Biology subjects

Park, W.

Publications and source records attributed to Park, W..

At least 19 recordsLinked to original sources

bioRxiv2026

Neoantigen-reactive CD8+ T cell engagement marks exceptional survivors of pancreatic cancer

Pancreatic cancer (PC) is largely refractory to immune checkpoint blockade (ICB), although homologous recombination-deficient (HRD) tumors may derive benefit. In the POLAR trial of maintenance pembrolizumab plus olaparib after platinum-based chemotherapy for metastatic PC, responses remained heterogeneous. To define determinants of productive antitumor immunity, we integrated longitudinal blood TCR sequencing with tumor single-cell and spatial profiling. Durable benefit was associated with rare tumor-infiltrating, peripherally expanding (TIE) CD8+ T cell clonotypes, a subset of which were functionally neoantigen-reactive. TIE patients showed markedly prolonged survival beyond established genomic and immune biomarkers. Conversely, resistance was associated with spatial T cell exclusion, myCAF-rich stromal remodeling, basal-like/KRAS-associated malignant-cell programs, and expansion of CTLA4 regulatory T cells linked to local immunosuppressive remodeling. These findings define a clonotype-resolved framework for immune monitoring and identify complementary stromal, tumor-intrinsic, and regulatory immune barriers that may guide rational combination immunotherapy in PC.

cancer biology↗

bioRxiv2026

Biologically Plausible Dopamine-Modulated STDP Model of Pavlovian Learning in Spiking Neural Networks

Spike-timing-dependent plasticity (STDP) and dopamine (DA) are fundamental to reward-based learning and memory formation. A widely used DA-modulated STDP model explains how neural networks associate stimuli with delayed dopaminergic rewards through an eligibility trace. However, we show that this model supports learning even at unrealistically high DA concentrations because DA simply scales the magnitude of STDP without changing its temporal profile. In contrast, experiments demonstrate that DA nonlinearly reshapes the STDP window, converting long-term depression (LTD) into long-term potentiation (LTP) at high DA levels. We therefore propose a DA-modulated STDP rule in which increasing DA progressively biases plasticity toward potentiation while receptor saturation limits further DA effects beyond a critical concentration. Simulations of recurrent networks of Izhikevich neurons show that the proposed rule supports robust conditioning only within a biologically realistic DA range (0.04-0.70 {micro}M). Successful learning produces a hybrid network architecture consisting of a strong feedforward backbone embedded within recurrent circuitry and generates enhanced burst responses selectively to reward-associated stimuli. At the upper limit of the biologically plausible DA range, the network passes through a narrow bistable regime, converging to one of two distinct stable configurations. At higher DA concentrations, conditioning fails altogether. These results provide a biologically grounded model of DA-dependent plasticity and offer new insight into how abnormal dopamine signaling can impair learning in neurological disorders.

neuroscience↗

bioRxiv2026

Lyophilization Maintains the Storage Stability and Bioactivity of Mesenchymal Stem Cell–Derived Extracellular Vesicles

BackgroundExtracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are emerging therapeutic candidates for bone regeneration, but their long-term stability remains a barrier to clinical translation. This study evaluated whether lyophilization supports the stable storage of extracellular vesicles derived from human epidural fat MSCs (hEF-MSCs) cell line. Research Design and MethodsEVs were isolated, lyophilized with 8.5 % sucrose and HEPES(4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and stored for 3 months at -80, -20, 4, and room temperature (20). After reconstitution in phosphate-buffered saline (PBS), structural characteristics were assessed using transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry. Functional activity was evaluated using MC3T3-E1 osteogenic differentiation assays. The main outcome measures included morphology, particle counts, marker expression, cytotoxicity, and sequencing profiles. ResultsLyophilization maintained EV morphology, structural integrity, and particle distribution across all storage temperatures. Marker expression remained comparable among the conditions. Reconstituted EVs promoted osteogenic differentiation of MC3T3-E1 cells without evidence of cytotoxicity. Sequencing profiles revealed no significant differences among storage conditions. ConclusionsLyophilized EVs from hEF-MSCs cell lines exhibited stable structural and functional properties across a range of storage temperatures, supporting their suitability for further development in bone regeneration applications.

