Interface Focus

Known for their rapid water surface locomotion, whirligig beetles (Gyrinidae) are also fast divers. They can transition from the surface to underwater swimming in under 100 ms. Diving requires them to generate sufficient hydrodynamic torque to break the water surface and reorient the body. However, the mechanism by which whirligigs create the necessary pitch-down torque is unknown. Here, we show that their diving is achieved by bending the abdomen. The inertial hydrodynamic pressure on the flexed abdomen generates the torque necessary for surface breaking and body rotation. The torque is maximized at an abdominal angle of 45{degrees}, whereas for effective diving, whirligigs display a preferential abdominal angle of around 28{degrees}, closely aligning with the angle that maximizes the torque-to-drag ratio. This abdominal angle is optimized by balancing torque generation with the drag penalty. Our findings inspire a mechanism for surface-to-underwater transition, offering design principles for multimodal robots capable of locomotion across the air-water interface.

In breast cancer, collagen fiber alignment and the second harmonic generation forward-to-backward ratio (F/B), an optical measurement sensitive to fibril-scale organization within collagen fibers, have each been associated with metastatic outcome. Although both are measured at the tumor-stromal interface, in vitro studies have largely examined them separately, because experimental systems have not allowed the two to be varied independently. To address this technical gap, we used a microfluidic biofabrication approach to generate paired aligned and unaligned regions within 3D collagen hydrogels via extensional strain and varied measured F/B across gels by changing the pre-gel pH. The resulting library covered alignment values reported in breast tumors and F/B values associated with poorer metastatic outcome in invasive ductal carcinoma at matched matrix stiffness, pore diameter, and fiber fraction. In MDA-MB-231 cells, alignment biased migration along the fiber axis without changing migration speed, as expected, but the magnitude of the contact guidance response varied with measured F/B. In contrast, MCF-7 cells showed little overall contact guidance, yet migrated ~30% faster in high F/B than low F/B gels in both aligned and unaligned regions. Together, these results show that cells migrated differently in matrices with similar fiber alignment. Thus, including measured F/B may provide additional information to help anticipate how cells migrate in collagen environments that appear similar based on alignment alone.

Background: The Central Brain Tumor Registry of the United States (CBTRUS) uses a histology grouping model based on the World Health Organization (WHO) to classify cancer registry records for clinically relevant statistical reporting. Methods: CBTRUS, together with its consulting neuropathologists, reviewed and aligned its histology groupings with the 2021 WHO Classification update. Obsolete histology nomenclature along with their International Classification of Diseases for Oncology 3rd edition (ICD-O-3) codes were identified, currently included histologies were reclassified as appropriate, and new codes found in 2021 WHO were incorporated. An evaluation was conducted of the frequency of histology codes affected in the reclassification process, as well as incidence and survival pre- and post- reclassification of CBTRUS Tumor Type Groupings. Results: After review of the CBTRUS grouping scheme, 205 codes were reclassified and 2 new codes were incorporated. Tumor type groups most affected were those classified as glioma. Reclassification resulted in 167,038 of 491,804 (34%) cases having a grouping reclassification or reporting change, indicating that the 2021 revision of the WHO Classification has significantly impacted the collection and reporting of primary b CNS tumors. Conclusion: This work demonstrates the need to be responsive to changes in classification and coding for statistics to reflect real-world clinical use. This will require collaboration from all parties to have the ability to reconcile clinical practices and surveillance requirements.

Solid tumors are known to be refractory to treatment due to a poorly perfused microenvironment that blocks immune cell infiltration and effective drug delivery. Histotripsy is a non-invasive focused ultrasound modality that generates cavitation microbubbles to mechanically disrupt solid tumors, resulting in decreased hypoxia and stimulation of tumor-targeted adaptive immune responses. In this study, we aim to investigate whether post-histotripsy hypoxia reduction and cytotoxic immune cell infiltration are associated with normalization of tumor vasculature and enhanced perfusion. Structural analysis of both treated and contralateral off-target tumors following partial histotripsy revealed significant vascular remodeling, enhanced vascular integrity, and decreased vascular leakage indicative of vascular normalization in multiple in vivo models of melanoma, hepatocellular carcinoma, and pancreatic adenocarcinoma. These effects appeared antigen-specific, as no remodeling was observed in contralateral off-target tumors of discordant pathology. Mechanistically, CXCR3+CD8+ T cells played a role in mediating these vascular changes, as genetic knockout of CD8 and pharmacological antagonism of CXCR3 impaired both remodeling and normalization. Additionally, significant changes in endothelial CXCR4 and Angiopoietin-1 and -2 expressions were observed following histotripsy, indicating their potential involvement in promoting vascular normalization and remodeling. Significant vascular remodeling was accompanied by enhanced tumor perfusion and intratumoral delivery of chemotherapeutic agents and therapeutic monoclonal antibodies. These findings suggest that histotripsy not only creates a more favorable microenvironment for immunotherapy, but also optimizes drug delivery into solid tumors. Combinatorial approaches using histotripsy with chemotherapy or immunotherapy may be a novel therapeutic strategy to overcome current limitations of conventional solid tumor therapies.

