Biology subjects

Shan, L.

Publications and source records attributed to Shan, L..

At least 19 recordsLinked to original sources

bioRxiv2026

A defined multi-kingdom microbial consortium naturalizes the immune system of laboratory mice

Dirty mice experiments suggest that laboratory mice fail to model certain human immune responses due to their limited microbial exposure. The identity, nature, and number of microbes required to normalize the immune system are unknown. Through systematic screening, we uncovered unexpected cross-kingdom microbial interactions and identified four enteric microbes spanning viruses, fungi, and protists sufficient to reproduce key immunological features of pet store mice and adult humans. This consortium also altered cancer outcomes, shifting tumorigenesis toward the anatomical pattern of human familial adenomatous polyposis in ApcMin/+ mice and recapitulating the imperfect response of many patients to chimeric antigen receptor (CAR) T cell therapy. Thus, surprisingly few microbes can profoundly reshape immune states and disease responses, providing a tractable approach to naturalize laboratory mouse immunity.

immunology↗

bioRxiv2026

FLASH X-rays preserve neutrophil chromatin to boost antitumor immunity

Ultrahigh dose-rate FLASH radiotherapy spares normal tissue while preserving antitumor activity, positioning it as a promising technology in radiation oncology, but its immunomodulatory mechanisms remain elusive. Here, using a bilateral subcutaneous tumor model in mice, we show that 8-Gy FLASH X-rays, versus an equal dose of conventional dose-rate (CONV) X-rays, elicit a robust abscopal antitumor effect with anti-PD-1 therapy. Single-cell transcriptome and T cell receptor (TCR) profiling reveal that FLASH X-rays plus anti-PD-1 enhance antitumor immune infiltration and TCR clonal expansion in both irradiated and abscopal tumor lesions. Mechanistically, the ultrashort temporal continuity of FLASH dose delivery conserves chromatin compaction in neutrophils during exposure and inhibits neutrophil extracellular trap (NET) formation, thereby dismantling NET-mediated CD8+ T-cell exclusion within primary tumors. Conversely, CONV or split-dose FLASH X-rays induce marked chromatin modifications prior to dose completion, leading to chromatin hyper-relaxation which promotes NETosis and consequently abrogates systemic antitumor immunity. Notably, this detrimental effect can be reversed by pharmacologically suppressing PARylation-mediated chromatin modification and decondensation in neutrophils. These findings identify radiation-induced aberrant chromatin relaxation in neutrophils as a critical determinant of radioimmunotherapy outcomes, providing a rationale for redefining the FLASH effect and advancing its clinical translation.

cancer biology↗

bioRxiv2026

Reverse genetics in humanized mice reveals CARD8-mediated pyroptosis causing pancytopenia in human DPP9 deficiency

Loss of function mutation in the human DPP9 gene causes Hatipoglu syndrome leading to severe inflammasomopathy. A key feature of the disease is pancytopenia and patients require bone marrow transplantation, but the mechanism of cell loss is unclear since Dpp9 mutant mice have normal hematopoiesis, suggesting that a distinct mechanism of disease occurs in humans. Here, we present a model of human DPP9 deficiency leveraging reverse genetics in the MISTRG6 humanized mice. We found that CRISPR editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to very efficient and persistent gene deletion in vivo. Human DPP9 deletion recapitulated cytopenia in peripheral blood and in the bone marrow, and cell loss was cell intrinsic. However, DPP9 deletion led to little transcriptional changes suggesting post-transcriptional regulation in human HSPCs. Mechanistically, DPP9 deficiency led to the activation of the CARD8 inflammasome resulting in HSPC pyroptosis, whereas NLRP1 was dispensable for cell death. Thus, our results reveal a unique human mechanism of disease and offer therapeutic insight for this inflammasomopathy.

immunology↗

bioRxiv2026

Prediction of Transcription Factor DNA Binding Affinity with High-Throughput Kd Measurements and Deep Learning