Molecular Biology↗

bioRxiv2026

Lyophilized Extracellular Vesicles Retain Regenerative Activity and Accelerate Wound Healing

Extracellular vesicles (EVs) are promising therapeutic agents for tissue regeneration because they regulate intercellular communication and modulate inflammatory responses. However, preserving EV bioactivity during long-term storage remains a major barrier to clinical application. We examined whether lyophilized EVs stored at -80 {degrees}C maintain their structural integrity and therapeutic efficacy in an in vivo wound-healing model. Mesenchymal stem cell-derived EVs were isolated and freeze-dried before storage at -80 {degrees}C. We evaluated EV physicochemical characteristics before and after lyophilization using nanoparticle tracking analysis, transmission electron microscopy, and EV marker-expression analysis. To assess regenerative efficacy, lyophilized EVs were applied topically to full-thickness ear wounds in CCR2-GFP mice. Wound-healing progression and CCR2-positive cell infiltration were monitored during tissue recovery using intravital microscopy. Lyophilized EVs preserved their characteristic morphology, particle-size distribution, and EV surface marker expression after storage. In vivo analysis showed that EV-treated wounds closed significantly faster than phosphate-buffered saline-treated controls. Additionally, lyophilized EV treatment reduced CCR2-positive inflammatory cell recruitment during healing, suggesting an immunomodulatory role in tissue regeneration. These findings show that EVs lyophilized and stored at -80 {degrees}C retain biological function and therapeutic potential in vivo. Lyophilized EVs may, therefore, provide a practical strategy for long-term storage and delivery of EV-based regenerative therapeutics.

Cell Biology↗

bioRxiv2026

Rhizo-PET: A Dedicated PET System for 4D Imaging of Carbon Dynamics in the Rhizosphere

Imaging carbon movements in the rhizosphere is fundamentally limited by high soil heterogeneity, low signal levels, and lack of methodology. We present Rhizo-PET, a dedicated positron emission tomography (PET) imaging and analysis framework designed to characterize the 4D spatiotemporal patterns of tracer distribution in intact plant-soil systems. The system achieved a global energy resolution of 11.93 {+/-} 0.02% FWHM at 511 keV and maintained stable performance over 8 h of continuous acquisition, with a coincidence rate variation of only 0.7%. Spatial resolution reached 1.06 mm near the center of the field of view, establishing a high-fidelity region for root-scale analysis. Dynamic datasets were acquired from live Phaseolus vulgaris plants (N= 3) over 180 min following 11CO2 pulse labeling and reconstructed into 3 min temporal frames. Quantitative analysis across 243 independent regions of interest (ROI) revealed that cumulative tracer accumulation decreases monotonically with radial distance from the root axis, while axial transport delays increase systematically in lower root segments (p <0.001). Hierarchical variability analysis showed that within-plant spatial organization (CVTTP = 0.03) is significantly more stable than inter-plant variation (CVTTP = 0.14), proving that the observed heterogeneity reflects biological spatial organization rather than experimental instability. These results establish Rhizo-PET as a robust, reproducible platform for the non-invasive, time-resolved analysis of carbon dynamics in the rhizosphere under realistic soil conditions.

plant biology↗

bioRxiv2026

POLQ-driven repair scars shape the immunogenic landscape of homologous recombination-deficient pancreatic cancer

Pancreatic cancer (PC) is broadly resistant to immune checkpoint blockade, although a subset of homologous recombination-deficient (HRD) tumors exhibits durable immune engagement. The genomic features that distinguish these immune-responsive tumors from immune-inert HRD tumors remain poorly understood. Here we identify a microhomology-mediated end joining (MMEJ) repair scar, the MMEJ Deletion Footprint (MDF), as a genomic readout of POLQ-associated error-prone repair that enriches for frameshift indels. Across the multi-omic discovery cohort integrating tumor genomics, single-nucleus transcriptomics and spatial immune profiling, MDF-high HRD PC exhibited increased frameshift-indel-derived neoantigens and interferon programs. MDF was further associated with remodeling of the myeloid compartment toward MHC II-high dendritic cell-like antigen-presenting macrophage states and the immune synapse architecture marked by increased spatial interaction between APC-like macrophages and cytotoxic CD8+ T cells. These tissue-level features aligned with a functional trajectory shift of CD8+ T cells, consistent with effective anti-tumor immunity and was associated with favorable clinical outcomes of patients. Together, our findings position MMEJ-linked repair scarring as actionable biology that connects an HRD genotype to immune organization and suggests rational immunotherapy combinations that may enhance antigen presentation and myeloid activation to extend durable benefit in HRD-lineage cancers.

