low pH environments, allowing targeted therapy only within the ischemic region. Here we map the 93-series compound binding site in the GluN1-GluN2B NMDA receptor amino terminal domain and show that the interaction of the N-alkyl group with a hydrophobic cage of the binding site is critical for pH-dependent inhibition. Mutation of residues in the hydrophobic cage alters pH-dependent potency, and remarkably, can convert inhibitors into potentiators. Our study provides a foundation for the development of highly specific neuroprotective compounds for the treatment of neurological diseases. Context-dependent inhibition of NMDA receptors has important therapeutic implications for treatment of neurological diseases. Here, the authors use structural biology and biophysics to describe the basis for pH-dependent inhibition for a class of allosteric NMDAR inhibitors, called the 93-series.

How the glioma immune microenvironment fosters tumorigenesis remains incompletely defined. Here, we use single-cell RNA-sequencing and multiplexed tissue-imaging to characterize the composition, spatial organization, and clinical significance of extracellular purinergic signaling in glioma. We show that microglia are the predominant source of CD39, while tumor cells principally express CD73. In glioblastoma, CD73 is associated with EGFR amplification, astrocyte-like differentiation, and increased adenosine, and is linked to hypoxia. Glioblastomas enriched for CD73 exhibit inflammatory microenvironments, suggesting that purinergic signaling regulates immune adaptation. Spatially-resolved single-cell analyses demonstrate a strong spatial correlation between tumor-CD73 and microglial-CD39, with proximity associated with poor outcomes. Similar spatial organization is present in pediatric high-grade gliomas including H3K27M-mutant diffuse midline glioma. These data reveal that purinergic signaling in gliomas is shaped by genotype, lineage, and functional state, and that core enzymes expressed by tumor and myeloid cells are organized to promote adenosine-rich microenvironments potentially amenable to therapeutic targeting. The components of the glioma immune microenvironment and their roles in promoting tumourigenesis remain poorly understood. Here, the use of single-cell RNA sequencing and multiplexed tissue-imaging in adult and pediatric high-grade gliomas reveals the activity and spatial organization of the immunomodulatory purinergic signaling pathway.

We report on James Webb Space Telescope (JWST) commissioning observations of the transiting exoplanet HAT-P-14 b, obtained using the Bright Object Time Series (BOTS) mode of the NIRSpec instrument with the G395H/F290LP grating/filter combination (3–5 μ m). While the data were used primarily to verify that the NIRSpec BOTS mode is working as expected, and to enable it for general scientific use, they yield a precise transmission spectrum which we find is featureless down to the precision level of the instrument, consistent with expectations given HAT-P-14 b’s small scale-height and hence expected atmospheric features. The exquisite quality and stability of the JWST/NIRSpec transit spectrum—almost devoid of any systematic effects—allowed us to obtain median uncertainties of 50–60 ppm in this wavelength range at a resolution of R = 100 in a single exposure, which is in excellent agreement with pre-flight expectations and close to the (or at the) photon-noise limit for a J = 9.094, F-type star like HAT-P-14. These observations showcase the ability of NIRSpec/BOTS to perform cutting-edge transiting exoplanet atmospheric science, setting the stage for observations and discoveries to be made in Cycle 1 and beyond.