Biology subjects

Liu, A.

Publications and source records attributed to Liu, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Transcriptional targets of SPEECHLESS and FAMA control guard cell division and expansion in the late stomatal lineage

Plant tissue development often relies on the specification of cell type initials with stem cell-like properties. These later undergo differentiation, losing division potential and acquiring specific identities and functions. In the stomatal lineage, protodermal cells develop into guard cells (GCs) through the action of bHLH transcription factors (TFs) SPEECHLESS (SPCH), MUTE and FAMA. Existing models support that these regulators act sequentially, but recent evidence indicates that SPCH expression and function are retained in late stomatal cells. Here, we combine transcriptomic and genetic approaches to define SPCHs function during the late stomatal lineage. We show that relative levels and activities of SPCH and FAMA control GC division and expansion. Through cell type-specific TF induction and mRNA sequencing, we identify late-lineage targets of both TFs, and through genetic perturbation of these targets, we demonstrate that their precise temporal regulation is required for proper GC morphology and function. Our findings reveal a previously unrecognized role for SPCH in late stomatal development and support a revised model in which the functions of stomatal bHLHs are not strictly separated in time.

plant biology↗

bioRxiv2025

The regulatory effect of Ca2+/calmodulin dependent protein kinase II mediated by receptor interacting protein kinase 3 on necroptosis of hypertrophic cardiomyocytes

Receptor interacting protein kinase 3 (RIPK3) plays a crucial role in the signaling pathway of necroptosis and calcium/calmodulin dependent protein kinase II (CaMKII) is a novel substrate for RIPK3 induced necroptosis. The aim of this study is to investigate the regulation and mechanism of RIPK3 on AngII induced cardiomyocyte hypertrophy.AngII was used to stimulate myocardial cells for 72 hours, inducingcardiomyocytehypertrophy; Use RIPK3 inhibitor GSK872 to reduce RIPK3 expression.Detect indicators related to myocardial hypertrophy, cell damage, necroptosis,CaMKII activation and gene expression, oxidative stress, mitochondrial membrane potential, etc. After AngII stimulation of cardiomyocytes, the expression of hypertrophy markers ANP and BNP increased, LDH release increased, ATP levels decreased, splicing factors ASF and SC35 expression increased, CaMKII oxidation and phosphorylation levels increased, and CaMKII{delta} alternative splicing was disrupted. However, treatment with GSK872 can alleviate myocardial dysfunction, inhibit CaMKII activation, correct CaMKII{delta} variant splicing disorder and ultimately alleviating myocardial hypertrophy. In addition, pretreatment with RIPK3 can reduce the accumulation of reactive oxygen species (ROS) induced by AngII, decrease the activity of ASF and SC35, and restore mitochondrial membrane potential. RIPK3 inhibitor GSK872 can inhibit the activation of CaMKII, alleviate necroptosis and oxidative stress to alleviate myocardial hypertrophy. It has a protective effect on myocardial hypertrophy and is expected to become a new targeted drug for clinical treatment of dilated cardiomyopathy and heart failure.

cell biology↗

bioRxiv2025

Cis-Regulatory Editing of Peptide Signaling Rewires Plant Growth-Defense Balance

Precise cis-regulatory control of gene expression is essential for plant growth. In Arabidopsis thaliana, PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY) peptides and their receptors (PSYRs) mediate growth-stress trade-offs, yet the transcriptional regulation of these genes remains poorly understood. Here, we mapped transcription factor (TF)-promoter interactions for nine PSY and three PSYR genes by combining high-throughput enhanced yeast one-hybrid screening with DNA Affinity Purification sequencing (DAP-seq) data, uncovering 1,207 interactions that reveal both shared and gene-specific regulatory relationships, defining the global TF-promoter interaction network of the PSY/PSYR pathway. Functional analysis of 25 TF mutants identified 12 regulators that significantly influence root growth, most acting as repressors. Of these, CYTOKININ RESPONSE FACTOR 10 (CRF10) emerged as a strong growth inhibitor. We identified a CRF10-binding motif in the PSYR3 promoter using DAP-seq data and validated it using eY1H. This motif is also located in the last 3' terminal exon of Topoisomerase 3A (TOP3A). Guided by these insights, we used CRISPR/Cas9-mediated promoter editing to delete a small region encompassing or flanking a functional TF-binding site (TFBS). Removal of this motif, or of its surrounding region, enhanced root growth, yielding variants that retained root length comparable to the crf10 mutant. Our results suggest that the observed root growth phenotype results either from disruption of the CRF10 binding motif or from the mutation in the TOP3A exon.

plant biology↗

bioRxiv2025

Modeling Fibrosis with MASH Patient Liver-Derived Organoids.

