Biology subjects

Lei, Y.

Publications and source records attributed to Lei, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

DNA replication errors drive genome-wide small inverted triplication dynamics

Structural variants (SVs) have a profound impact on phenotype and diversity and are associated with human diseases. To explore the origination of SVs, we have analyzed 1,340 cancer genomes with annotation of 4,608 novel small inverted triplication (SIT) events and found that FEN1 is strongly associated with SIT incidence. Then, we performed long-read sequencing and developed PacBioR to annotate SITs in yeast FEN1 mutant cells. We found that SIT structures mimic classic inverted triplications but with a smaller DUP/IN/DUP structure of 184/160/184 bp on average, with a spacer sequence of 30 bp and breakpoint junction of 6 bp. We further showed that breakpoints of SITs preferentially occurred at nucleosome midpoints, aligned with Okazaki fragment termini, and those harbored in plasmids were precisely eliminated via DNA polymerase slippage over SIT-derived hairpin structures. This study provides mechanistic insight into SIT origination and offers practical tools for future studies on genome rearrangements.

genetics↗

bioRxiv2025

Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

Maintaining sustained deoxyribonucleotide triphosphate (dNTP) pools is essential for DNA replication fidelity and genome stability. In EGFR-mutant non-small cell lung carcinoma (NSCLC), we find that disruption of dNTP homeostasis plays a critical role in determining sensitivity to the EGFR inhibitor osimertinib and in shaping mechanisms of acquired resistance. Transcriptomic and biochemical analyses revealed that osimertinib suppresses RRM2 expression, a key regulator of dNTP synthesis, through the downregulation of the transcription factor MYBL2. In response to osimertinib-mediated replication stress and dNTP depletion, cells activated a compensatory pathway involving the stress-inducible ribonucleotide reductase subunit RRM2B via a novel transcriptional regulator, TNNT3. We further identified CHK2 signaling as essential for TNNT3 nuclear translocation and RRM2B transcriptional activation. Inhibition of CHK2 or combined CHK1/2 blockade impaired RRM2B induction, exacerbated replication stress, and delayed the development of osimertinib resistance both in vitro and in xenograft models. Collectively, our findings reveal that EGFR-mutant NSCLC cells rely on a dynamic EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory axis to maintain dNTP pool balance under therapeutic pressure. Disruption of this axis sensitizes tumors to osimertinib and impairs the acquisition of resistance, highlighting dNTP metabolism as a critical vulnerability and actionable target in EGFR-driven lung cancer. STATEMENT OF SIGNIFICANCETargeting dNTP metabolism via the EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory axis sensitizes EGFR-mutant NSCLC to osimertinib treatment and delays drug resistance, revealing a novel and actionable therapeutic vulnerability in EGFR-driven lung cancer.

cancer biology↗

bioRxiv2025

A Causal Role for the Hippocampus in Gating Rapid Motor Memory Consolidation

How humans acquire motor skills is a fundamental biological question. Early learning involves rapid gains during practice and brief rest, yet the causal contribution of the hippocampus remains unclear. Here, we applied individualized, MRI-guided transcranial ultrasound stimulation (TUS) to modulate the posterior hippocampus in healthy adults. To establish a physiological benchmark, we first showed that this TUS protocol produced a sustained reduction in corticospinal excitability when applied to the primary motor cortex. When applied to the posterior hippocampus, electroencephalography revealed reduced frontocentral gamma-band activity, providing physiological evidence of hippocampal-frontal network modulation. Behaviorally, TUS altered the temporal dynamics of skill acquisition. Compared with sham, hippocampal TUS attenuated micro-offline gains during rest while increasing performance improvements during active practice. This shift from offline to online learning was replicated in an independent cohort with comparable baseline motor performance. Importantly, hippocampal TUS enhanced overall performance during learning, and this higher level of performance remained evident 24 hours later. Together, these findings provide causal evidence that the human hippocampus contributes to how motor learning is distributed between active practice and brief rest, showing that this temporal organization of learning can be altered by non-invasive neuromodulation.

neuroscience↗

bioRxiv2025

Dissociable Roles of Primary Motor and Supplementary Motor Cortex in Shaping the Neural Drive to Muscle

