Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model.

Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. Osmotic pressure gradients are considered the drivers of lumen expansion across development, but their role in human epiblasts is unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based epiblast model using engineered hydrogels. We find that leaky junctions prevent osmotic pressure gradients in early epiblasts and, instead, forces from apical actin polymerization drive lumen expansion. Once the lumen reaches a radius of ∼12 µm, tight junctions mature, and osmotic pressure gradients develop to drive further growth. Computational modeling indicates that apical actin polymerization into a stiff network mediates initial lumen expansion and predicts a transition to pressure-driven growth in larger epiblasts to avoid buckling. Human epiblasts show transcriptional signatures consistent with these mechanisms. Thus, actin polymerization drives lumen expansion in the human epiblast and may serve as a general mechanism of early lumenogenesis.

Scoping review of magnetic resonance motion imaging phantoms.

To review and analyze the currently available MRI motion phantoms. Publications were collected from the Toronto Metropolitan University Library, PubMed, and IEEE Xplore. Phantoms were categorized based on the motions they generated: linear/cartesian, cardiac-dilative, lung-dilative, rotational, deformation or rolling. Metrics were extracted from each publication to assess the motion mechanisms, construction methods, as well as phantom validation. A total of 60 publications were reviewed, identifying 48 unique motion phantoms. Translational movement was the most common movement (used in 38% of phantoms), followed by cardiac-dilative (27%) movement and rotational movement (23%). The average degrees of freedom for all phantoms were determined to be 1.42. Motion phantom publications lack quantification of their impact on signal-to-noise ratio through standardized testing. At present, there is a lack of phantoms that are designed for multi-role as many currently have few degrees of freedom.

MEA-NAP compares microscale functional connectivity, topology, and network dynamics in organoid or monolayer neuronal cultures.

Microelectrode array (MEA) recordings are commonly used to compare firing and burst rates in neuronal cultures. MEA recordings can also reveal microscale functional connectivity, topology, and network dynamics-patterns seen in brain networks across spatial scales. Network topology is frequently characterized in neuroimaging with graph theoretical metrics. However, few computational tools exist for analyzing microscale functional brain networks from MEA recordings. Here, we present a MATLAB MEA network analysis pipeline (MEA-NAP) for raw voltage time-series acquired from single- or multi-well MEAs. Applications to 3D human cerebral organoids or 2D human-derived or murine cultures reveal differences in network development, including topology, node cartography, and dimensionality. MEA-NAP incorporates multi-unit template-based spike detection, probabilistic thresholding for determining significant functional connections, and normalization techniques for comparing networks. MEA-NAP can identify network-level effects of pharmacologic perturbation and/or disease-causing mutations and, thus, can provide a translational platform for revealing mechanistic insights and screening new therapeutic approaches.

Structured information extraction from scientific text with large language models.

Extracting structured knowledge from scientific text remains a challenging task for machine learning models. Here, we present a simple approach to joint named entity recognition and relation extraction and demonstrate how pretrained large language models (GPT-3, Llama-2) can be fine-tuned to extract useful records of complex scientific knowledge. We test three representative tasks in materials chemistry: linking dopants and host materials, cataloging metal-organic frameworks, and general composition/phase/morphology/application information extraction. Records are extracted from single sentences or entire paragraphs, and the output can be returned as simple English sentences or a more structured format such as a list of JSON objects. This approach represents a simple, accessible, and highly flexible route to obtaining large databases of structured specialized scientific knowledge extracted from research papers.

Use of Novel National Data Sets to Monitor Chemotherapy Use and Outcomes: A Retrospective Cohort Study of Non-Small-Cell Lung Cancer in Aotearoa New Zealand.