Devireddy et al. [1] previously showed that an enthalpy-based, macroscale model simulated tissue freezing histories are in close agreement with a coupled model incorporating cellular water transport and intracellular ice formation (IIF). That comparison, however, was performed for a limited set of tissue geometries, cooling conditions, and biophysical parameters. Here, we revisit that conclusion by reimplementing the published model from its governing equations and systematically expanding the parameter space to determine when microscale processes alter the predicted macroscale freezing response. We incorporate both surface catalyzed nucleation (SCN) and volume catalyzed nucleation (VCN) of intracellular ice with cellular osmotic dehydration and examine a broad range of tissue dimensions, cooling rates, convective boundary conditions, membrane permeabilities, cell sizes, activation energies, intracellular water fractions, and nucleation rates. Linearization of the cellular water transport equation about osmotic equilibrium yields an osmotic relaxation timethat can be compared with the local residence time of tissue undergoing phase change. Their ratio defines a dimensionless group, that organizes the transition between regimes in which the uncoupled enthalpy formulation and the coupled micro and macroscale formulation produce similar or substantially different results. Across the one-dimensional parameter space examined here, the two models remain within a prescribed thermal-error bound for the dimensionless group below approximately 0.1, whereas the discrepancy increases systematically above this threshold. Model divergence also depends on intracellular ice nucleation, i.e., when nucleation is sufficiently slow, cellular water transport can become rate limiting for latent heat release. Conversely, sufficiently rapid intracellular ice formation reduces the macroscopic discrepancy by providing an additional pathway for latent-heat release. Multidimensional simulations further show that geometry alone can move a nominally conventional freezing protocol into the coupled regime. In particular, multidirectional cooling can reduce the local residence time sufficiently to produce substantial differences in predicted frozen volume even when the corresponding thermal histories appear similar. Finally, we formulate an inverse approach for estimating IIF parameters from tissue scale thermal histories. Synthetic simulations indicate that parameter recovery is feasible within a finite range of cooling conditions, providing a testable strategy for future experimental measurements. Together, these results provide a mechanistic criterion for deciding when microscale biophysical processes must be retained in macroscale tissue-freezing models.

Healing of the gastrointestinal (GI) tract following surgical interventions is governed by coupled mechanobiological processes and may lead to severe complications such as perforation or stricture formation. A first comprehensive mechanobiological modeling framework of GI healing is herein proposed. The framework addresses two clinically relevant applications of bioprinting: endoscopic treatment of GI tissue defects and bioprinting-assisted surgical anastomosis. Tissue remodeling is based upon a hyperelastic material model with fiber dispersion coupling the spatiotemporal reaction diffusion dynamics of fibroblasts and cytokines with collagen deposition, reorientation, and plastic deformation. An active stress formulation drives fibroblasts and myofibroblast contraction while a multiplicative decomposition approach is adopted for collagen remodeling as a permanent change. The governing equations are discretised in time with an implicit Euler scheme, finite element in space and solved by a staggered algorithm implemented in the open-source platform FEniCS. Numerical simulations show that the initial collagen fibers content and orientation strongly influence fibroblast infiltration and scar contracture formation. The study quantifies the advantage of having a collagen bioprinted architecture aligned with tissue fibers, to reduce structural remodeling distortion. The model also predicts a strong correlation between collagen deposition and luminal burst pressure during the recovery of mechanical integrity in GI anastomosis.