The primary motor cortex (M1) and supplementary motor area (SMA) are critical for motor execution and planning, yet their distinct causal contributions to modulating the neural drive to muscles remain incompletely understood. To dissociate their roles, we applied inhibitory transcranial magnetic stimulation (TMS) over M1, SMA, or a sham condition in 72 healthy participants and characterized the activity of single motor units from high-density EMG recorded during a sustained isometric contraction. Our results revealed a clear functional divergence. M1 inhibition produced a direct failure of motor output, causing a rapid force decline compared to sham, which was strongly correlated with a reduction in motor unit firing rates. Conversely, SMA inhibition did not impair net force. Instead, it altered the fundamental structure of the motor command, compelling a compensatory strategy characterized by a reliance on smaller-amplitude motor units with lower firing rates and a marked degradation of the low-frequency (delta-band) coherence that organizes stable output. These results provide direct causal evidence that M1 directly dictates the magnitude of motor output via population firing rates, while SMA orchestrates the composition and temporal structure of the active motor unit pool to generate an efficient and stable command. Significance StatementUsing causal brain stimulation, we provide the first direct evidence that the primary motor cortex (M1) and supplementary motor area (SMA) serve distinct, non-redundant roles in controlling motor unit populations. We show that inhibiting M1 directly impairs force magnitude by reducing motor unit firing rates. In contrast, inhibiting SMA spares net force but disrupts the underlying motor plan, compelling a compensatory strategy that uses smaller motor units and degrades temporal firing organization via a loss of delta-band coherence. By demonstrating that M1 governs motor power while SMA organizes motor strategy, this study opens new avenues for personalizing neuro-rehabilitation to address the specific cortical origin of a patients motor deficits.

neuroscience↗

bioRxiv2025

XPF mediates 3' flap processing for FEN1-independent Okazaki fragment maturation

Okazaki fragment maturation (OFM), the process that removes RNA-DNA primers, is a major source of DNA replication stress and mutations. It involves Pol{delta}-mediated DNA strand displacement synthesis that produces 5 flaps, FEN1-mediated 5 flap cleavage, and LIG1-catalyzed nick ligation. Recently, we discovered that under stress conditions, yeast cells convert 5 flaps into 3 flaps, which are degraded by 3 flap nucleases to produce ligatable DNA nicks. However, little is known about this stress-induced, 3 flap-based OFM in human cells. Here, we report that 3 flaps frequently form in various human cancer cells, and that FEN1 deficiency significantly enhances 3 flap levels. XPF1 is recruited to the replication forks in FEN1 mutant or FEN1-chemically inhibited cells. Notably, XPF deficiency or inhibition in those defective cells leads to accumulation of 3 flaps, replication-related DNA strand breaks, and unique mutation signatures. Furthermore, XPF and FEN1 inhibitors show synergistic effects in killing human cancer cells. In summary, we demonstrate that 3 flap-based OFM is an important alternative of 5 flap-based OFM in mammalian cells. XPF is a key nuclease to degrade 3 flaps and complete OFM for survival. Targeting this compensatory mechanism could provide new therapeutic strategies to selectively impair cancer cell survival under pre-existed replication stress. HighlightsO_LIXPF is recruited to DNA replication forks in mouse or human cells of functional deficiency of FEN1 due to genetic mutations or chemical inhibition. C_LIO_LIXPF gene deficiency or chemical inhibition results in accumulation of 3 flaps and replicative DNA single-stranded breaks. C_LIO_LIFunctional deficiency of XPF and FEN1 displays a synergy in inducing genome instability and cell death in human cancer cells. C_LI

cell biology↗

bioRxiv2025

Correlative 4D-STEM Ptychography and EELS for Cryogenic Single-Particle Analysis

Cryogenic electron microscopy has enabled atomic resolution structural determination of biological macromolecules. However, phase contrast imaging alone does not reveal elemental composition or other valuable chemical information. Electron energy loss spectroscopy (EELS) provides rich vibrational and chemical details, integrating it with high-resolution structural imaging for correlative analysis of biological specimens remains a major challenge, as conventional acquisition modes are often mutually exclusive. Here we propose two optical geometries that enable correlative 4D-STEM with EELS: (1) a sequential acquisition with a post-specimen beam deflector, and (2) a simultaneous acquisition with a hollow detector. We compared the two setups for biological specimens, and focused on the hollow detector geometry which allows simultaneous acquisition of diffraction patterns and EELS spectra in a single dose budget. Using an apoferritin dataset, we demonstrate sub-nanometre 3D resolution in single particle analysis, even with a hollow angle as of 55% of the convergence angle and approximately 30% of the total dose allocated to EELS. Finally, we propose a workflow that integrates ptychography with EELS for correlative, energy-resolved 3D mapping of biological macromolecules, paving the way toward unified structural, chemical, and vibrational analysis of biological specimens.

