Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

A rapid and efficient red-light-activated Cre recombinase system for genome engineering in mammalian cells and transgenic mice

The Cre-loxP recombination system enables precise genome engineering; however, existing photoactivatable Cre tools suffer from several limitations, including low DNA recombination efficiency, background activation, slow activation kinetics, and poor tissue penetration. Here, we present REDMAPCre, a red-light-controlled split-Cre system based on the {Delta}PhyA/FHY1 interaction. REDMAPCre enables rapid activation (1-second illumination) and achieves an 85-fold increase in recombination efficiency. We demonstrate its efficient regulation of DNA recombination in mammalian cells and mice, as well as its compatibility with other inducible recombinase systems for Boolean logic-gated DNA recombination. Using a single-vector adeno-associated virus (AAV) delivery system, we successfully induced REDMAPCre-mediated DNA recombination in mice. Furthermore, we generated a REDMAPCre transgenic mouse line and validated its efficient, light-dependent recombination across multiple organs. To explore its functional applications, REDMAPCre transgenic mice were crossed with the relative Cre-dependent reporter mice, enabling optogenetic induction of insulin resistance and hepatic lipid accumulation via Cre-dependent overexpression of ubiquitin-like with PHD and ring finger domains 1 (UHRF1), as well as targeted cell ablation through diphtheria toxin fragment A (DTA) expression. Collectively, REDMAPCre provides a powerful tool for achieving remote control of recombination and facilitating functional genetic studies in living systems.

synthetic biology↗

bioRxiv2025

Fecal microbiota transplantation mitigates cardiac remodeling and functional impairment in mice with chronic colitis

BackgroundInflammatory bowel disease (IBD) is a chronic inflammatory disorder with significant extraintestinal manifestations, including cardiovascular derangements. However, the molecular mechanisms underlying the cardiac remodeling and dysfunction remain unclear. MethodsWe investigated the effects of chronic colitis on the heart using two mouse models: DSS-induced colitis and Il10-/-spontaneous colitis. Echocardiography was employed to assess heart function and molecular characterization was performed using bulk RNA-sequencing, RT-qPCR, and western blot. ResultsBoth models exhibited significant cardiac impairment, including reduced ejection fraction and fractional shortening as well as increased collagen deposition, inflammation, and myofibril reorganization. Molecular analyses revealed upregulation of fibrosis markers (i.e. COL1A1, COL3A1, Fibronectin) and {beta}-catenin reactivation, indicating a pro-fibrotic cardiac environment. Each model yielded common upregulation of eicosanoid-associated and inflammatory genes (Cyp2e1, Map3k6, Pck1, Cfd), and model-specific alterations in pathways regulating cAMP- and cGMP-signaling, arachidonic and linoleic acid metabolism, Cushing syndrome-related genes, and immune cell responses. DSS colitis caused differential regulation of 232 cardiac genes, while Il10-/- colitis yielded 105 dysregulated genes, revealing distinct molecular pathways driving cardiac dysfunction. Importantly, therapeutic fecal microbiota transplantation (FMT) restored heart function in both models, characterized by reduced fibrosis markers and downregulated pro-inflammatory genes (Lbp and Cdkn1a in Il10-/- mice and Fos in DSS mice), while also mitigating intestinal inflammation. Post-FMT cardiac RNA-sequencing revealed significant gene expression changes, with three altered genes in DSS mice and 67 genes in Il10-/- mice. Notably, Il10-/- mice showed relatively less cardiac recovery following FMT, highlighting IL-10s cardioprotective and anti-inflammatory contribution. ConclusionsOur findings elucidate novel insights into colitis-induced cardiac remodeling and dysfunction and suggest that FMT mitigates cardiac dysfunction by attenuating systemic inflammation and correcting gut dysbiosis. This study underscores the need for further evaluation of gut-heart interactions and microbiome-based therapies to improve cardiovascular health in IBD patients. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/643179v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@13d79eaorg.highwire.dtl.DTLVardef@10f28a7org.highwire.dtl.DTLVardef@35f4b0org.highwire.dtl.DTLVardef@538d7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

RAMA: an Integrative Approach to Identify Hot Spots and Guide the Directed Evolution of High Molecular Weight Enzyme

