Biology subjects

Kim, H.

Publications and source records attributed to Kim, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

A self-limiting orexin-habenula circuit for stress resilience

Resilience requires neural systems that mobilise active coping during stress while limiting its persistence to preserve homeostasis under sustained challenge1-4. Here we identify a self-limiting orexin-habenula circuit in which lateral hypothalamic orexin neurons engage aromatic L-amino acid decarboxylase-expressing D-neurons (encoded by Ddc) in the lateral habenula via orexin receptor type 2 (OX2R)5-7. Activation of this pathway increased nucleus accumbens dopamine and promoted active coping and positive valence. Optotagging revealed rapid stress-evoked recruitment followed by post-stress suppression. In lateral habenula neurons, orexin peptides exerted dissociable effects: orexin-A engaged an OX2R-dependent inhibitory programme superimposed on a parallel inward current, whereas orexin-B did not reproduce this inhibitory profile and instead exerted a distinct membrane effect. Chronic stress disrupted this buffering system through coordinated inflammatory activation, promoter methylation and erosion of D-neuron identity and orexin responsiveness. Restoring orexin-A reversed behavioural and molecular deficits through OX2R-dependent suppression of nuclear factor kappa B signalling, preservation of Tet2 expression, and demethylation-linked maintenance of the Ddc programme. Together, these findings define a self-limiting orexin-habenula resilience circuit that enables adaptive coping while constraining stress-induced vulnerability.

neuroscience↗

bioRxiv2026

Amplification-Free Detection of Antibiotic Resistance in Enterococcus faecium using PNA-FISH

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen, with antibiotic resistance mediated by the vanA and vanB operons. Rapid and accurate detection of antibiotic resistance is critical for the timely treatment of bacteremia and sepsis. Although imaging-based approaches using fluorescence in situ hybridization (FISH) provide a potential diagnostic solution, detecting mRNAs of antibiotic resistance genes (ARGs) in individual cells remains particularly challenging due to their low copy number and transient expression. Here, we present a peptide nucleic acid (PNA)-FISH method for direct detection of vanA- and vanB-associated resistance in individual VREfm cells. A universal probe targeting the conserved region across vancomycin resistance genes and a set of probes exclusively targeting the vanB gene were designed. The universal probe showed increased fluorescence in the vanA-genotype strain upon vancomycin or teicoplanin treatment, and in the vanB-genotype strain upon vancomycin treatment. In contrast, vanB-specific probes showed increased fluorescence exclusively from the vanB-genotype strain upon vancomycin treatment, confirming their specificity to the vanB gene. Efficient cellular penetration and strong hybridization of PNA probes enabled efficient and accurate detection of antibiotic-resistant bacterial cells, even under a wide-field fluorescence microscope. No detectable signals above background were observed in other major bacterial species associated with bacteremia and sepsis. These findings demonstrate robust detection of antibiotic-resistant cells in mixed microbial populations. When integrated with microbe-capturing techniques, this method may support culture-free detection of antibiotic resistance without nucleic acid amplification or sequencing, with the potential to reduce diagnostic turnaround time.

microbiology↗

bioRxiv2026

RNA exosome-mediated RNA surveillance governs developmental timing in the human cerebellum

Defects in RNA metabolism are a defining feature of neurodevelopmental disease, yet how RNA decay pathways contribute to human brain development remains poorly understood. Mutations in ubiquitously expressed RNA surveillance factors often cause highly tissue-selective disease, highlighting a central paradox in human biology. The RNA exosome is a conserved ribonuclease complex traditionally viewed as a housekeeping machine for RNA turnover, yet recessive mutations in genes encoding structural subunits of the complex disproportionately cause neurological disease, suggesting an instructive role in nervous system development. Here, we show that the RNA exosome regulates the temporal progression of gene expression programs during human cerebellar differentiation. Using CRISPR-engineered human cerebellar organoids modeling EXOSC3 variants, we find that RNA exosome dysfunction does not broadly alter transcript abundance, but instead disrupts transitions between developmental states. Mutant organoids exhibit incomplete and mis-timed resolution of early transcriptional programs, altered lineage specificity, and impaired coordination of maturation-associated gene expression programs, with pronounced effects in neuronal lineages, particularly Purkinje cells and rhombic lip-derivatives. These defects are accompanied by disorganized laminar architecture and reduced coordination of neuronal activity, despite preserved intrinsic excitability. Together, our findings establish RNA surveillance as a key regulator of developmental timing, lineage fidelity, and neurodevelopmental disease.

