Biology subjects

Lin, P.-Y.

Publications and source records attributed to Lin, P.-Y..

15 recordsLinked to original sources

bioRxiv2026

Dodecagon light-sheet fluorescence microscopy for large-volume imaging without striping artifacts

Light-sheet fluorescence microscopy (LSFM) has revolutionized biological imaging by enabling high spatial and temporal resolution with minimal photodamage. However, conventional LSFM techniques often suffer from striping artifacts in the resulting images due to light scattering and absorption within samples, leading to uneven illumination that negatively impacts the accuracy of subsequent image analyses. To address this limitation, we introduce dodecagon light-sheet fluorescence microscopy (dodecaLSFM), a novel approach that maximizes angular diversity to achieve homogeneous illumination and suppress striping artifacts. dodecaLSFM employs diffraction optics and cylindrical lenses to generate twelve light sheets, providing 360{degrees} omnidirectional illumination that significantly enhances illumination uniformity compared to traditional mSPIM, mDSLM, and ultramicroscopy systems, which use only one or two illumination planes. We demonstrate the effectiveness of dodecaLSFM by achieving high-resolution imaging of whole mouse brain vasculature following tissue clearing, allowing precise morphometric analysis of vascular networks without striping artifacts. Furthermore, we show that combining dodecaLSFM with expansion microscopy (ExM) enables whole-organ 3D imaging at cellular resolution. This novel approach provides an advanced, scalable solution for large-volume imaging, facilitating detailed structural and functional studies across diverse biological applications.

bioengineering↗

bioRxiv2026

Cellular remodeling during photosymbiosis establishment revealed by single-cell dual proteomics

Endosymbiosis of phytoplankton in heterotrophic hosts is ecologically important and has led to key evolutionary innovations. However, the dynamic molecular processes underlying endosymbiosis establishment remain poorly understood. Here, using large-particle sorting and liquid chromatography-tandem mass spectrometry, we unravel heterogeneous changes in proteomes of the cosmopolitan ciliate Paramecium and algal endosymbiont Chlorella from engulfment to stable endosymbiosis. The initial digestion sees a sharp decline of intracellular Chlorella cells, along with host cellular reorganization involving a reduction of the cortex-localized defensive organelles, trichocysts, and proteins for intracellular transport and recycling. The remaining Chlorella cells enter a bottleneck stage characterized by energy production and cell cycle commitment before active proliferation. Comparison of Paramecium with successful and failed endosymbiosis further identifies a solute carrier transporter that potentially mediates metabolic homeostasis of the endosymbiotic system. Our study reveals inter-organismal coordination during the transition from predator-prey to host-endosymbiont relationships. The approach of time-course single-cell dual proteomics can be useful for investigating diverse interactions between microbial eukaryotes.

microbiology↗

bioRxiv2025

MEX3A control of mitochondrial fitness is essential for ovarian clear cell carcinoma tumorigenesis and liver metastasis

Ovarian cancer (OC) is highly metastatic and chemoresistant. Due to heterogeneity among OC subtypes, the mechanisms underlying OC malignancy and metastasis remain largely unknown. Ovarian clear cell carcinoma (OCCC) accounts for 5-25% of OC and its incidence rate is rising. Liver metastasis is particularly high in OCCC patients and leads to significantly reduced median survival. Why OCCC metastasizes to liver at such high frequency remains elusive. We previously identified MEX3A as a key factor that promotes OCCC tumorigenesis in part by circumventing p53-mediated ferroptosis. Here, we report that MEX3A control of mitochondrial fitness, which occurs independently of p53, is essential for OCCC primary tumor growth and liver metastasis. MEX3A depletion resulted in chronic mitochondrial fragmentation and accumulation of non-functional mitochondria. MEX3A-depleted cells had decreased mitochondrial membrane potential, increased superoxide and decreased NAD+/NADH ratio, resulting in inhibition of oxidative phosphorylation (OXPHOS) and decreased ATP levels. In an environment enriched with mitophagy stressors, such as the liver, MEX3A-depleted OCCC cells had greatly reduced survival due to failure to recover from mitophagy. Consistent with these observations, MEX3A knockdown greatly reduced liver metastasis. Together, these data demonstrate that MEX3A-mediated mitochondrial fitness is a major factor underlying its p53-independent promotion of OCCC tumorigenesis and liver metastasis. Thus, targeting MEX3A will be a promising strategy to inhibit OCCC progression. Statement of significanceUnexpected effects of MEX3A on mitochondrial fitness indicate that the need for high MEX3A expression is an OCCC vulnerability that can be exploited to uncover new treatment strategies.

