Biology subjects

Yan, X.

Publications and source records attributed to Yan, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Hierarchical taxonomic constraints shape bacterial nitrogen cycling across functional and molecular scales

Microbes are essential for global nitrogen cycling, yet the extent to which taxonomic identity constrains functional potential remains poorly quantified. Using 73,472 representative bacterial genomes, we establish a multi-scale quantitative framework revealing systematic, hierarchical relationships between taxonomic identity and nitrogen cycling functional potential. Hierarchical variance decomposition reveals that taxonomy explains 20-46% of functional variation across six nitrogen pathways, with the strongest constraints for dissimilatory nitrite reduction to ammonium (46.1%) and nitrogen fixation (44.2%), both negatively correlated with functional prevalence. K-means clustering identifies four class-level functional archetypes (Functionally Inactive, Diversified, Integrated, and Specialized) among 77 bacterial classes. 1,281 genera resolve into five ecological strategies differentiated by their nitrogen retention versus loss capabilities, exhibiting strong phylogenetic signals. Cross-scale validation demonstrates 78.7% genus-level conformity to class-level archetypes, confirming hierarchical functional organization. Molecular evolutionary analysis of 13 genes reveals sequence conservation as a dimension partially independent of pathway-level functional constraints. Paradoxically, the functionally most constrained pathway exhibits only moderate sequence conservation (nrfA: 9th/13 genes), while a moderately constrained pathway shows exceptional conservation (napA). Four constraint-conservation patterns demonstrate that gene-specific structural and ecological factors generate evolutionary rate variation independently of taxonomic associations. Our results establish a hierarchical framework in which taxonomic constraints set baseline functional potential, ecological trade-offs shape strategy diversification, and molecular evolution modulates gene-level conservation patterns across biological scales. This framework establishes quantitative baselines that enable probabilistic inference of nitrogen cycling capabilities from taxonomic composition, with potential applications in amplicon-based community analysis, targeted cultivation, and biogeochemical modeling. ImportanceA fundamental challenge in microbial ecology is inferring functional potential from taxonomic data--a relationship widely assumed but never rigorously quantified. Resolving this is critical because while amplicon sequencing provides cost-effective taxonomic profiling, functional characterization requires expensive metagenomics, limiting large-scale biogeochemical studies. We provide the first systematic quantification demonstrating that taxonomic identity explains 20-46% of nitrogen cycling functional variation, operating hierarchically from class to genus level. Crucially, we reveal that taxonomic constraints operate at both functional distribution and molecular evolution levels as partially independent dimensions, indicating distinct evolutionary mechanisms. This work establishes quantitative foundations for taxonomy-based functional prediction, enabling researchers to extract functional insights from readily available taxonomic surveys. As reference databases expand, this framework will enhance predictive capabilities for nitrogen cycling and broader biogeochemical processes.

genomics↗

bioRxiv2025

Computational design of metalloproteases

Although significant progress has been made in creating de novo metalloenzymes that hydrolyze activated esters, the energetically demanding cleavage of amide bonds has remained a major challenge for enzyme design: amide bonds are significantly more stable than ester bonds, the amine leaving groups in proteins are not activated, and peptide substrates are flexible making them difficult to bind precisely. Here, we report the de novo design of zinc proteases from minimal catalytic motifs using RoseTTAFold Diffusion 2 for Molecular Interfaces, optimized for both enzyme and protein-protein interaction design. Of 135 computational designs experimentally tested, 36% had activity and cleaved precisely at the intended site. The most active design accelerates peptide bond hydrolysis by more than 10^8-fold relative to the uncatalyzed reaction, and by over 10^10-fold following the introduction of four point mutations that enhanced active-site preorganization4. Building on these capabilities, we designed metalloproteases that specifically cleave human TDP-43, the amyloid-{beta} peptide, and serum amyloid A with rate accelerations up to 9.2x10^8-fold over background. We illustrate the potential of our approach for bio-orthogonal control over cell state by generating caged cytokines and caged receptor antagonists that are selectively unmasked by our designed proteases. These results demonstrate that de novo enzyme design has advanced well beyond model reactions with activated substrates and open the door to design of proficient metallohydrolases for a wide range of applications in medicine and bioremediation.