Metabolic dysfunction-associated steatohepatitis (MASH) can lead to liver fibrosis and cirrhosis ultimately leading to liver transplantation or death. Therapeutic options for MASH-associated fibrosis are limited in part because of the lack of good model systems. To address this challenge, we developed a 3D MASH liver fibrosis model by using organoids derived from MASH patient liver co-cultured with human liver-derived hepatic stellate cells (HSC) and human peripheral blood monocytes (MC). Spontaneous self-assembly resulted in fibrotic scar-like 3D structures with senescent parenchymal cells, proliferating collagen secreting myofibroblasts (MFB) and proinflammatory TREM2+ scar-associated macrophages (MP). Single cell RNA sequencing suggested high similarity with MASH patient liver fibrotic scars. Lipid nanoparticles (LNPs) formulated with anti-YAP1siRNA could specifically and efficiently knockdown YAP1 in the MFBs, resulting in MFB senescence, a desirable therapeutic goal. This MASH patient liver-derived fibrosis model opens novel avenues towards testing treatments for MASH-associated liver fibrosis with reduced adverse effects.

bioengineering↗

bioRxiv2025

Polysialic Acid-Functionalized MAP Scaffolds Promote Regulatory Immune Responses After Ischemic Stroke

Glycosylation regulates immune and neural functions within the central nervous system (CNS), yet biomaterials rarely leverage glycans due to their structural complexity. Polysialic acid (PSA), comprising 2,8-linked sialic acid residues, is a promising candidate owing to its potent immunomodulatory interactions with inhibitory Siglec receptors. Systematic screening of multiple sialic acid derivatives identifies PSA as uniquely effective in inducing anti-inflammatory polarization of bone marrow-derived macrophages (BMDMs). Based on these findings, an injectable microporous annealed particle (MAP) scaffold presenting PSA covalently via its reducing end (MAP-PSA) is engineered, recapitulating physiological glycan orientation. MAP-PSA exhibits robust mechanical properties, stable glycan immobilization, and resistance to enzymatic degradation. Using ischemic stroke as a CNS injury model, MAP-PSA significantly reduces neutrophil infiltration and inflammatory activation while enhancing reparative macrophage and microglial phenotypes. These immunomodulatory effects persist into subacute stages, characterized by sustained reductions in inflammation and enhanced microglial homeostasis. Overall, MAP-PSA scaffolds demonstrate a novel therapeutic paradigm for CNS injuries such as stroke, with translational potential for broader neuroinflammatory and regenerative applications.

bioengineering↗

bioRxiv2025

VAMP8 function reveals tight linkage between endocytic recycling and endocytosis

Clathrin-mediated endocytosis (CME) is a multistage process that involves the initiation and stabilization of clathrin-coated pits (CCPs) that invaginate and finally detach from the plasma membrane to form clathrin-coated vesicles (CCVs). Given that SNARE proteins are essential for downstream vesicle targeting and fusion events, their recruitment into nascent CCVs has been suggested to be a prerequisite for CME progression. However, which and how SNARE proteins regulate CME remains to be explored. Here, we showed that siRNA-mediated knockdown of the R-SNARE, VAMP8 impairs CCP initiation, stabilization and invagination and strongly inhibits CME. Mechanistically, recruitment of VAMP8 to CCVs is not required for CME. Instead, depletion of VAMP8 inhibits recycling of endocytic cargoes and as exemplified here by transferrin receptor, skews their trafficking toward lysosomal degradation. VAMP8 depletion therefore indirectly impairs CCV formation and inhibits CME by depleting endocytic cargo. Overall, our study provides new insights into the crosstalk between endocytosis and endocytic recycling of CME cargo and demonstrates the critical role for cargo recruitment in stabilizing nascent CCPs to regulate CME. Significance StatementWe demonstrate that VAMP8 depletion inhibits recycling of endocytic cargo and reroutes transferrin receptors toward lysosomal degradation, thereby impairing endocytic vesicle formation and inhibiting clathrin-mediated endocytosis. Thus, the work uncovers a critical link between endocytic recycling and endocytosis, highlighting the importance of recycling pathways in maintaining membrane dynamics. However, the recruitment of VAMP8 into nascent clathrin-coated pits by CALM is not required for CME. While SNARE recruitment is essential for formation of COPI and COPII vesicles, our findings suggest that this requirement is not generalizable to endocytic CCVs.