Directed evolution has revolutionized enzyme engineering but remains challenging for large, multidomain proteins due to their complex dynamics and vast combinatorial sequence space. Traditional NMR spectroscopy, while powerful for identifying functional residues, is limited by the requirement for complete resonance assignments that become intractable in high molecular weight systems. Here, we present a hybrid NMR-computational framework that integrates chemical shift perturbation mapping with AlphaFold3-docked distance constraints, enabling the identification and assignment of ligand-proximal hotspots without the need for full resonance assignment. By analyzing side chain 1H-13C correlation spectra, probabilistic amino acid typing, and structural proximity filtering, we mapped functional hotspots in the [~]90 kDa Pyrococcus furiosus DNA polymerase. Targeted saturation mutagenesis of these sites resulted in variants exhibiting multi-fold increases in catalytic efficiency while sampling less than 0.6% of total residues. The identified hotspots revealed spatially distinct yet cooperative contributions to catalysis, as further supported by combinatorial mutagenesis and kinetic analyses. This hybrid strategy provides a practical pipeline for narrowing mutagenesis targets in large enzymes under catalytic conditions, overcoming the size, assignment, and screening limitations of conventional NMR-guided evolution, offering a powerful route toward efficient biocatalyst optimization by prioritizing high-probability target residues.

molecular biology↗

bioRxiv2025

Tumor cell villages define the co-dependency of tumor and microenvironment in liver cancer

Spatial cellular context is crucial in shaping intratumor heterogeneity. However, understanding how each tumor establishes its unique spatial landscape and what factors drive the landscape for tumor fitness remains significantly challenging. Here, we analyzed over 2 million cells from 50 tumor biospecimens using spatial single-cell imaging and single-cell RNA sequencing. We developed a deep learning-based strategy to spatially map tumor cell states and the architecture surrounding them, which we referred to as Spatial Dynamics Network (SDN). We found that different tumor cell states may be organized into distinct clusters, or villages, each supported by unique SDNs. Notably, tumor cell villages exhibited village-specific molecular co-dependencies between tumor cells and their microenvironment and were associated with patient outcomes. Perturbation of molecular co-dependencies via random spatial shuffling of the microenvironment resulted in destabilization of the corresponding villages. This study provides new insights into understanding tumor spatial landscape and its impact on tumor aggressiveness.

cancer biology↗

bioRxiv2025

Sequencing-free whole genome spatial transcriptomics at molecular resolution in intact tissue

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, compositions, interactions, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution. Leading spatial-capture or spatial-tagging transcriptomics techniques that rely on in-vitro sequencing offer whole-transcriptome coverage, in principle, but at the cost of lower spatial resolution compared to image-based techniques. In contrast, high-performance image-based spatial transcriptomics techniques, which rely on in situ hybridization or in situ sequencing, achieve single-molecule spatial resolution and retain sub-cellular morphologies, but are limited by probe libraries that target only a subset of the transcriptome, typically covering several hundred to a few thousand transcript species. Together, these limitations hinder unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here we develop a new image-based spatial transcriptomics technology termed Reverse-padlock Amplicon Encoding FISH (RAEFISH) with whole-genome level coverage while retaining single-molecule spatial resolution in intact tissues. We demonstrate image-based spatial transcriptomics targeting 23,000 human transcript species or 22,000 mouse transcript species, including nearly the entire protein-coding transcriptome and several thousand long-noncoding RNAs, in single cells in cultures and in tissue sections. Our analyses reveal differential subcellular localizations of diverse transcripts, cell-type-specific and cell-type-invariant tissue zonation dependent transcriptome, and gene expression programs underlying preferential cell-cell interactions. Finally, we further develop our technology for direct spatial readout of gRNAs in an image-based high-content CRISPR screen. Overall, these developments provide the research community with a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNA species in many biomedical contexts.