Transcription factors (TFs) regulate gene expression through specific interactions with genomic DNA. While TF binding motifs from public databases describe sequence preferences, quantifying genome-wide affinity (Kd) is highly desirable for a more accurate thermodynamic description. Here, we report ivtFOODIE (in vitro FOOtprinting with DeamInasE), an assay that leverages deaminase-mediated cytosine-to-uracil conversion to measure Kd values for a given TF across accessible genomic regions from human cells. By pre-training on TF binding sites from JASPAR and fine-tuning with our ivtFOODIE data from 46 TFs representing 13 different DNA-binding domains (DBDs), we developed Seq2Kd, a deep learning model capable of predicting a TFs absolute binding affinity on DNA sequences. Seq2Kd enables de novo motif discovery of [~]500 previously uncharacterized human TFs and reveals the effects of genetic variation both in TF-coding regions and DNA-binding sites on gene expression and disease susceptibility. By correlating predicted affinity changes with the sign and magnitude of expression quantitative trait locus (eQTL) effects, we stratified TFs into activator-like and repressor-like groups. Compared to clinically benign variants, pathogenic single-nucleotide variants (SNVs) within regulatory and protein-coding regions show significantly larger predicted shifts in Kd. We provide an interactive web portal, the ENcyclopedia of Transcription-factor Interactions with Regulatory Elements (ENTIRE), which integrates the Seq2Kd model with the ivtFOODIE dataset. This resource offers thermodynamic prediction for TF-DNA interactions for functional genomics and human disease.

biophysics↗

bioRxiv2025

MFAID-Net: A Multi-modal Feature Fusion Deep Learning Network for Robust Adaptive Introgression Detection Across Diverse Evolutionary Scenarios

Adaptive introgression (AI), the beneficial genetic transfer between species, is key to adaptation, yet its genomic identification is challenging. Existing methods are inconsistent and often require unavailable donor population data. We propose MFAID-Net, a novel multi-modal network for adaptive introgression detection. MFAID-Net employs a dual-encoder architecture, combining a Convolutional Neural Network for local genomic features with a Multi-Layer Perceptron for population genetic statistics and structure. A Transformer-like attention mechanism adaptively fuses modalities, dynamically weighting their importance. Multi-task learning differentiates AI from confounding signals like neutral introgression and selective sweeps. Comprehensive simulations demonstrate MFAID-Nets superior performance across challenging scenarios (e.g., strong/weak selection, ancient migration), often outperforming current methods. It is also robust to donor data availability. Ablation and attention analyses confirm component contributions and provide interpretability. MFAID-Net offers a more robust, accurate, and adaptable tool for discovering AI events.

bioinformatics↗

bioRxiv2025

Increased numbers of CD4+ T-cells in hypocretin/orexin region of Narcolepsy Type 1

Narcolepsy type 1 (NT1) is proposed to be an autoimmune disorder targeting hypothalamic hypocretin (orexin, Hcrt) neurons. Hcrt reactive T-cells have been identified in blood and cerebrospinal fluid (CSF) of NT1 patients. However, it remains unknown whether T-cells infiltrate the brain. Since T-cells can be retained for a lifetime in tissues as tissue resident memory T-cells after primary antigen exposure, we now systematically assess the presence of CD4+ and CD8+ T-cells in NT1 brains to determine their regional distribution and potential autoimmune involvement in NT1 neuropathology. We immunohistochemically stained and quantified CD4+ and CD8+ T-cells in post-mortem brain tissue of NT1 patients (n=5) and matched controls (n=5) in the Hcrt region, paraventricular nucleus (PVN) and median eminence (ME) as well as in the substantia nigra (SN) and locus coeruleus (LC). To phenotypically characterize CD4+ T-cells, we performed double staining with CD49a or C-X-C chemokine receptor type 6 (CXCR6). Furthermore, we stained for fibrinogen to estimate blood-brain barrier integrity, as well as microglia markers and an astrocyte marker to evaluate acute immune reactivity in the Hcrt region of NT1 brains. In NT1 there was an 11-fold increase in total number of CD4+ T-cells in the Hcrt region, but not in the PVN, ME, nor SN or LC. These CD4+ T-cells exhibited tissue residential memory features with double staining with CD49a or CXCR6. There were no changes in blood-brain barrier integrity, microglia and astrocyte staining intensities between NT1 and controls. In addition, the total number of CD4+ T-cells in the Hcrt region showed significant negative correlations with mean sleep latency in NT1 cases. Our findings suggest an enrichment of CD4+ T-cells specifically in the Hcrt region of NT1 indicating prior local antigen engagement. Moreover, greater CD4+ T-cell presence in the Hcrt region of NT1 was associated with increased symptom severity, as reflected by shorter sleep latency. These data support the hypothesis that CD4+ T-cells infiltrate the Hcrt region, where they may contribute to the autoimmune process that initiates NT1.