Lumens populate most of our organs and their size, which dynamically changes throughout the day and lifetime, can influence their function. During mammalian development, the first lumen appears in the blastocyst. Surface epithelial cells pump ions to build an osmotic gradient that draws water, which inflates the embryo and stretches the epithelium as well as the zona pellucida, an elastic shell protecting the embryo. Blastocyst expansion is not steady and instead undergoes cycles of expansion interrupted by collapses during which the lumen leaks out. Early studies identified the critical role of osmotic pressure difference in regulating the expansion of the blastocyst whereas recent studies proposed that mechanical stresses would set the size of the embryo. However, the relative contributions of osmotic and mechanical stresses to lumen expansion dynamics remain unclear. Here, we investigate this using microfluidics, biophysics, machine learning and modelling on mouse embryos. Breaking down the expansion dynamics of mouse embryos, we discovered that fluid pumping rate accelerates, compensating for the increased frequency of collapses interrupting episodes of expansion. We reasoned that fluid transport could be rate limited by osmotic pumping, water permeability and/or mechanical constraints. Removing the mechanical constraint from the zona pellucida only marginally affected these expansion dynamics. Forcing fluid exchange with osmotic shocks we measured that water permeability is large enough not to restrict fluid accumulation rates. Finally, we estimate ion pumping for mouse blastocysts and interfere with it using inhibitors to measure how it quantitatively sets blastocyst expansion rates. Together, we propose that osmotic pressure differences, regulated by fluid transport properties, dominate over mechanical stresses in controlling blastocyst expansion dynamics.

A densely vascularized tissue can still be starved of oxygen. Tissue oxygenation depends heavily on vessel arrangement, blood flow distributions, and cellular metabolism. Resolving these effects from individual constituent vessels to whole organs has historically been computationally prohibitive. We introduce the Cardiovascular Analysis and Simulation of Circulatory Advection, Diffusion, and Exchange (CASCADE) toolkit, which makes vessel-resolved oxygen transport tractable at the organ scale through a novel screened Green's-function formulation. On laptop-class hardware, CASCADE simulates networks containing thousands of vessels in under one second and a full biventricular human heart model in about two minutes. We validate spatial oxygen and cell-survival predictions against experiments in perfused engineered tissues and matched finite-element simulations. In engineered tissues, sweeps across thousands of network designs and physiological conditions reveal when vessel density, oxygen supply, or network architecture limits viability, establishing design rules for sustaining thick constructs. In a human heart model, CASCADE translates coronary occlusion, anemia, polycythemia, and hypoxemia into distinct myocardial oxygenation patterns, revealing where oxygen delivery fails and how much tissue is at risk.

Epithelial cells of the female reproductive tract (FRT) form a protective barrier and serve as the first line of defense against sexually transmitted infections (STIs). They recognize pathogens, release cytokines and initiate innate immune responses. Sex hormones 17{beta}-estradiol (E2) and progesterone (P4) fluctuate throughout the menstrual cycle and influence epithelial cell functions. Generally, E2 confers protection against viral STIs like HIV-1 and HSV-2, whereas P4 increases susceptibility. However, the underlying mechanisms remain incompletely understood. MicroRNAs, small non-coding RNAs that regulate gene expression by inhibiting translation of target mRNAs, significantly influence viral infections, by promoting or restricting viral pathogenesis. Here, we examined the effect of sex hormones on microRNA expression in vaginal epithelial cells (VECs), the first responders to pathogens in the FRT. Using next generation sequencing analysis, the effect of physiological concentrations of E2 or P4 on microRNA transcription profile of VECs grown in air-liquid interface cultures was characterized. Thirteen miRNAs were uniquely regulated by E2 and 102 by P4, with 29 miRNAs that overlapped between both. The top four differentially expressed miRNAs from both treatments were validated by RT-qPCR. The mRNA targets of unique E2-miRNAs were involved in adherens junction, tight junction and chemokine signaling pathways; whereas the mRNA targets of unique P4-miRNAs were involved in MAPK signaling pathway. Interestingly, several miRNAs regulated by E2- (eg. hsa-miR-9-3p, hsa-miR-200c- 5p, hsa-miR-548c-5p) and by P4 (eg. hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-27b-3p) targeted genes related to cell cycle pathway. Together, these findings indicate that microRNAs regulated by E2 and P4 influence mRNAs in the pathways associated with cell adhesion, cell growth and development and innate immune responses, critical for maintenance of epithelial barrier integrity and regulating pathogen susceptibility in the vaginal tract. These hormone-regulated miRNAs may serve as potential targets for therapeutic interventions.

