Biology subjects

Hsu, C.-H.

Publications and source records attributed to Hsu, C.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

StainAI-Web: An Interactive Platform for Whole-Slide Microglial Morphology Analysis

Microglial morphology analysis has emerged as a critical approach in neuroinflammation research. The increasing availability of high-resolution and whole-slide histology images has created strong demand for robust, scalable analytical methods capable of efficiently processing these datasets. Existing approaches may require code-based local computation or prolonged processing times for whole-slide images, while manual morphology quantification remains labor-intensive. To address these limitations, we introduce StainAI-Web, a server that performs rapid, automated microglial morphology detection across whole-slide immunohistochemistry images in the cloud. The system provides interactive grayscale preprocessing and supports ROI-based regional quantification, allowing users to define specific tissue regions and systematically compare microglial populations across regions. An interactive visualization module dynamically updates quantitative summaries of morphological distributions as ROIs are selected or modified. Analysis results, including structured JSON files and morphology-annotated overlays, are downloadable for downstream analysis or integration into external computational pipelines. These integrated features provide an accessible framework for efficient and reproducible whole-slide microglial morphology analysis. The StainAI-Web is available at https://stainaiviewer.azurewebsites.net/.

bioinformatics↗

bioRxiv2026

Tubulin polyglutamylation modulates Golgi morphodynamics and neurite branching during neuronal morphogenesis

Neurons establish functional networks through morphological remodeling during neuronal differentiation. Microtubule polyglutamylation is a key microtubule post-translational modification that is highly enriched during this process and plays an important role in differentiation. However, how remodeling of organelle features such as morphology, distribution and interactions depend on tubulin polyglutamylation during neuronal differentiation remain unclear. Here, we employed multispectral imaging combined with quantitative 3D organelle analysis to comprehensively profile eight organelles simultaneously in human induced pluripotent stem cell-derived neurons. We discovered that depletion of tubulin polyglutamylation induces pronounced alterations in somatic Golgi morphology and associated organelle interactions. In addition, Golgi-derived compartments in proximal neurites exhibited altered morphology and dynamics, namely decreased retrograde directionality. These changes were accompanied by increased neurite branching and tortuosity. Together, our findings reveal a previously unrecognized role for tubulin polyglutamylation in coordinating organelle organization with neurite architecture, providing a mechanistic link between tubulin post-translational modification, Golgi morphology, dynamics, and neuronal morphogenesis.

cell biology↗

bioRxiv2026

A Pan-Cancer Single-Cell Atlas to Evaluate Tumor Identity, Cell Line Concordance, and Dependency Mapping

Bulk RNA sequencing enables pan-cancer transcriptional analyses, but obscures cancer cell-specific programs due to admixture with nonmalignant cells, thereby limiting direct comparison between experimental models and primary tumors. Single-cell RNA sequencing (scRNA-seq) overcomes these limitations; however, the biological interpretability of public datasets is often compromised by variable data quality, inconsistent annotation, and atlas-scale aggregation strategies that prioritize data volume over biological coherence. We therefore developed a stringent integration framework that prioritizes representative malignant transcriptional states. Using Mahalanobis distance-based selection within batch-corrected latent space, we constructed a pan-cancer atlas comprising 135,424 high-quality malignant cells from 499 samples across 36 adult and pediatric cancers. Atlas-derived cancer signatures were used to determine tumor-cell line concordance and project ElasticNet models trained on DepMap CRISPR screens to infer cancer-specific gene dependencies. The scTumor Atlas establishes a scalable framework for tumor identity inference, cancer cell line benchmarking, and systematic identification of genetic vulnerabilities.

cancer biology↗

bioRxiv2026

Tubulin acetylation governs organelle remodeling and lysosomal reformation during neuronal differentiation