cancer biology↗

bioRxiv2025

VEGFR2 blockade overcomes acquired KRAS G12D inhibitor resistance driven by PI3Kγ activation

KRAS G12D mutation is a key oncogenic driver in many solid tumors, including pancreatic, gastric, and colorectal cancers. While recent studies have characterized features associated with primary and acquired resistance to KRAS inhibitors, strategies to overcome such resistance, particularly in the context of gastrointestinal cancers, remain underexplored. Here, we have generated nine human gastrointestinal cancer models, including three patient-derived organoids (PDOs), with acquired resistance to the KRAS G12D-selective inhibitor MRTX1133. Using single-cell RNA sequencing analysis, we identified the enrichment of angiogenesis, hypoxia, and epithelial-to-mesenchymal transition (EMT) signatures in the resistant model compared to the parental PDO. Across all resistant models, VEGFA expression and VEGFR2 phosphorylation were uniformly elevated, which were driven by AKT activation and SP1 nuclear translocation. Mechanistic investigations uncovered increased PI3K{gamma} activity in MRTX1133-resistant models via complex formation of KRAS with p110{gamma} and p101. This leads to an autocrine VEGFA-VEGFR2 signaling loop formation and EMT induction. Therapeutically, the disruption of VEGFA-VEGFR2 signaling restored MRTX1133 sensitivity and inhibited EMT. Furthermore, cancer-endothelial paracrine signaling amplified angiogenesis, hypoxia, and EMT signatures in cancer cells and simultaneously promoted endothelial cell proliferation, reinforcing an adaptive feedback mechanism. In a mouse model of MRTX1133-resistant tumor xenograft, a combination of anti-VEGFR2 therapy and MRTX1133 more effectively reduced tumor growth, angiogenesis, and proliferation markers than monotherapy without significant body weight change. These findings establish VEGFA-VEGFR2 signaling by PI3K{gamma} activation as a key driver of acquired resistance to KRAS G12D inhibition and provide a rationale for combining VEGFA-VEGFR2 inhibition with KRAS blockade in KRAS-mutant cancers. HighlightO_LIVEGFA-VEGFR2 signaling activation is a common feature of MRTX1133 resistance in KRASG12D cancer cells C_LIO_LINuclear translocation of SP1 by AKT activation promotes VEGFA transcription in MRTX1133-resistant models C_LIO_LIInteraction of p110{gamma}-p101 with KRAS activates PI3K{gamma} in the resistant models C_LIO_LIVEGFA-VEGFR2 inhibition reverses MRTX1133 resistance in vitro and in vivo C_LI

cancer biology↗

bioRxiv2025

Transcranial Direct Current Stimulation Modulates Resting Brain Hemodynamics and Autonomic Function: A Multimodal fNIRS-HRV Study

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique that can influence brain activity and physiological function. We conducted a sham-controlled multimodal study to examine the effects of low-intensity (0.375 mA) bifrontal tDCS at rest in healthy adult males. The tDCS, with the anode placed over the left dorsolateral prefrontal cortex (DLPFC) and the cathode over the right DLPFC, was applied for twelve minutes, and outcomes were measured with functional near-infrared spectroscopy (fNIRS) for cortical hemodynamics and connectivity, heart rate variability (HRV), photoplethysmography (PPG) for autonomic function, and subjective surveys for emotional stress. The results showed that, compared to sham, active tDCS produced a decrease in oxyhemoglobin (HbO) concentration in the left DLPFC during stimulation, indicating reduced cortical oxygenation in the stimulated region. Functional connectivity analysis of fNIRS signals further revealed altered network connectivity, including modulation of intra- and inter-frontal connections, in the tDCS condition relative to sham. Concurrently, tDCS induced an increase in HRV indices such as RMSSD, SDNN, pNN50, and Poincare plot measures SD1 and SD2, reflecting enhanced parasympathetic activity and autonomic regulation. Participants in the active tDCS group also reported selective reductions in both state and trait anger after stimulation, whereas the sham group showed minimal changes in mood. These findings demonstrate that even at a subthreshold intensity, bifrontal tDCS at rest engaged neurovascular and neurovisceral mechanisms, coupling changes in prefrontal cortical activity with autonomic outflow and mood. This study provides new evidence of cortical-autonomic coupling during neuromodulation and suggests that low-intensity frontal tDCS may promote a calm physiological and emotional state. The results have implications for future research on brain stimulation in emotion regulation and for developing neuromodulation-based interventions to improve autonomic balance and mood in both healthy individuals and clinical populations.