biochemistry↗

bioRxiv2025

Copper-Free Click Chemistry Enables High-Fidelity Engineering of Mitochondria Targeted Brain-Derived Exosomes

Mitochondrial dysfunction is a hallmark of neurodegenerative and neuroinflammatory disorders, including hypertension and cardiovascular disease, yet strategies for safe and precise mitochondrial-targeted delivery remain limited. Here, we establish strain-promoted azide-alkyne cycloaddition (SPAAC) as a biocompatible, high-fidelity chemical platform for engineering mitochondria-targeted brain-derived exosomes (BR-EVs). Copper-free click conjugation of a mitochondrial-targeting ligand (e.g. Cy5-DBCO) under mild aqueous conditions preserved vesicle morphology (30-150 nm core; 120-200 nm hydrodynamic), proteomic composition, and uptake dynamics. Time-course imaging and fluorescence recovery after photobleaching (FRAP) revealed unaltered endocytic kinetics, >75 % mitochondrial colocalization, and intact organelle architecture. In vivo neuroinflammation and biodistribution analyses demonstrated immunological neutrality, strong central nervous system retention, and minimal peripheral dispersion following intracerebroventricular administration. Proteomic profiling of unlabeled Sprague-Dawley (SD) and hypertensive Dahl salt-sensitive (DSS) BR-EVs uncovered hypertension-driven enrichment of oxidative and complement pathways correlating with mitochondrial fragmentation and reactive oxygen species generation in neuronal cultures. These findings establish SPAAC-mediated ligand conjugation as a biocompatible and chemically precise approach for generating mitochondria-targeted exosomes that preserve exosomal identity, biodistribution, and signaling fidelity--advancing a foundational platform for organelle-specific delivery and mechanistic imaging in the central nervous system.

neuroscience↗

bioRxiv2025

A Reversible Mitochondrial ROS Probe for Monitoring Mitophagy Dynamics: Development and Application of MitoFlare

Mitochondrial dysfunction and defective mitophagy are defining features of numerous neurodegenerative and metabolic disorders, yet existing tools provide limited ability to quantify mitophagy dynamics in real time within living, post-mitotic cells. Here we present MitoFlare, a mitochondria-targeted, reversible mtROS-responsive fluorogenic probe that enables continuous, non-genetic visualization of mitochondrial oxidative activation and turnover. MitoFlare incorporates dual TEMPO nitroxide quenchers into a long-wavelength rhodamine scaffold, producing >95% basal quenching and rapid, fully reversible fluorescence activation in response to mitochondrial superoxide, hydroxyl radicals, lipid-derived peroxyl species, and peroxynitrite. When combined with LysoTracker Green, MitoFlare forms a dual-probe imaging platform that resolves the entire mitophagy cascade with high spatial and temporal fidelity in intact PC12 neuronal cells. Using this platform, we established a quantitative framework comprising three mechanistically distinct metrics: (i) a proximity index that reports early mitochondrial engagement with lysosomes, (ii) Manders M1 coefficient that captures mid-stage mitochondria-lysosome fusion and mitophagosome formation, and (iii) a quenching/swelling index that resolves terminal lysosomal degradation. Nutrient deprivation induced a complete, temporally ordered mitophagy program, including mtROS priming, Parkin-OPTN-associated fusion, and efficient acidification-dependent cargo degradation. In contrast, inhibition of v-ATPase with bafilomycin A1 arrested mitophagy at the fusion stage, resulting in persistent redox-active mitochondrial cargo that failed to undergo lysosomal digestion. Importantly, MitoFlares reversible redox chemistry uniquely revealed accumulation of undegraded, oxidatively active mitochondrial remnants within non-acidified vesicles--pathological intermediates that are undetectable using irreversible ROS dyes or genetically encoded reporters. These findings demonstrate that mitophagy proceeds through discrete, redox-regulated and lysosome-dependent phases that can be quantitatively mapped in real time. By enabling synchronized measurement of oxidative activation, organelle trafficking, fusion, and degradation, the MitoFlare-LysoTracker system establishes a new benchmark for dynamic mitophagy analysis in physiologically relevant models. This platform provides a powerful foundation for mechanistic interrogation of mitochondrial quality control and for accelerating the discovery of therapeutic strategies aimed at restoring mitophagic fidelity in neurodegenerative, cardiovascular, and metabolic diseases.