cell biology↗

bioRxiv2025

fSuSiE enables fine-mapping of QTLs from genome-scale molecularprofiles

Molecular quantitative trait locus (QTL) studies seek to identify the causal variants affecting molecular traits like DNA methylation and histone modifications. However, existing fine-mapping tools are not well suited to high-dimensional molecular traits, and so analyses of these traits typically proceed by considering each variant and each molecular measurement independently, ignoring the LD among variants and the spatial correlation in effects between nearby sites. This severely limits accuracy in identifying causal variants and quantifying their molecular trait effects. Here, we introduce fSuSiE ("functional Sum of Single Effects"), a fine-mapping method that addresses these challenges by explicitly modeling the spatial structure of genetic effects on molecular traits. fSuSiE integrates wavelet-based functional regression with the computationally efficient "Sum of Single Effects" framework to simultaneously finemap causal variants and identify the molecular traits they affect. In simulations, fSuSiE identified causal variants and affected CpGs more accurately than methods that ignore spatial structure. In applications to DNA methylation and histone acetylation (H3K9ac) data from the ROSMAP study of the dorsolateral prefrontal cortex, fSuSiE achieved dramatically higher resolution than existing methods (e.g., identifying 6,355 single-variant methylation credible sets compared to only 328 from an existing approach). Applied to Alzheimers disease (AD) risk loci, fSuSiE identified potential causal variants colocalizing with AD GWAS signals for established genes, including CASS4 and CR1/CR2, suggesting specific potential regulatory mechanisms underlying these AD risk loci.

genetics↗

bioRxiv2025

Molecular insights into ligand recognition and signaling of GPR99/OXGR1

GPR99/OXGR1 is a G protein-coupled receptor (GPCR) with two endogenous agonists, the tricarboxylic acid cycle derivative oxoglutarate and the inflammatory lipid mediator leukotriene E4 (LTE4). How GPR99/OXGR1 recognizes two distinct ligands is a biologically important question relevant to therapeutic development due to its established roles in asthma, allergy and inflammation. Here we present cryo-EM structures of GPR99/OXGR1-Gq complex with oxoglutarate and LTE4, respectively. The oxoglutarate-bound structure shows a binding pocket surrounded by the transmembrane domains (TM), with a primary site and an accessory site for simultaneous binding of two oxoglutarate molecules for full activation of the receptor. The TM binding pocket, however, is too small to accommodate the "Y" shaped structure of LTE4, a cysteinyl leukotriene. Alanine substitution of key residues for oxoglutarate binding had little impact on LTE4-induced signaling. An alternative site in between TM3/4/5 just above intracellular loop 2 was identified in the cryo-EM structure of GPR99/OXGR1-Gq complex formed with LTE4, but the densities were less well defined. Alanine substitution of amino acids potentially involved in LTE4 interaction at this site abrogated LTE4-induced receptor activation, with no effect on oxoglutarate-induced signaling. Both ligands activated GPR99/OXGR1 primarily through the Gq pathway, but LTE4 also induced inhibition of cAMP accumulation that was sensitive to pertussis toxin. These findings illustrate the structural basis for GPR99/OXGR1 interaction with oxoglutarate and suggest the presence of a distinct binding site for LTE4.

biochemistry↗

bioRxiv2025

A modular method for rapidly prototyping targeted gas vesicle protein nanoparticles

Gas vesicles (GVs) are air-filled protein nanoparticles which are proving to be useful in a number of biomedical applications. We hypothesized that it could be possible to develop a modular method for creating rapidly prototyped GVs by modifying their surface chemistry to include targeting peptides in an orientation-specific manner. Here, we describe a modular method to create targeted GVs using His-tagged antibody fragments, ensuring that the antibody fragments are connected to the GV in an orientation-specific manner. This is achieved via the functionalization of the GVs with nickel-nitrilotriacetic acid (Ni-NTA) group. First, we validated that these functionalized GVs can bind His-tagged green fluorescent protein and characterized the particle size and surface charge of functionalized GVs. Then, GVs targeted to prostate-specific membrane antigen (PSMA) using a minibody were validated using a knockout validation in vitro.

bioengineering↗

bioRxiv2025

Spatially distributed and regionally unbound cellular resolution brain-wide processing loops in mice

Until recently, brain activity could be examined either globally, through regional averaging, or locally, at cellular resolution--but not both at once. As a result, regions were characterized as functionally homogeneous units while single neurons were often characterized as heterogeneously and randomly tuned. Here, we overcome this long-standing divide by leveraging the unprecedented density of International Brain Laboratory electrophysiological recordings in mice performing a sensorimotor decision-making task. Computationally integrating these recordings and ordering neurons by functional similarity reveals directly to the eye the full temporal arc of computations unfurling over the phases of a trial and across the brain at cellular and millisecond resolution, providing a dynamical map of the architecture of brain-wide computation. We report that one of the largest shares of brain-wide variance is explained not by task-related sensory responses, integration, or decision related signals, but by neurons encoding internally generated, trial-timed broadcast sequences that carry contextual information and are biased toward hippocampal and entorhinal involvement. We find that individual neurons reliably specialize in interpretable functions, yet neurons with similar functional roles are widely dispersed across regions. Each region contains cells spanning nearly the full repertoire of response types, rendering functional tuning largely unpredictable from cytoarchitectural boundaries or spatial location alone. In particular, even primary visual cortex contains a broad diversity of response types beyond stimulus-locked responses. The dominant discernible organizational distinction is instead between cortical and subcortical processing. Subcortical neurons carry the bulk of internal computations in expert mice, including integration, decision-making, and movement initiation. These patterns support a model of broadcast rather than staged computation, in which signals are widely shared and transformed rather than serially processed. Finally, we find that despite widespread regional mixing, representations at the single neuron level are not randomly mixed. Neurons combine specific features in highly correlated groupings, contradicting the hypothesis of random mixed selectivity at the cellular level. Together, our findings suggest a revised architecture of brain computation: typically interpretable and non-random selectivity at the level of single neurons, coupled to continual brain-wide broadcast rather than staged processing, in which internally structured activity dominates neural dynamics and scaffolds cognition.