biophysics↗

bioRxiv2025

ACE2 Phosphorylation Modulates Angiogenesis via the Activator Protein-1

BackgroundAngiogenesis plays a crucial role in organ development. However, aberrant blood vessel growth is involved in various diseases, including tumors and neovascular eye diseases. Angiotensin-converting enzyme 2 (ACE2) is a critical enzyme regulating the health of cardiovascular system, and its post-translational modifications (PTMs) are crucial to determine ACE2 expression level and activity. Here, we studied how the PTM of ACE2 in vascular endothelial cells (ECs) affect pathological retinal neovascularization and tumor angiogenesis. MethodsThe angiogenic capabilities of ECs were assessed by tube formation, sprouting assays, and 5-ethynyl-2-deoxyuridine (EdU) and filopodia staining. EC angiogenesis was examined by poteome profiler array and aortic ring assays in vitro and by the oxygen-induced retinopathy (OIR) and tumor angiogenesis models in vivo. High-throughput screening involving data from RNA-seq, ATAC-seq, and ChIP-seq were used to explore the epigenetic and transcriptional regulations of pro-angiogenic genes regualtged by ACE2 PTMs. ResultsACE2 Ser-680 dephosphorylation in connection with Lys-788 ubiquitination increased EC angiogenic phenotype, which were manifested by aberrant vascularization in mouse OIR and tumor models. ACE2 Ser-680 dephosphorylation led to the activation of activator protein-1 (AP-1), which transactivated multiple genes involved in angiogenesis. AP- 1 inhibition mitigated such angiogenesis in vivo. ConclusionOur findings show a novel PTM mechanism of ACE2 involved in pathological angiogeneis. Specifically, ACE2 Ser-680 dephosphorylation facilitated AP-1 transactivation of the downstream pro-angiogenic genes in ECs.

cell biology↗

bioRxiv2025

Chronic kidney disease exacerbates hyperlipidemia and atherosclerosis via miR-148a-3p targeting SIK1/AMPKα1 in liver

BackgroundCardiovascular complications are the leading cause of death in patients with chronic kidney disease (CKD). As a major risk factor for atherosclerotic cardiovascular disease (ASCVD), hyperlipidemia is a common sequela of CKD, which is linked to increased lipid synthesis, impaired lipoprotein clearance, and maladapted reverse cholesterol transport. Because exosomes can transport miRNAs to modulate intercellular and tissue communication, we hypothesized that exosomal miRNAs can act as endocrine-like molecules to modulate the CKD-associated hyperlipidemia and ASCVD. MethodsSmall RNA-seq and qPCR were used to determine the differential plasma level of exosomal miR-148a-3p in ASCVD patients with and without CKD. RNA-seq and in silico analysis were used to establish the regulation of lipid metabolism via miR-148a-3p/salt-inducible kinase 1 (SIK1)/AMP-activated protein kinase alpha 1 subunit (AMPK1) in the liver. Inhibition of miR-148a-3p or overexpression of SIK1/AMPK1 in ApoE-/- mice with 5/6 nephrectomy was used to elucidate the role of the miR-148a-3p/SIK1/AMPK1 axis in CKD-related hyperlipidemia and atherosclerosis. ResultsThe plasma level of exosomal miR-148a-3p was significantly elevated in patients with ASCVD and CKD (ASCVD/CKD) and ApoE-/- mice with 5/6 nephrectomy. Via circulating exosomes, kidney-generated miR-148a-3p was delivered to the liver, where it targeted SIK1/AMPK1 transcripts, thereby upregulating SREBP2/PCSK9 and reciprocally downregulating LDLR. Levels of SIK1 and AMPK1 were decreased in hepatocytes transfected with miR-148a-3p or treated with plasma exosomes from ASCVD/CKD patients, with attendant SREBP2 activation and attenuated LDL-C binding. The decreased LDL-C binding was rectified by SIK1/AMPK1 or LNA-miR-148a-3p overexpression. LNA-miR-148a-3p or AAV8-SIK1/AMPK administration in 5/6 nephrectomy/ApoE-/- mice significantly ameliorated hyperlipidemia and atherosclerosis. ConclusionIn ASCVD/CKD patients and nephrectomy/ApoE-/- mice, kidney-originating miR-148a-3p targeted hepatic SIK1/AMPK1 mRNA. This exosome-mediated endocrine effect upregulated SREBP2/PCSK9 and reciprocally reduced LDLR level, which elevated plasma LDL-C level and exacerbated ASCVD. These findings underscore a kidney-liver-artery axis involved in cardiovascular-kidney-metabolic syndrome.