neuroscience↗

bioRxiv2025

Clade V MLO proteins are bona fide host susceptibility factors required for powdery mildew pathogenesis in Arabidopsis

Obligate biotrophic powdery mildew (PM) fungi strictly require living host to survive. To search for host factors or processes essential for PM pathogenesis, a tailored genetic screen was conducted with the immuno-compromised eds1-2/pad4-1/sid2-2 (eps) triple Arabidopsis mutant. This led to the identification of five allelic disruptive mutations in Mildew Locus O 2 (MLO2) to be responsible for the compromised-immunity-yet-poor infection (cipi) mutant phenotype upon challenge from an adapted PM isolate. Moreover, the eds1/pad4/sid2/mlo2/mlo6/mlo12 (eps3m) sextuple mutant display near complete immunity to the adapted PM fungus without sign of defense activation, demonstrating that these three clade V MLOs in Arabidopsis are bona fide host susceptibility factors of PM fungi. Confocal imaging revealed focal accumulation of MLO2-GFP in the peri-penetration peg membranous space, implicating MLO2 in repairing and stabilizing the damaged host plasma membrane, which may be co-opted by PM fungi for haustorium differentiation. Results from domain-swapping analysis between MLO1 and MLO2 suggest a bipartite functional configuration for MLO2: its C-terminus determines where and when MLO2 functions, while its N-terminal seven transmembrane domain region executes the cellular function that is critical for PM pathogenesis. Genetic studies further demonstrate that, unlike MLO7 in synergids, focal accumulation of MLO2 does not depend on FERONIA (FER) and its five other family members, nor does it require phosphatidylinositol 4,5-bisphosphate produced from phosphatidylinositol 4-phosphate 5-kinase 1 (PIP5K1) and PIP5K2. Together, these findings define clade V MLOs as host factors co-opted by obligate biotrophic PM fungi for successful host colonization.

plant biology↗

bioRxiv2025

New alleles of Arabidopsis BIK1 reinforce its predominant role in pattern-triggered immunity and caution interpretations of other reported functions

The receptor-like cytoplasmic kinase BIK1 and its close homolog PBL1 have been widely recognized as central components of plant immunity. However, most genetic studies of BIK1 and PBL1 functions were carried out with single T-DNA insertional mutant alleles. Some phenotypes observed in these mutants, e.g. autoimmunity, have been difficult to reconcile with the proposed role of BIK1 and PBL1 in pattern-triggered immunity. In this study, we generated multiple new alleles of bik1 and pbl1 by CRISPR-Cas9-based gene editing and systematically analyzed these mutants alongside existing T-DNA insertional lines. These analyses reinforced the central role of BIK1 and PBL1 in pattern-triggered immunity mediated by both receptor kinases and receptor-like proteins. At the same time, however, we revealed several pleiotropic phenotypes associated with T-DNA insertions that are not necessarily linked to loss of BIK1 or PBL1 function. Further analyses of newly generated bik1 pbl1 double mutants uncovered an even greater contribution of these kinases to immune signaling and disease resistance than previously appreciated. These findings clarify longstanding ambiguities surrounding BIK1 and PBL1 functions.

plant biology↗

bioRxiv2025

Establishment of a Reverse Genetics System for the Study of Human Immune Functions in Mice