neuroscience↗

bioRxiv2025

De novo design of high-affinity miniprotein binders targeting Francisella tularensis virulence factor

Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella-like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well-defined binding pockets and structural information on native interactions has hindered structure-guided ligand discovery against Flpp3. Here, we used a combination of physics-based and deep-learning methods to design high-affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24-110 nM. For the second site, an initial binder showed a dissociation constant (KD) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub-nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X-ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (C root-mean-square deviation: 0.9 [A]). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

biochemistry↗

bioRxiv2025

A Universal RT-qPCR Method for DNA Aptamer Quantification

Aptamers are widely used in various applications; however, their quantification methods remain underdeveloped. In this study, we established a universal RT-qPCR-based method for DNA aptamer quantification. By incorporating reverse transcription, primers and probes could be added to aptamers, enabling their detection via qPCR (Figure 1). We developed a software tool for primer and probe design and optimized the reverse transcription process. Two aptamers, CD9-aptamer and CD63-aptamer, were selected as model systems representing two distinct aptamer types: the CD9-aptamer contains an intrinsic primer-binding site within its sequence, whereas the CD63-aptamer does not. Using this method, the limit of detection (LOD) for CD9-aptamer reached 10-14 M, while the LOD for CD63-aptamer was 10-12 M. Compared to existing quantification methods, this approach significantly improves accuracy and cost-effectiveness. Additionally, the method supports SYBR Green, TaqMan, and one-step RT-qPCR assays, broadening its applicability and enhancing precision for various aptamer-based applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/662296v1_fig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@44ec4corg.highwire.dtl.DTLVardef@f59184org.highwire.dtl.DTLVardef@2480fdorg.highwire.dtl.DTLVardef@906845_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Schematic illustration of RT-qPCR-Based Aptamer Quantification. C_FIG

molecular biology↗

bioRxiv2025

Thermally controlled state switches for macrophage immunotherapy

Advances in cellular immunotherapy promise new treatments for conditions such as cancer, autoimmune disease, and heart disease. While engineered cells have the ability to recognize clinically relevant signals, traffic to disease sites and interface with the host immune system, their activity must be tightly controlled to minimize undesirable effects in healthy tissues. One approach to obtaining specificity is to activate the cells spatially using externally applied energy, such as ultrasound-delivered heating. To facilitate such control, we designed and characterized a genetic circuit that enables stable transcriptional activation of macrophages after a brief thermal stimulus, resulting in the expression of reporters or secretion of the cytokine IL-12. We demonstrate that in vivo activation of a mouse macrophage cell line containing this bioswitch results in spatially localized gene expression for at least 14 days after ultrasound heating. This thermal bioswitch provides a precise control element for cell-therapeutic agents.

bioengineering↗

bioRxiv2025

Bacterial extracellular vesicles as a tunable platform for vaginal drug delivery

There is a critical gap in the development of new therapeutic platforms designed to treat gynecologic and obstetric diseases. Compared to systemic drug delivery, vaginal drug administration of nanoparticle formulations limits off-target side effects while increasing therapeutic concentration in target tissues, showing promise for clinical translation. However, these formulations suffer from limited scalability, high-cost reagents, and long optimization timelines. Recent work highlights the potential of bacterial extracellular vesicles (bEVs) as a low-cost, tunable platform for therapeutic applications. Here, we evaluate bEVs as a therapeutic carrier for vaginal drug delivery. We demonstrate the loading of the model protein moxNeonGreen into Escherichia coli Nissle 1917 bEVs. By optimizing growth parameters, we increase protein loading into bEVs. We evaluate the effect of bEVs on the vaginal microenvironment, and observe no negative impact on vaginal epithelial cells, endocervical cells, or vaginal bacteria in vitro. Additionally, we observe the retention of bEVs in the murine female reproductive tract for more than six hours. This study provides a framework for using genetically engineered bEVs to rapidly generate customizable therapies for a range of gynecologic and obstetric conditions, addressing longstanding challenges in womens health therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/658669v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e483d2org.highwire.dtl.DTLVardef@8a6d2corg.highwire.dtl.DTLVardef@5756borg.highwire.dtl.DTLVardef@5fdb67_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Microengineered transplantation of human solid tumors for in vitro studies of CAR T immunotherapy