PURPOSE: Te Aho o Te Kahu, the New Zealand Cancer Control Agency, is establishing a systemic anticancer therapy (SACT) database (Anti-Cancer Therapy-Nationally Organized Workstream [ACT-NOW]) which can be linked to other national health data collections. In this article, we explore the application of ACT-NOW data in the monitoring of uptake and outcomes after the public funding of pemetrexed in Aotearoa New Zealand. METHODS: We used the ACT-NOW collection to identify patients with advanced nonsquamous non-small-cell lung cancer, who were treated with first-line platinum-based doublet chemotherapy over an 8-year period. Data were extracted for a period of 4 years before and 4 years after the national funding of pemetrexed (November 1, 2017). Treatments were classified as historical platinum doublet (cisplatin or carboplatin with gemcitabine, vinorelbine, paclitaxel, or docetaxel) or platinum pemetrexed doublet (cisplatin or carboplatin with pemetrexed). The primary outcome was the proportion of patients receiving each treatment type, before and after November 1, 2017. To prototype linkage to outcomes data, we evaluated hospitalization and 1-year overall survival (OS) rates by treatment. RESULTS: A total of 331 patients were included from four cancer centers. All patients (116 of 116) who were treated with first-line platinum-based doublet chemotherapy between November 2013 and November 2017 received historical platinum doublet chemotherapy. After the introduction of pemetrexed, between November 2017 and November 2021, 94% (203 of 215) were treated with platinum pemetrexed doublet chemotherapy and 6% (12 of 215) with historical platinum doublet chemotherapy. Linkage to outcomes data for 1-year OS, hospitalization rates, and lengths of stay outcome data were achievable. CONCLUSION: The ACT-NOW data set has the potential to facilitate evaluation of the impact of national-level SACT funding decisions on prescribing practice and specific patient outcomes. Our results support the use of these data to inform resource planning and quality improvement.

Content-aware frame interpolation (CAFI): deep learning-based temporal super-resolution for fast bioimaging.

The development of high-resolution microscopes has made it possible to investigate cellular processes in 3D and over time. However, observing fast cellular dynamics remains challenging because of photobleaching and phototoxicity. Here we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo and Depth-Aware Video Frame Interpolation, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series post-acquisition. We show that CAFI is capable of understanding the motion context of biological structures and can perform better than standard interpolation methods. We benchmark CAFI's performance on 12 different datasets, obtained from four different microscopy modalities, and demonstrate its capabilities for single-particle tracking and nuclear segmentation. CAFI potentially allows for reduced light exposure and phototoxicity on the sample for improved long-term live-cell imaging. The models and the training and testing data are available via the ZeroCostDL4Mic platform.

Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes.

Cryogenic-electron tomography (cryo-ET) has provided an un-precedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET.

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context.

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

PDZ Domains from the Junctional Proteins Afadin and ZO-1 Act as Mechanosensors.

Intercellular adhesion complexes must withstand mechanical forces to maintain tissue cohesion, while also retaining the capacity for dynamic remodeling during tissue morphogenesis and repair. Most cell-cell adhesion complexes contain at least one PSD95/Dlg/ZO-1 (PDZ) domain situated between the adhesion molecule and the actin cytoskeleton. However, PDZ-mediated interactions are characteristically nonspecific, weak, and transient, with several binding partners per PDZ domain, micromolar dissociation constants, and bond lifetimes of seconds or less. Here, we demonstrate that the bonds between the PDZ domain of the cytoskeletal adaptor protein afadin and the intracellular domains of the adhesion molecules nectin-1 and JAM-A form molecular catch bonds that reinforce in response to mechanical load. In contrast, the bond between the PDZ3-SH3-GUK (PSG) domain of the cytoskeletal adaptor ZO-1 and the JAM-A intracellular domain becomes dramatically weaker in response to ∼2 pN of load, the amount generated by single molecules of the cytoskeletal motor protein myosin II. These results suggest that PDZ domains can serve as force-responsive mechanical anchors at cell-cell adhesion complexes, and that mechanical load can enhance the selectivity of PDZ-peptide interactions. These results suggest that PDZ mechanosensitivity may help to generate the intricate molecular organization of cell-cell junctions and allow junctional complexes to dynamically remodel in response to mechanical load.

Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.