neuroscience↗

bioRxiv2025

Dynamic interactions in human amygdala subregions support hierarchical processing of fearful faces

Fearful-expression recognition is critical for adaptive responses to potential threats and relies on both rapid threat detection and fine-grained face encoding. Yet how human amygdala subregions differentially support these distinct cognitive components remains unclear. Here, we recorded intracranial EEG from lateral and medial amygdala in epilepsy patients performing an emotional face-matching task and combined multivariate decoding, time-frequency and directed-connectivity analyses with intracranial stimulation. The lateral amygdala exhibited early fear-specific responses, characterized by higher decoding accuracy and increased theta/alpha-band (4-12 Hz) power, and transmitted this fear-related information to the medial amygdala, which showed delayed and sustained activation. By contrast, the medial amygdala encoded face-specific information at later stages in the 2-16 Hz band with superior decoding accuracy and then relayed this information back to the lateral amygdala. Intracranial stimulation produced a double dissociation in behavior, with lateral amygdala stimulation disrupting fear detection, whereas medial amygdala stimulation selectively accelerated neutral-face recognition. Together, these findings reveal a temporal hierarchy in the human amygdala, whereby dynamic bidirectional interactions between subregions implement distinct components of fearful-expression recognition, providing a circuit-level framework for understanding social threat processing.

neuroscience↗

bioRxiv2025

Shared neural geometries for bilingual semantic representations

The human brain has the remarkable ability to comprehend and express similar concepts in multiple languages. To understand how it does so, we examined responses of hippocampal neurons during passive listening, directed speaking, and spontaneous conversation, in both English and Spanish, in a small group of balanced bilinguals. We find a small number of putative cross-language neurons, whose responses to equivalent words (e.g., "tierra" and "earth") are correlated. However, neurons semantic tunings differed substantially by language, suggesting language-specific neural implementations. Instead, the crucial driver of translation was a preserved geometric organization of neural responses between the two languages, one that did not depend on neuron level functional overlap. Indeed, that geometry was implemented by a common set of neurons along distinct readout axes; this difference in readout may help prevent cross-language interference. Together, these results suggest that hippocampus encodes a language-independent internal model for meaning.

neuroscience↗

bioRxiv2025

Multimodal AI Decodes Extreme Environment Functional Dark Matter Beyond Homology

Functional annotation of proteins from extreme environments represents a major bottleneck for bioresource discovery, as a vast reservoir of functional dark matter defies existing homology-based methods. We demonstrate that environmental pressures impart conserved physicochemical energy signatures that co-determine protein function with sequence and structure. Here we developed ACCESS, a multimodal graph neural network employing hierarchical contrastive learning with a tailored label-sample co-embedding to fuse energy, sequence, and structural information and overcome homology scarcity. ACCESS surpasses state-of-the-art methods including BLASTp and CLEAN in annotating low-identity enzymes. Applied to extreme environmental metagenomics, we constructed a function map of extremophile enzymes to expand the biocatalyst library, pinpointed functionally critical residues to guide rational design, and enabled large-scale, function-based macro-evolutionary analyses. This paradigm transcends the limitations of homology, illuminating protein dark matter and accelerating the exploration of the biospheres functional diversity for applications in biotechnology and therapeutic development.

bioinformatics↗

bioRxiv2025

Spatiotemporal control of PIWI compartmentalization by mitochondrial scaffolds defines pachytene piRNA pathway organization

Pachytene piRNAs are the least understood class of piRNAs in the mammalian male germ line. During meiosis, their biogenesis occurs near mitochondrial outer membrane in germ granules known as intermitochondrial cement (IMC). However, how mitochondrial factors regulate the trafficking of PIWI proteins into and out of the IMC remain poorly understood. Here we show that the cytoplasmic PIWI proteins MILI and MIWI are recruited for pachytene piRNA biogenesis via distinct mitochondrial membrane proteins. Loss of the mitochondrial scaffold protein ASZ1 during meiosis in mice disrupts multiple downstream biogenesis steps, leading to misregulation of MILI and MIWI, failure of IMC formation, and a near-complete loss of mature pachytene piRNAs. Strikingly, despite the drastic depletion of pachytene piRNAs, LINE1 transposon silencing remains unaffected. We identify three classes of pachytene piRNA pathway components that coordinate piRNA production and compartmentalization. Our findings reveal that chromatoid body precursors serve as a central hub for the accumulation of pachytene PIWI-piRNA complexes, thus establishing a connection between IMC-based biogenesis and downstream piRNA function.