Treatment of solid malignancies using chimeric antigen receptor (CAR) T cells remains a significant challenge, but current efforts to advance this therapy are challenged by our limited capacity to probe and understand cancer-immune interactions in human solid tumors. Here, we present a microengineered platform for in vitro modeling of malignant solid tumors during CAR T therapy. This system makes it possible to vascularize human tumor explants and perfuse them with blood-borne immune cells in a controlled manner. We first present a microphysiological model of human lung adenocarcinomas infused with CAR-T cells and show how this system can be used to simulate, visualize, and interrogate tumor-directed trafficking and effector function of CAR T cells. We then demonstrate the proof-of-principle of testing a chemokine-directed CAR T cell engineering strategy in a model of malignant pleural mesothelioma and validating our in vitro assessment using a matching in vivo mouse model. Finally, we describe a potential therapeutic target discovered by single-cell RNA sequencing that can be pharmacologically modulated to increase the efficacy of CAR T cells for lung adenocarcinoma, for which we also present specific biomarkers identified by global metabolomics analysis. We believe that the bioengineering principle demonstrated here will make important contributions to developing new capabilities for preclinical studies of adoptive cell therapies for cancer and other complex diseases.

bioengineering↗

bioRxiv2025

Tracing the stemness and malignant transition in a heritable colorectal cancer Lynch Syndrome by single-cell RNA-seq analysis