Motivation: Retrobiosynthesis tools use different reaction rules, search strategies and biochemical priors, so each can recover transformations missed by others. Integrating their outputs could broaden pathway coverage, but ensembling can also worsen ranking. Results: SynBioCrow runs independent retrosynthesis engines, normalizes predictions into a provenance-preserving reaction graph, and separates route generation from biochemical evidence, sequence design and bounded learning. On the frozen 65-path Galaxy-SynBioCAD literature benchmark used for SynBioCrow 2.3, connectivity-level partial recovery was 19/65 for the ensemble, 13/65 for RetroBioCat2, 9/65 for RetroPath and 0/65 for DORAnet; the ensemble preserved two exact RetroPath routes but worsened their ranks. SynBioCrow 2.4 subsequently repaired structured-reaction parsing and target-connected Candidate generation and prospectively locked two new external targets, 1,3-propanediol and 1,4-butanediol, before pathway truth was examined. RetroBioCat2 produced 3 and 2 backend-internal SOLVED search outcomes, respectively, whereas DORAnet produced none; however, neither backend recovered any predeclared explicit literature intermediate by exact structure. The negative prospective result was frozen without post-reveal tuning. Production ranking in 2.4 is therefore NONE and the release remains Candidate-stage; validated complete-pathway recovery is not claimed. Availability: SynBioCrow 2.4.0 is archived at DOI 10.5281/zenodo.23173173 with source code and frozen supporting evidence.

The human pancreas secretes digestive fluid that flows through an extensive ductal network before emptying into the duodenum. However, little is known about how hydrodynamic forces influence pancreatic function and development. We hypothesized that pancreatic cells respond to physiological levels of fluid shear stress. In this study, we developed a microfluidic system featuring reversible sealing between the device layer and the cell culture substrate, enabling (1) conventional open-well cell culture prior to fluid shear stress application and (2) selective patterning of proteins and cells for migration studies. Channel dimensions were chosen to support 24-hour perfusion using standard syringe pumps. Using this platform, we investigated effects of physiological shear stress levels representing basal and activated pancreatic function (2, 8 and 33 mPa) on human iPSC-derived pancreatic progenitor cells. Pancreatic progenitor cells exhibit graded sensitivity to shear stress at the transcriptomic level. Specifically, shear stress upregulated expression of cell cycle-related gene sets; endocrine-associated genes including NEUROG3; and Notch receptor ligands (DLL1, DLL3) in a magnitude-dependent manner. The exposure to unidirectional fluid flow also resulted in a reduction in YAP1 protein content, decreased WWTR1 gene expression, and downregulation of YAP/TAZ target genes. Furthermore, barrier-based migration assays under shear stress revealed that cell patches expanded their boundaries both along and against the flow, with greater distance in the direction of flow. Our system and findings may help lay the foundation for understanding biophysical regulation of developmental processes in a physiologically important, yet hard-to-access, epithelial model system.

Meteorite impact structures on earth represent unique terrestrial analogues of extreme planetary environments where fractured lithologies, hydrothermal alteration, elevated concentrations of radionuclides, and prolonged water-rock interactions create ecological niches for specialized microbial communities. The Palaeoproterozoic Dhala impact structure in north-central India is characterized by uranium-bearing impactites and possesses a very high concentration of Uranium (up to 99.5 ppm) that makes it a unique natural laboratory to investigate microbial adaptation to radioactive and metal-rich geological environments. In addition, the Dhala structure harbors specialized radiotoxic hydrothermal mineral phases such as coffinite and pitchblende, which show a globally distinct and biologically unexplored niche. The present investigation is the first in-depth, whole-genome metagenomic study of soil and water samples of uranium-rich sites from the Dhala structure, exploring the whole microbial community (Bacteria, Archaea, Eukaryotes, and Viruses) up to the species level present in the samples. The cross-domain metagenomics analysis identified a highly unique and resilient microbiome that consists of bacteria, archaea, eukaryotes, and viruses, coevolved under prolonged radiological and multiple metal stress. Functional metagenomic analysis revealed numerous metabolic adaptations responsible for their survival, homeostasis, and biomineralization in situ. The deep sequencing method further enabled the identification of 58 functional genes directly involved in uranium bioremediation. Furthermore, this study revealed important pathways for radionuclide reduction, cellular efflux, and biotransformation. Novel findings of the present study point out the in-depth evolutionary relations of biosphere-geosphere interaction within an ancient hypervelocity impact-generated ecosystem. In a nutshell, the Dhala extremophiles with novel genetic makeup present an untapped and promising reservoir for the development of eco-friendly, next-generation microbial bioremediation strategies to efficiently tackle the global anthropogenic contamination of uranium and the challenges of nuclear waste management. Thus, the present study establishes Dhala structure as an unparalleled site for environmental geomicrobiology, microbial evolution, and astrobiology.

