Biology subjects

Fu, Y.

Publications and source records attributed to Fu, Y..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

ISKNV-pORF71 hijacks outer mitochondrial membrane-localized VDAC2 to the nucleus, enhancing apoptosis by disrupting mitochondrial membrane permeability

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus within the family Iridoviridae, is one of the most devastating pathogens affecting global teleost populations. Our previous study confirmed that ISKNV-pORF71 (p71/VP71) is a viral virulence factor by constructing a recombinant virus with ISKNV orf071 deletion (ISKNV-{Delta}71). In the present study, we further found that compared with ISKNV-WT, the ISKNV-{Delta}71 mutant is more capable of maintaining mitochondrial membrane potential, alleviating mitochondrial membrane permeabilization, preserving mitochondrial membrane integrity, sustaining lower cytochrome c release, and thereby reducing the apoptosis rate of infected cells. Further investigations demonstrated a strong interaction between p71 and the outer mitochondrial membrane (OMM)-localized voltage-dependent anion channel 2 (VDAC2). At the subcellular level, p71 and mandarin fish VDAC2 (mfVDAC2) are originally localized to the nucleus and mitochondria, respectively. However, in cells co-transfected with p71 and mfVDAC2, robust nuclear translocation of mfVDAC2 was verified. Endogenous mfVDAC2 was also confirmed to undergo nuclear translocation upon ISKNV-WT infection. In the zebrafish infection model, zebrafish VDAC2 (zfVDAC2)--but not zfVDAC-1 or zfVDAC-3--was observed to translocate into the nucleus through interaction with p71. Using CRISPR/Cas9 technology, VDAC2-knockout zebrafish were successfully constructed. Infection experiments showed that ISKNV-{Delta}71 exhibits significantly reduced lethality to both wild-type zebrafish and zfVDAC2-knockout zebrafish. In conclusion, our findings reveal that p71 exerts its function during ISKNV infection by hijacking VDAC2 into the nucleus, which alters mitochondrial membrane permeability and enhances ISKNV-induced apoptosis. The phenotypic characteristics of VDAC2 knockout (vdac-/-) zebrafish were also thoroughly analyzed and discussed. Author summaryVDAC2, a well-characterized cellular protein localized to the outer mitochondrial membrane (OMM), plays critical roles in viral pathogenesis and antiviral immune escape. Previous studies have shown that VDAC2 interacts directly or indirectly with viral proteins or functions as a functional receptor to modulate viral pathogenesis; however, few studies have demonstrated that VDAC2 is hijacked from its native OMM to other organelles. Our study here provides robust evidence that cellular VDAC2 is rerouted from its original OMM to the nucleus through interaction with ISKNV p71, thereby altering viral pathogenesis. This work offers novel insights into the role of VDAC2 in pathogen virulence. As far as we know, this is the first report demonstrating that host VDAC2 is hijacked into the nucleus by a viral protein to subsequently modulate the viral life cycle. Collectively, these findings advance our understanding of the active regulation of host functional proteins by iridoviruses.

microbiology↗

bioRxiv2025

Exploring the Conformational Landscape of Adenylate Kinase and Beyond: A Benchmark of Protein Folding Models

Protein folding models have revolutionized structure prediction but struggle to capture conformational flexibility. Recent studies perturb inputs or parameters to sample alternative conformations, while diffusion-based approaches generate conformational ensembles directly. Although the former have been benchmarked to some extent, the latter have yet to be evaluated, and sub-domain dynamics validation remains limited. Here, we present a systematic benchmark of nine methods across 20 monomeric proteins with active and inactive states. We extend the pairwise aligned error metric to ensembles and reveal that protein identity exerts a non-negligible influence on model performance. Focusing on Adenylate Kinase, a well-studied enzyme with extensive molecular dynamics (MD) data, we find that Chai-1 performs the best in recovering known conformations, identifying mobile regions, and capturing transition trajectories. These results highlight the potential of generative models as efficient alternatives to MD for exploring protein conformational dynamics and provide a rigorous benchmark for dynamic structure prediction.

bioinformatics↗

bioRxiv2025

Multiplex design and discovery of proximity handles for programmable proteome editing