A functional nervous system depends on neuronal morphology established during differentiation. The microtubule (MT) cytoskeleton supports neuronal differentiation by organizing organelle positioning and facilitating transport. The dynamics and properties of MTs are regulated by a variety of post-translational modifications (PTMs), with many organelle interactions occurring preferentially on modified MTs. Here we find that tubulin acetylation is enriched at specific subcellular locations during differentiation of human induced neurons. We apply a quantitative multispectral imaging pipeline to simultaneously analyze eight membrane-bound organelles and define how tubulin acetylation reshapes organelle architecture and interaction networks during neuronal differentiation. We find that loss of tubulin acetylation broadly alters organelle morphology, spatial distribution, and inter-organelle interactions, with lysosome-organelle interactions most affected. Loss of acetylated MTs leads to enlarged, highly acidified lysosomes, impaired lysosomal fission, and accumulation of autolysosomes, consistent with defective lysosomal reformation. Super-resolution microscopy further reveals that lysosome-endoplasmic reticulum (ER) contacts preferentially associate with acetylated MTs. Together, our data support a model in which tubulin acetylation coordinates lysosome-ER interactions to facilitate lysosome remodeling and turnover. This work establishes tubulin acetylation as a key cytoskeletal regulator that links organelle interactions to organelle homeostasis important for neuronal differentiation.

cell biology↗

bioRxiv2026

Organelle communication networks rewire to support lipid metabolism during neuronal differentiation

Cell fate transitions require coordinated remodeling of intracellular organelles, but how organelle morphology and interactions rewire during neurogenesis remains unclear. Here we combine multispectral imaging with quantitative organelle signature analysis to simultaneously map eight organelles as human induced pluripotent stem cells differentiate into forebrain-like neurons. We find compartment and time-specific rescaling of organelles and a progressive increase in higher-order membrane contacts, with mitochondria emerging as an early interaction hub. Later, endoplasmic reticulum (ER)-organelle contacts dominate with ER-peroxisome contacts promoting ether lipid biosynthesis, membrane homeostasis and synapse formation. Disrupting this contact impairs plasmalogen production, synaptic organization, and neuronal activity, identifying the ER-peroxisome axis as a key regulator of neuronal maturation.

cell biology↗

bioRxiv2026

Mechanisms of disrupted neurodevelopment after Zika virus infection in infancy

Congenital and early-life Zika virus (ZIKV) infection can result in neurologic deficits. Precise mechanisms of injury, especially in the more subtle presentation of postnatal infection, are not fully elucidated. Here, we defined the effects of ZIKV on the developing brain using single cell transcriptomics, histopathology and design-based stereology, diffusion MRI, and neurobehavioral assessments in infant rhesus macaques. ZIKV upregulated interferon-stimulated genes in activated microglia and cell death pathways in neurons and downregulated metabolism and differentiation genes in mature oligodendrocytes. Abnormal micro-organization of the corpus collosum and limbic white matter tracts was seen on diffusion weighted imaging. A curated gene set associated with autism spectrum disorder risk was negatively enriched in inhibitory and excitatory neurons from ZIKV-infected infants, with increased emotional reactivity already evident two weeks following infection. From single cells to organism-level behaviors, these results define the pathways and processes disrupted by early-life ZIKV infection.

neuroscience↗

bioRxiv2025

Innate Immune Remodeling Drives Therapy Resistance via Macrophage-NK Cell Crosstalk.

The dynamic evolution of the immune tumor microenvironment (TME) during targeted therapy is a critical yet poorly understood determinant of treatment response and resistance. While most studies compare immune states before and after treatment, temporal immune changes during therapy remain largely uncharacterized, limiting development of effective combination strategies. Here, we investigated immune dynamics throughout targeted therapy using mouse melanoma models that recapitulate human therapeutic responses. Single-cell RNA sequencing (scRNA-seq) identified a previously unrecognized inflection point where the inflamed TME during tumor regression, characterized by robust NK cell infiltration, transitions to an immune-excluded state upon onset of drug-tolerant residual disease. We uncovered a unique macrophage subset (F4/80hiCCL5MHCIICD63) that orchestrates NK cell recruitment through CCR2/5 signaling during regression. Depletion of these macrophages using LysM-cre;iDTR mice significantly reduced NK cell infiltration. Specifically during residual disease, pharmacological inhibition of Ptpn22, a phosphatase that negatively regulates immune activation, reprogrammed macrophages, restored NK cell recruitment and enhanced therapeutic efficacy. Extending these findings to human cancer, longitudinal scRNA-seq analysis of melanoma and lung cancer patient samples revealed dynamic NK cell infiltration during targeted therapy, establishing a direct link between innate immune remodeling and treatment outcome. Unlike prior prognostic studies assessing immune states at single time points, our results provide mechanistic evidence of a temporal relationship between NK cell infiltration and therapeutic efficacy. Together, these findings position immune evolution as a driver of acquired resistance and identify macrophage-NK cell crosstalk as a therapeutically actionable axis to overcome immune exclusion and improve targeted therapy across multiple cancer types. One Sentence SummaryCancer therapy resistance emerges from a dynamic evolution of the tumor microenvironment, characterized by macrophage-driven NK cell infiltration during initial tumor regression, followed by exclusion of NK cells during residual disease, highlighting macrophage-NK cell interactions as a promising therapeutic target to improve clinical outcomes.