molecular biology↗

bioRxiv2026

Lack of effect of physiological oxidative stress on N-terminal cysteine dependent proteolysis

Oxidative post-translational modifications on the sulfhydryl group of cysteines can occur spontaneously or enzymatically. The dioxygenation of N-terminal cysteines has emerged as a new oxygen sensing paradigm, catalysed by 2-aminoethanethiol dioxygenase (ADO) in mammals. Conflicting evidence has been reported in recent years on whether this reaction can occur in the absence of ADO. Here we sought to address whether physiological oxidative stress can interfere with ADO-catalysed N-terminal dioxygenation. Using a system to produce titratable intracellular levels of H2O2, we demonstrate that the stability of RGS4 and 5 is not affected by oxidative stress, whether ADO is present or not. However, cytotoxic levels of oxidative stress did induce an increase in RGS4/5 protein levels that occurred independently of the Cys N-degron pathway. This effect of tBHP was reduced by Fe2+ chelation and perturbations of lysosomal function, suggesting the possible involvement of ferroptosis. We conclude that N-terminal cysteine dependent proteolysis of RGS4/5 is not sensitive to physiological oxidative stress, but these proteins can be stabilised during the process of oxidative stress-induced cell death through an N-terminal cysteine independent mechanism.

cell biology↗

bioRxiv2026

High-variance phenome database reveals important roles of WD40 proteins in the plant pathogenic fungus Fusarium graminearum

WD40 is a highly conserved protein domain in eukaryotes, playing a critical role in various cellular process. We conducted genome-wide functional analysis of WD40 genes in Fusarium graminearum-a phytopathogenic fungus that causes severe yield loss and mycotoxin contamination in major cereal crops. Comprehensive phenome analysis of 119 WD40 gene deletion mutants across 22 distinct phenotypic traits revealed phenotypic divergence within the phenome, establishing a strong correlation between virulence and sexual reproduction. Notably, 21 core WD40 genes were identified, offering valuable insights into divergent biological processes. Pilot interactome studies of Fgwd101 and Fgwd133 provided further insights into their potential pathobiological functions. Our investigation contributes to broadening our knowledge of the biological mechanisms underlying fungal pathogenesis and may assist in the identification of targets for antifungal agents.

molecular biology↗

bioRxiv2026

Redox-Triggered Coupling Network Mediates Long-Range Energy Trans-duction in Respiratory Complex I

Complex I is a gigantic redox-driven proton pump that powers oxidative phosphorylation by a unique long-range (>200 [A]) proton-coupled electron transfer process. To elucidate the molecular principles underlying this intricate action-at-a-distance effect, we combine here multiscale quantum/classical (QM/MM) simulations with site-directed mutagenesis, proteoliposome experiments, and cryo-electron microscopy (cryo-EM). We find that quinol binding at a distinct site within a local membrane cavity, triggers a long-range protonation cascade over water-mediated proton wires along a conserved carboxylate pathway (E-channel). We identify a central mechanical switch point, comprising the conserved Tyr156H, the mutation of which impedes conformational changes along conserved loops, but not the proton transfer reaction itself. Using our integrative multi-disciplinary approach, we reveal central coupling sites along the redox-driven proton transport process mediating energy conversion in Complex I, and illustrate the power of theory-guided experiments.

biophysics↗

bioRxiv2026

KIF18A Maintains Kinetochore-Microtubule Attachments in CIN Cells by Limiting Microtubule Polymerization

