Biology subjects

Shen, P.

Publications and source records attributed to Shen, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Dietary tryptophan rescues growth impairment during giardiasis independently of parasite burden

Giardiasis is an intestinal disease caused by the parasite Giardia duodenalis. Intestinal barrier dysfunction during Giardia infection has been linked to childhood growth stunting, particularly in communities with prevalent malnutrition and repeated exposure to Giardia. In pediatric cohort studies, growth stunting has been strongly associated with asymptomatic Giardia detection in fecal samples. Several studies have described potential mechanisms by which Giardia disrupts the epithelial barrier, but less is known about factors that may support epithelial homeostasis in response to infection. Microbiota-derived tryptophan metabolites that activate aryl hydrocarbon receptor (AHR) signaling are a key pathway involved in epithelial barrier homeostasis. We tested whether dietary tryptophan availability influenced host growth outcomes and aryl hydrocarbon receptor activity during giardiasis. We used established murine Giardia malnutrition models and compared host growth, a correlate of barrier dysfunction, with a tryptophan-supplemented diet group. Giardia-infected mice fed a low-protein diet failed to gain weight whereas infected mice fed an isocaloric regular protein diet gained weight similar to diet-matched controls. Tryptophan supplementation of the low-protein diet rescued this diet-dependent and infection-induced weight defect in C57BL/6J and BALB/c mice. This growth rescue was associated with increased intestinal Cyp1a1 expression, a canonical AHR target, but without a reduction in infection burden. Glycine supplementation, used as a non-essential amino acid control that is not expected to generate AHR ligands, did not produce the same effect, suggesting that the growth rescue observed with tryptophan supplementation was not due to general amino acid supplementation. Measurements of circulating tryptophan metabolites in plasma showed limited and inconsistent differences across experimental groups, which may suggest that the growth rescue phenotype may be dependent on local intestinal or mucosal AHR-associated signaling rather than an increase in circulating metabolites. Our findings show that dietary tryptophan availability shapes host growth outcomes during giardiasis. This may also be important as it highlights that host nutrition may be a key contributor to post-infection growth impairment in young children.

microbiology↗

bioRxiv2026

TLR-mediated activation of synovial fibroblasts from osteoarthritis patients promotes chondrocyte dysfunction

Objective: Toll like receptor (TLR) activation by cartilage derived damage-associated molecular patterns contributes to osteoarthritis (OA) pathogenesis, but the role of synovial fibroblasts in this process remains incompletely understood. We investigated the TLR responsiveness of human OA synovial fibroblasts and determined how TLR activation influences fibroblast-mediated regulation of chondrocyte function. Design: Primary synovial fibroblasts isolated from OA patients were characterized for TLR expression and stimulated with agonists targeting TLR1/2 to TLR9. Inflammatory mediator production, matrix metalloproteinase expression, mitochondrial respiration, and transcriptomic responses were assessed. Functional effects on cartilage homeostasis were evaluated using autologous chondrocyte spheroid co-cultures. Results: Approximately 50% of cells within OA synovial membranes were fibroblasts (CD45-CD31-PDPN+). Synovial fibroblasts expressed multiple TLRs at both the transcript and protein levels. Activation of TLR1/2, TLR4, TLR5, and TLR2/6 induced robust expression of IL-6, IL-8, G-CSF, MMP3, and MMP10, with minimal effects on mitochondrial respiratory function. Transcriptomic analysis revealed activation of inflammatory pathways together with enrichment of antigen presentation and protein translation programs. In autologous co-culture, TLR1/2-activated synovial fibroblasts promoted inflammatory and catabolic gene expression, suppressed anabolic gene expression, and impaired chondrocyte spheroid growth. Conclusions: Human OA synovial fibroblasts are highly responsive to TLR activation and acquire a pro-inflammatory, cartilage-degrading phenotype that directly impairs chondrocyte homeostasis. These findings identify synovial fibroblasts as key effectors of innate immune signalling within the OA joint and support targeting shared TLR-mediated pathways in joint-resident cells as a potential disease-modifying therapeutic strategy.

immunology↗

bioRxiv2026

Divergent activation of the RXFP1 relaxin receptor by protein and small molecule agonists