cancer biology↗

bioRxiv2025

Coro1A and TRIM67 collaborate in netrin-dependent neuronal morphogenesis

Neuronal morphogenesis depends on extracellular guidance cues accurately instructing intracellular cytoskeletal remodeling. Here, we describe a novel role for the actin binding protein Coronin 1A (Coro1A) in neuronal morphogenesis, where it mediates responses to the axon guidance cue netrin-1. We found that Coro1A localizes to growth cones and filopodial structures and is required for netrindependent axon turning, branching, and corpus callosum development. We previously discovered that Coro1A interacts with TRIM67, a brain enriched E3 ubiquitin ligase that interacts with a netrin receptor and is also required for netrin-mediated neuronal morphogenesis. Loss of Coro1A and loss of TRIM67 shared similar phenotypes, suggesting that they may function together in the same netrin pathway. A Coro1A mutant deficient in binding TRIM67 was not able to rescue loss of Coro1A phenotypes, indicating that the interaction between Coro1A and TRIM67 is required for netrin responses. Together, our findings reveal that Coro1A is required for proper neuronal morphogenesis, where it collaborates with TRIM67 downstream of netrin.

cell biology↗

bioRxiv2025

The Kelch 3 motif on gigaxonin mediates the interaction with NUDCD3 and regulates vimentin filament morphology

Gigaxonin is an intermediate filament (IF)-interacting partner belonging to the Kelch-like (KLHL) protein family. Gigaxonin is encoded by the KLHL16 gene, which is mutated in Giant Axonal Neuropathy (GAN). The lack of functional gigaxonin in GAN patient cells impairs IF proteostasis, leading to focal abnormal accumulations of IFs and compromised neuronal function. We hypothesized that gigaxonin forms molecular interactions via specific sequence motifs to regulate IF proteostasis. The goal of this study was to examine how distinct Kelch motifs on gigaxonin regulate IF protein degradation and filament morphology. We analyzed vimentin IFs in HEK293 cells overexpressing wild type (WT) gigaxonin, or gigaxonin lacking each of the six individual Kelch motifs: K1 (aa274-326), K2 (aa327-374), K3 (aa376-421), K4 (aa422-468), K5 (aa470-522), and K6 (aa528-574). All six gigaxonin deletion mutants ({Delta}K1-{Delta}K6) promoted the degradation of soluble vimentin. The {Delta}K3 gigaxonin mutant exhibited soluble vimentin degradation and promoted the bundling of vimentin IFs relative to WT gigaxonin. Using mass spectrometry proteomic analysis we found that, relative to WT gigaxonin, {Delta}K3 gigaxonin had increased associations with ubiquitination-associated and mitochondrial proteins and lost the association with the NudC domain-containing protein 3 (NUDCD3), a molecular chaperone enriched in the nervous system. Collectively, our cell biological data show the induction of an abnormal GAN-like IF phenotype in cells expressing {Delta}K3-gigaxonin, while our mass spectrometry profiling links the loss of gigaxonin-NUDCD3 interactions with defective IF proteostasis, revealing NUDCD3 as a potential new target in GAN.

cell biology↗

bioRxiv2025

Preclinical evaluation of the efficacy of α-Difluoromethylornithine and Sulindac against SARS-CoV-2 infection