Although we now have a rich toolset for genome editing, an equivalent framework for manipulating the proteome with a comparable flexibility and specificity remains elusive. A promising strategy for "proteome editing" is to use bifunctional molecules (e.g. PROteolysis-Targeting Chimeras or PROTACs1) that bring a target protein into proximity with a degradation or stabilization effector, but their broader application is constrained by a limited repertoire of well-characterized target or effector "handles". We asked whether coupling de novo protein design to a multiplex screening framework could address this gap by accelerating the discovery of effector handles for intracellular protein degradation, stabilization, or relocalization. Using LABEL-seq2, a sequencing-based assay that enables multiplex, quantitative measurement of protein abundance, we screened 9,715 de novo designed candidate effector handles for their ability to recruit a target protein to components of the ubiquitin-proteasome system3 (UPS) (FBXL12, TRAF2, UCHL1, USP38) or the autophagy pathway4 (GABARAP, GABARAPL2, MAP1LC3A). In a single experiment, we discovered hundreds of de novo designed effector handles that reproducibly drove either intracellular degradation (n = 277) or stabilization (n = 204) of a reporter protein. Validation of a subset of these hits in an orthogonal assay confirmed that sequencing-based measurements from the primary screen reliably reflected changes in intracellular abundance of the target protein. Successful effector handles were discovered for both the UPS (n = 194) and autophagy (n = 287) pathways, which provide complementary routes for programmable proteome editing. Autophagy-recruiting effector handles generalized to endogenous targets, as substituting the reporter-specific target handle with a high-affinity MCL1 binder5 reduced endogenous levels of this intracellular oncoprotein6. Moreover, directing autophagy-recruiting effector handles to the outer mitochondrial membrane dramatically perturbed mitochondrial networks in a manner consistent with synthetic tethering and sequestration7,8. Beyond generating a diverse repertoire of protein abundance or localization effector handles, our results establish a scalable, low-cost platform that links deep learning-guided protein design to functional cellular readouts, and chart a course toward a general framework for programmable proteome editing.

synthetic biology↗

bioRxiv2025

Design of TCR-mimicking binders for pHLA with high potency

The rational design of high-specificity binders to peptide-HLA (pHLA) complexes remains a major challenge in personalized immunotherapy, particularly for shared neoantigens with single-point mutations. To address this, we have developed an integrated framework that combines knowledge-based deep learning with physics-based simulation for the design of highly specific pHLA binders. Applied to p53 R175H-HLA-A*02:01, molecular-dynamics-guided electrostatic filtering yielded nanomolar binders with >10-fold selectivity over wild type. Motivated by the challenge of KRAS G12V-HLA-A*03:01, which lacks electrostatic cues, we implemented message-passing neural network (MPNN)-based optimization strategies to broaden binder design beyond charge-dependent interfaces. When reapplied to p53 R175H, these strategies improved success rates from 2/5 to 5/5, with most candidates exhibiting minimal or no binding to the wild-type complex. Chimera-based and residue-retention approaches within the MPNN optimization further expanded the sequence-structure search space while preserving critical hotspot interactions. This integrated framework thus enables the design of nanomolar, mutation-selective binders to pHLA complexes, advancing next-generation personalized cancer immunotherapies.

biophysics↗

bioRxiv2025

Comprehensive benchmarking of somatic structural variant detection at ultra-low allele fractions

Postzygotic mosaicism gives rise to somatic structural variants (SVs) at ultra-low variant allele fractions (VAFs), which pose challenges for detection due to the high-coverage sequencing required and noise introduced by sequencing artifacts. Although somatic SV detection has been extensively studied in cancer, these studies are not directly applicable to the study of tissue mosaicism, as they rely on matched normals, target higher VAF ranges, and are enriched for different types of SVs. We present comprehensive benchmark data and best practices for non-cancer somatic SV detection. We created a synthetic mosaic sample by combining six HapMap individuals at varying proportions, generating allele fractions as low as 0.25%. This sample was sequenced to [~]2,300x total coverage using Illumina, PacBio, and Nanopore technologies across multiple sequencing centers. A high-confidence benchmark SV set containing over 21,000 pseudo-somatic insertions and deletions [≥]50bp was derived from haplotype-resolved assemblies. We evaluated 12 SV discovery pipelines and identified caller-specific strengths and sequencing platform-specific shortcomings. We find that short read-based approaches show reduced recall for insertions and repeat-associated SVs, whereas long-read sequencing achieves high accuracy throughout the genome, increasing linearly with coverage. The best algorithms sensitivity exceeded 80% for VAFs [≥]4% and 15% for VAFs of 0.5-1% with 60x coverage. The publicly available benchmarking data and comparative analysis of current methods provide a foundation for robust discovery of SV mosaicism in non-cancer tissues..