Chromosomal instability (CIN) generates vulnerabilities that can be therapeutically exploited, including sensitivity to inhibition of the kinesin motor KIF18A. However, the mechanistic basis for why a subset of CIN tumor cells depend on KIF18A remains unclear. Here, we compare mitotic phenotypes across KIF18A-sensitive and -insensitive cell models. In sensitive CIN cells, KIF18A inhibition leads to formation of polar chromosomes with unattached kinetochores, recruitment of spindle assembly checkpoint proteins, and prolonged mitotic arrest. Although KIF18A loss reduces kinetochore-microtubule stability in all cell lines, sensitive cells exhibit lower baseline attachment stability and heightened microtubule polymerization rates, predisposing them to attachment failure. Acute KIF18A inhibition disrupts maintenance of attachments after metaphase alignment, while reducing microtubule polymerization suppresses mitotic defects. These findings support a model in which CIN tumor cells rely on KIF18A to restrain excessive microtubule dynamics and maintain attachment.

cancer biology↗

bioRxiv2026

Loss of Mast cells and histaminergic signaling link diet to platelet-mediated NETosis and mammary cancer recurrence

Breast cancer recurrence remains a clinical challenge. The period after the treatment of the primary tumor while cancer cells that evaded initial treatment lay dormant, provides a unique window of opportunity for interventions to prevent recurrence. Specific modifiable factors such as consumption of high fat diets or elevated circulating cholesterol are associated with decreased time to recurrence. Mechanistically, oxidized cholesterol and lipid species have been implicated in the regulation of the tumor microenvironment. This suggests that consumption of food prepared under oxidizing conditions such as pan-frying, may be an underappreciated risk. Using murine models of mammary cancer dormancy, we found that a diet enriched with fat from fried, cured bacon (cfBF) decreased dormancy latency times. Resulting lesions had fewer mast cells (MCs). Loss of MCs alone resulted in reemergence from dormancy. Elevated expression of a MC gene signature in breast tumors was associated with improved progression free and overall survival, highlighting the human relevance of these findings. MCs are a major source of tissue histamine, and lesions from mice fed cfBF had decreased concentrations. Importantly, antagonists of the histamine receptor 2 (H2R) sparked reemergence from dormancy. H2R antagonists are over-the-counter drugs are taken to alleviate gastroesophageal reflux disease. Chronic treatment of mice with H2R-antagonists sensitized platelets towards activation and crosstalk with neutrophils, and subsequent formation of neutrophil extracellular traps (NETs). The loss of platelet or NETosis activity mitigated the H2R-antagonist stimulated reemergence from dormancy. Therefore, we establish a novel metastatic axis which links diet to recurrence via MCs, histaminergic signaling and NETosis: Diet -- MC -- H2R -- (decreased) Platelet Activity -- (decreased) Neutrophil-NETosis -- (decreased) Reemergence from Dormancy. Our data reveal several potential intervention strategies: lifestyle, MC stabilization, histaminergic signaling, and neutrophil and platelet activity.

cancer biology↗

bioRxiv2026

Label free multimodal optical imaging of metabolic heterogeneity in aging by integrating SRS, MPF, FLIM, and SHG

Cellular metabolism is governed by the coordinated organization of macromolecules, including lipids and proteins, together with redox-active cofactors such as NADH and FAD. However, resolving these biochemical features quantitatively and spatially at subcellular resolution remains challenging because no single imaging modality can capture molecular composition, redox state, and tissue architecture simultaneously without labeling. Here, we present MANIFEST (Multimodal Analysis of Nonlinear Imaging for Functional Endogenous Spatial correlaTion), a label-free imaging platform that integrates stimulated Raman scattering (SRS), second harmonic generation (SHG), multiphoton fluorescence (MPF), and fluorescence lifetime imaging microscopy (FLIM) with their spatial correlation. MANIFEST combines chemical imaging of lipids with autofluorescence- and lifetime-based quantification of NADH and FAD metabolism, enabling spatially resolved analysis of metabolic heterogeneity at organelle and tissue-compartment levels. We apply this framework to four distinct aging or disease models: amyloid-beta-treated tri-cultured brain cells, high-fat diet mouse liver, human non-ischemic cardiomyopathy tissue, and aging mouse retina. Across these systems, MANIFEST reveals disease-associated lipid remodeling, redox imbalance, disrupted metabolic zonation, collagen reorganization, and layer-specific metabolic changes. By integrating complementary nonlinear optical modalities into a single label-free platform, MANIFEST provides an extensible, proof-of-concept platform for high-resolution metabolic phenotyping in complex biological systems and offers new opportunities for studying disease mechanisms, aging biology, and metabolism-driven tissue pathology.