developmental biology↗

bioRxiv2025

A semantotopic map in human hippocampus

The hippocampus plays a central role in encoding abstract conceptual and semantic information. However, little is known about the topography of that encoding. We leveraged the rare opportunity to examine neural responses densely along a small portion of the human hippocampus, specifically, the mediolateral axis of the anterior body. We collected responses to passive language listening using Neuropixels probes in three anesthetized patients during clinically indicated neurosurgical procedures. We computed semantic tuning functions for each recording site by regressing threshold crossing events and single unit responses against semantic embeddings from GPT-2, Word2Vec, and SBERT. We find that tuning functions of more distant recording sites are more dissimilar, supporting the hypothesis semantotopic organization. Multiple semantic features showed systematic changes along that axis, including animacy, concreteness, and familiarity; notably, effects were individual-specific. Surprisingly, we also found a small but significant increase in semantic similarity as a function of distance between recording sites, on a shorter spatial scale, suggesting a modest periodic organization. Together, these results demonstrate the presence of a multiscale functional organization of semantics in the hippocampus.

neuroscience↗

bioRxiv2025

Inhibition of CGRP receptor ameliorates AD pathology by reprogramming lipid metabolism through HDAC11/LXRβ/ABCA1 signaling

The Calcitonin gene-related peptide (CGRP) receptor has gained attention in Alzheimers Disease (AD) research due to its involvement in regulating neuroinflammation. However, its role and mechanism in AD pathology remain unclear. Here, we demonstrate that CALCRL, a core component of the CGRP receptor, is upregulated in the hippocampus of AD dementia patients and 5xFAD mice. Knockout of the CGRP receptor ligand Calca or pharmacological blockade using Rimegepant (Rim), reduces soluble A{beta}1-42 oligomer-induced neuronal death and glial inflammation. Rim treatment also rescues neurobehavioral impairments, neurodegeneration, and lipid metabolism dysfunction in 5xFAD mice. Mechanistically, these effects are mediated through HDAC11 inhibition, which enhances LXR{beta} acetylation and ABCA1 expression, promoting the reprogramming of neuronal lipid metabolism. Importantly, this CALCRL/HDAC11/LXR{beta}/ABCA1 axis is conserved across both humans and mice. Our findings uncover a novel mechanism underlying AD pathogenesis and highlight the therapeutic potential of targeting CGRP signaling in AD. HIGHLIGHTSInhibition of CGRP receptor ameliorates disease pathology in models of AD HDAC11 mediates CGRP receptor function in AD HDAC11 is a pivotal regulator in lipid metabolism by LXR{beta}/ABCA1 signaling The HDAC11/LXR{beta}/ABCA1 axis is conserved in AD humans and mice

neuroscience↗

bioRxiv2025

Elevated Temperatures Representing Heatwave Conditions Shift Active Nitrifying Communities and Their Viruses in Tidal Flats and Agricultural Soils

Global heatwave intensification associated with climate change will impact the nitrogen cycle, yet its effect on specific nitrifier groups or their interactions with viruses remains unclear. Using 13CO2-DNA-based stable isotope probing (SIP) coupled with metagenomics, we show that elevated temperatures associated with heatwave conditions restructure active nitrifying communities and their viruses in Yangtze River estuary tidal flats and adjacent agricultural soils. In tidal flats, high temperatures shifted active ammonia-oxidizing archaea and bacteria (AOA and AOB), and nitrite-oxidizing bacteria (NOB) from marine to terrestrial ecotypes. In contrast, heatwave conditions stimulated terrestrial ecotypes of AOA but suppressed AOB in agricultural soils. 13C-labeled nitrifier-infecting viruses also showed temperature-driven shifts in activity, lifestyle, and auxiliary metabolic genes in concert with hosts. Notably, AOA viruses carried the plastocyanin gene, potentially augmenting host metabolism. These findings demonstrate heatwaves drive complex shifts in nitrifier communities and their interactions with viruses, impacting global nutrient cycling under climate extremes.