Response of lymphatic endothelial cells to combined spatial and temporal variations in fluid flow.

One-way valves within lymphatic vessels are required for the efficient drainage of lymphatic fluids. Fluid flow is proposed to be a key cue in regulating both the formation and maintenance of lymphatic valves. However, to our knowledge, no previous study has systematically examined the response of LECs to the complex combination of spatially and temporally varying fluid flows that occur at lymphatic valves in vivo. We built an in vitro microfluidic device that reproduces key aspects of the flow environment found at lymphatic valves. Using this device, we found that a combination of spatially and temporally varying wall shear stresses (WSSs) led to upregulated transcription of PROX1 and FOXC2. In addition, we observed that combined spatial and temporal variations in WSS-modulated Ca2+ signaling and led to increased cellular levels of NFATc1. These observations suggest that the physical cues generated by the flow environment present within lymphatic valves may act to activate key regulatory pathways that contribute to valve maintenance.

Wildebeest herds on rolling hills: Flocking on arbitrary curved surfaces.

The collective behavior of active agents, whether herds of wildebeest or microscopic actin filaments propelled by molecular motors, is an exciting frontier in biological and soft matter physics. Almost three decades ago, Toner and Tu developed a continuum theory of the collective action of flocks, or herds, that helped launch the modern field of active matter. One challenge faced when applying continuum active matter theories to living phenomena is the complex geometric structure of biological environments. Both macroscopic and microscopic herds move on asymmetric curved surfaces, like undulating grass plains or the surface layers of cells or embryos, which can render problems analytically intractable. In this paper, we present a formulation of the Toner-Tu flocking theory that uses the finite element method to solve the governing equations on arbitrary curved surfaces. First, we test the developed formalism and its numerical implementation in channel flow with scattering obstacles and on cylindrical and spherical surfaces, comparing our results to analytical solutions. We then progress to surfaces with arbitrary curvature, moving beyond previously accessible problems to explore herding behavior on a variety of landscapes. This approach allows the investigation of transients and dynamic solutions not revealed by analytic methods. It also enables versatile incorporation of new geometries and boundary conditions and efficient sweeps of parameter space. Looking forward, the paper presented here lays the groundwork for a dialogue between Toner-Tu theory and data on collective motion in biologically relevant geometries, from drone footage of migrating animal herds to movies of microscopic cytoskeletal flows within cells.

Chromatin accessibility dynamics of neurogenic niche cells reveal defects in neural stem cell adhesion and migration during aging.

The regenerative potential of brain stem cell niches deteriorates during aging. Yet the mechanisms underlying this decline are largely unknown. Here we characterize genome-wide chromatin accessibility of neurogenic niche cells in vivo during aging. Interestingly, chromatin accessibility at adhesion and migration genes decreases with age in quiescent neural stem cells (NSCs) but increases with age in activated (proliferative) NSCs. Quiescent and activated NSCs exhibit opposing adhesion behaviors during aging: quiescent NSCs become less adhesive, whereas activated NSCs become more adhesive. Old activated NSCs also show decreased migration in vitro and diminished mobilization out of the niche for neurogenesis in vivo. Using tension sensors, we find that aging increases force-producing adhesions in activated NSCs. Inhibiting the cytoskeletal-regulating kinase ROCK reduces these adhesions, restores migration in old activated NSCs in vitro, and boosts neurogenesis in vivo. These results have implications for restoring the migratory potential of NSCs and for improving neurogenesis in the aged brain.

Co-opting signalling molecules enables logic-gated control of CAR T cells.

Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.