bioengineering↗

bioRxiv2026

ZDHHC17 Links S-Acylation, Huntington Disease, VCP-associated Multisystem Proteinopathy, and Amyotrophic Lateral Sclerosis.

Protein mislocalization is an important contributor to neurodegeneration. We have identified disrupted S-acylation as a driver of mislocalization. S11acylation, the addition of long-chain fatty acids to cysteine residues, is mediated by the ZDHHC family of S-acyltransferases, and regulates protein localization by modulating protein hydrophobicity. Among these enzymes, we have identified ZDHHC17 as a central node in neurodegenerative disease. Although decreased ZDHHC17 activity is correlated with Huntington disease (HD) pathology, its broader contributions to neurodegeneration remain poorly defined. Here, we present new evidence that ZDHHC17 S-acylates or interacts with multiple proteins implicated in amyotrophic lateral sclerosis (ALS), including Valosin-containing protein (VCP) and TAR DNA Binding Protein (TDP1143). We further confirm that VCP, TDP-43, FUS, C9ORF72, and SQSTM1 are S-acylated across rodent models of multiple neurodegenerative diseases, including HD, VCP-associated multisystem proteinopathy, and ALS. Using biochemical S-acylation assays and confocal microscopy, we show that ZDHHC17 S-acylates VCP, depletes the nuclear localization of both VCP and TDP-43, and modulates VCP-dependent toxicity via ER-stress. We additionally demonstrate that TDP-43 is S-acylated by ZDHHC9. Motivated by these findings, we investigated the functional significance of motor-neuronal (MN) ZDHHC17 by studying dHip14 (the fly analog of ZDHHC17) in Drosophila melanogaster. Here, we found that motor neuron-specific dHip14 knockdown (KD) in flies resulted in impaired motor function, whereas ubiquitous depletion led to pharate-adult lethality. Together, these results highlight a shared mechanism across neurodegenerative diseases. Collectively, our findings position ZDHHC17 as a critical enzyme that links fatty acylation, protein homeostasis, and neurodegeneration. Understanding how ZDHHC17 orchestrates S-acylation across different cellular pathways may reveal new therapeutic strategies to restore proteostasis in neurodegenerative disease.

molecular biology↗

bioRxiv2026

Mapping the mammalian dark metabolome by in vivo isotope tracing

Despite decades of biochemical study, a comprehensive map of the mammalian metabolome remains elusive. Mass spectrometry-based metabolomics detects thousands of small molecule-associated signals in mammalian tissues, but it is currently unclear how many of these reflect products of endogenous metabolism. Here, we leverage systematic in vivo isotope tracing to infer the biosynthetic origins of unidentified metabolites. We administered 26 different isotopically labelled nutrients to mice, measured circulating and tissue metabolite labelling by mass spectrometry, and developed a statistical framework to infer the number of carbon atoms incorporated from each of these precursors into more than 4,000 putative metabolites. We show this information can be harnessed for biosynthesis-aware structure elucidation using a multimodal AI model that co-embeds isotopic labelling patterns with chemical structures. This approach revealed several previously unrecognized families of mammalian metabolites, including cysteine-derived alkylthiazolidines, dithioacetal mercapturic acid derivatives, short-chain N-acyltaurines, acylglycyltaurines, and N-oxidized taurines. It further uncovered a family of mevalonate-derived isoprenoid metabolites that includes 2,3-dihydrofarnesoic acid, which is markedly depleted in both mouse and human aging. Age-related depletion of these isoprenoids is driven by impaired coenzyme A synthesis. Our work establishes the biosynthetic precursors for thousands of unidentified metabolites and reveals multiple previously unrecognized branches of mammalian metabolism.