genetics↗

bioRxiv2025

Salmonella effector kinase SteC is activated by host-mediated phosphorylation

The pathogen Salmonella, which causes significant human morbidity and mortality, encodes an effector kinase, SteC, which mediates actin polymerisation and cell migration. Given the minimal nature of its kinase domain, it remains unclear how SteC is catalytically active and how this activity is regulated. Here, we show that SteC is activated following the phosphorylation of the highly conserved S379 residue by a host kinase. Phosphorylation of S379 dramatically increases nucleotide binding affinity of SteC, enabling substrate phosphorylation and promoting actin polymerisation. Further mutational analysis identified the functional role of HD and DGD motifs that likely mimic the HxD and DFG motifs of eukaryotic kinases. Meanwhile, the C-tail of SteC, encompassing amino acids 429-457, is essential for function following translocation from Salmonella, but dispensable for catalysis in vitro. Overall, our findings uncover two previously unappreciated mechanisms that mediate the activity of the only Salmonella effector kinase within the host. HIGHLIGHTSSteC is phosphorylated at S379 in mammalian cells SteC phosphorylation is critical for kinase activity and actin polymerisation Phosphorylation at S379 promotes nucleotide binding The C-tail of SteC is not required for catalysis but is essential for activity in infected cells

microbiology↗

bioRxiv2025

Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes

With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.

biophysics↗

bioRxiv2025

Integrative Analysis of Single-cell and Bulk RNA Sequencing Reveals Macrophage Heterogeneity in Lyme Arthritis

IntroductionLyme arthritis (LA) is a common late-stage manifestation of Lyme disease caused by Borrelia burgdorferi (Bb) infection. Macrophages play a crucial role in LA pathogenesis, yet their heterogeneity and functional states within the arthritic microenvironment remain poorly understood. MethodsWe integrated single-cell RNA sequencing data from ankle joint tissues of Bb-infected C57BL/6 mice with bulk RNA sequencing data from both infected ankle joints and bone marrow-derived macrophages. We developed a LA mouse model and validated key findings through histopathological examination and RT-qPCR analysis. ResultsWe identified nine distinct cell types in the joint microenvironment with significant compositional shifts during disease progression. Analysis of 16,617 Macrophages/Monocytes revealed seven functionally distinct subpopulations with differential responses to infection. Pseudo-time trajectory analysis demonstrated three principal differentiation pathways from monocyte-derived macrophages toward anti-inflammatory, TNF+, and IL1b+ inflammatory phenotypes. Cell-cell communication analysis revealed significantly altered TGF-{beta} signaling networks in LA, with the Tgfb1-(Tglbr1+TgIbr2) axis playing a critical role. GSVA and pathway analyses showed metabolic reprogramming from oxidative phosphorylation toward glycolysis in macrophages during infection. Through integrated analysis and LASSO regression, we identified four characteristic genes (SIRPB1C, FABP5, MMP14, and EGR1) as potential LA biomarkers and developed a diagnostic nomogram with high predictive accuracy. ConclusionOur integrative analysis provides comprehensive insights into macrophage heterogeneity and functional plasticity in LA, identifying characteristic biomarkers and potential therapeutic targets. The metabolic reprogramming and altered TGF-{beta} signaling networks we identified may contribute to disease pathogenesis and offer new avenues for intervention strategies.

genomics↗

bioRxiv2025

A single-cell transcriptomic atlas of developing inhibitory neurons reveals expanding and contracting modes of diversification.

The cerebral cortex relies on vastly different types of inhibitory neurons to compute. How this diversity emerges during development remains an open question. The rarity of individual inhibitory neuron types often leads to their underrepresentation in single-cell RNA sequencing (scRNAseq) datasets, limiting insights into their developmental trajectories. To address this problem, we developed a computational pipeline to enrich and integrate rare cell types across multiple datasets. Applying this approach to somatostatin-expressing (SST+) inhibitory neurons--the most diverse inhibitory cell class in the cortex--we constructed the transcriptomic maps, Dev-SST-v1 and Dev-SST-v2, a comprehensive resource containing mouse scRNAseq data of over 51,000 SST+ neurons. We identify three principal groups--Martinotti cells (MCs), non-Martinotti cells (nMCs), and long-range projecting neurons (LRPs)--each following distinct diversification trajectories. MCs commit early, with distinct embryonic and neonatal clusters that map directly to adult counterparts. In contrast, nMCs diversify gradually, with each developmental cluster giving rise to multiple adult cell types. LRPs follow a unique contracting mode. Initially, two clusters are present until postnatal day 5 (P5), but by P7, one type is eliminated through programmed cell death, leaving a single surviving population. This transient LRP type is also found in the fetal human cortex, revealing an evolutionarily conserved feature of cortical development. Together, these findings highlight three distinct modes of SST+ neuronal diversification--invariant, expanding, and contracting--offering a new framework to understand how the large repertoire of inhibitory neurons emerges during development.