Piconewton forces mediate GAIN domain dissociation of the latrophilin-3 adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-Autoproteolysis Inducing (GAIN) domain, but the two resulting fragments remain associated on the cell surface. It is thought that force-mediated dissociation of the fragments exposes a peptide that activates G-protein signaling of aGPCRs, but whether GAIN domain dissociation can occur on biologically relevant timescales and at physiological forces is unknown. Here, we show using magnetic tweezers that physiological forces dramatically accelerate the dissociation of the latrophilin-3 GAIN domain. Forces in the 1-10 pN range were sufficient to dissociate the GAIN domain on a seconds-to-minutes timescale, and the GAIN domain fragments reversibly reassociated after dissociation. Thus, mechanical force may be a key driver of latrophilin signaling during synapse formation, suggesting a physiological mechanism by which aGPCRs may mediate mechanically-induced signal transduction.

Multi-level Force-dependent Allosteric Enhancement of αE-catenin Binding to F-actin by Vinculin.

Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via ß-catenin and α-catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in αE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell-cell junctions, which contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell-cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (-) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (-) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization.

Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors.

The visualization of mechanical stress distribution in specific molecular networks within a living and physiologically active cell or animal remains a formidable challenge in mechanobiology. The advent of fluorescence-resonance energy transfer (FRET)-based molecular tension sensors overcame a significant hurdle that now enables us to address previously technically limited questions. Here, we describe a method that uses genetically encoded FRET tension sensors to visualize the mechanics of cytoskeletal networks in neurons of living animals with sensitized emission FRET and confocal scanning light microscopy. This method uses noninvasive immobilization of living animals to image neuronal ß-spectrin cytoskeleton at the diffraction limit, and leverages multiple imaging controls to verify and underline the quality of the measurements. In combination with a semiautomated machine-vision algorithm to identify and trace individual neurites, our analysis performs simultaneous calculation of FRET efficiencies and visualizes statistical uncertainty on a pixel by pixel basis. Our approach is not limited to genetically encoded spectrin tension sensors, but can also be used for any kind of ratiometric imaging in neuronal cells both in vivo and in vitro.

Driving quality improvement through better data: The story of New Zealand's radiation oncology collection.

Aotearoa/New Zealand is one of the first nations in the world to develop a comprehensive, high-quality collection of radiation therapy data (the Radiation Oncology Collection, ROC) that is able to report on treatment delivery by health region, patient demographics and service provider. This has been guided by radiation therapy leaders, who have been instrumental in overseeing the establishment of clear and robust data definitions, a centralised database and outputs delivered via an online tool. In this paper, we detail the development of the ROC, provide examples of variation in practice identified from the ROC and how these changed over time, then consider the ramifications of the ROC in the wider context of cancer care quality improvement. In addition to a review of relevant literature, primary data were sourced from the ROC on radiation therapy provided nationally in New Zealand between 2017 and 2020. The total intervention rate, number of fractions and doses are reported for select cancers by way of examples of national variation in practice. Results from the ROC have highlighted areas of treatment variation and have prompted increased uptake of hypofractionation for curative prostate and breast cancer treatment and for palliation of bone metastases. Future development of the ROC will increase its use for quality improvement and ultimately link to a real time cancer services database.

Multiomic characterization and drug testing establish circulating tumor cells as an ex vivo tool for personalized medicine.

Matching the treatment to an individual patient's tumor state can increase therapeutic efficacy and reduce tumor recurrence. Circulating tumor cells (CTCs) derived from solid tumors are promising subjects for theragnostic analysis. To analyze how CTCs represent tumor states, we established cell lines from CTCs, primary and metastatic tumors from a mouse model and provided phenotypic and multiomic analyses of these cells. CTCs and metastatic cells, but not primary tumor cells, shared stochastic mutations and similar hypomethylation levels at transcription start sites. CTCs and metastatic tumor cells shared a hybrid epithelial/mesenchymal transcriptome state with reduced adhesive and enhanced mobilization characteristics. We tested anti-cancer drugs on tumor cells from a metastatic breast cancer patient. CTC responses mirrored the impact of drugs on metastatic rather than primary tumors. Our multiomic and clinical anti-cancer drug response results reveal that CTCs resemble metastatic tumors and establish CTCs as an ex vivo tool for personalized medicine.

Improved immunoassay sensitivity and specificity using single-molecule colocalization.

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.