bioinformatics↗

bioRxiv2025

Single-cell Genomic Copy Number Evolution Reveals Frequent Loss of the Y chromosome in Esophageal Adenocarcinoma

The incidence of esophageal adenocarcinoma (EAC), an aggressive cancer, is rising in Western countries, and incidence is higher in men. To better understand the roles of DNA copy number evolution, tumor heterogeneity, and tumor microenvironment in EAC and its origins, we evaluated EAC, Barretts Esophagus (BE), and normal adjacent tissue using our previously developed ultra-sensitive, single-nucleus DNA copy number analysis (12,186 nuclei, 20 specimens) and single-nucleus RNA-sequencing (snRNA-seq; 87,027 nuclei, 32 specimens). Loss of Y (LOY) chromosome was rare in BE but markedly enriched in EAC. In genome unstable cells, complete LOY chromosome often coincided with X-chromosome duplication. We did not detect X-chromosome inactivation via XIST expression in cells with LOY, suggesting increased expression of X-chromosome genes and potential effects on tumor immune evasion. When considering patients clinical features, we identified distinct differences in tumor microenvironment by sex, age, and obesity status, particularly massively reduced immune cell composition in obese patients. Finally, our cell line models recapitulate LOY, and we identify the first candidate anti-LOY agent. Our findings highlight how LOY affects tumor subpopulations and shapes the tumor microenvironment during EAC progression.

cancer biology↗

bioRxiv2025

Dual checkpoint blockade of glioblastoma with Anti-PD-1 and Anti-LAG-3 promotes expansion of tumor-reactive T cell clones along a unique pathway of differentiation

IDH-wildtype grade IV glioblastoma is the most aggressive adult primary brain tumor and remains refractory to anti-PD-1 monotherapy despite evidence of limited tumor-specific T cell induction. To determine the impact of immune checkpoint inhibitors (ICIs) on glioblastoma T cell transcriptional landscape and repertoire, we conducted paired single-cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (TCR-seq) of tumor-infiltrating lymphocytes (TILs) from patients with untreated, newly diagnosed glioblastoma and from recurrent glioblastoma treated with dual checkpoint blockade targeting PD-1 and LAG-3. Using a validated transcriptional signature, we found that predicted tumor-reactive T cells (TRC) in untreated glioblastomas reside almost exclusively in a clonally expanded GZMKhi population with developmental plasticity, affording them the potential to differentiate into both tissue-resident and terminal effector T cells. Dual ICI therapy induced substantial clonal remodeling, characterized by the recruitment of new TRC from the periphery into the tumor microenvironment (TME) and differentiation into transitional effectors and ultimately terminal effectors along a gradient characterized by simultaneous acquisition of cytotoxic and exhaustion genes, regulated by specific transcriptional, metabolic, and epigenetic programs. Longitudinal clonal tracking in peripheral blood confirmed that with ICI treatment, most TRC expand transiently in circulation prior to tumor infiltration, with peripherally derived clones becoming the major contributor to the GZMKhi TRC that further expand in the tumor. Our study provides the first comprehensive map of T cell clonal dynamics and differentiation in glioblastoma following dual ICIs and highlights a potential mechanism of immune activation and peripheral recruitment of TRC in glioblastoma not previously described. Our results suggest that therapeutic strategies to sustain these GZMKhi early effector and transitional effector T cells may further enhance ICI therapeutic efficacy in glioblastoma.

immunology↗

bioRxiv2025

Enteric ChAT-expressing neurons as new target for Lactobacillus plantarum ameliorates inflammatory bowel diseases