neuroscience↗

bioRxiv2025

Discovery of first-in-class inhibitors of the TRF1-TIN2 protein-protein interaction by fragment screening

TRF1 is a subunit of the shelterin complex that binds to and protects the linear ends of chromosomes known as telomeres. Both genetic deletion and chemical inhibition of TRF1 have been shown to block the growth of lung carcinoma, glioblastoma, and renal cell carcinoma in mice without affecting mouse survival or tissue function, making TRF1 a potential therapeutic target in cancer1-3. Here, we report the discovery of a series of fragment hits that bind at the interface between the TRFH domain of TRF1 (TRF1TRFH) and a peptide of TIN2 (TIN2TBM), an interaction essential for the recruitment of TRF1 to shelterin, using X-ray crystallography (XChem) and ligand-observed NMR (LO-NMR) fragment screening. We discovered a first-in-class inhibitor of the TRF1-TIN2 interaction (compound 40) that binds to TRF1TRFH with a KD of 29 M (95% CI: 20 - 41 M), displaces a TIN2 probe with an IC50 of 67 {+/-} 28 M, and expels TRF1 from purified shelterin. Aided by a novel crystal system of TRF1TRFH, we characterised fragments binding in a hotspot at the TRF1-TIN2 interface which will serve as a starting point for the structure-guided development of potent inhibitors of TRF1 protein-protein interactions to disrupt shelterin complex assembly.

biochemistry↗

bioRxiv2025

Single-Cell RNA Sequencing Reveals Hemocyte Heterogeneity, Differentiation Trajectories, and Viral Tropism in Shrimp (Macrobrachium rosenbergii) Infected with Decapod Iridescent Virus 1

Decapod Iridescent Virus 1 (DIV1) is a highly pathogenic virus threatening crustacean aquaculture. Hemocytes, the primary immune cells in shrimp, play crucial roles in host defense, yet their transcriptional heterogeneity and differentiation dynamics under viral infection remain poorly understood. Here, we characterize hemocyte subpopulations in Macrobrachium rosenbergii before and after DIV1 infection at single-cell resolution. We identified 12 distinct hemocyte clusters with unique molecular signatures. DIV1 infection induced significant shifts in hemocyte composition, with clusters 0, 5, 6, and 8 expanding, while clusters 9 and 10 exhibited resistance. Clusters 0, 5, and 8 were highly susceptible, showing elevated viral gene expression. Pseudo-time analysis revealed that DIV1 accelerates hemocyte differentiation, driving prohemocytes and semi-granulocytes toward terminally differentiated granulocytes and hyalinocytes, particularly the immune-active HC4 subpopulation. Functional enrichment analysis showed that susceptible hemocytes were associated with viral processes, oxidative stress, and phagocytosis, while resistant clusters exhibited distinct immune signatures. Furthermore, knockdown experiments confirmed the antiviral roles of PPO2, RAB11B, LAMP1, and Dorsal, as their silencing led to increased viral loads, higher MCP protein levels, and reduced shrimp survival. Taken together, our study provides the first single-cell resolution atlas of shrimp hemocytes under DIV1 infection, revealing their transcriptional heterogeneity, differentiation trajectories, and immune responses. These findings offer novel insights into shrimp antiviral immunity and lay the foundation for disease-resistant breeding and immunostimulatory strategies in aquaculture. ImportancesDecapod Iridescent Virus 1 (DIV1) is a highly pathogenic virus threatening crustacean aquaculture, causing severe economic losses. Hemocytes, the primary immune cells in shrimp, play crucial roles in antiviral defense, yet their diversity, differentiation, and responses to viral infection remain poorly understood. In this study, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively characterize hemocyte subpopulations in Macrobrachium rosenbergii before and after DIV1 infection. We identified 12 transcriptionally distinct hemocyte clusters and revealed significant changes in hemocyte composition following infection. Notably, we discovered that specific clusters (0, 5 and 8) were highly susceptible to DIV1, while others (9 and 10) exhibited resistance. Additionally, we reconstructed hemocyte differentiation trajectories and found that DIV1 infection drives hematopoiesis, accelerating the transition of progenitor cells into terminally differentiated immune-active hemocytes. Functional analysis highlighted key antiviral effectors, including PPO2, RAB11B, LAMP1, and Dorsal, which were significantly upregulated in response to infection. These findings provide novel insights into shrimp antiviral immunity and viral pathogenesis, offering potential targets for disease-resistant breeding and immunostimulatory interventions.