Clinical records preserve patient findings and final recommendations but rarely capture how specialists select evidence, develop alternative interpretations, disagree, and converge on a management strategy. We describe a dataset derived from ten retrospectively selected, completed cases of histologically confirmed colorectal cancer. For each case, an administrator uploaded de-identified textual clinical records to an authenticated web-based annotation platform. A medical oncologist, surgical oncologist, and radiation oncologist, each with more than ten years of clinical experience, independently linked relevant source fragments to patient-specific interpretations and documented clinical problems, differential hypotheses, uncertainty, and diagnostic and treatment strategies. After the three records were locked, a large language model generated candidate questions for review by the tumour-board chair. The four physicians then discussed each case online; recordings were automatically transcribed and verified by two medical residents. The dataset comprises 30 source-linked specialist reasoning records, 10 verified case-level discussion transcripts, 10 final tumour-board decisions, and associated provenance and quality-control metadata. It supports research on evidence-grounded reasoning, cross-specialty disagreement, multidisciplinary decision-making, and multi-agent clinical decision support.

Objectives Contemporary oncology has achieved remarkable advances in biomedical treatment while increasingly emphasizing patient-centered care. Nevertheless, routine oncology consultations must address multiple competing clinical priorities, potentially limiting opportunities for patients' experiences to become visible. To address this gap, we established a Dialogue-Based Outpatient Clinic, a complementary patient-directed consultation model embedded within routine oncology care. This study explored the dimensions of patients' experiences that became visible through this consultation model. Methods We conducted a qualitative study of 44 consecutive patients who attended a Dialogue-Based Outpatient Clinic embedded within routine oncology care between 2021 and 2023. Patients attended the clinic either on their own initiative or following a recommendation from a healthcare professional who recognized that they were struggling. Regardless of how they came to the clinic, patients independently determined the consultation agenda. Results Of the 44 patients, 13 (29.5%) sought biomedical information regarding diagnosis, treatment, or adverse effects, whereas 31 (70.5%) discussed concerns extending beyond biomedical issues. Reflexive thematic analysis of these 31 patients identified four interconnected themes: (1) making sense of a cancer diagnosis within one's own life; (2) negotiating treatment in relation to personal values and life priorities; (3) being treated for disease but not seen as a person; and (4) reconstructing life and identity through the cancer journey. Across all themes, patient-directed dialogue consistently revealed how patients understood cancer within the broader context of their lives rather than as an isolated biomedical condition. Conclusions Patient-directed dialogue within routine oncology care revealed dimensions of patients' experiences that may be less readily recognized in conventional consultations. A complementary consultation model that allows patients to determine the agenda may provide opportunities for these experiences to become clinically recognizable.