biochemistry↗

bioRxiv2026

Coupling between sterol and sphingolipid structure in ordered membrane domains

A hallmark of eukaryotic membranes is the pairing of lineage-specific sterols with characteristic sphingolipid species. Mammalian cell membranes are enriched in both cholesterol and long-chain sphingolipids like sphingomyelin, whereas fungi synthesize ergosterol and very long-chain sphingolipids with sugar-containing head groups. It has been proposed that these two lipid classes co-evolved to support membrane structure and organization. Here we investigated how sterol structure and sphingolipid chain length together control membrane order and phase behavior. In the yeast Saccharomyces cerevisiae, loss of very long-chain C26 sphingolipids disrupted formation of liquid-ordered (Lo) domains in the vacuole membrane. Similarly, substitution of ergosterol synthesis for that of cholesterol also prevented vacuole Lo domains. To determine a possible physical basis of these effects, we investigated synthetic membranes of defined composition containing either ergosterol or cholesterol and sphingomyelin with different chain lengths. In membranes containing egg sphingomyelin with C16 chains, ergosterol only sparsely supported Lo domains, in contrast to cholesterol. Membranes containing sphingomyelin with C26 chains displayed a different pattern. Cholesterol mixtures were largely homogeneous across most compositions, with only a limited region that supported fluid domains. Ergosterol mixtures exhibited a distinct compositional window that supported fluid domains positioned between regimes of uniform membranes and gel phases. This window corresponded to stoichiometric changes in the vacuole as it phase-separates during nutritional restriction. Measurements of membrane order showed that cholesterol strongly increased membrane packing compared to ergosterol in membranes containing egg sphingomyelin, whereas this difference was lost in membranes containing C26 sphingomyelin. The results suggest that sphingolipid chain length can tune sterol interactions needed for membrane organization. SignificanceMembrane phase separation into coexisting ordered and disordered fluid domains has largely been investigated using characteristic mammalian lipid components, cholesterol and long-chain saturated lipids like sphingomyelin. Under nutrient limitation, vacuole membranes in yeast organize into micron-scale domains that are important for their physiology. Compared to mammals, yeast synthesize an alternative sterol, ergosterol, and sphingolipids with very long-chains. We show that vacuole membrane domains are sensitive to both these features, which also show preferential interactions in liposomes that support membrane ordering and phase properties. In lipid mixtures containing very long-chain sphingomyelins, stoichiometric regimes that support phase separation of fluid domains are similar to those of the vacuole lipidome under nutrient limitation. This finding supports a model in which sterols and sphingolipids co-evolved to support membrane structure.

biophysics↗

bioRxiv2026

Epigenomic methylome landscape of promoters in vertebrate genomes

Genomic promoters are crucial gene regulatory elements1,2. Yet, comparative analyses of promoter architecture have been constrained by the limited resolution of GC-rich regions in short-read-based genome resources3-6. The Vertebrate Genomes Project (VGP) provides more complete long-read-based assemblies7, which further detect 5-methylcytosine signals directly from PacBio HiFi circular consensus reads8,9. Here, we developed a scalable computational framework to characterize DNA methylomes from HiFi data on high-quality Phase I VGP assemblies with RefSeq gene annotations for 82 vertebrate species spanning seven major taxonomic classes: mammals, birds, reptiles, amphibians, lobe-finned fishes, ray-finned fishes, and cartilaginous fishes. We observed a conserved, transcription start site-centered hypomethylation signature in promoters across all vertebrates, and an unexpected hypermethylation signature near gene boundaries that is discordant with transcripts. In addition to this conserved pattern, there were lineage-specific differences in promoter methylation profiles, with birds showing the most diverse patterns. These epigenetic landscapes track phylogenetic relationships more closely than tissue-type methylation differences and infer lineage-dependent widths of core promoters and broader promoters across major vertebrate classes. Our findings establish a comparative epigenomic framework for profiling promoter methylomes from long-read sequencing data.