The gut microbiota plays a crucial role in inflammatory bowel diseases (IBD), yet how specific microbial components influence disease progression remains incompletely understood. Our study reveals that decreased abundance of Lactobacillus species correlates with ulcerative colitis severity in patients. Among tested strains, L. plantarum A736, isolated from traditional fermented foods, demonstrated remarkable efficacy in ameliorating dextran sulfate sodium (DSS)-induced colitis in a mouse model. Strikingly, multi-omics analysis revealed that L. plantarum A736 uniquely enhances choline acetyltransferase-expressing (ChAT+) neurons in the enteric nervous system and elevates acetylcholine (ACh) levels. Further analysis identified that L. plantarum A736 metabolizes tryptophan to produce indole-3-lactic acid (ILA), and supplementation with ILA significantly mimics this strains anti-inflammatory effects by activating ChAT+ neurons. Crucially, selective ablation of colonic ChAT+ neurons completely abolished the therapeutic benefits of both L. plantarum A736 and ILA, establishing their essential role in mediating these effects. These findings demonstrate for the first time that microbial metabolites can directly modulate enteric neuronal circuits to regulate gut immunity. The identified ILA-ChAT+ neuron axis represents a novel neuro-immune pathway that could be targeted for IBD treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/673902v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1e0d3c5org.highwire.dtl.DTLVardef@16f5481org.highwire.dtl.DTLVardef@152283org.highwire.dtl.DTLVardef@18cfad0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Mating-Type Imputation (MTI) provides an efficient tool for the mating-type inference of tetrapolar fungi

Mating-type identification is fundamental for the genetic diversity and genetic breeding study in fungi, especially for tetrapolar basidiomycetes, whose mating-types are determined by two multiallelic loci A and B. Traditional mating-type identification of monokaryons relies on manual inference based on their hybridization experiments; however, the process is rather complex, time-consuming and error-prone for large-scale hybridization experiments. In this study, we developed the Mating-Type Imputation (MTI) software for automatic, rapid and accurate monokaryon mating-type inference for tetrapolar fungi using a combinatorial pruning traversal algorithm. Taking the compatibility matrix from 435 hybridization experiments of 30 Flammulina velutipes monokaryons as an example, MTI takes a few minutes to infer the accurate mating-types of all monokaryons, which might take several days for manual inference by experienced investigators. Furthermore, MTI enables us to investigate how false-positives and false-negatives influence mating-type inference results. Using simulated compatibility matrix, we found that MTI could accurately detect potential false-negatives in compatibility and successfully infer the true mating-type combination with limited false-negatives, but the tool could be misled by any false-positives to result in a wrong mating-type combination, indicating that false-positives records in hybridization experiments should be strictly obviated for mating-type inferences. In summary, MTI provides an efficient tool for mating-type inference of tetrapolar fungi, offering technical support for the research paradigm in mating-type studies of edible and medicinal fungi and holding significant theoretical value and broad application prospects in the fields of fungal genetic diversity and breeding studies.

genetics↗

bioRxiv2025

Deciphering Cell Fate and Clonal Dynamics via Integrative Single-Cell Lineage Modeling

Through natural or synthetic lineage barcodes, single-cell technologies now enable the joint measurement of molecular states and clonal identities, providing an unprecedented opportunity to study cell fate and dynamics. Yet, most computational methods for inferring cell development and differentiation rely exclusively on transcriptional similarity, overlooking the lineage information encoded by lineage barcodes. This limitation is exemplified by T cells, where subtle transcriptional differences mark divergent fates with distinct biological activity. Single-cell RNA and matched TCR sequencing is now ubiquitous in the analysis of clinical samples, where the TCR sequence provides an endogenous clonal barcode and could reveal clonal T cell responses. We present Clonotrace, a computational framework that jointly models gene expression and clonotype information to infer cell state transitions and fate biases with higher fidelity. While motivated by challenges in analyzing T cell populations, especially in the tumor microenvironment and immunotherapy settings, Clonotrace is broadly applicable to any lineage-barcoded single-cell dataset. Across diverse systems including T cells, hematopoietic differentiation, and cancer therapy resistance models, Clonotrace reveals differentiation hierarchies, distinguishes unipotent from multipotent states, and identifies candidate fate-determining genes driving lineage commitment.

bioinformatics↗

bioRxiv2025

Genome-edited retinal organoids restore host bipolar connectivity in the primate macula

Retinal organoids (ROs) represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but whether host cone bipolar cells (BCs) in the primate macula can rewire with transplanted photoreceptors remains unresolved. Here, we transplanted genome-edited human retinal organoids lacking ON-BCs (Islet-1-/- ROs) into a non-human primate macular degeneration model. Remarkably, host rod and cone BCs extended dendrites toward grafted photoreceptors, forming functional synapses confirmed by immunohistochemistry, ultrastructural imaging, and focal macular electroretinography. Both ON- and OFF-pathway connectivity was rebuilt, providing the first demonstration of host-graft synaptic integration in the primate macula. These results establish that primate cone circuits retain a surprising capacity for rewiring and highlight genome-edited ROs as a powerful platform for vision restoration. Our findings represent a critical translational step toward stem cell-based therapies capable of repairing central vision in patients with advanced macular degeneration.