cancer biology↗

bioRxiv2025

BMAL2 is a druggable target for ARID1A-wildtype ovarian clear cell carcinoma (OCCC)

Ovarian clear cell carcinoma (OCCC) is highly chemo-resistant and has worse clinical outcome at advanced stages than other ovarian cancer subtypes. The most frequent ([~]50%) alterations in OCCC are AT-rich interactive domain 1A gene (ARID1A) mutations which lead to ARID1A deficiency. However, OCCC that retains ARID1A function differs substantially from ARID1A mutated OCCC. Particularly, targeted therapies that sensitize ARID1A-deficient OCCC to DNA damage are largely ineffective against OCCC with wild-type (wt) ARID1A. Thus, it is important to identify druggable targets and develop targeted therapies specifically for ARID1A-wt OCCC. We identified BMAL2 as a critical OCCC oncogene that promotes tumorigenesis by preventing DNA damage from endogenous origins. BMAL2 depletion altered expression of genes in DNA damage repair pathways, including RAD51, a core enzyme of the homologous recombination (HR) pathway. This led to double-stranded break accumulation, decreased cell viability and reduced tumor growth. This dependence on BMAL2 to maintain DNA integrity and cell viability can be a new route to suppress ARID1A-wt OCCC. Consistent with this idea, we found that GW833972A, a cannabinoid receptor agonist, bound BMAL2 with high affinity and facilitated its degradation. This in turn reduced RAD51 expression, leading to an accumulation of DNA damage and decreased cell viability. Xenograft models further demonstrated that GW833972A treatment alone inhibited ARID1A-wt OCCC tumor growth. Together, our findings reveal an essential oncogenic role of BMAL2 and demonstrate that it is an appealing therapeutic target, especially for ARID1A-wt OCCC. Statement of significanceBMAL2 depletion, or degradation by a small molecule, led to DNA damage accumulation, decreased cell viability and reduced tumorigenesis of ARID1A-wildtype ovarian clear cell carcinoma, indicating that BMAL2 is an appealing therapeutic target for treating this malignant disease.

cancer biology↗

bioRxiv2025

Ex Vivo Expanded Regulatory T Cells Inhibit AAA Progression by Limiting CD4+ and CD8+ T Cell Accumulation in Aortic Tissue

BackgroundRegulatory T cells (Tregs) play a crucial role in the pathophysiology of abdominal aortic aneurysms (AAA), a chronic inflammatory condition with few treatment options for patients with early-stage disease. Treg therapy for AAA is potentially beneficial but its specific mechanism requires further investigation for clinical applications. MethodsAfter identifying the critical role of T-cells in AAA using human and mouse AAA single-cell RNA sequencing data, we investigated the influence of Tregs on immune cell infiltration within mouse AAA--specifically CD3+ T cells--using congenic transfer of Thy1.1 allelic donor mice Tregs into AAA-induced wild-type C57BL/6J mice. AAA progression was quantified with ultrasound and image micrometry. Tissues obtained on postoperative days 7-42 were analyzed with flow cytometry, qRT-PCR, Verhoeff-van Gieson staining, hematoxylin-eosin staining, and immunohistochemistry. ResultsCD3+ T cell population was profoundly elevated in the elastase induced AAA mouse model which was further used in the study. The AAA mice that received Treg cell therapy had less elastin degradation and aortic wall enlargement than their control counterparts. Donor Tregs were detected in draining lymph nodes even after five weeks, with characteristic expression of FOXP3 and CD25. Although donor Tregs were not detected in the aortic microenvironment, the pro-inflammatory cell population including CD4 and CD8 cells was reduced, compared to control mice. ConclusionElevated T cell population aggravates inflammation and promotes AAA progression. Treg therapy impedes the recruitment of T cells into AAA tissue by colonizing the draining lymph nodes, thereby mitigating AAA progression. This study deepens our understanding of Treg stability, function, and potential as a promising therapy for early-stage aneurysms.