immunology↗

bioRxiv2025

Novel Pipelines to Extract Differences in Proteome Dynamics Based on Health Status

Understanding dynamics and co-regulatory patterns in the human proteome is a promising path for unraveling the molecular basis of health and disease. Nevertheless, there remains an open challenge in extracting concise information from high-throughput proteomic data that can effectively characterize and predict health. We develop novel statistical and computational pipelines to tackle this problem in a longitudinal saliva proteomics data set collected throughout the awakening response in six healthy controls and six subjects with severe mitochondrial disease (MitoD), a clinical condition caused by genetic mitochondrial defects that affects cellular energy transformation and alters multiple dimensions of health. We undertook three independent unsupervised approaches to characterize proteome dynamics and assessed their ability to separate MitoD individuals from controls. First, we designed a permutation test to detect the global difference in the proteomic co-regulation structure between healthy and unhealthy subjects. Second, we performed non-linear embedding and cluster analysis on elasticity to capture a more complicated relationship between health and the proteome. Third, we developed a machine learning algorithm to extract low-dimensional representations of the proteome dynamic and use them to cluster subjects into healthy and unhealthy groups without any knowledge of their true status. All three methods showed clear differences between MitoD individuals and controls. Our results revealed a significant and consistent association between MitoD status and the saliva proteome at multiple levels during the awakening response, including its dynamic change, co-regulation structure, and elasticity. This connection is not restricted to a few MitoD-specific proteins but spreads over a wide range of proteins from many body functions and pathways. Pipelines such as those shown here are the first step toward establishing interpretable and accurate prediction rules for health based on proteome dynamics.

bioinformatics↗

bioRxiv2025

Neoadjuvant anti-4-1BB confers protection against spontaneous metastasis through low-affinity intratumor CD8+ T cells in triple-negative breast cancer

Neoadjuvant immunotherapy seeks to harness the primary tumor as a source of relevant tumor antigens to enhance systemic anti-tumor immunity through improved immunological surveillance. Despite having revolutionized the treatment of patients with high-risk early-stage triple-negative breast cancer (TNBC), a significant portion of patients remain unresponsive and succumb to metastatic recurrence post-treatment. Here, we found that optimally scheduled neoadjuvant administration of anti-4-1BB monotherapy was able to counteract metastases and prolong survival following surgical resection. Phenotypic and transcriptional profiling revealed enhanced 4-1BB expression on tumor-infiltrating intermediate (Tint), relative to progenitor (Tprog) and terminally exhausted (Tterm) T cells. Furthermore, Tint was enriched in low-affinity T cells. Treatment with anti-4-1BB drove clonal expansion of Tint, with reduced expression of tissue-retention marker CD103 in Tprog. This was accompanied by increased TCR clonotype sharing between paired tumors and pre-metastatic lungs. Further interrogation of sorted intratumor T cells confirmed enhanced T cell egress into circulation following anti-4-1BB treatment. In addition, gene signature extracted from anti-4-1BB treated Tint was consistently associated with improved clinical outcomes in BRCA patients. Combinatorial neoadjuvant anti-4-1BB and ablation of tumor-derived CXCL16 resulted in enhanced therapeutic effect. These findings illustrate the intratumor changes underpinning the efficacy of neoadjuvant anti-4-1BB, highlighting the reciprocity between local tissue-retention and distant immunologic fortification, suggesting treatment can reverse the siphoning of intratumor T cells to primary tumor, enabling redistribution to distant tissues and subsequent protection against metastases.