BackgroundLynch Syndrome (LS) is an autosomal dominant disease characterized by germline heterozygous mutations in DNA mismatch repair (MMR) genes. High-risk LS patients may proceed to colorectal cancer (CRC). However, the drivers or biomarkers of LS benign colon tissue approaching malignant CRC are not completely understood. The similarity and difference between LS-related and nonLS-related CRC are also not well interrogated (LS-CRC vs. nonLS-CRC). This study aimed to understand the cellular changes during malignant transition in LS. MethodsSingle-cell RNA sequencing (scRNA-seq) was used to analyze paired biopsy samples from 3 patients with LS (cancer tissues vs. adjacent normal tissues, n=3). Single-nuclear RNA sequencing (snRNA-seq) was used to analyze a frozen biopsy sample from a patient with LS (n=1). scRNA-seq or snRNA-seq datasets from CRC were downloaded from the open source. Integrative computational analysis was performed to conclude the distinct pattern in the single-cell atlas. Immuno-histo-fluorescence staining (IHF) were also performed for three key markers. ResultsIn the single-cell atlas, we observed an increase in primitive cancer stem-cell-like cells with high expression of a general cancer biomarker CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 5 (CEACAM5) in the epithelium of the LS. Infiltration of immune cells and DNA repair biological activity are dramatically increased in LS carcinoma. The burden in LS is fundamentally elevated compared to that in CRC or healthy donors when the mutations are in the coding-sequence-wide range. Furthermore, T cell and macrophage-related tumor immunity is readily mobilized in carcinomas compared to paraCArcinomas. ConclusionsThis study provides single-cell transcriptomic resource using affected tissues from patients with Lynch Syndrome and describes an integrative profile covering the alterations (cancer stem cell markers, mutation burden, and tumor immunity) during the malignant transition from healthy to Lynch Syndrome and to colorectal cancer at the single-cell level. Highlights and Figure/Table Index (Take-Home Messages)O_LIColon tissues from HD (n=4), patients with LS (n=6) and with CRC (n=3) were collected for snRNA-seq or scRNA-seq analysis; Diagnosis of the 6 LS patients are clearly supported by our pedigree documentations (Fig. 1 and Supp. Fig. 1) and briefed in Table 1; C_LIO_LIAmong the 6 patients with LS, 1 for snRNA-seq analysis (a carcinoma tissue only), 3 for scRNA-seq analysis (3 paired para-carcinoma and carcinoma tissues) and 3 for experimental validations; (Fig. 1) C_LIO_LIFollowing studies are focused on the comparisons between HD, LS and CRC; In addition, comparisons between paired carcinoma and para-carcinoma tissues from patients with LS were also performed; (Fig. 2-7) C_LIO_LIWhen HD and LS are compared, a cancer stem cell marker CEACAM5 is readily detected by snRNA-seq analysis; (Fig. 5) C_LIO_LIWhen carcinoma and para-carcinoma from LS patients are compared, increased immune cell infiltration of and enhanced DNA repair activity in the malignant tissues is readily detected by scRNA-seq analysis; in addition, three upregulated markers in LS carcinoma (BACE2, GRPC5A and OLFM4) were identified and validated by immunohistoinfloresnce; (Fig. 3 and Fig. 4) C_LIO_LISComatic algorithm-based mutation calling analysis suggests a comparable mutation burden between carcinoma and para-carcinoma from LS patients, but such mutation burdens are grossly greater than that from HD or that from patients with CRC; (Fig. 6) C_LI O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@174af7dorg.highwire.dtl.DTLVardef@1ad3e71org.highwire.dtl.DTLVardef@dea1e4org.highwire.dtl.DTLVardef@568f91_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1C_FLOATNO Enrollment of LS patients and overall design of the study. (A) Three family pedigrees of the LS patients (LS-CRC) who donated the samples for single-cell transcriptomic analysis. Cropped pedigree information is provided in the Main text. See the Supp. Fig. 1 for the complete pedigree information. (B) Two different platforms were used in the study: snRNA-seq and scRNA-seq. (C) Various types of comparison were performed to characterize the malignant transition of LS. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@eb8e4eorg.highwire.dtl.DTLVardef@cbafaaorg.highwire.dtl.DTLVardef@79ca83org.highwire.dtl.DTLVardef@1de7544org.highwire.dtl.DTLVardef@1e4fcbc_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONClinical information of enrolled patients with LS. An Excel version of the table is provided in the Supp. Material C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig2.gif" ALT="Figure 2"> View larger version (56K): org.highwire.dtl.DTLVardef@8cd71corg.highwire.dtl.DTLVardef@7e75b3org.highwire.dtl.DTLVardef@61757eorg.highwire.dtl.DTLVardef@68ca5f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 2C_FLOATNO snRNA-seq analysis of a frozen colon biopsy sample from a LS patient reveals a cluster of cancer stem cells (A) Frozen colon biopsy samples were subjected for snRNA analysis (Healthy donors, N=4; Lynch Syndrome, N=1) and epithelial cells were filtered out for further study. Data were integrated by the Seurat and Harmony standard procedure. Clusters 0-9 were identified in a total of 11,353 cells (the Healthy Controls have 7,373 cells; the LS patient has 3,980 cells). The malignancy of the epithelial cells was inferred by the inferCNV and SCEVAN algorithm (two major compartments: tumor and no-tumor). The portion of tumor or non-tumor cells in each cluster was also illustrated (right panel). Note that the major contributor in Cluster 4, 6, and 7 is the tumor-like cells from the LS patient. (B) Each cluster of the epithelial cells was further annotated according to the combination of Seurat cluster identity and SCEVAN malignant identity. Mix_1 to 5 contain both tumor-like cells and non-tumor-like cells; Other clusters are marked as BEST4+-Enterocytes, Enterocytes and Goblet cells where much fewer tumor-like cells were assigned. Note that the epithelial cells from the LS patient are mainly classified into Mix_2, 4, and 5 tumor-like cells. (C) Three clones were inferred by SCEVAN algorithm. In the phylogenetic tree, Clone_1 and Clone_2 are closer and probably originated from Clone_3. (D) A Sankey plot showing overall belonging relationships in the epithelial snRNA-seq dataset. Note that the Mix_2 pool is dominated by cells from Lynch_01 and that in the Mix_2 pool Clone_3 is the most dominant clone. (E) The Mix_2 cells specifically express numerous colon epithelial stem cells signature markers such as SMOC2, RGBM, OLFM4, AXIN2 and LGR5. Of note, all these intestine stem cell signature genes were reported as marker genes for cancer stem cells, especially LGR5. (F) Pseudotime trajectory analysis of the Lynch Syndrome epithelium cells indicates the Mix_2 cells are the most primitive in development. (G) Top 36 differentially expressed genes (DEGs, both upregulated and downregulated) were highlighted when the non-tumor-like cells were compared with tumor-like cells in the Mix2. (H) The Mix_2, 4, 5 epithelial cells all have enriched signaling pathways involved in DNA repair response and MYC targets (fonts in red) while Mix_4 and 5 manifest Interferon alpha or gamma response (fonts in blue). C_FIG O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig7.gif" ALT="Figure 7"> View larger version (54K): org.highwire.dtl.DTLVardef@1be2c6aorg.highwire.dtl.DTLVardef@55f267org.highwire.dtl.DTLVardef@9d60fborg.highwire.dtl.DTLVardef@4a2d37_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 7C_FLOATNO Distinct tumor immunity microenvironment