Despite numerous research efforts and several effective vaccines and therapies developed against COronaVIrus Disease 2019 (COVID-19), drug repurposing remains an attractive alternative to identify new treatments for SARS-CoV-2 virus variants and other viral infections that may emerge in the future. Cellular polyamines support viral propagation and tumor growth. Here we tested the antiviral activity of an irreversible inhibitor of polyamine biosynthesis, -difluoromethylornithine (DFMO) and a non-steroidal anti-inflammatory drug (NSAID) Sulindac, which have been previously evaluated for colon cancer chemoprevention. The drugs were tested as single agents and in combination in human Calu-3 lung adenocarcinoma and Caco-2 colon adenocarcinoma cell lines and the K18-hACE2 transgenic mouse model of severe COVID-19. DFMO/Sulindac combination significantly suppressed SARS-CoV-2 N1 Nucleocapsid mRNA and ACE2 mRNA levels in the infected human cell lines by interacting synergistically when cells were pretreated with drugs and additively when treatment was applied to the infected cells. The antiviral activity of DFMO and Sulindac was tested in vivo as prophylaxis (drug supplementation at the doses equivalent to the human chemoprevention trial started 7 days before infection) or as treatment (drug supplementation started 24 hours post-infection). Prophylaxis with DFMO and Sulindac as single agents significantly increased survival rates in the young male mice (p=0.01, and p=0.027, respectively), and the combination was effective in the aged male mice (p=0.042). Young female mice benefited the most from the prophylaxis with Sulindac alone (p=0.001) and DFMO/Sulindac combination (p=0.018), while aged female mice did not benefit significantly from any interventions. The treatment regime was ineffective in suppressing SARS-CoV-2 infection in K18-hACE2 mice. Overall, animal studies demonstrated the protective age- and sex-dependent antiviral efficacy of DFMO and Sulindac against SARS-CoV-2.

cell biology↗

bioRxiv2025

Muscle Activation of Upper Body in Different-Angle Suspension Push-Ups: An Analysis of Angle-Specific Muscle Engagement

The aim of this study was to understand the effect on upper body muscle activation of bodily angle during push-ups performed with TRX suspension training. Nineteen men (age: 21.1 {+/-} 1.2 years; height: 174.1 {+/-} 4.9 cm; weight: 70.7 {+/-} 7.2 kg) with resistance training experience participated in this study. The participants were required to perform five push-ups on a stable surface and with TRX at five angles (+30{degrees}, +15{degrees}, 0{degrees}, -15{degrees}, and -30{degrees}, where 0{degrees} indicates that the shoulder joints were at the same height as the ankle joints when the arms were extended). The recovery period between the sets at each angle was 3-5 minutes. Stable-surface and TRX push-ups were separated by at least 48 hours. During push-ups, electromyography (EMG) data from the pectoralis major (PM), anterior deltoids (AD), triceps brachii (TRI), upper trapezius (UT), and serratus anterior (SA) were recorded. Muscle activation was indicated by the percentage of maximum voluntary isometric contraction (%MVIC). The %MVIC of each muscle group was then categorized. Repeated-measured two-way analysis of variance was used to determine differences in the muscle group activation between the push-up surface types and five angles. Statistical significance was set at p < .05. The activation of the PM, AD, and TRI during TRX push-ups was categorized as medium to extremely high. Compared with that for stable-surface push-ups, the activation of the PM during TRX push-ups was significantly higher (p < .05). Furthermore, +30{degrees}, +15{degrees}, and 0{degrees} push-ups produced greater PM activation than push-ups at lower angles (p < .05). Both TRX and stable-surface push-ups resulted in greater anterior deltoids, TRI, UT, and SA activation during -30{degrees} push-ups. This study indicates the appropriate push-up practices for different muscle groups, as determined by quantifying the muscle activation.

physiology↗

bioRxiv2025

Massive Chromosome Fission and Speciation in Asian River Chub: Integrating Kinetochore Reproduction Theory with Transposable Element Dynamics

Chromosomal speciation poses a paradox: if rearrangements cause postmating isolation, how do they persist and spread? We propose that centromere remodeling, driven by transposable element accumulation, provides a mechanistic solution. In Opsariichthys, a hyper-diverse lineage of Asian river chub with 2N = 76-78 chromosomes, we identify [~]15 fission events coinciding with TE-fueled centromeric DNA expansion, potentially linked to a mutation in the PIWI1 protein. These expansions enable neokinetochore formation, allowing fissioned chromosomes to segregate faithfully, while subsequent rearrangements disrupt pairing and reduce gene flow. Comparative genomics reveals that fissioned chromosomes exhibit lower migration and greater divergence, acting as reproductive barriers. Our findings show that TE-mediated kinetochore duplication can facilitate extensive chromosome fission without meiotic disruption, providing a viable and previously underappreciated path to rapid speciation.