immunology↗

bioRxiv2025

Dietary Menaquinone-9 Supplementation Does Not Influence Bone Tissue Quality or Bone Mineral Density in Mice

Vitamin K has been implicated in skeletal health because vitamin K-dependent proteins are present in bone. While there are multiple forms of vitamin K, most research has focused on phylloquinone, which is found mainly in plant-based foods, and its metabolite menaquinone-4 (MK4). However, there are additional forms of vitamin K that are bacterially produced that appear to influence bone health but have not yet been studied extensively. Herein, we evaluated the effects of menaquinone-9 (MK9), a bacterially produced form of vitamin K on bone tissue quality and density in young mice. Four-week-old male (n=32) and female (n=32) C57BL/6 mice were supplemented with 0.06 mg/kg diet or 2.1 mg/kg diet of MK9 for 12 weeks. During week 11, a sub-group of mice (n=7/sex/group) received daily deuterium-labeled MK9 to trace its metabolic fate in bone. Liver MK4 and MK9 were significantly higher in mice fed 2.1 mg MK9/kg compared to those receiving 0.06 mg MK9/kg, regardless of sex (all p [≤] 0.017). MK4 was the only vitamin K form detected in bone, with 63-67% of skeletal MK4 in mice fed 2.1 mg MK9/kg derived from deuterium-labeled MK9. Femoral tissue strength, maximum bending moment, section modulus, and bone mineral density did not differ significantly across diet groups in either sex (all p[≥]0.083). Cross-sectional area (p=0.003) and moment of inertia (p=0.001) were lower in female mice receiving 2.1 mg MK9/kg compared to those receiving 0.06 mg MK9/kg, but no differences were found in male mice. Higher bone MK4 concentrations did not correlate with higher bone tissue quality or density. Despite dietary MK9 being a dietary precursor to MK4 in bone, dietary MK9 supplementation did not affect bone tissue quality or bone mineral density. Lay summaryMost research about vitamin K and bone health has focused on phylloquinone, the plant-based vitamin K form, and its metabolite menaquinone-4. Because interest in bacterially produced forms of vitamin K, which are abundant in the intestinal microbiome, is growing, we evaluated the effect of menaquinone-9 (a bacterially-produced form of vitamin K) on skeletal health. We supplemented mice with low and high doses of menaquinone-9 and also used stable-isotope labeled menaquinone-9 to trace its conversion to menaquinone-4 in bone. We found menaquinone-9 served as a precursor to menaquinone-4 in bone, but menaquinone-9 supplementation did not improve bone health.

physiology↗

bioRxiv2025

fCUT&Tag-seq: An Optimized CUT&Tag-based Method for High-Resolution Histone Modification and Chromatin-Binding Protein Profiling in both Model and Plant Pathogenic Fungi

Histone modifications and chromatin-binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used ChIP-Seq method. Here, we present an optimized CUT&Tag-Seq protocol (fCUT&Tag-Seq) specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein-DNA binding efficiency. We then successfully applied fCUT&Tag-Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including Verticillium dahliae, Neurospora crassa, Fusarium graminearum, and Sporisorium scitamineum, showing good signal-to-noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin-binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag-Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity. Impact StatementWe developed a faster, lowLJinput method to study how genes are turned on and off in fungi, even in tough species that are difficult to analyze with standard methods. Our new approach, named fCUT&Tag-Seq, requires only 10,000 cells and can be completed in just two days. It delivers clearer, more reliable results and has already been successfully applied to multiple fungi, including crop pathogens. By revealing the molecular "control switches" that govern fungal development and virulence, we expect it will accelerate basic research and help identify new targets for controlling destructive plant diseases.

microbiology↗

bioRxiv2025

Viscoelastic HyA Hydrogel Promotes Recovery of Muscle Quality and Vascularization in a Murine Model of Delayed Rotator Cuff Repair