microbiology↗

bioRxiv2025

Plant diversity and water regime have distinct effects on soil fungal communities that are linked to plant productivity

The soil microbiome both shapes and is shaped by the ecological consequences of drought and plant diversity loss. Sequencing soil fungal communities in a biodiversity experiment, we found that water addition, plant species richness, and plant phylogenetic diversity all impacted soil fungal composition, and increased the arbuscular mycorrhizal fungi (AMF) richness in particular. The richness of different fungal functional guilds all increased with plant functional trait diversity but were related to different above- and below-ground plant functional traits -- AMF richness was negatively associated with root traits that indicate mycorrhizal dependence, whereas diverse soil pathogens generally co-occurred with plant traits linked to faster life histories. Unexpectedly, the effect of plant diversity loss on fungal diversity did not interact with the effects of either the watering treatment or the community weighted mean of plant traits. Shifts in AMF richness and soil fungal taxa due to plant diversity loss were further associated with reduced plant productivity and diversity effects. Overall, our results indicate that changes in water input and loss in plant taxonomic, phylogenetic, and functional diversity can negatively impact soil fungal communities, and through changes in soil microbiome, may also reduce plant productivity. This negative effect of plant diversity loss is consistent across water regimes and plant community-level traits, suggesting a general vulnerability of soil microbiome and ecosystem productivity towards the biodiversity crisis.

ecology↗

bioRxiv2025

Genetic elements promote retention of extrachromosomal DNA in cancer cells

Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer1,2. Circular megabase-sized ecDNAs lack centromeres and segregate stochastically during cell division3-6 yet persist over many generations. EcDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei over 40 years ago with unknown mechanism3,7. Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. We develop Retain-seq, a genome-scale assay that reveals thousands of human retention elements conferring generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromatin conformation capture show that retention elements physically interact with mitotic chromosomes at regions which are mitotically bookmarked by transcription factors and chromatin proteins, intermolecularly recapitulating promoter-enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated; targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, suggesting that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, revealing the principles of episome immortality intrinsic to the human genome.

cancer biology↗

bioRxiv2025

Synchronous spatio-temporal control of autophagy and organelle trafficking is necessary for appressorium-mediated plant infection by Magnaporthe oryzae

The blast fungus Magnaporthe oryzae infects plants using a specialised infection structure called an appressorium that generates physical force to break the rice leaf cuticle. Appressorium development follows a cell cycle-controlled morphogenetic program, requiring autophagy-associated cell death of the fungal spore from which the infection cell develops. How proliferative growth of the fungus is regulated at the same time as programmed cell death, however, is unknown. In this study, we provide evidence that each cell of the conidium undergoes a separate developmental program, which is necessary for plant infection. Using quantitative live-cell imaging, we monitored trafficking of ten organelle types during appressorium morphogenesis in a wild-type M. oryzae strain and isogenic {Delta}atg8 autophagic mutant. High-resolution microscopy using a photoactivatable green fluorescent protein revealed that organelle trafficking occurs from a single conidium cell into the appressorium, while the remaining two cells undergo autophagy. Organelle inheritance operates independently of cell cycle checkpoints but is always associated with spore germination. We furthermore defined the temporal sequence of organelle movement and de novo organelle biogenesis in the incipient appressorium using photoconvertible fluorescent localisation microscopy. Taken together, our study reveals how synchronous spatiotemporal control of autophagy and organelle trafficking is necessary for rice blast infection.

molecular biology↗

bioRxiv2025

Mirror manifolds: partially overlapping neural subspaces for speaking and listening

We utilize internal representations of meaning for two purposes: to understand the words we hear and to generate our own speech. This dual requirement necessitates abstract, modality-agnostic representations. Building on work identifying it as a hub for relational mapping, we hypothesized that the hippocampus supports abstract, cross-person representations, and uses shared semantic geometries to do so. We tested this hypothesis by examining hippocampal activity in a remarkable single-neuron dataset derived from conversational speech. Neurons robustly encoded meanings of both spoken and heard words, and used common geometric embeddings for both, leading to abstract meaning performance. Speaker identity was aligned with meaning via partial subspace alignment, which affords speaker-meaning binding by partitioning meaning by speaker while maintaining cross-speaker generalization. Degrees of subspace rotation varied on a single word level and depended systematically on semantic category. Together, these findings indicate how geometric principles allow for abstract cross-personal meanings while preserving binding to speaker identity.