Vector flow mapping (VFM) reconstructs left-ventricular (LV) blood velocity from color-Doppler echocardiography by combining the measured beamwise component with physical and regularizing constraints. Analysis of the discrete VFM formulation shows that the inverse problem is intrinsically ill posed: the occurrence of singular modes can be predicted from the geometry of the segmented blood-pool domain, the imposed boundary conditions, and the degree of smoothing. These modes can propagate uncertainty along entire transverse bands of the reconstructed velocity field, yet conventional VFM neither quantifies this uncertainty nor allows for correcting the blood-pool segmentation. We introduce Bayesian VFM (B--VFM), a hierarchical framework that jointly infers radial and transverse velocities, a probabilistic blood-pool mask, their spatially resolved uncertainties, and hyperparameters weighting Doppler and segmentation fidelity, mass conservation, boundary conditions, and smoothness. The discretized posterior admits a closed-form gradient and exact Hessian, enabling computationally efficient, gradient-based MAP estimation, sampling, and direct analysis of ill-posed modes. Posterior inference combines Gibbs sampling of conjugate Gamma-distributed hyperparameters with conditional maximum-a-posteriori estimation and a Laplace approximation for the high-dimensional velocity and mask fields. To accommodate systematic departures from planar mass conservation, B-VFM can learn the covariance of the planar divergence residual from an ensemble of flows and incorporate it as a structured model-discrepancy prior. Independent chains converged reproducibly, while covariance priors learned from flow ensembles illustrated how model discrepancies can be incorporated into the inference. B--VFM was evaluated using Lamb-Chaplygin dipoles under ideal conditions and with Doppler corruption, Doppler voids, and segmentation defects, and using the Hicks-Moffatt family of spherical vortices to assess violations of planar mass conservation. The method produced smooth reconstructions, localized uncertainty near unreliable measurements and regions of model inconsistency, and used flow information to correct segmentation errors. Within the tested vortex family, the data-informed planar divergence prior reduced velocity bias and mask distortion. B--VFM thus provides an uncertainty-aware reconstruction method and a flexible foundation for future VFM formulations incorporating additional priors, observations, and physical models. Future work will evaluate the method using clinical data and more complex three-dimensional benchmark flows.

The chick embryo is a widely used vertebrate model for developmental biology, cardiovascular, and bioengineering research. We present an automated laser speckle contrast imaging (LSCI) platform for non-invasive, longitudinal, label-free imaging of the developing heart and extraembryonic vasculature of the chick embryo through the intact eggshell. The platform can detect blood vessels as small as 70 +/- 17 um in diameter and enable repeated imaging from day 0 to day 8 of incubation without disturbing normal embryonic development. It provides both structural (a map of growing extraembryonic blood vessels) and functional (blood flow dynamics and heart rate) information. Using this system, we generated the first continuous, non-invasive, time-lapse recordings of the early functioning chick heart and its extraembryonic vessels. Longitudinal imaging was performed on 15 embryos from different chicken breeds with eggshell colors white, brown and green. Analysis revealed slight variability in early cardiac and vascular morphogenesis across breeds and shell types and shows its potential for automated developmental staging. Finally, for the first time, the growing vasculature of the yolk sac and the chorioallantoic (CAM) membrane was imaged from day 4 to 8. The heart rate and growth of the extraembryonic blood vessels were quantified automatically during imaging.

Nanoplastic interactions with cell membranes can influence particle uptake, translocation, and biological effects, making their molecular description important for assessing the environmental and biological consequences of plastic pollution. However, molecular simulations generally represent nanoplastics using monodisperse polymer chains, whereas nanoplastics formed by environmental degradation are polydisperse. Here, we construct 4 nm polyethylene terephthalate (PET), polyethylene (PE), and polystyrene (PS) nanoparticles with lognormal molecular-weight distributions reported for degraded polymers, and with melt-like chain packing and entanglement. Using Martini coarse-grained molecular dynamics, we simulate their interactions for 10 microseconds with three lipid bilayers of increasing complexity. PE and PS insert rapidly and remain largely intact, whereas PET remains surface-associated and progressively releases individual chains. Fluid, polyunsaturated lipid environments undergo greater deformation and promote substantially more chain release, up to 56% of the chains for PET and 43% for PS. Release begins with the shortest chains and progressively extends to longer chains, while semicrystalline PE remains largely intact. In contrast, a size-matched monodisperse PS particle releases no chains and minimally perturbs the membrane. These results identify the molecular-weight distribution as a key determinant of nanoplastic-membrane interactions, and indicate that uniform model particles underestimate the release of polymer chains into biological membranes.