Reverse genetics approaches in mice are widely utilized to understand gene functions and their aberrations in diseases. However, limitations exist in translating findings from animal models to human physiology. Humanized mice provide a powerful bridge to understanding human physiology and mechanisms of diseases pathogenesis while maintaining the feasibility of working with small animals. Methods for generating humanized mouse models that allow scientists to probe contributions of particular genes have been rudimentary. Here, we established an efficient method for generating genetically modified human cord blood derived CD34+ cells for transplantation, resulting in humanized mice with near-complete loss of specific gene expression by the human immune system. Mice transplanted with Cas9-edited human CD34+ cells recapitulate functional consequences of specific gene losses in the human immune system. This advancement enables the development of humanized mouse models with targeted gene knockouts, offering a valuable research tool for human gene function studies in vivo.

immunology↗

bioRxiv2025

The dichotomous impacts of semaphorin3E deficiency on exacerbating airway hyperresponsiveness, remodelling, and inflammation in type-2 low and type-2 high asthma models

Semaphorin3E has shown promise in alleviating the severity of asthma in preclinical studies; however, its role in the chronic features of type 2-low asthma remains unclear. Therefore, we aimed to investigate the role of Sema3E in a mouse model of severe asthma that exhibits a mix of granulocytic inflammation with neutrophils dominance and compared the results with those from the type-2 high eosinophilic asthma model. Sema3E knockout (KO) and wild-type (WT) mice were subjected to type-2 low and type-2 high regimens using house dust mite (HDM) combined with cyclic-di-GMP or HDM alone, respectively. Airway hyperresponsiveness parameters were measured using the FlexiVent ventilator. Bronchoalveolar lavage fluid cell phenotyping was performed by flowcytometry. Additionally, cytokines and antibodies were quantified using Mesoscale and ELISA. Mucus overproduction and goblet cell hyperplasia were visualized by Periodic-acid-Schiff staining. In comparison to WT mice, Sema3E KO mice exhibited an enhanced tissue resistance and tissue elastance in the type 2-low asthma model. Concurrently, Sema3E KO mice that were subjected to the type-2 low asthma model demonstrated an elevated presence of pulmonary neutrophils, dendritic cells, CD4 T cells, as well as increased levels of IL-17, TNF, IL-1{beta}, CXCL-8, and MCP-1/CCL2 in comparison to their WT counterparts. However, in the type-2 high model, Sema3E KO mice exhibited a significant increase in goblet cell numbers and mucus overproduction, as well as enhancements in the number of eosinophils, IgE-producing B cells, and IL-4 levels compared to WT mice, highlighting the homeostatic role of Sema3E in the distinct immune niche of type-2 low and type-2 high asthma. Overall, our data showed that Sema3E is critical in modulating AHR, airway inflammation, and tissue remodelling in type 2 low and type 2 high phenotypes of asthma. The Sema3E regulatory network varies depending on the immunization regimen, affecting distinct parameters in type-2 low and type-2 high asthma models.

immunology↗

bioRxiv2025

Endocytosis of the damage-associated molecular pattern receptor PEPR1 is BAK1-dependent.

After cellular damage caused by wounding or pathogens, Arabidopsis thaliana endogenous elicitor peptides (Peps) are released into the apoplast, enhancing innate immunity by directly binding to the membrane-localized leucine-rich repeat receptor kinase PEP RECEPTOR1 (PEPR1). Ligand binding induces PEPR1 heterodimerization with the co-receptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1), followed by PEPR1 internalization, both essential for a subset of Pep1-induced responses. However, the role of BAK1 in Pep1-triggered PEPR1 endocytosis remains unclear. Here, we show that the ligand-induced PEPR1 endocytosis depends on its kinase activity and requires BAK1 C-terminal tail phosphorylation, which is equally indispensable for immune signaling and BAK1 internalization. Using a GFP insertional mutagenesis approach, we generated a partially functional GFP-tagged BAK1 to demonstrate that, following Pep1 elicitation, BAK1 and PEPR1 are endocytosed together with similar dynamics. Our findings identify the BAK1 function as a prerequisite for PEPR1 internalization.

plant biology↗

bioRxiv2025

CD34 serves as an intrinsic innate immune guardrail protecting stem cells from replicating retroviruses