immunology↗

bioRxiv2025

P-KNN: Maximizing variant classification evidence through joint calibration of multiple pathogenicity prediction tools

Clinical guidelines for Mendelian disease diagnosis require that outputs from variant pathogenicity prediction tools be converted into well-calibrated probabilities. However, the existing calibration process is only valid when pre-committing to a specific tool, preventing clinicians from using multiple tools with complementary strengths. To lift this restriction, we introduce Pathogenicity K-Nearest Neighbors (P-KNN), a simple, flexible method that jointly calibrates any set of tools. P-KNN scores each variant by the fraction of pathogenic variants among those with the most similar scores across all underlying tools. We compared P-KNN to single-tool calibration over 13 real tools, including two meta-predictors. Compared to BayesDel, the best-performing tool, P-KNN produced better-calibrated probabilities and stronger evidence strength, with mean log likelihood ratios of 2.56 vs. 2.24 for pathogenic variants, 2.29 vs. 1.56 for benign variants, and 2.28 vs. 2.07 for variants of uncertain significance. We also evaluated P-KNN at four historical time points to assess scalability and robustness, finding that it improved steadily as new tools became available, while remaining well-calibrated. It also correctly integrated computational predictions with experimental measurements, whereas guidelines-based evidence summation systematically overestimated pathogenicity. In summary, P-KNN allows full flexibility to work with any set of tools in a reliable and robust manner. It is consistent with clinical variant classification guidelines, making it well positioned to improve diagnostic yield for rare genetic diseases. P-KNN is available via command line (https://github.com/Brandes-Lab/P-KNN) and precomputed scores (Dataset: https://huggingface.co/datasets/brandeslab/P-KNN, User Interface: https://huggingface.co/spaces/brandeslab/P-KNN-Viewer).

bioinformatics↗

bioRxiv2025

Genomic heterogeneity inflates the performance of variant pathogenicity predictions

Recent studies have reported unprecedented accuracy predicting pathogenic variants across the genome, including in noncoding regions, using large AI models trained on vast genomic data. We present a comprehensive evaluation of these frontier models, showing that performance is inflated by differences in the prevalence of pathogenic variants across genomic contexts. We identify the best-performing models for each variant type and establish a benchmark to guide future progress.

bioinformatics↗

bioRxiv2025

Plasma membrane mediated GLUT10 mitochondrial targeting regulates intracellular ascorbic acid homeostasis

Regulation of intracellular ascorbic acid (AA) levels is critical for connective tissue development and maintenance, but the underlying mechanisms remain unclear. In this study, we uncover a novel regulatory role of glucose transporter 10 (GLUT10) in AA homeostasis through a noncanonical trafficking pathway from the endomembrane system to mitochondria, traditionally considered as separate entities. We demonstrate that GLUT10 transit from ER to mitochondria through the plasma membrane (PM) and endosomes increases under stress conditions. This PM localization enhances the transport of dehydroascorbic acid (DHA), the oxidized form of AA, thereby maintaining intracellular AA levels. Disruption of this trafficking impairs AA homeostasis. Our findings reveal a previously unrecognized localization of GLUT10 at the PM and endosomes, highlighting a novel communication between endomembrane system and mitochondria for proetin redistribution, which is essential for maintaining intracellular AA homeostasis and facilitating environmental adaptation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/632083v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@17ac1d1org.highwire.dtl.DTLVardef@4aa2f3org.highwire.dtl.DTLVardef@101849org.highwire.dtl.DTLVardef@aa9c26_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Du-IN-v2: Unleashing the Power of Vector Quantization for Decoding Cognitive States from Intracranial Neural Signals