Nanobubbles (NBs) have been widely investigated as ultrasound contrast and therapeutic agents, yet whether submicron gas bubbles can directly generate measurable ultrasound signals remains controversial. Because acoustic scattering scales with bubble size, several studies have recently attributed nanobubble echogenicity primarily to larger bubbles. A recent Science Advances publication, attributed the loss of ultrasound signal after filtration to the removal of residual larger bubbles [1]. This interpretation assumes that filtration acts solely as a size-selection process. Here, we show that filtration also alters the shell properties and stability of lipid-coated NBs, independent of size. Filtered NBs (F-NBs) show significant lipid loss and accumulation on the filters themselves, resulting in decreased bubble stability and greater susceptibility to destruction under acoustic excitation. To decouple size from processing effects, we generated NB populations size-matched to filtered samples using increased centrifugation (IC-NBs), a comparatively gentler separation method. Despite comparable size distributions, IC-NBs retained sustained ultrasound scattering, whereas filtered NBs showed significantly reduced acoustic persistence. Filtration through 5 m pores, ~5-fold larger than the largest bubbles, preserved acoustic persistence but still reduced bubble concentration by ~30%, suggesting that membrane contact may contribute to bubble loss beyond size exclusion. Complementary lipid and gas-content assays provided further evidence of filtration-induced changes. The 0.45 m filtration removed substantially more total lipid than could be attributed to free-lipid removal alone, while the lipid associated with the larger bubbles removed during filtration accounted for only ~5% of the additional lipid loss. Encapsulated gas content remained comparable to that of size-matched centrifuged controls, suggesting that the reduced acoustic stability of filtered NBs is more closely associated with changes in lipid content than with loss of the gas core. These results indicate that membrane filtration is not a purely size-selective process: it removes substantially more lipid than size exclusion predicts and yields bubbles that are less stable under ultrasound, most likely through changes in shell lipid content or in the surrounding lipid reservoir, underscoring the importance of processing-induced effects in interpreting nanobubble echogenicity.

Lymphatic endothelial cell (LEC) function is strongly tied to transitions between button- and zipper-like junctions that control fluid flow into vessels. Published methods quantifying junction morphology are limited to single cell analysis or use few quantitative metrics to describe junctions across LEC monolayers. Therefore, a novel 3-step image analysis pipeline was developed to identify new metrics that quantify differences in button- and zipper-like junctions in LECs. The pipeline used Cellpose, CellProfiler, and Fiji to analyze VE-cadherin expression across lymphatic vessels within a 3D microfluidic chip and quantify VE-cadherin radial intensity, number of objects, object size, branchpoints, end-points, and junction thickness. Tumor necrosis factor (TNF)- exposure produced but-ton-like junctions, and unexposed cells were zippered. Radial intensity was heightened at the cell membrane in zipper-like junctions while perinuclear intensity increased for but-ton-like junctions. Increased branchpoints, endpoints, objects per cell, and junction thick-ness were associated with junction buttoning, while increased object size was associated with junction zippering. Principle component analysis results reinforced these trends and identified metric combinations that represented each junctional state. Overall, this semi-automated pipeline quantified differences in VE-cadherin junction morphology across LEC monolayers and generated new quantitative metrics to expand the tools available for studying junction morphology in lymphatic vasculature.

PURPOSE: To demonstrate an end-to-end research workflow combining a large electronic health record (EHR) resource and a multi-agent artificial intelligence (AI) system governed by Human-Guided Orchestration (HGO), using sequential sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) orders in breast cancer as the clinical use case. METHODS: A June 2026 EHR release contained 4.95 million patients, including 156,051 with breast cancer codes. Muse Spark supported cohort construction, executable statistical analysis, data-quality checks, literature retrieval, and reporting executed through Symbiotic Agent Teaming (SAT) under investigator direction. The main clinical measure was the second-drug record span, from first to last order. Analyses were descriptive and unadjusted; orders did not confirm administration or progression. RESULTS: The workflow identified 106 patients: 73 had T-DXd ordered first and 33 had SG ordered first. Median second-drug spans were 63 days (interquartile range [IQR], 29-116) for SG after T-DXd and 49 days (IQR, 21-126) for T-DXd after SG; the median difference was -14 days (95% bootstrap CI, -41 to 21; distributional P = 0.42). Reported outputs additionally included cohort definitions, aggregate analyses, evidence context, figures, and a preliminary draft manuscript. CONCLUSION: This study provides a concrete model for integrating large EHR resources, agentic AI, and human expertise across the research cycle. The ADC use case illustrates how computational execution and Expert-in-the-Loop (EITL) adjudication can produce interpretable research outputs and refine their scientific meaning, providing a foundation for extending and evaluating this approach across clinical questions.