Stem cells are highly resistant to viral infection compared to their differentiated progeny, and this resistance is associated with stem cell-specific restriction factors and intrinsic interferon stimulated genes (ISGs). In HIV infection, proviral DNA has been detected in certain bone marrow hematopoietic stem cells, yet widespread stem cell infection in vivo is restricted. Intriguingly, exposing bone marrow stem cells to HIV in vitro led to viral replication selectively only in the CD34- population, but not in the CD34+ cells. The mechanism dictating this CD34-based HIV restriction remained a mystery, especially since HIV has a capacity to antagonize restriction factors and ISGs. CD34 is a common marker of hematopoietic stem and progenitor cells. Here, we report the intrinsic antiviral properties of CD34. Expression of CD34 in HIV-1 producer cells results in the loss of progeny virion infectivity. Conversely, removal of CD34 using CRISPR/Cas9 knockout or stem cell differentiation cytokines promotes HIV-1 replication in stem cells. These results suggest that in addition to restriction factors and intrinsic ISGs, CD34 serves as a host innate protection preventing retrovirus replication in stem cells. Mechanistically, CD34 does not block viral entry, integration, and release. Instead, it becomes incorporated onto progeny virions, which inactivates virus infectivity. These findings offer new insights into innate immunity in stem cells, and highlight intriguing retrovirus-host interactions in evolution.

immunology↗

bioRxiv2024

Pre-rRNA spatial distribution and functional organization of the nucleolus

The self-organized nucleolus is where the pre-ribosomal RNA (pre-rRNA) processing and pre-ribosomal ribonucleoprotein (RNP) assembly take place. Here we present the spatiotemporal distribution of pre-rRNA intermediates in the human nucleolus, revealing a segregated spatial distribution of small subunit (SSU) and large subunit (LSU) precursors. Notably, the 5 external transcribed spacer (5 ETS)-containing SSU pre-rRNAs are retained across FC-DFC-PDFC regions, while the internal transcribed spacer 2 (ITS2)-containing LSU pre-rRNAs move to PDFC-GC regions for processing. Inhibiting 5 ETS processing disrupts the SSU pre-rRNA distribution and the nested FC-DFC sub-nucleolar structure. Cells in amniotes possess a multi-layered nucleolus, whereas anamniotes have only a bipartite structure with a merged FC-DFC. Kinetic labeling of pre-rRNA outflow shows a 7-fold higher pace in tetrapartite nucleoli over that in bipartite nucleoli, indicating that the emergence of the nested FC-DFC may facilitate an efficient SSU pre-rRNA processing over the course of evolution. Collectively, depicting the spatiotemporal distribution of pre-rRNAs reveals a key role of processing steps in organizing the multi-layered nucleolus and suggests a possible evolutionary advantage of the multi-layered structure in amniotes.

molecular biology↗

bioRxiv2024

MAPK signaling modulates the partition of DCP1 between P-bodies and stress granules in plant cells

Processing bodies (PBs) and stress granules (SGs) are membrane-less cellular compartments consisting of ribonucleoprotein complexes. Whereas PBs are more ubiquitous, SGs are assembled mainly in response to stress. PBs and SGs are known to physically interact and molecules exchange between the two have been documented in mammals. However, the molecular mechanisms underpinning these processes are virtually unknown in plants. We have reported recently that tandem CCCH zinc finger 1 (TZF1) protein can recruit MAPK signaling components to SGs. Here we have found that TZF1-MPK3/6-MKK4/5 form a protein-protein interacting network in SGs. The mRNA decapping factor 1 (DCP1) is a core component of PBs. MAPK signaling mediated phosphorylation triggers a rapid reduction of DCP1 partition into PBs, concomitantly associated with an increase of DCP1 assembly into SGs. Furthermore, we have found that plant SG marker protein UBP1b (oligouridylate binding protein 1b) plays a role in maintaining DCP1 in PBs by suppressing the accumulation of MAPK signaling components. Together, we propose that MAPK signaling and UBP1b mediate the dynamics of PBs and SGs in plant cells.

cell biology↗

bioRxiv2024

The multi-stage plasticity in the aggression circuit underlying the winner effect