profiles in LS carcinoma (A) T cells from LS-CA and LS-paraCA patients were filtered for further annotation. The LS-paraCA group had 1410 T cells, whereas the Lynch CA group had 2125 T cells. Six major T cell subtypes were identified: Naive CD4-T, NKT, Treg, IL7R+CD8_T, GZMA+CD8_T, and GZMK+CD8_T. Note that GZMA+CD8_T cells appear to be more abundant in Lynch CA (arrows). (B) The pseudotime trajectory indicating the T cells developed towards two branches: the Naive CD4-T and Treg branches, and GZMA+CD8_T and GZMK+CD8_T branches. GZMA+CD8_T cells appear to be more mature in LS-CA than in LS-paraCA (arrows). (C-F) Biological function-instructed scoring of the paired single-cell datasets of T cells from the three LS patients, including T cell cytotoxicity (B), T cell exhaustion (C), T cell immune escape (D), and T cell surveillance (E). (G) Circle plots showing interaction profiles across indicated cells in the paired LS samples. The direction of the lines indicates the direction of the cell-cell communication, the width of the lines indicates the interaction strength, and the color of the lines indicates the cell types of the senders. (H) Summary of the Study. In this study, we profiled Lynch Syndrome biopsy samples using single-cell or single-nucleus RNA-seq analysis. Paired carcinoma and adjacent para-carcinoma samples were included to facilitate the analysis, including tracing tumor clones, calculating mutation burden, identifying biomarkers for cancer stem cells, and tumor immune microenvironment changes during the transition from pre-carcinoma to carcinoma in the inheritable cancer Lynch Syndrome. See the text in the Discussion section for further detail. C_FIG O_FIG O_LINKSMALLFIG WIDTH=148 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig5.gif" ALT="Figure 5"> View larger version (44K): org.highwire.dtl.DTLVardef@1de7f42org.highwire.dtl.DTLVardef@74e8ddorg.highwire.dtl.DTLVardef@1642847org.highwire.dtl.DTLVardef@907a23_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 5C_FLOATNO Distinct pre-cancer signs in epithelial and immune cells of LS para-carcinoma (A) The scRNA-seq datasets of LS-paraCA were integrated with those of the colons from healthy donors (HD) (N=3, respectively; the HD group had 7885 cells in total, whereas the LS-paraCA group had 4766 cells). A dominance of CEACAM5high epithelial cells were identified in LS-paraCA. The sample from the patient with relapsed LS is marked with an arrow in the bar plot (left panel). (B) The portion of CEACAM5high epithelial cells, infiltrated CD8_T cells and macrophages was quantified. The sample from the relapsed LS patient is marked with arrows. (C, E) Top marker genes co-expressed in CEACAM5high epithelial cells and the enriched pathways are illustrated in the gene expression heatmap. Cells from the Enterocytes, Tufts, and Goblet subtypes were used as controls. (D, F) Quantification of the expression of CEACAM5 in epithelial cells of HD and LS-paraCA cells. C_FIG O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig3.gif" ALT="Figure 3"> View larger version (48K): org.highwire.dtl.DTLVardef@142c991org.highwire.dtl.DTLVardef@1bf5d99org.highwire.dtl.DTLVardef@99edd5org.highwire.dtl.DTLVardef@1cd5c15_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 3C_FLOATNO Distinct infiltration of immune cells in carcinoma and para-carcinoma tissues of LS (A) Fresh paired carcinoma (LS-CA) and adjacent para-carcinoma (LS-paraCA) biopsies of colon tissue was subjected to scRNA-seq analysis (N=3). Colorectal cancer (nonLS-CRC, they are not diagnosed as LS) tumor samples from the public dataset were also included as controls (N=4). Cells were first grossly annotated as stromal, epithelial, or immune cells. Note that the LS-CA group showed increased infiltration of myeloid cells compared with LS-paraCA group, but T cell infiltration was comparable between the two groups. (B) Expression of classic markers for annotating the major 5 cell types. (C) Detailed annotations of 16 cell types in paired LS-CA and LS-paraCA samples. (D) Stacked bar plot showing the contribution of sample sources in each cell type. (E) Quantification of immune cell infiltration in LS-CA compared with LS-paraCA. Although no significant differences were observed, the fold-changes were approximately 2-10. Although the overall T cell infiltration was comparable, infiltration of GZMA+CD8_T and GZMK+CD8_T cells appeared to be higher in LS-CA than in LS-paraCA. Of the three LS patients enrolled in the study, one was a relapsed patient (gray dashed line, surgery treatment performed; whose son donated the frozen sample in Figure 2), while the other two were treatment-naive (gray solid lines). (F) Boxplots show DNA repair score quantification per sample (above) or per epi-cell. (bottom). C_FIG O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig4.gif" ALT="Figure 4"> View larger version (51K): org.highwire.dtl.DTLVardef@1588458org.highwire.dtl.DTLVardef@58db3borg.highwire.dtl.DTLVardef@1a12084org.highwire.dtl.DTLVardef@15de70e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 4C_FLOATNO Prioritizing novel epithelial biomarkers for tracing the transition between para-carcinoma and carcinoma in LS (LS-paraCA vs. LS-CA) (A) Epithelial cells were filtered out for further analysis and annotated as clusters 0-9 in the UMAP plot. (B, C) Bar and pie plots show the contribution of sources to epithelial clusters. Cluster 0 contained cells largely from Lynch CA_02, whereas Cluster 9 was composed of Lynch paraCA_02. The patient who donated Lynch CA_02 and paraCA_02 is identified as a relapsed LS. (D) KEGG enrichment analysis showing the enriched bioprocesses in the four epithelial cell clusters. (E) Five genes with upregulated expression in LS carcinoma epithelial cells were prioritized based on the scRNA-seq and snRNA-seq datasets. (F) Expression of BACE2, GPRC5A and OLFM4 in the paired LS-paraCA and LS-CA groups. Epithelial cells are circled by dashed lines. Note that the expression of genes in epithelial cells from nonLS-CRC samples was also very strong. (G) Verification of the low-to-high transition in the expression of BACE2, GPRC5A and OLFM4. Paraffin sections from paired LS-paraCA and LS-CA samples were prepared, and immunohistochemical (IHF) staining was performed. The expression of the indicated proteins is labeled in green, whereas the nuclei are stained with DAPI in blue. In total, biopsy samples from 4 patients with LS were used for IHF verification. C_FIG O_FIG O_LINKSMALLFIG WIDTH=148 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig6.gif" ALT="Figure 6"> View larger version (28K): org.highwire.dtl.DTLVardef@1ec5210org.highwire.dtl.DTLVardef@1ffd6f9org.highwire.dtl.DTLVardef@17a7a8forg.highwire.dtl.DTLVardef@4819e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 6C_FLOATNO Increased mutation burden in the colon tissues from Lynch Syndrome (A) The schematic diagram for calculating mutation burden based on the scRNA-seq datasets using the SComatic algorithm. Only single-base substitutions (SBS) were called in this study. Once mutation calls were achieved, the mutation burden value was quantified by normalizing the total mutation calls to the total cell number in the samples or cell types (See Methods section for details). (B) The total SBS mutation burden in the four groups of samples was indicated. (C) The six different SBS mutation burdens in the four groups of samples as indicated. (D) The SBS mutation burden in the cell types of the four groups is indicated. Note that the The mutation burden appears to be higher in mast, epithelial, plasma, myeloid, and Treg cells in the LS-CA group than in that the LS-paraCA group. C_FIG