RXFP1 is an unusual G protein-coupled receptor (GPCR) that mediates physiological adaptations to pregnancy and is a therapeutic target due to its benefits in treatment of fibrosis and heart failure. Protein and small-molecule agonists are currently in mid-stage clinical trials. Here, we present three cryo-electron microscopy structures of RXFP1: ligand-free, bound to the native agonist relaxin-2, and bound to the small-molecule drug candidate AZD5462. These structures show that relaxin-2 engages the receptors ectodomain while AZD5462 binds within transmembrane domain. Together with hydrogen-deuterium exchange mass spectrometry, we show that relaxin-2 induces conformational reorganization of the linker domain into a helical secondary structure. In contrast, AZD5462 stabilizes a unique active conformation capable of inducing {beta}-arrestin recruitment. Both mechanisms are distinctive from the "push-pull" mechanism of glycoprotein hormone receptors. Altogether, our findings define divergent activation mechanisms for protein and small-molecule agonists of RXFP1 and provide structural framework for next-generation drug discovery targeting relaxin receptors and their relatives.

biochemistry↗

bioRxiv2026

MeCP2 is Necessary in Cerebellar Purkinje Cells for Precise Network Dynamics During Associative Motor Learning

Loss-of-function variants of MECP2 cause Rett syndrome; however, their impact on cerebellar computations for learning remains poorly understood. Here, we show that Mecp2 deletion specifically in Purkinje cells does not broadly disrupt cerebellar-dependent behaviors but selectively compromises those that require precise timing, coordination, and associative updating in the cerebellar cortex. Mecp2 loss altered the emergence of learning-related Purkinje cell activity in vivo and disrupted their intrinsic and synaptic properties that support adaptive cerebellar output. These findings identify MeCP2 as a critical player in Purkinje cell function to regulate cerebellar learning signals and suggest that Rett syndrome-related motor dysfunction reflects impaired adaptive computation rather than a generalized loss of motor capacity.

neuroscience↗

bioRxiv2026

RNAP-seq: in vitro genome-scale transcription reveals preferential RNA polymerase pausing on Clostridioides difficile antisense DNA

Mechanistic dissection of gene transcription and its regulation in diverse organisms remains a central challenge for molecular biology. In vivo analyses using high-throughput DNA sequencing readouts (e.g., RNA-seq, Term-seq, and NET-seq) have revealed that transcriptional pausing by RNA polymerase (RNAP) underpins the regulation of gene transcription. However, these in vivo methods are difficult to apply in non-model organisms and lack the power of bottom-up biochemical analysis that can deconvolute the confounding effects of the cellular environment. Here we report a method for unbiased transcription of entire genomes in vitro called RNAP-seq, which is capable of defining pause, arrest, and termination sites with single-nucleotide resolution and of defining the regulatory contributions of cis-acting RNA and DNA sequences and dissociable transcription factors with complete control over transcription conditions. Using RNAP-seq to compare transcription by recombinant RNAPs from Clostridioides difficile (Cdf) and Escherichia coli (Eco), we discovered that CdfRNAP exhibits pausing signatures distinct from EcoRNAP. CdfRNAP pauses more frequently and more strongly at T-rich sequences, particularly on antisense regions of genes. We also defined the action of CdfNusG at genome scale, revealing its preferential action in riboswitch control regions. These findings demonstrate lineage-specific pausing features and suggest that genome composition and RNAP co-evolve to shape gene expression. RNAP-seq is broadly adaptable to diverse bacterial species and offers a powerful framework for uncovering fundamental principles of transcription that govern gene expression. HighlightsO_LIRNAP-seq enables unbiased transcription of every base-pair in an organisms genome C_LIO_LIRNAP-seq reveals distinct E. coli (Eco) and C. difficile (Cdf) pause sequences C_LIO_LICdfNusG broadly enhances pausing and termination, notably in riboswitch-encoding DNA C_LIO_LICdfRNAP but not EcoRNAP pauses more on the T-rich antisense strand of Cdf genes C_LI