Rotator cuff tears are among the most common musculotendinous injuries with high risk of permanent functional disability. Following surgical repair, sub-optimal patient outcomes are directly correlated with poor muscle quality; namely, injury site fatty infiltration (FI), fibrosis, and muscle atrophy. Muscle resident fibro-adipogenic progenitor cells (FAPs) have been identified as key regulators of post-injury skeletal muscle regeneration and repair by maintaining a promyogenic environment. In this work, human-derived FAPs (hFAPs) were encapsulated into hyaluronic acid (HyA)-based hydrogels functionalized with bsp-RGD(15) cell adhesion peptide, heparin, and a matrix metalloproteinase (MMP)-cleavable crosslinker. Hydrogel-encapsulated hFAPs increased expression of the promyogenic marker UCP1 and production of the anti-inflammatory cytokine IL-10, while downregulating the expression of the fibrotic marker SMA over time. A murine model of unilateral rotator cuff transection, denervation, and delayed repair was treated with the HyA hydrogel or PBS and compared to a contralateral, non-injured control limb. Muscle histology 6 weeks post-repair revealed that the hydrogel reduced fibrosis, FI, and muscle atrophy while supporting vascularization of the injured tissue region. Collectively, these results suggest that the hydrogel alone can promote muscle regeneration in a clinically relevant delayed repair model of rotator cuff tear, which we hypothesize is due to controlled FAP differentiation into promyogenic lineages.

bioengineering↗

bioRxiv2025

Flaviviruses Hijack HuR to Suppress CNPY3: A Novel Pathway for Immune Evasion and Therapeutic Intervention

Flaviviruses represent a serious global health threat, infecting millions annually with high rates of cross-infection among different flaviviruses, yet no broad-spectrum antiviral therapy currently targets these pathogens. Through biological big data analysis, we observed a significant downregulation of Canopy FGF Signaling Regulator 3 (CNPY3), a positive regulator of defense responses, in flavivirus-infected patients, with the level of downregulation correlating with infection severity. This observation was validated in cellular and animal models. Mechanistically, we demonstrate that flaviviruses hijack the RNA-binding protein Human antigen R (HuR) to destabilize CNPY3 mRNA via adenine-uridine-rich elements (AREs), thus downregulating CNPY3 expression and promoting viral replication. Our findings identify CNPY3 as a novel antiviral mediator essential for the TLR-mediated type I interferon (IFN-I) pathway in defense against flaviviruses. In vitro, CNPY3 overexpression reduced DENV and ZIKV replication, confirming its antiviral role. To explore therapeutic potential, we developed lipid nanoparticles (LNPs) encapsulating CNPY3 mRNA (LNP-CNPY3), and early administration in flavivirus-infected mice improved survival, reduced viral loads, and attenuated neuroinflammation by enhancing antiviral and interferon responses. Together, these findings establish CNPY3 as a critical antiviral target and support LNP-CNPY3 as a promising therapeutic approach against flavivirus infections.

microbiology↗

bioRxiv2024

Characterizing Tyrosine Ring Flips in Proteins by 19F NMR

Aromatic ring-flip dynamics are hallmarks of concerted protein "breathing" motions that are essential for biological function. Ring flips occur on a broad range of timescales (ns-s) and have been primarily studied by NMR spectroscopy, typically requiring expensive isotope labeling of aromatic side chains, thereby limiting such studies to a few proteins. Here, we report two novel di-fluorotyrosine probes, 3,5-F2Y and 2,6-F2Y, that can be incorporated into proteins cost-effectively for characterizing and modulating tyrosine ring-flip dynamics. We show that 19F rotating-frame (R1{rho}) relaxation dispersion is powerful for quantifying ring flips on the {micro}s-ms timescale. Importantly, the tyrosine ring-flip rates (kflip) for 3,5-F2Y in GB1 and HPr are comparable to those measured by 1H and 13C NMR studies, validating 3,5-F2Y as a largely non-perturbing, native-like ring-flip probe. In contrast, 2,6-F2Y acts as an effective "brake" on ring flips, enabling direct visualization and quantitative characterization of previously undetected tyrosine ring flips in ubiquitin via 19F lineshape analysis. Furthermore, we use 2,6-F2Y to assess the environmental effects on ring-flip dynamics in crowding reagents and in living X. laevis oocytes. Collectively, our study opens a new avenue for measuring and modulating ring-flip dynamics in vitro and in living cells. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/630691v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@dcf8e1org.highwire.dtl.DTLVardef@1578887org.highwire.dtl.DTLVardef@1c481d0org.highwire.dtl.DTLVardef@268ae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