neuroscience↗

bioRxiv2025

Immune Niche Formation in Engineered Mouse Models Reveals Mechanisms of Tumor Dormancy

Residual tumor cells can persist in a dormant state during clinical remissions that may last decades. The mechanisms that lead to such growth control vs. eventual reactivation and macroscopic tumor outgrowth remain unclear. Here, we report data from a mouse model that reveals a key role of host immunity and the cellular and molecular mechanisms that control tumor dormancy. Abrogation of myeloid-specific TGF-{beta}RII expression (T{beta}RIImyeKO) resulted in an IFN-{gamma} rich immune microenvironment. IFN-{gamma} in turn elevated KLF4-mediated SLURP1 production in malignant cells, which is critical to the tumor cell quiescent state through interruption of fibronectin-integrin signaling pathways. The dormant tumor lesions were located in spatially localized immune niches rich in NK cells, cDCs, monocytes, and neutrophils, concomitant with tumor cell inactivation of NK cell immune surveillance through a CD200-CD200R1 mechanism. Our studies identify the IFN-{gamma}-KLF4-SLURP1 and CD200-CD200R1 axes as critical molecular drivers in tumor dormancy regulated by immune-tumor crosstalk. These insights provide enhanced mechanistic understanding of tumor dormancy in a mouse model suitable for further investigation of cancer treatment resistance and prevention of metastatic spread.

cancer biology↗

bioRxiv2025

Transcriptomic plasticity is a hallmark of metastatic pancreatic cancer

Metastasis is the leading cause of cancer deaths; nonetheless, how tumor cells adapt to vastly different organ contexts is largely unknown. To investigate this question, we generated a transcriptomic atlas of primary tumor and diverse metastatic samples from a patient with pancreatic ductal adenocarcinoma who underwent rapid autopsy. Unsupervised archetype analysis identified both shared and site-specific gene programs, including lipid metabolism and gastrointestinal programs prevalent in peritoneum and stomach wall lesions, respectively. We developed a probabilistic approach for inferring clonal phylogeny from single-cell and matched whole-exome data. Distantly related genetic clones in the peritoneum express the lipid metabolism program, likely due to signaling by the adipocyte-rich peritoneum environment, and cells in most clones express multiple programs, suggesting that transcriptomic plasticity is a prevalent feature of metastatic cells. These deeply annotated analyses using a patient-centric platform provide a model for investigating metastatic mechanisms and plasticity in advanced cancer.

bioinformatics↗

bioRxiv2024

Genomic evolution of pancreatic cancer at single-cell resolution

We adapted a previously developed targeted single-nucleus DNA sequencing (snDNA-seq) method and constructed a new suite of computational analysis tools to study 137,491 single-nucleus DNA libraries from 24 pancreatic cancers collected under a variety of clinical scenarios including early and late diagnoses, different metastatic sites and before- and after-treatment. We refined the mutational landscape of pancreatic cancer by capturing events missed by bulk sequencing, and validated the evolution pattern of early fixation of driver single-nucleotide variants (SNVs) followed by generation of intratumoral heterogeneity for copy number variations (CNVs). Intertumoral convergent evolution was common, including subclonal inactivation of TGF-{beta} pathway by mutating various components of it; intratumoral convergence was rarely observed, likely due to strong selective force in pancreatic cancer development. Continuous evolution was frequently seen manifesting as CNVs. In the context of non-targeted treatments, no particular pattern was found across metastases or through treatment. In six pancreatic cancers with germline BRCA2 mutation, we discovered varied timing of biallelic inactivation of BRCA2, which sculpted different evolutionary trajectories and could presumably contribute to differential response to treatment. As the first large-scale application of targeted snDNA-seq on pancreatic cancer, this work provides a sample processing and computational analysis pipeline that warrants further clinical utility.

cancer biology↗

bioRxiv2024

Growth of soil ammonia-oxidizing archaea on air-exposed solid surface.