Winning increases the readiness to attack and the probability of winning, a widespread phenomenon known as the "winner effect". Here, we reveal a transition from target-specific to generalized aggression enhancement over 10 days of winning in male mice, which is supported by three stages of plasticity in the ventrolateral part of the ventromedial hypothalamus (VMHvl), a critical node for aggression. Over 10-day winning, VMHvl cells experience monotonic potentiation of long-range excitatory inputs, a transient local connectivity strengthening, and a delayed excitability increase. These plasticity events are causally linked. Optogenetically coactivating the posterior amygdala (PA) terminals and VMHvl cells potentiates the PA-VMHvl pathway and triggers the cascade of plasticity events as those during repeated winning. Optogenetically blocking PA-VMHvl synaptic potentiation eliminates all winning-induced plasticity. These results reveal the complex Hebbian synaptic and excitability plasticity in the aggression circuit during winning that ultimately leads to an increase in "aggressiveness" in repeated winners.

neuroscience↗

bioRxiv2024

Chemical genetics reveals cross-activation of plant developmental signaling by the immune peptide-receptor pathway

Cells sense and integrate multiple signals to coordinate development and defence. A receptor-kinase signaling pathway for plant stomatal development shares components with the immunity pathway. The mechanism ensuring their signal specificities remains unclear. Using chemical genetics, here we report the identification of a small molecule, kC9, that triggers excessive stomatal differentiation by inhibiting the canonical ERECTA receptor-kinase pathway. kC9 binds to and inhibits the downstream MAP kinase MPK6, perturbing its substrate interaction. Strikingly, activation of immune signaling by a bacterial flagellin peptide nullified kC9s effects on stomatal development. This cross-activation of stomatal development by immune signaling depends on the immune receptor FLS2 and occurs even in the absence of kC9 if the ERECTA-family receptor population becomes suboptimal. Furthermore, proliferating stomatal-lineage cells are vulnerable to the immune signal penetration. Our findings suggest that the signal specificity between development and immunity can be ensured by MAP Kinase homeostasis reflecting the availability of upstream receptors, thereby providing a novel view on signal specificity.

plant biology↗

bioRxiv2024

Engineering anti-amyloid antibodies with transferrin receptor targeting improves safety and brain biodistribution

Although the first generation of immunotherapies for Alzheimers disease (AD) are now clinically approved, amyloid-related imaging abnormalities (ARIA) remain a major safety problem for this class of drugs. Here, we report an antibody transport vehicle (ATV) targeting the transferrin receptor (TfR) for brain delivery of amyloid beta (A{beta}) antibodies that significantly reduced ARIA-like lesions and improved plaque target engagement in a mouse model of amyloid deposition. Asymmetrical Fc mutations (ATVcisLALA) allowed the molecule to selectively retain effector function only when bound to A{beta} while mitigating TfR-related hematology liabilities. Mice treated with ATVcisLALA:A{beta} exhibited broad brain parenchymal antibody distribution; in contrast, anti-A{beta} IgG was highly enriched at arterial perivascular spaces where vascular A{beta} localizes and likely plays a role in induction of ARIA. Importantly, ATVcisLALA: A{beta} almost completely eliminated ARIA-like lesions and vascular inflammation associated with anti-A{beta} treatment. Taken together, ATVcisLALA has the potential to significantly improve both safety and efficacy of A{beta} immunotherapy through enhanced biodistribution mediated by transport across the blood-brain barrier.

neuroscience↗

bioRxiv2024

Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton

Xanthomonas spp. employ transcription activator-like effectors (TALEs) to promote pathogenicity by activating host susceptibility (S) genes. Cotton GhSWEET10 is an S gene targeted by a TALE in an early isolate of Xanthomonas citri pv. malvacearum (Xcm), but not by recent field Xcm isolates. To understand the pathogenicity shift in Xcm and its adaptation to cotton, we assembled the whole genome and the TALE repertoire of three recent Xcm Texas field isolates. A newly evolved TALE, Tal7b, activated different GhSWEET genes, GhSWEET14a and GhSWEET14b. Simultaneous activation of GhSWEET14a and GhSWEET14b resulted in pronounced water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identified a pectin lyase as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9-based gene editing supported the function of GhSWEETs as S genes in cotton bacterial blight and the promise of disrupting the TALE-binding site in these genes to control the disease. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes simultaneously to promote pathogenicity.

plant biology↗