genomics↗

bioRxiv2024

Exploring The Formation And Permeability Of Plasmodesmata In The Liverwort, Marchantia polymorpha

Plasmodesmata are cell-wall-embedded channels that evolved in the common ancestor of land plants to increase cell-to-cell communication. Whether all the fundamental properties of plasmodesmata emerged and were inherited in all land plants at the same time is unknown. Here we show that the bryophyte Marchantia polymorpha (a non-vascular plant) forms mostly simple plasmodesmata in early-developing gemmae. The complexity of plasmodesmata increases during gemma maturation, and complex plasmodesmata with enlarged cavities are majorly observed in thalli. In contrast to vascular plants, whose simple plasmodesmata can transport monomeric fluorescent proteins, plasmodesmata in Marchantia polymorpha limited their permeability before the juvenile-to-adult transition. In support, callose, a known polysaccharide regulating plasmodesmata permeability in vascular plants, accumulated in most of the Marchantia polymorpha tissues examined. Furthermore, we found that in the apical meristematic region, plasmodesmata allowed the transport of monomeric fluorescent proteins, and this relaxation might correlate with the lower accumulation of callose. Taken together, our study suggests that certain plasmodesmata properties, such as complexity progression and callose accumulation, may have evolved before the divergence between vascular and non-vascular plants.

plant biology↗

bioRxiv2024

Neurons and astrocytes have distinct organelle signatures and responses to stress

Neurons and astrocytes play critical yet divergent roles in brain physiology and neurological conditions. Intracellular organelles are integral to cellular function. However, an in-depth characterization of organelles in live brain cells has not been performed. Here, we used multispectral imaging to simultaneously visualize six organelles - endoplasmic reticulum (ER), lysosomes, mitochondria, peroxisomes, Golgi, and lipid droplets - in live primary rodent neurons and astrocytes. We generated a dataset of 173 Z-stack and 99 time-lapse images accompanied by quantitative analysis of 1418 metrics (the "organelle signature"). Comparative analysis revealed clear cell-type specificity in organelle morphology and interactions. Neurons were characterized by prominent mitochondrial composition and interactions, while astrocytes contained more lysosomes and lipid droplet interactions. Additionally, neurons displayed a more robust organelle response than astrocytes to acute oxidative or ER stress. Our data provide a systems-level characterization of neuron and astrocyte organelles that can be a reference for understanding cell- type-specific physiology and disease.

cell biology↗

bioRxiv2024

The spread of pESI-mediated extended-spectrum cephalosporin resistance in Salmonella serovars - Infantis, Senftenberg, and Alachua isolated from food animal sources in the United States

The goal of this study is to investigate the origin, prevalence, and evolution of the pESI megaplasmid in Salmonella isolated from animals, foods, and humans. We queried 510,097 Salmonella genomes under the National Center for Biotechnology Information (NCBI) Pathogen Detection (PD) database for the presence of potential sequences containing the pESI plasmid in animal, food, and environmental sources. The presence of the pESI megaplasmid was confirmed by using seven plasmid-specific markers (rdA, pilL, SogS, TrbA, ipf, ipr2 and IncFIB(pN55391)). The plasmid and chromosome phylogeny of these isolates was inferred from single nucleotide polymorphisms (SNPs). Our search resolved six Salmonella clusters carrying the pESI plasmid. Four were emergent Salmonella Infantis clusters, and one each belonged to serovar Senftenberg and Alachua. The Infantis cluster with a pESI plasmid carrying blaCTX-M-65 gene was the biggest of the four emergent Infantis clusters, with over 10,000 isolates. This cluster was first detected in South America and has since spread widely in United States. Over time the composition of pESI in United States has changed with the average number of resistance genes showing a decrease from 9 in 2014 to 5 in 2022, resulting from changes in gene content in two integrons present in the plasmid. A recent and emerging cluster of Senftenberg, which carries the blaCTX-M-65 gene and is primarily associated with turkey sources, was the second largest in the United States. SNP analysis showed that this cluster likely originated in North Carolina with the recent acquisition of the pESI plasmid. A single Alachua isolate from turkey was also found to carry the pESI plasmid containing blaCTX-M-65 gene. The study of the pESI plasmid, its evolution and mechanism of spread can help us in developing appropriate strategies for the prevention and further spread of this multi-drug resistant plasmid in Salmonella in poultry and humans.