bioinformatics↗

bioRxiv2026

Programmable domestication of thermophilic bacteria through removal of non-canonical defense systems

Thermophilic bacteria offer major advantages for industrial biotechnology, yet most remain genetically intractable because cellular defense systems block efficient DNA acquisition. Here, we present a programmable domestication strategy that converts wild Geobacillus strains into genetically tractable thermophilic hosts. We developed the Domestication of Non-Model Bacteria (DNMB) Suite, a multi- omics-guided computational framework that systematically identifies genetic barriers to transformation. DNMB analysis revealed that non-canonical nuclease-based defense systems, including Wadjet II, constitute dominant barriers to DNA uptake in previously intractable Geobacillus strains. Targeted deletion of these loci increased transformation efficiency by up to six orders of magnitude. We further established a hierarchical thermophilic engineering toolkit that integrates plasmid artificial modification, conjugation-assisted DNA delivery, and genome editing using an endogenous CRISPR-Cas9 system. The resulting domesticated strains support stable heterologous expression and tunable genetic control at elevated temperatures. Together, these results establish a generalizable framework for transforming genetically intractable thermophiles into programmable industrial chassis.

bioengineering↗

bioRxiv2026

ABI5-mediated ABA signaling enhances aliphatic glucosinolates biosynthesis by transcriptionally suppressing BR signaling factors in Arabidopsis

Brassinosteroids (BRs) are key regulators of plant growth and have been implicated in suppressing glucosinolates (GSLs) biosynthesis in Brassicaceae species, including Arabidopsis thaliana. However, the molecular mechanism linking BR signaling to transcriptional control of the aliphatic GSL pathway remains unclear. Here, we provide genetic and molecular evidence that the BR-responsive transcription factor BZR1 negatively regulates aliphatic GSL biosynthesis through interaction with TOPLESS (TPL) family corepressors and the histone deacetylase HISTONE DEACETYLASE 19 (HDA19). The gain-of-function mutant bzr1-1D exhibited reduced accumulation of aliphatic glucosinolates (GSLs), whereas disruption of TPL family genes (tpl, tpr1, tpr4) or HDA19 resulted in elevated GSL levels, supporting a repressive role for the BZR1-associated complex in the regulation of GSL biosynthetic gene expression. Furthermore, we uncover a functional connection between ABA and BR signaling in this process. The ABA-responsive transcription factor ABI5 reduces the expression of UBP12 and UBP13, which encode deubiquitinases known to influence BZR1 stability. Genetic and transcriptional analyses indicate that ABI5-mediated attenuation of the UBP12/13-BZR1 pathway contributes to enhanced aliphatic GSL accumulation under ABA treatment. Collectively, our findings delineate a regulatory framework linking ABA and BR signaling pathways and suggest that modulation of the UBP12/13-BZR1 module by ABI5 integrates growth and defense responses by fine-tuning aliphatic GSLs biosynthesis in Arabidopsis. One sentence summaryan ABA signaling factor, ABI5 acts to suppress expression of brassinosteroids (BR) hormone signaling genes like UBP12, UBP13, and BZR1 to promote biosynthesis of defensive secondary metabolites, glucosinolates (GSLs) in Arabidopsis.

plant biology↗

bioRxiv2026

Characterizing Physicochemical Selection in Protein Evolution with Property-Informed Models (PRIME)

