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Microgravity produces a headward fluid shift that alters venous hemodynamics and has been associated with stagnant or retrograde flow in the internal jugular vein (IJV), raising concern for thrombosis risk during spaceflight. Lower body negative pressure (LBNP) has emerged as a promising countermeasure to partially restore hydrostatic gradients, yet its physiological effects in true microgravity remain poorly characterized. In this case study, we applied graded LBNP (0, -20, and -30 mmHg) during parabolic flight in a single female participant and obtained matching supine baseline measurements on the ground. Bilateral IJV cross-sectional area and flow were quantified using ultrasound, and IJV pressure was measured using compression sonography. To enhance characterization of venous flow, we developed the continuous flow directionality index (FDI), integrating time spent in antegrade, retrograde, and stagnant flow. Compared with 1 g baseline, microgravity was associated with a larger IJV cross-sectional area and lower IJV pressure. Increasing LBNP reduced both variables across gravity conditions. In microgravity, IJV flow at LBNP of 0 mmHg included periods of stagnation, whereas -20 and -30 mmHg progressively improved venous return, reflected by higher FDI values. FDI provided greater granularity than traditional qualitative grading, revealing differences in flow quality otherwise difficult to detect. These findings demonstrate the feasibility and physiological relevance of graded LBNP during true microgravity to mitigate cephalad fluid shift-related alterations in jugular venous hemodynamics and introduce a sensitive quantitative approach for evaluating venous flow. This work establishes a foundation for future multisubject studies aimed at optimizing countermeasures for long-duration spaceflight.NEW & NOTEWORTHY Microgravity alters jugular venous flow, which in turn may increase thrombosis risk. In this graded lower body negative pressure experiment performed in true microgravity conditions during parabolic flight, we show that increasing negative pressure improves venous drainage in a dose-dependent manner. A new flow directionality index reveals subtle changes in flow not captured by existing grading systems, supporting lower body negative pressure as a practical countermeasure to mitigate stagnant internal jugular vein blood flow during future space missions.
BackgroundSpatial transcriptomics (ST) technologies are reshaping our understanding of tissue organization and cellular context in health and disease. However, technical benchmarking across platforms remains limited, particularly in formalin-fixed, paraffin-embedded (FFPE) clinical samples, which represent the most common tissue format in oncology.ResultsHere, we systematically benchmark five commercial ST platforms (Visium v1, Visium v2/CytAssist, Visium HD, Xenium, and CosMx) using matched FFPE human tumor sections from six cancer types. Uniquely, our study includes both sequencing-based and imaging-based platforms profiled on the same samples, enabling direct technical comparisons across spatial capture modalities. We evaluate platform performance across multiple dimensions, including transcript and UMI detection, gene-histology concordance, cell type recovery, and integration with a targeted protein panel (Visium v2, 30 proteins), enabling spatial multi-omics. We also quantify the impact of sampling strategies and area coverage on cell type estimation, revealing trade-offs in spatial resolution versus tissue context. Notably, we present the first same-sample comparison of Xenium Multi-Tissue (377 genes) and Xenium Prime (5,000 genes), highlighting key differences in transcript recovery and spatial signal despite shared chemistry and imaging infrastructure. Finally, we integrate Visium targeted protein data with matched RNA profiles, uncovering widespread RNA-protein decoupling and spatial heterogeneity in concordance.ConclusionsCollectively, this work provides a harmonized dataset and technical reference for the spatial transcriptomics community, offering insight into the relative strengths, limitations, and design considerations associated with high-throughput spatial profiling of FFPE tumors.
Developmental transcriptional programs are increasingly recognized as key drivers of paediatric gliomagenesis. Here, we unexpectedly find that the forebrain-specific doublesex- and mab-3-related transcription factor a2 (DMRTA2), despite being highly expressed in H3G34-mutant diffuse hemispheric glioma (DHG-H3G34) and implicated in tumour initiation, is not required for the maintenance of established patient-derived tumour cells. These findings support a stage-specific framework in which transcriptional dependencies are dynamically rewired during tumour progression in paediatric brain tumours.
DNA ends generated by double-strand breaks are vulnerable intermediates that must be rapidly recognized, protected, and resolved to preserve genome integrity. We present optical tweezers (OT)-Curtains, a single-molecule method inspired by DNA curtains that uses a custom branched DNA substrate containing multiple accessible ends for simultaneous observation on dual-trap OT coupled to confocal fluorescence microscopy. Eliminating DNA surface anchoring, facilitating rapid protein and buffer exchange, and offering the possibility for force-free experiments, OT-Curtains overcomes common limitations of flow-stretch-based methods. OT-Curtains allows real-time visualization and quantification of end recognition, protection, resection, and cleavage at several DNA ends in parallel. We demonstrate compatibility with well-studied DNA-binding systems by monitoring Ku-mediated DNA break recognition, AddAB-mediated DNA break resection, ParB-mediated DNA condensation, and KpnI-mediated DNA cleavage. We show that kinetic and mechanistic parameters can be extracted from the data under defined forces and solution conditions. OT-Curtains offers an accessible and multiplexed route to interrogate DNA-end transactions central to double-stranded DNA break repair pathways and telomere biology, as well as a general framework for benchmarking proteins acting at DNA ends.
BACKGROUND:Pathologic response is an emerging surrogate marker for therapeutic efficacy and long-term patient outcomes. Updated guidelines for pan-tumor pathologic response assessment have recently been adopted for ongoing clinical trials and routine clinical care. The goal of this study was to prospectively evaluate the interobserver variability among pathologists in assessments of treatment response across tumor types, anatomic locations, and specimen types. MATERIALS AND METHODS:A multi-institutional, international study led by the Society for Immunotherapy of Cancer was carried out to assess the concordance of pathologic response assessment using the pan-tumor criteria in neoadjuvant-treated resection specimens and on-treatment biopsies. Online lecture-based modules for scoring were developed, and 14 pathologists from multiple institutions were trained. The pathologists then assessed 42 specimens representing 12 different tumor types (total of n = 362 hematoxylin-eosin-stained slides) for the three tissue classes that are assessed: %residual viable tumor (RVT), %regression, and %necrosis. Pathologists were also surveyed regarding interest in and barriers related to pan-tumor pathologic response assessment, as well as quality and effectiveness of the training materials. RESULTS:Scoring of pathologic response using the pan-tumor system was highly reproducible, irrespective of disease location (primary tumor versus lymph node) or specimen type (resection versus biopsy), with intraclass correlation coefficients (ICCs) > 0.8 for %RVT, %regression, and %necrosis. Subset analyses also showed strong reproducibility of %RVT within almost all individual tumor types evaluated (ICC all ≥ 0.86). The poststudy survey completed by the participating pathologists was used to refine the training materials, and the revised modules are provided as a resource to the wider pathology and oncology communities. CONCLUSIONS:This highly reproducible scoring system enables quantitative assessment of treatment response in pathology specimens across multiple tumor types, anatomic sites, disease stages, and therapies, akin to RECIST for radiographic assessment. We identified potential barriers to implementation and highlight strategies to overcome these challenges.