pathology↗

bioRxiv2025

JMJD1B-mediated FEN1 demethylation allows timely switching of Okazaki fragment maturation core enzymes to avoid mutagenic flap ligation by PARP1-LIG3

Efficient and faithful Okazaki fragment maturation (OFM) depends on the PCNA-coordinated actions of core enzymes Pol{delta}, FEN1, and LIG1. We demonstrate that Pol{delta}, FEN1, and LIG1 sequentially but not simultaneously bind to PCNA in mammalian cells. The association of FEN1 with PCNA, which lies at the center of this orderly program, is mediated by FEN1 R192 methylation, which is also crucial for preventing premature loading of LIG1. Conversely, FEN1 demethylation by the recently identified arginine demethylase JMJD1B promotes FEN1 dissociation from PCNA and LIG1 recruitment. Disruption of the sequential PCNA binding program in R192Q or Jmjd1b-/- cells results in unprocessed 5 flaps that prevent OFM and induction of PARP1-dependent recruitment of LIG3, which has flap ligation activity to join incompletely processed OFs. This alternative OFM process supports cell survival but causes duplications and other DNA mutations. Our findings define fundamental and alternative DNA replication processes underlying mutagenesis and cell survival. HighlightsO_LIThe core enzymes involved in Okazaki fragment maturation (OFM), DNA polymerase {delta} (Pol{delta}), flap endonuclease 1 (FEN1), and ligase 1 (LIG1), sequentially bind to the scaffold protein PCNA for their concerted actions C_LIO_LIFEN1 R192 methylation enables FEN1 binding to PCNA; JMJD1B-mediated FEN1 arginine demethylation provokes FEN1 exit from PCNA and LIG1 recruitment C_LIO_LIFEN1 R192Q mutation and JMJD1B deficiency oppositely alter dynamic PCNA interactions with FEN1 and LIG1 and result in distinct OFM DNA intermediate structures C_LIO_LIActivation of PARP1 at replication forks due to disruption of the sequential PCNA binding program induces LIG3-mediated alternative mutagenic 5 flap ligation for survival. C_LI

cell biology↗

bioRxiv2025

Neuronal HDAC9: A key regulator of cognitive and synaptic aging, rescuing Alzheimer's disease-related phenotypes

Epigenetic regulation is a key determinant of the aging process, and its dysregulation contributes to cognitive aging and increased vulnerability to Alzheimers disease (AD). As major regulators of epigenetic processes, histone deacetylases (HDACs) have emerged as potential therapeutic targets for cognitive enhancement in neurodegenerative diseases. However, the distinct roles of individual HDAC isoforms remain to be defined. Here, we report that HDAC9 is specifically expressed in neurons of human and mouse brains, and its expression declines with age. HDAC9 deficiency impairs cognitive function and synaptic plasticity in young mice. Selective deletion of HDAC9 in hippocampal CA1 neurons also induces cognitive impairment. In contrast, overexpression of HDAC9 in forebrain glutamatergic neurons preserves cognitive function in aged mice. Moreover, HDAC9 is also downregulated in the brain of AD mouse models, whereas neuronal overexpression of HDAC9 alleviates AD-related cognitive and synaptic deficits and reduces A{beta} deposition. Together, these findings suggest neuronal HDAC9 is necessary and sufficient for maintaining cognitive and synaptic functions in the context of aging and AD.