genomics↗

bioRxiv2023

Contact-FP: a dimerization-dependent fluorescent protein toolkit for visualizing membrane contact site dynamics

Membrane contact sites (MCSs) are sites of close apposition between two organelles used to exchange ions, lipids, and information. Cells respond to changing environmental or developmental conditions by modulating the number, extent, or duration of MCSs. Because of their small size and dynamic nature, tools to study the dynamics of MCSs in live cells have been limited. Dimerization-dependent fluorescent proteins (ddFPs) targeted to organelle membranes are an ideal tool for studying MCS dynamics because they reversibly interact to fluoresce specifically at the interface between two organelles. Here, we build on previous work using ddFPs as sensors to visualize the morphology and dynamics of MCSs. We engineered a suite of ddFPs called Contact-FP that targets ddFP monomers to lipid droplets (LDs), the endoplasmic reticulum (ER), mitochondria, peroxisomes, lysosomes, plasma membrane, caveolae, and the cytoplasm. We show that these probes correctly localize to their target organelles. Using LDs as a test case, we demonstrate that Contact-FP pairs specifically localize to the interface between two target organelles. Titration of LD-mitochondria ddFPs revealed that these sensors can be used at high concentrations to drive MCSs or can be titrated down to minimally perturb and visualize endogenous MCSs. We show that Contact-FP probes can be used to: (1) visualize LD-mitochondria MCS dynamics, (2) observe changes in LD-mitochondria MCS dynamics upon overexpression of PLIN5, a known LD-mitochondrial tether, and (3) visualize two MCSs that share one organelle simultaneously (e.g., LD-mitochondria and LD-ER MCSs). Contact-FP probes can be optimized to visualize MCSs between any pair of organelles represented in the toolkit.

cell biology↗

bioRxiv2023

A Weakly Supervised U-Net Model for Precise Whole Brain Immunolabeled Cell Detection

Cell segmentations low precision due to the intensity differences hinders widespread use of whole brain microscopy imaging. Previous studies used ResNet or CNN to account for this problem, but are unapplicable to immunolabeled signals across samples. Here we present a semiauto ground truth generation and weakly-supervised U-Net-based Deep-learning precise segmentation pipeline for whole brain immunopositive c-FOS signals, which reveals the distinct neural activity maps with different social motivations.

neuroscience↗

bioRxiv2021

A photo-switchable assay system for dendrite degeneration and repair in Drosophila melanogaster

Neurodegeneration arising from aging, injury or disease has devastating health consequences. Whereas neuronal survival and axon degeneration have been studied extensively, much less is known about how neurodegeneration impacts dendrites. To develop an assay for dendrite degeneration and repair in the Drosophila peripheral nervous system, we used photo-switchable caspase-3 (caspase-LOV) to induce neuronal damage with tunable severity by adjusting illumination duration, thereby revealing cell type-specific responses to caspase-3 induced dendrite degeneration in dendrite arborization (da) neurons. To ask whether mechanisms underlying axon degeneration also govern dendrite degeneration, we tested the involvement of the Wallerian degeneration pathway by examining the effects of expressing the mouse Wallerian degeneration slow (WldS) protein and knockdown of the Drosophila sterile alpha/Armadillo/Toll-Interleukin receptor homology domain protein (dSarm1) and Axundead (Axed) in class 4 da neurons. Here we report WldS expression or knockdown of dSarm1 improved dendrite repair following caspase- 3 induced dendrite degeneration. Whereas both dSarm1 and Axed were required for thermal nocifensive behavior in uninjured animals, WldS expression improved the recovery of thermal nocifensive behavior that was impaired by chronic low-level of caspase-LOV activity. By establishing ways to induce graded dendrite degeneration, we uncover a protective role of WldS in caspase-3 induced dendrite degeneration and repair.

neuroscience↗