neuroscience↗

bioRxiv2025

High-precision Biomedical Text Corpora for Multi-Entity Recognition: A CoDiet study

We present here five biomedical, multi-entity corpora that can be used as benchmarks for named-entity recognition (NER), targeted to literature on metabolic syndrome. The CoDiet-Gold corpus contains annotations for 500 full-text publications and 348,406 annotations. It is divided into CoDiet-Gold-public (450 documents) and CoDiet-Gold-private (50 documents). Each document was independently annotated by two human experts, with disagreements fully adjudicated by a third expert. The CoDiet-Electrum corpus (2,998,273 annotations) contains 4,423 publications that were annotated using case-insensitive matching of the surface forms with punctuation ignored, found in CoDiet-Gold-public. Finally, for the same 4,423 documents, two fully machine annotated corpora CoDiet-Bronze (2,938,738 annotations) and CoDiet-Silver (2,298,988 annotations), were created by utilising existing NER algorithms to annotate these. These corpora contain categories (organisms, disease, genes, proteins, metabolites) that add depth to existing corpora, as well as new categories that do not appear in other corpora (food, dietary methods, sample types, computational methods, study methodology, population characteristics, data types, and microbiome).

bioinformatics↗

bioRxiv2025

scYeast: a Biological-knowledge-guided Foundation Model on Yeast Single-Cell Transcriptomics

Though large-scale pre-trained models are vital for foundational cell modeling, most focus on human or mouse systems, neglecting model organisms like yeast (Saccharomyces cerevisiae) and often failing to use existing biological prior knowledge effectively. To address this, we present scYeast, the first foundational cell model tailored for yeast that seamlessly embeds biological priors. scYeast employs a novel asymmetric parallel architecture to infuse transcriptional regulatory information directly into the Transformers attention mechanism, leveraging established biological knowledge during training. Pre-trained on large-scale yeast single-cell transcriptomics data, scYeast demonstrates strong generalization and biological interpretability. It excels in zero-shot tasks, such as inferring regulatory relationships and identifying critical cell states. After fine-tuning, scYeast performs exceptionally across a diverse set of tasks from cell type classification to predicting growth doubling time and gene perturbation response. Additionally, using transfer learning, scYeast can be adapted to other omics datasets, such as proteomics, thus broadening its utility in big data analysis. Overall, scYeast is a powerful tool for yeast single-cell biology research and sets a new standard for integrating foundational models with biological prior knowledge, dramatically accelerating the pace of discovery in yeast synthetic and systems biology and providing a replicable framework for other organisms.

bioinformatics↗

bioRxiv2025

Formulation and Characterization of Primary Tissue-Derived Microfluidic Droplet-Engineered Organoids

Organoid technology offers a powerful platform for modeling human tissues, studying disease mechanisms, and developing personalized therapies. However, widespread clinical application is hindered by challenges in scalability, reproducibility, and the handling of ultra-small tissue samples typical of clinical biopsies. Here, we present a comprehensive and automated protocol for the formulation and characterization of microfluidic droplet-engineered organoids (DEOs) derived from primary tissue samples. This protocol integrates 3 key stages: (1) Extraction and purification of viable primary cells from ultra-small tissue specimens using the small-Tissue Extraction Device (sTED); (2) High-throughput fabrication of uniform cell-laden microspheres using an integrated microfluidic bioprinter (OrgFab), capable of generating over 100 organoids from just 10 L of bioink; and (3) Rapid organoid characterization using lamination-based processing for single-cell analysis while preserving spatial context. The automated workflow minimizes manual intervention, reducing variability and enhancing reproducibility, making it suitable for high-throughput applications such as drug screening and disease modeling. Our method allows for the generation of patient-derived organoids that closely mimic the native tissue microenvironment, including diverse cell types and structural features, within a significantly reduced timeframe. This 3 stage protocol enhances the use of organoids in personalized medicine by enabling the rapid assessment of drug efficacy in patient-specific models. Here, the integration of advanced techniques supports existing organoid protocols, providing a valuable resource for researchers and clinicians seeking to improve patient outcomes through more efficient and precise organoid-based applications.

bioengineering↗