cancer biology↗

bioRxiv2025

Epidermal Resident Memory T Cell Fitness Requires Antigen Encounter in the Skin

CD8+ tissue resident memory T cells (TRM) develop from effectors that seed peripheral tissues where they persist providing defense against subsequent challenges. TRM persistence requires autocrine TGF{beta} transactivated by integrins expressed on keratinocytes. TRM precursors that encounter antigen in the epidermis during development outcompete bystander TRM for TGF{beta} resulting in enhanced persistence. ScRNA-seq analysis of epidermal TRM revealed that local antigen experience in the skin resulted in an enhanced differentiation signature in comparison with bystanders. Upon recall, TRM displayed greater proliferation dictated by affinity of antigen experienced during epidermal development. Finally, local antigen experienced TRM differentially expressed TGF{beta}RIII, which increases avidity of the TGF{beta}RI/II receptor complex for TGF{beta}. Selective ablation of Tgfbr3 reduced local antigen experienced TRM capacity to persist, rendering them phenotypically like bystander TRM. Thus, antigen driven TCR signaling in the epidermis during TRM differentiation results in a lower TGF{beta} requirement for persistence and increased proliferative capacity that together enhance epidermal TRM fitness.

immunology↗

bioRxiv2025

Population and adaptation history of 739 Thlaspi arvense natural accessions

Pennycress (Thlaspi arvense) is a promising intermediate oilseed crop, producing oil suitable for conversion to biofuels--including aviation fuels. While domestication efforts are ongoing, a deeper understanding of the genetic architecture of traits is crucial for informing future breeding efforts. Here, we conducted the largest genomic and phenotypic survey of pennycress to date, analyzing 739 accessions collected across four continents. Leveraging whole-genome sequencing and field-collected phenotypes, we characterized the standing genetic variation underlying key agronomic traits and climate resilience. Our findings revealed multiple independent migration events to North America, with substantial genetic admixture. We identified homologs of Arabidopsis thaliana flowering-time genes that contribute to adaptation and demonstrated the agronomic benefits of winter-type pennycress. Furthermore, through multi-season field trials, we identified a genomic region containing a cluster of mTERF genes strongly associated with green canopy coverage, a critical trait for biomass retention and yield stability. These insights provide a genomic roadmap for accelerating pennycress domestication and improving its resilience to climate variability.

genomics↗