Soil microorganisms often thrive as microcolonies or biofilms within pores of soil aggregates exposed to the soil atmosphere. However, previous studies on the physiology of soil ammonia-oxidizing microorganisms (AOM), which play a critical role in the nitrogen cycle, were primarily conducted using freely suspended AOM cells (planktonic cells) in liquid media. In this study, we examined the growth of two representative soil ammonia-oxidizing archaea (AOA), Nitrososphaera viennensis EN76 and "Nitrosotenuis chungbukensis" MY2, and an ammonia-oxidizing bacterium, Nitrosomonas europaea ATCC 19718 on polycarbonate membrane filters floated on liquid media to observe their adaptation to air-exposed solid surfaces. Interestingly, ammonia oxidation activities of N. viennensis EN76 and "N. chungbukensis" MY2 were significantly repressed on floating filters compared to the freely suspended cells in liquid media. Conversely, the ammonia oxidation activity of N. europaea ATCC 19718 was comparable on floating filters and liquid media. N. viennensis EN76 and N. europaea ATCC 19718 developed microcolonies on floating filters. Transcriptome analysis of N. viennensis EN76 floating filter-grown cells revealed upregulation of unique sets of genes for cell wall and extracellular polymeric substance biosynthesis, H2O2-induced oxidative stress defense, and ammonia oxidation, including ammonia monooxygenase subunit C (amoC3) and the multicopper oxidases. These genes may play a pivotal role in adapting AOA to air-exposed solid surfaces. Furthermore, the floating filter technique resulted in the enrichment of distinct soil AOA communities dominated by the "Ca. Nitrosocosmicus" clade. Overall, this study sheds light on distinct adaptive mechanisms governing AOA growth on air-exposed solid surfaces.

ecology↗

bioRxiv2024

Loss of p21-activated kinase 4 (PAK4) suppresses pancreatic tumor progression and metastasis through regulating E-cadherin

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a poor prognosis with early and frequent metastasis. While p21-activated kinase 4 (PAK4) has been implicated in cell migration, and invasion, the molecular mechanisms in PDAC remain unknown. In this study, we found that PAK4 overexpression was correlated with poor survival in PDAC patients through analysis of TCGA data. PAK4-amplified PDAC cells showed enhanced mobility in contrast with wild-type. PAK4 knockdown in PAK4 amplified cells inhibited cell migration, invasion, and displacement by increased and stabilized E-cadherin, which was attributed to decreased activity of Cdc42. PAK4 knock-in in PAK4 wild-type models enhanced cell migration, invasion, and displacement by reduced E-cadherin through elevated Cdc42 activity. PAK4 bounded to E-cadherin, Cdc42, and p120ctn in immunoprecipitation. In confocal imaging, the colocalization of PAK4, E-cadherin, p120ctn, and Cdc42 was also identified. In an orthotopic PDAC mouse model, PAK4 knockdown decreased primary tumor size and occurrence of malignant ascites by activation of E-cadherin. Notably, in patients tissue specimens, inverse correlation on expression of PAK4 and E-cadherin were also shown. In conclusion, our study highlights that PAK4 promotes invasive and metastatic behavior by regulating E-cadherin in PDAC. PAK4 could be a potential therapeutic target for PDAC patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/594599v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1755a23org.highwire.dtl.DTLVardef@170b2a1org.highwire.dtl.DTLVardef@1df96edorg.highwire.dtl.DTLVardef@2dc46b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

HTLV-1 infected T cells cause bone loss via small extracellular vesicles

Adult T cell leukemia (ATL), caused by infection with human T cell leukemia virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T lead to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone. To study the direct effect of HTLV/T and ATL-PDX on osteoclasts, supernatants were added to murine and human osteoclast precursors. ATL-PDX supernatants from hypercalcemic patients promoted formation of mature osteoclasts, while those from HTLV/T were variably stimulatory, but had largely consistent effects between human and murine cultures. Interestingly, this osteoclastic activity did not correlate with expression of osteoclastogenic cytokine RANKL, suggesting an alternative mechanism. HTLV/T and ATL-PDX produce small extracellular vesicles (sEV), known to facilitate HTLV-1 infection. We hypothesized that these sEV also mediate bone loss by targeting osteoclasts. We isolated sEV from both HTLV/T and ATL-PDX, and found they carried most of the activity found in supernatants. In contrast, sEV from uninfected activated T cells had little effect. Analysis of sEV (both active and inactive) by mass spectrometry and electron microscopy confirmed absence of RANKL and intact virus. Viral proteins Tax and Env were only present in sEV from the active, osteoclast-stimulatory group, along with increased representation of proteins involved in osteoclastogenesis and bone resorption. sEV injected over mouse calvaria in the presence of low dose RANKL caused more osteolysis than RANKL alone. Thus, HTLV-1 infection of T cells can cause release of sEV with strong osteolytic potential, providing a mechanism beyond RANKL production that modifies the bone microenvironment, even in the absence of overt leukemia.