AbstractStandard probabilistic models of coding sequence evolution effectively identify where and when selection acts but remain agnostic to the mechanistic realization of these forces. We introduce PRIME (PRoperty Informed Models of Evolution), a framework of codon-level maximum likelihood methods--including global (G-PRIME), episodic (E-PRIME), and site-specific (S-PRIME) implementations--that explicitly model amino acid exchange-ability as a function of physicochemical properties. By parameterizing attributes such as molecular volume, hydropathy, and secondary structure propensities, PRIME resolves the biophysical basis of selective constraint across both the sequence and the phylogeny. At the site level, S-PRIME leverages an explicit biophysical taxon-omy to precisely categorize residues as conserved, neutral, or changing for specific properties, resolving selective signals that remain invisible to traditional rate-based metrics. Our analysis of a benchmark of 24 diverse datasets and a genome-wide screen of 18,944 mammalian genes demonstrates that biophysical realism yields substantial improvements in model fit, acting synergistically with rate variation to explain complex evolutionary patterns. We find that power to detect physicochemical constraints at individual sites is fundamentally governed by simple informational redundancy (substitutions per unique amino acid; AUC = 0.91), with sensitivity exceeding 90% in data-rich alignments. E-PRIME reveals a distinct biophysical hierarchy: while core packing and beta-sheet scaffolds are rigidly conserved, alpha-helix propensity and surface electrostatics serve as the primary substrates for adaptive tuning. Furthermore, PRIME importance weights align with aspects of the primary semantic axes of deep learning representations (ESM-2) and capture key features of experimental fitness landscapes. By trans-forming abstract evolutionary rates into interpretable biophysical rules, PRIME provides a useful framework for characterizing the mechanistic drivers of protein diversity.

evolutionary biology↗

bioRxiv2026

Fixative eXchange (FX)-seq: Scalable Single-nucleus RNA Sequencing Analysis of PFA-fixed or FFPE Tissue

Single-nucleus RNA sequencing (snRNA-seq) of clinical formalin-fixed, paraffin-embedded (FFPE) samples has long been a challenge due to low reverse transcription (RT) yields. Here, we present Fixative-eXchange (FX)-seq, a highly scalable snRNA-seq method for heavily paraformaldehyde (PFA)-fixed and/or FFPE samples. We employ an organocatalyst to facilitate the removal of PFA crosslinks to increase RT yield and additional regiospecific Pt(II)-based crosslinking of RNA molecules to prevent leakage. FX-seq reveals cellular heterogeneity across multiple fixed samples by analyzing 321,710 nuclei, including PFA-fixed tissue, FFPE blocks, thin FFPE and hematoxylin and eosin (H&E)-stained sections from mouse brain and human cancer specimens such as gastrointestinal stromal tumor and colorectal cancer. FX-seq enables integrated analysis with pathologist annotation to label tumor and non-tumor regions of H&E-stained sections. FX-seq can also be applied to PFA-perfusion-based animal studies, large human cohort studies, and personalized drug treatment through precision medicine.

genomics↗

bioRxiv2026

PAVR: High-Resolution Cellular Imaging via a Physics-Aware Volumetric Reconstruction Framework

The rapid convergence of advanced microscopy and deep learning is transforming cell biology by enabling imaging systems in which optical encoding and computational inference are jointly optimized for volumetric information capture and interpretation. However, broadly accessible three-dimensional imaging at high spatiotemporal resolution remains constrained by volumetric reconstruction throughput, susceptibility to artifacts, and the burden of collecting modality-matched training data. Here, we introduce PAVR, a physics-aware light-field imaging platform that integrates single-shot volumetric acquisition with fast, end-to-end volumetric reconstruction. PAVR is trained entirely using in silico system responses, avoiding reliance on external high-resolution ground-truth modalities and enabling sample-independent reconstruction across diverse biological contexts. Using fixed and live mammalian cells, we demonstrate multicolor volumetric imaging of subcellular organelles, three-dimensional tracking of autofluorescent particles, and high-speed visualization of organelle remodeling and interactions. We further extend PAVR to quantify coupled morphological and functional dynamics in beating human induced pluripotent stem cell-derived cardiomyocytes under pharmacological perturbation. Together, PAVR establishes a scalable hardware-software platform for high-throughput volumetric imaging and quantitative analysis of dynamic cellular systems in both basic and translational settings.

bioengineering↗