neuroscience↗

bioRxiv2025

Large-Scale Neural Network Compensation Underlying Camouflaging in Trait Autism and Its Potential Mental Health Costs

Social camouflaging refers to strategies to hide or compensate for social difficulties, often at a significant mental health cost, and is particularly prevalent in autism. The large-scale neural network mechanisms underlying this adaptation remain poorly understood. This study aimed to identify these neural underpinnings and their link to other potential mental health issues. Using a dimensional approach, we recruited 110 healthy young adults who completed self-report questionnaires measuring autistic traits and camouflaging as well as depression and anxiety, and underwent resting-state fMRI scans. Using the 300-node Seitzman atlas encompassing 13 functional networks, we examined the interaction between camouflaging and autistic traits on brain network connectivity. Results showed that among individuals with higher autistic traits, greater camouflaging was associated with hyperconnectivities between the Default Mode (DMN) and Cingulo-Opercular Networks (CON), and within the CON. Crucially, DMN-CON hyperconnectivity mediated the relationship between camouflaging and potential mental health cost (i.e., depression and anxiety scores) but only in individuals with higher autistic traits. These findings reveal a specific neuro-compensatory mechanism underlying autistic camouflaging involving self-referential and executive control systems and provide a neurobiological explanation for its potential mental health burden, highlighting the need for societal changes that reduce the pressure for such adaptations. Lay AbstractSocial camouflaging refers to strategies autistic people often use to hide their social differences to better adapt to everyday social contexts. While it may help in the short term, this constant effort can be mentally exhausting and is often linked to severe anxiety and depression. However, little is known about how different brain networks work together to enable social camouflaging and might contribute to a greater risk of anxiety and depression. In this study, autistic traits were measured in 110 young healthy adults in addition to questionnaire-based measures of social camouflaging, anxiety, and depression. Additionally, brain activity was recorded using magnetic resonance imaging while they were resting and not doing any task. We found that, in people with higher autistic traits, social camouflaging was linked to stronger connections between the brain network involved in thinking about oneself (self-reflection; Default Mode Network) and the one in charge of maintaining goal-directed behavior (action control; Cingulo-Opercular Network), as well as stronger connections within the latter brain network. These stronger connections between the self-reflection network and action control network partly explained why camouflaging may be associated with higher levels of anxiety and depression. In short, autistic camouflaging seems to rely on a cognitively demanding neural compensatory process, which can take a toll on mental health. Understanding this underscores the importance of creating a more accepting society to support the well-being of autistic individuals.

neuroscience↗

bioRxiv2025

Genetically encoded assembly recorder temporally resolves cellular histories in cellulo and in vivo

Mapping cellular activity with high spatiotemporal precision in complex tissues is essential for understanding organ physiology, pathology, and regenerative processes. Here, we introduce Granularly Expanding Memory for Intracellular Narrative Integration (GEMINI), an in cellulo recording platform that leverages a computationally designed protein assembly as an intracellular memory device to record individual cells activity histories. GEMINI grows predictably within live cells with minimal interference to cellular functions, capturing cellular activities as tree-ring-like fluorescent patterns in the expanding scaffolds for imaging-based retrospective readout. Absolute chronological information of activity histories was attainable with hour-level accuracy through the integration of fiducial timestamps. GEMINI effectively resolved differential NF{kappa}B-mediated transcriptional changes, distinguishing fast dynamics of 15 minutes, and providing quantifiable signal amplitudes. In a xenograft model, GEMINI recorded inflammation-induced signaling dynamics across tissue with cellular resolution, revealing spatial heterogeneity linked to vascular density. When expressed in the mouse brain, GEMINI exhibited negligible impact on neuronal survival, with animals maintaining normal motor and cognitive behaviors. In physiological contexts, GEMINI successfully resolved both transcriptional changes and activity patterns of neurons in the brain. Together, GEMINI provides a robust and generalizable means for spatiotemporal mapping of cell dynamics underlying physiological and pathological processes in both culture and intact tissues.

bioengineering↗

bioRxiv2025

Single-cell spatial map of cis-regulatory elements for disease-related genes in the macaque cortex