cancer biology↗

bioRxiv2023

Altitude-dependent agro-ecologies impact the microbiome diversity of scavenging indigenous chicken in Ethiopia

Scavenging indigenous village chickens play a vital role in sub-Saharan Africa, sustaining the livelihood of millions of farmers. These chickens are exposed to vastly different environments and feeds compared to commercial chickens. In this study, we analysed the caecal microbiota of 243 Ethiopian village chickens living in different altitude-dependent agro-ecologies. Differences in bacterial diversity were significantly correlated with differences in specific climate factors, topsoil characteristics, and supplemental diets provided by farmers. Microbiota clustered into 3 enterotypes, with one particularly enriched at high altitudes. We assembled 9,977 taxonomically and functionally diverse metagenome-assembled genomes, the vast majority of which were not found in a dataset of previously published chicken microbes, or in the Genome Taxonomy Database. The wide functional and taxonomic diversity of these microbes highlights their importance in the local adaptation of indigenous poultry, and the significant impacts of environmental factors on the microbiota argues for further discoveries in other agro-ecologies.

microbiology↗

bioRxiv2022

Architecture and dynamics of a novel desmosome-endoplasmic reticulum organelle

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signaling, and lipid transfer. Using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometer proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization and mobility. These findings indicate that desmosomes and the keratin cytoskeleton pattern the distribution of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction. One-Sentence SummaryThe desmosome adhesive junction regulates the organization and dynamics of the endoplasmic reticulum network.

cell biology↗

bioRxiv2021

Label-free monitoring of 3D cortical neuronal growth in vitro using optical diffraction tomography

The highly complex central nervous systems of mammals are often studied using three-dimensional (3D) in vitro primary neuronal cultures. A coupled confocal microscopy and immunofluorescence labeling are widely utilized for visualizing the 3D structures of neurons. However, this requires fixation of the neurons and is not suitable for monitoring an identical sample at multiple time points. Thus, we propose a label-free monitoring method for 3D neuronal growth based on refractive index tomograms obtained by optical diffraction tomography. The 3D morphology of the neurons was clearly visualized, and the developmental processes of neurite outgrowth in 3D spaces were analyzed for individual neurons. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/454602v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@b858baorg.highwire.dtl.DTLVardef@c946caorg.highwire.dtl.DTLVardef@468168org.highwire.dtl.DTLVardef@da022c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

Automatic whole cell organelle segmentation in volumetric electron microscopy

Cells contain hundreds of different organelle and macromolecular assemblies intricately organized relative to each other to meet any cellular demands. Obtaining a complete understanding of their organization is challenging and requires nanometer-level, threedimensional reconstruction of whole cells. Even then, the immense size of datasets and large number of structures to be characterized requires generalizable, automatic methods. To meet this challenge, we developed an analysis pipeline for comprehensively reconstructing and analyzing the cellular organelles in entire cells imaged by focused ion beam scanning electron microscopy (FIB-SEM) at a near-isotropic size of 4 or 8 nm per voxel. The pipeline involved deep learning architectures trained on diverse samples for automatic reconstruction of 35 different cellular organelle classes - ranging from endoplasmic reticulum to microtubules to ribosomes - from multiple cell types. Automatic reconstructions were used to directly quantify various previously inaccessible metrics about these structures, including their spatial interactions. We show that automatic organelle reconstructions can also be used to automatically register light and electron microscopy images for correlative studies. We created an open data and open source web repository, OpenOrganelle, to share the data, computer code, and trained models, enabling scientists everywhere to query and further reconstruct the datasets.

cell biology↗