neuroscience↗

bioRxiv2025

Dynamic uterine microenvironment drives endometrial adenocarcinoma carcinogenesis and progression

Endometrial cancer (EC) development is driven by the interaction between the tumor and the microenvironment. However, the immune microenvironment dynamics during this process are not clear. Here, we applied single-cell RNA sequencing (scRNA-seq) to uterine blood samples collected at hysterectomy from 15 individuals encompassing four groups--benign controls, endometrial intraepithelial neoplasia (EIN), the transition from EIN to carcinoma, and EC. Uterine blood, obtained without prior enrichment, provides a liquid biopsy of the local tumor milieu, enabling high-resolution profiling of both immune and stromal cells in a minimally invasive manner. Our analysis revealed simultaneous immune and stromal remodeling in early premalignant lesions. Notably, even in the EIN, we observed significant immune cell reprogramming alongside the emergence of protumorigenic stromal-epithelial interactions. Importantly, we discovered predictive single-cell transcriptomic signatures derived from neutrophils that stratify patients according to disease state, highlighting the potential of tumor-educated innate immune cells as biomarkers. These findings yield candidate cellular and molecular signatures, particularly from neutrophils, that could enhance early EC detection and guide therapeutic strategies. Our work provides a proof-of-concept for leveraging local liquid biopsies in single-cell oncology, offering new insights into EC initiation and paving the way for noninvasive diagnostics on the basis of single-cell transcriptomic profiles.

cancer biology↗

bioRxiv2025

In vivo systematic detection of the outcomes of CRISPR/Cas9 mediated DNA repair in skeletal muscle stem cells

CRISPR/Cas9 has revolutionized genome editing with broad therapeutic applications, yet its repair patterns in vivo remain poorly understood. Here, we systematically profiled CRISPR/Cas9 editing outcomes at 95 loci using our established CRISPR/Cas9/AAV9-sgRNA system in skeletal muscle stem cells (MuSCs). Through comprehensive characterization of the repair outcomes, our findings demonstrate that the general rules governing CRISPR/Cas9-mediated editing in vivo largely align with those observed in vitro but with reduced editing precision. Additional to the anticipated small editing indels such as MMEJ mediated deletions and NHEJ mediated templated insertions, we uncovered a prevalent occurrence of large on-target modifications, including large deletions (LDs) characterized by microhomology (MH) and large insertions (LIs). Notably, the LIs comprise not only exogenous AAV vector integrations but also endogenous genomic DNA fragments (Endo-LIs). Endo-LIs preferentially originate from active genomic regions, with their integration shaped by three-dimensional chromatin architecture. By disrupting key components of the NHEJ and MMEJ repair pathways in vivo, we identified their distinct roles in regulating the large on-target modifications. Together, our work for the first time systematically profiles the CRISPR/Cas9 repair outcomes in vivo and offers valuable guidance for improving the safety of CRISPR/Cas9-based gene therapies.

genetics↗

bioRxiv2025

In Vivo Evolution of Monoclonal Antibody CR3022 to Achieve Cross-Neutralization of SARS-CoV-2 and Implications for Vaccine Strategies Against SARS-Related Viruses

The epitope that monoclonal CR3022 binds to represents a promising target for broad protection against a wide range of human and zoonotic coronaviruses. We developed a powerful model to evaluate antibody affinity maturation in vivo using immunoglobulin (Ig)-humanized mice that express the predicted germline heavy chain of antibody CR3022. SARS-CoV/SARS-CoV-2 sequential immunization led to the convergent evolution of the germline CR3022 through somatic hypermutation (SHM) that resembled the affinity-matured CR3022 from a human, but now also adapted to key variants and divergent sarbecoviruses. While simple prime-boost strategies drove CR3022-epitope targeting, an intensive vaccination protocol elicited dominant responses to other epitopes. X-ray crystal structures revealed that SARS-CoV-2-neutralizing CR3022-like antibodies exhibit enhanced affinity by increasing polar and electrostatic interactions. Overall, these findings show CR3022-like clones can be readily adapted through SHM to increase breadth and potency to sarbecoviruses by relatively minor shifts in affinity with appropriate vaccination strategies.