Single-cell spatial transcriptomes have demonstrated molecular and cellular diversity in the brain1, 2, 3, 4, 5, 6, 7, 8, but gene regulatory mechanisms underlying transcriptomic profiles and disease pathogenesis remain largely unknown in primates. Here we performed single-nucleus Assay for Transposase-Accessible Chromatin followed by sequencing (snATAC-seq) for [~]1.6 million cells from 142 cortical regions of two male cynomolgus monkeys (Macaca fascicularis), and identified distinct chromatin accessibility profiles of cis-regulatory elements (CREs) for various cell types. By integrative analysis with large-scale spatial transcriptome data, we found that these CREs showed laminar and regional preferences, with their regional accessibility exhibiting striking dependence on the regions hierarchical level. Cross-species comparison of snATAC-seq data revealed human/macaque-enriched layer-4 glutamatergic neurons and LAMP5/LHX6-expressing GABAergic neurons as well as human/macaque-biased CREs for genes related to neurodevelopment and psychiatric diseases. Importantly, risk single-nucleotide polymorphisms for many brain disorders strongly associated with human/macaque-biased CREs in glutamatergic neuronal types and those for Alzhemers disease strongly associated with CREs exclusively in microglia. Our results provided the basis for understanding the spatial gene regulatory mechanisms underlying cellular diversity and disease pathogenesis in the primate cortex.

neuroscience↗

bioRxiv2025

Stage-resolved gene regulatory network analysis reveals developmental reprogramming and genes with robust stem-preferred expression in sorghum

BackgroundSorghum bicolor is a deep-rooted, heat- and drought-tolerant crop that thrives on marginal lands and is increasingly valued for its applications in biofuel, bioenergy, and biopolymer production. The sorghum stem, which can reach 4-5 meters in length, serves as the primary reservoir of both lignocellulosic biomass and soluble sugars, making it a promising bioenergy feedstock. Although recent advances in genetic, genomic, and transcriptomic resources have improved our understanding of sorghum biology, comprehensive genome-wide analyses of functional dynamics across diverse organ types and developmental stages remain limited. In particular, candidate genes with stem preferred expression pattern or their associated cis-regulatory elements, which may program key stem-related functions and enable organ- or tissue-specific engineering, have not yet been identified. ResultsTo address these gaps, we reanalyzed a published RNA-seq dataset to identify genes with organ-preferential expression and to infer representative organ functions across major developmental stages. Our analysis revealed that the sorghum stem exhibits distinct temporal functional signatures, which correlate with the developmental dynamics of stem-specific genes and their associated regulatory elements. We further identified a set of genes with ubiquitous stem-specific expression across diverse sorghum genotypes, suggesting their universal importance and broad potential for genetic engineering applications. Among them, SbTALE03 and SbTALE04 emerged as stem hub transcription factors (TF). Both genes were empirically validated for their stem specificity across stages. Gene regulatory network analysis further indicated that these TFs participate in stage-specific transcriptional programs that maintain and regulate stem development. ConclusionsThis study presents a genome-wide analysis of organ-specific gene expression, functions, and regulatory networks in sorghum, with a focus on genes preferentially-expressed in stems and their promoter motifs. We identified a set of core stem-specific genes with ubiquitous expression across genotypes and developmental stages, including two experimentally validated transcription factors with potential roles in stem development. These findings offer valuable candidates for further functional characterization and genetic engineering aimed at improving sorghum stem biomass and composition.

plant biology↗

bioRxiv2025

Alternating 2D and 3D Culture Reduces Cell Size and Extends the Lifespan of Placenta-Derived MSCs

Mesenchymal stem cells (MSCs) offer significant therapeutic potential, but traditional 2D culture on rigid substrates results in progressive cell enlargement and senescence, reducing proliferative capacity and therapeutic potency. This poses a major challenge for widespread clinical application. We explored a novel strategy for placenta-derived MSCs combining 2D expansion with 3D spheroid culture to address these limitations. Our research shows that culturing MSCs as 3D spheroids significantly reduces individual cell size and size distribution compared to 2D culture. Spheroid formation is feasible in chemically defined media with minimal cell death, and is enhanced by extracellular matrix protein supplementation. Although MSCs do not proliferate in 3D suspension, an alternating 2D/3D culture protocol, transitioning cells between 2D flasks and 3D spheroids after each passage, effectively slows MSC enlargement and senescence over long periods. This alternating method also preserves MSC immunomodulatory function, unlike continuous 2D culture which leads to its loss. For scalability, we developed an RGD-functionalized alginate hydrogel tube (AlgTube) system that mimics this alternating environment, supporting both adherent growth and chemically triggered spheroid formation. This alternating 2D/3D culture strategy and the scalable AlgTube platform provide a foundation for developing next-generation MSC manufacturing technologies to meet future clinical demands.