While invasive brain-computer interfaces have shown promise for high-performance speech decoding under medical use, the potential of intracranial stereoElectroEn-cephaloGraphy (sEEG), which causes less damage to patients, remains underex-plored. With the rapid progress in representation learning, leveraging abundant pure recordings to further enhance speech decoding becomes increasingly attractive. However, some popular methods pre-train temporal models based on brain-level tokens, overlooking the brains desynchronization nature; others pre-train spatial-temporal models based on channel-level tokens, yet fail to evaluate them on more challenging tasks, e.g., speech decoding, which demands intricate processing in specific brain regions. To tackle these issues, we introduce a general pre-training framework for speech decoding - Du-IN-v2, which can extract contextual embeddings based on region-level tokens through discrete codex-guided mask modeling. To further push its limits, we propose Decoupling Product Quantization (DPQ), where different codexes are designed to extract different parts of brain dynamics. Our model achieves SOTA performance on both the 61-word classification task and the 49-syllable sequence classification task, surpassing all baselines. Model comparison and ablation studies reveal that our design choices, including (i) temporal modeling based on region-level tokens by utilizing 1D depthwise convolution to fuse channels in vSMC and STG regions and (ii) self-supervision by discrete decoupling codex-guided mask modeling, significantly contribute to these performances. Collectively, our approach, inspired by neuroscience findings, capitalizing on region-level representations from specific brain regions, is suitable for invasive brain modeling. It marks a promising neuro-inspired AI approach in BCI.

bioengineering↗

bioRxiv2024

TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1,954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

biochemistry↗

bioRxiv2023

Vegetation of Chamaecyparis montane cloud forest in Lalashan Forest Dynamics Plot

To understand the vegetation-environment relationships within the Chamaecyparis montane mixed cloud forest in Taiwan, we established a 1-ha Lalashan Forest Dynamics Plot (LFDP) in northern Taiwan (24{degrees}42D N, 121{degrees}26D E). We established the plot in July 2019 and finished the first census of all woody species in August 2020. We collected environmental factors related to the topography and soil properties, measured microclimate and soil moisture within the plot, and collected further microclimatic data with a nearby weather station. In total, we recorded 5220 individuals belonging to 65 species, 42 genera and 29 families, with a basal area of 69.1 m2 ha-1, dominated by Chamaecyparis obtusa var. formosana, Rhododendron formosanum and Quercus sessilifolia. Modified TWINSPAN classified vegetation into three types (ridge, east-facing slope and valley). Unconstrained ordination showed that the main gradients behind compositional changes are related to windwardness and convexity. The prevailing wind direction in the area is from the northeast, linked to the winter monsoon. Both east-facing slope type and valley type have relatively lower temperatures than ridge type, especially during summer. Convexity is related to soil moisture gradient (from dryer convex to wet concave sites). Fog frequency is seasonal, with the highest values during autumn and winter months. From soil properties, pH is negatively and phosphorus is positively related to topographical convexity. Litter decomposition is linked to both topographical, soil and biotic variables. Collected data will serve as a baseline for future resurveys and monitoring changes within this montane cloud forest.

ecology↗

bioRxiv2023

Zaxinone Synthase overexpression modulates rice physiology and metabolism, improving growth and productivity under normal and low phosphate supply

The rice Zaxinone Synthase (ZAS) gene encodes a carotenoid cleavage dioxygenase (CCD) that forms the apocarotenoid growth regulator zaxinone. Here, we generated and characterized constitutive ZAS-overexpressing rice lines, to better understand ZAS role in determining zaxinone content and regulating growth and architecture. ZAS overexpression enhanced endogenous zaxinone level, promoted root growth and meristem size, and increased the number of productive tillers, leading to an up to 30% higher grain yield per plant. Hormone analysis revealed a decrease in strigolactone (SL) content, which we confirmed by rescuing the high-tillering phenotype through application of a SL analog. Metabolomics analysis revealed that ZAS overexpressing plants accumulate higher amounts of monosaccharide sugars, in line with transcriptome analysis. Moreover, transgenic plants showed higher carbon (C) assimilation rate and elevated root phosphate, nitrate and sulfate level, enhancing the tolerance towards low phosphate (Pi) and indicating a generally better nutrient uptake. Our study shows that ZAS regulates hormone homeostasis and a combination of physiological processes to promote growth and grain yield, which makes this gene an excellent candidate for sustainable crop improvement. TeaserZaxinone Synthase overexpression modulates rice metabolism and physiology and improves growth and phosphate uptake.