immunology↗

bioRxiv2025

Reengineering the antigen optimization process for superior neoantigen vaccine design

Identifying effective neoantigen sequences is essential for enhancing anti-tumor immunity. However, the vast sequence space (>109 possible peptides) and limited accuracy of existing immunogenicity predictors hinder efficient vaccine design for patient-specific human leukocyte antigens (HLAs). We present AlphaVacc, a deep reinforcement learning framework that integrates Monte Carlo Tree Search with a Transformer-based network to optimize antigenic peptides. AlphaVacc outperforms previous generative models in binding-affinity prediction. Experimental validation of 12 AlphaVacc-generated variants of the BING-4 peptide confirmed that 11 showed increased HLA-A*02:01 binding and 7 elicited significant T cell responses. Further testing of 16 single-mutation peptides confirmed computational predictions for 15 candidates, exhibiting a remarkable success rate. AlphaVacc thus provides a powerful tool for designing neoantigen-based cancer vaccines and may accelerate personalized immunotherapies.

bioinformatics↗

bioRxiv2025

Histone Acetylation Differentially Modulates CTCF-CTCF Loops and Intra-TAD Interactions

The cohesin complex structures the interphase genome by extruding loops and organizing topologically associating domains (TADs). While cohesin engages chromatin in context- dependent modes, the regulatory influence of chromatin state on these interactions remains unclear. Here, we show that histone hyperacetylation, induced by the histone deacetylase inhibitor trichostatin A (TSA), preferentially disrupts short-range interactions within TADs but spares CTCF-anchored loops, despite reduced cohesin occupancy at these sites. These findings point to two functionally distinct cohesin populations: a TSA- sensitive pool within TADs, likely representing extruding, non-topologically bound cohesin, and a TSA-resistant population at CTCF-CTCF anchors that maintains loops through topological entrapment. Using a semi-in vitro system with TEV-cleavable RAD21, we show that TSA-resistant cohesin at CTCF sites becomes TSA-sensitive after proteolytic cleavage that opens the cohesin ring, showing that it is the topological engagement with DNA that makes cohesin, and CTCF-CTCF loops, TSA-resistant. Notably, we also detect TSA-sensitive cohesin at CTCF sites, suggesting the presence of transient, non-encircling cohesin that either precedes conversion to the stable form or is halted by pre-existing encircling cohesin. Together, our results suggest that cohesin exists in distinct biochemical states: an extruding form found within TADs and at CTCF sites, that is sensitive to hyperacetylation, and a topologically bound form specifically at CTCF-CTCF loops that is insensitive. The former may allow dynamic changes in chromatin loops, while latter ensures robustness of CTCF-anchored loops in response to chromatin state changes.

genomics↗

bioRxiv2025

Dynamic Retinal Pathology in Glaucoma Progression Revealed by High-Resolution Functional Imaging in Vivo

Glaucoma, a complex and multifactorial neurodegenerative disease, is commonly associated with elevated intraocular pressure and primarily characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Despite its prevalence, our understanding and treatment of this disease remain challenging due to the intricate interplay of various pathophysiological factors and the limited capability for in vivo functional study of the disease. In this work, we investigated the dynamic retinal pathology of glaucoma from onset to late stages through longitudinal in vivo high-resolution imaging in a silicone oil-induced ocular hypertension glaucoma mouse model. We developed an optimized adaptive optics two-photon excitation fluorescence microscopy (AO-TPEFM) technique for both morphological and functional assessments of pathological changes in the retina across three distinct glaucoma phenotypes with varying progression rates. Our AO-TPEFM technology visualized the complete process of functional and structural changes in the three most important retinal components related to glaucoma: microvascular vessels, microglia, and RGCs during disease progression. Notably, our functional imaging revealed pathological alterations in microvascular circulation, microglia state, and RGC functionalities at a very early stage of disease development when retinal morphology still appeared normal, providing critical insights into glaucoma pathogenesis. This research also demonstrates that AO-TPEFM is a powerful tool for the in vivo study of general retinal diseases.

bioengineering↗

bioRxiv2025

Genome-Wide Investigation of Transcription Factor Occupancy and Dynamics Using cFOOT-seq

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences. Notably, cFOOT-seq, combined with FootTrack analysis, predicts TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.

molecular biology↗