bioengineering↗

bioRxiv2025

Scalable Culture of Mammalian Cells in RGD Peptide-Modified Alginate Hydrogel Microtubes

Traditional livestock farming is resource-intensive and environmentally unsustainable, necessitating alternative methods for meat production. Cell cultured meat, produced by expanding and differentiating animal cells, offers great potential for substituting for conventional animal meat. Nevertheless, it is still limited by the scalability and efficiency of current cell culture technologies. In this study, we developed an RGD peptide-modified alginate hydrogel microtube microbioreactor (AlgTubes) to support the scalable culture of anchor-dependent cells, such as myoblasts and adipocytes, for cell-cultured meat production. AlgTubes provide a cell-friendly 3D microenvironment that enhances cell viability, growth, and yield while overcoming limitations of conventional bioreactors, such as shear stress, aggregation, and diffusion constraints. We successfully expanded mouse (C2C12) and quail (QM7) myoblasts in AlgTubes, achieving cell densities exceeding 1.0 x 10 cells/mL, far surpassing traditional stirred-tank bioreactors. Differentiation resulted in the formation of mature myotubes. Co-culturing myoblasts with mesenchymal stem cells or fibroblasts further improved yield and viability, particularly under differentiation conditions. By significantly increasing cell culture density, AlgTubes can substantially reduce culture volume, lowering labor requirements, reagent costs, equipment needs, facility space, and manufacturing expenses.

bioengineering↗

bioRxiv2025

Collagen Nanofiber Reinforced Alginate Hydrogel Tube Microbioreactors for Cell Culture

The large-scale production of mammalian cells is pivotal for various applications; however, current bioreactor technologies encounter significant technical and economic challenges. Scaling up cell cultures remains problematic due to excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates. Nevertheless, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. To address these limitations, this study reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Utilizing a novel micro-extruder, we efficiently produced cell-loaded ColAlgTubes. Collagen formed a dense nanofiber network interwoven with the alginate mesh, enhancing the hydrogels mechanical properties while providing cell adhesion sites. ColAlgTubes protected cells from hydrodynamic stress and maintained cell mass within a 400 m diameter, ensuring efficient nutrient exchange and waste removal. This optimized microenvironment resulted in high cell viability, rapid proliferation, and exceptional yields of 5x108 cells/mL - 200 times higher than conventional culture methods. With their scalability, cost-effectiveness, and efficiency, ColAlgTubes offers a transformative solution for large-scale cell production with broad applications in biotechnology, regenerative medicine, and therapeutic manufacturing.

bioengineering↗

bioRxiv2025

Imputation Disparities Driven by Recent Selectionand Their Impact on Disease Risk Estimation in East and Southeast Asian Populations

Using genotype data consisting of 8,316 individuals, we systematically evaluated imputation performance across six state-of-the-art reference panels for Chinese and Thai populations. A substantial proportion of variants identified through whole-genome sequencing, especially low-frequency variants, remained undetected by existing reference panels. In the Chinese population, the TOPMed panel required an R2 threshold of 0.60-0.70 to achieve comparable imputation accuracy of the ChinaMAP panel without R2 filtering, challenging the standard practice of applying a fixed R2 threshold for downstream analyses. Regional analysis highlighted the role of recent selection in imputation discrepancies and revealed an enrichment of immune-related genes in poorly imputed regions. In addition, we showed that the selection of reference panels and R2 thresholds could significantly influence estimation of polygenic risk score for disease prediction. These findings underscore the importance of developing ancestrally diverse reference panels and provide valuable guidelines for improving genotype imputation in East and Southeast Asian populations.

genomics↗