plant biology↗

bioRxiv2020

Chromatin Fiber Folding Represses Transcription and Loop Extrusion in Quiescent Cells

Determining the conformation of chromatin in cells at the nucleosome level and its relationship to cellular processes has been a central challenge in biology. We show that in quiescent yeast, widespread transcriptional repression coincides with the local compaction of chromatin fibers into structures that are less condensed and more heteromorphic than canonical 30-nanometer forms. Acetylation or substitution of H4 tail residues decompacts fibers and leads to global transcriptional de-repression. Fiber decompaction also increases the rate of loop extrusion by condensin. These findings establish a role for H4 tail-dependent local chromatin fiber folding in regulating transcription and loop extrusion in cells. They also demonstrate the physiological relevance of canonical chromatin fiber folding mechanisms even in the absence of regular 30-nanometer structures.

molecular biology↗

bioRxiv2020

Non-canonical, potassium-driven cerebrospinal fluid clearance

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation is deleterious for perinatal neurodevelopment, but how CSF leaves the brain during this critical period is unknown. We found in mice a postnatal neurodevelopmental transition phase featuring precipitous CSF K+ clearance, accompanied by water, through the choroid plexus (ChP). The period corresponds to a human fetal stage when canonical CSF clearance pathways have yet to form and congenital hydrocephalus begins to manifest. Unbiased ChP metabolic and ribosomal profiling highlighted this transition phase with increased ATP yield and activated energy-dependent K+ transporters, in particular the Na+-K+-Cl- and water cotransporter NKCC1. ChP-targeted NKCC1 overexpression enhanced K+-driven CSF clearance and enabled more permissive cerebral hydrodynamics. Moreover, ventriculomegaly in an obstructive hydrocephalus model was improved by ChP-targeted NKCC1 overexpression. Collectively, we identified K+-driven CSF clearance through ChP during a transient but critical neurodevelopmental phase, with translational value for pathologic conditions.

neuroscience↗

bioRxiv2017

Bioengineered intestinal muscularis complexes with long-term spontaneous and periodic contractions

Although critical for studies of gut motility and intestinal regeneration, the in vitro culture of intestinal muscularis with peristaltic function remains a significant challenge. Periodic contractions of intestinal muscularis result from the coordinated activity of smooth muscle cells (SMC), the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). Reproducing this activity requires the preservation of all these cells in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal muscularis cells for months. In this system, SMC expressed the mature marker myosin heavy chain, and multipolar/dipolar ICC, uniaxonal/multipolar neurons and glial cells were present. Furthermore, drugs affecting ENS, ICC or SMC altered the contractions. Combining this method with scaffolds, contracting cell sheets were formed with organized architecture. With the addition of intestinal epithelial cells, this platform enabled at least 9 types of cells from mucosa and muscularis to coexist and function. The method constitutes a powerful tool for mechanistic studies of gut motility disorders and the regeneration of full-thickness engineered intestine.\n\nIn the small intestine, the mucosa processes partially digested food and absorbs nutrients while the muscularis actuates the peristaltic flow to transport luminal content aborally. Gut motility is central to its digestive and absorptive function. The intestinal muscularis contains various types of cells: of these, smooth muscle cells, the enteric nervous system (ENS)1,2, and the pacemaker interstitial cells of Cajal (ICC)3 are three important players involved in the development of gut motility. Recent studies on intestinal tissue engineering have highlighted the importance of regenerating the functional intestinal muscularis4-9. A variety of systems derived from different cell sources, including pluripotent stem cells (PSC)4-6, embryonic stem cells (ESC)7 and primary tissue8,9, have been established to accomplish this goal and different contractile activities were developed in these systems. Notably, spontaneous contractions have been generated in culture systems that contained both ICC and smooth muscle cells4,6,10-13. In addition, electrical-induced neurogenic contractions were also successfully produced4,5,8 when ENS was introduced into culture. In one of the most recent studies, both spontaneous contractions and electrical-induced neurogenic contractions were developed in a PSC-based culture system4.

bioengineering↗