genomics↗

bioRxiv2026

Analysis of in vivo infection dynamics using bioluminescent Giardia duodenalis

Giardia duodenalis is an intestinal protozoan transmitted through contaminated food and water and is a major cause of diarrheal disease worldwide and has been linked to post-infectious sequelae and growth impairment in children. Quantifying parasite burden in vivo is essential for studying infection dynamics and host responses, yet commonly used methods (stool PCR, antigen detection, and terminal trophozoite counts from segments of the intestine) are limited by intermittent stool shedding, uneven parasite distribution, sampling error, and lack of longitudinal data. We developed a bioluminescent reporter system for the assemblage B strain GS to enable noninvasive tracking of infection in mice without antibiotic pretreatment. Using in vivo bioluminescent imaging (BLI), we observed peak signal in immunocompetent C57BL/6J and BALB/c mice at days 6-8 post-infection, followed by return to baseline by ~day 10, consistent with the self-limiting nature of giardiasis. In immunodeficient mice, radiance increased and persisted over time, demonstrating stable luciferase expression during chronic infection in vivo without continuous antibiotic selection. Whole-animal radiance strongly correlated with direct trophozoite counts from excised intestinal segments (r = 0.915, p < 0.001), validating BLI as a useful alternative for determining parasite burden. Together, these findings establish BLI as a robust platform for longitudinal studies of Giardia infection dynamics and for future applications that may examine host immunity, the microbiota, nutrient-dependent effects, and drug testing.

microbiology↗

bioRxiv2026

Enhancing Patient Lymphocyte Response to Peritoneal Malignancies Using a Personalized Immunocompetent Microfluidic Co-Culture Platform

Harnessing patient immune cells via adoptive cellular therapy is a promising cancer therapeutic strategy. However, major challenges remain for advanced solid malignancies, including difficulty isolating sufficient tumor infiltrating lymphocytes (TILs) and limited targeting of diverse neoantigens in heterogenous tumors. To address this, we have developed a tumor-on-a-chip platform with co-cultured patient-derived tumor cells, autologous peripheral blood mononuclear cells (PBMCs), and lymphoid tissue-derived antigen presenting cells. This approach generates organoid interacting lymphocytes (OILs) with enhanced anti-tumor activity. In peritoneal malignancies, OIL-induced cytotoxicity of patient-matched tumor cells surpasses both TILs and static-expanded PBMCs. We find that this improved performance was linked to increased CD8+ T and NK cells among OILs, and increased effector cytokine polyfunctionality, particularly Granzyme A. Our platform represents a versatile and scalable approach to generate patient-specific therapeutic lymphocytes even when TILs are insufficient, offering a promising avenue to treat diverse solid tumors associated with poor outcomes under current immunotherapies. TeaserA tumor-on-a-chip device primes patient immune cells with tumor recognition for personalized immunotherapy applications.

cancer biology↗

bioRxiv2025

CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders

Transcriptional machinery often operates in the configuration of biomolecular condensates, presenting emerging opportunities to develop therapies targeting transcriptionally addicted malignancies. In this study, we discovered that cyclin-dependent kinase 12 (CDK12), a key regulator of transcription elongation, formed dynamic liquid-like droplets within nuclear speckles, a process enhanced by high CDK12 expression and CDK12 inhibitor treatment. Recognizing this unique property, we rationally designed ZSQ253, a modular cyclin K molecular glue degrader that coupled binding moieties for both CDK12 and DDB1. By promoting phase separation-driven assembly and subsequent proteasomal degradation of the CDK12-cyclin K complex, ZSQ253 interfered with oncogenic transcription and demonstrated potent antitumor capability. Further structure-guided optimization yielded HQY1428, a derivative with a multi-site gluing effect and improved drug activity. Given the frequent genomic co-amplification of CDK12 and ERBB2 across diverse human cancers, we showed that combining HQY1428 with HER2 inhibitors provided synergistic therapeutic benefits. Collectively, our findings establish CDK12 condensation within nuclear speckles as an exploitable vulnerability, and introduce a novel mechanism of action underlying cyclin K molecular glue degraders.

cancer biology↗

bioRxiv2025

Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies

Chimeric antigen receptor-engineered macrophages (CAR-Ms) hold great promise for solid tumor immunotherapy. The intracellular domains (ICDs) of CARs determine the phenotypic output of therapeutic macrophages but remain largely unexplored. Here, we constructed a CAR library containing 131 unique signaling domains derived from native immune receptors and identified 17 ICDs that enhance macrophage phagocytosis, inflammatory responses, or tumor infiltration in vitro and in vivo. We further developed a scalable 3 barcode technology, CARode, to uniquely label and trace ICD variants within large-scale combinatorial CAR library and applied it to single-cell RNA sequencing and single-cell CAR analysis to assess the synergetic effects of ICD combinations on macrophage activation. Our approach uncovered a novel CD40-LY9-FCRL1 chimeric receptor that modulates the tumor microenvironment and improves solid tumor clearance. In conclusion, our findings demonstrate that pooled screening can accelerate the discovery of complex ICD constructs, providing a powerful platform for engineering macrophage-based immunotherapies.

bioengineering↗

bioRxiv2025

Mechanism of nascent chain removal by the Ribosome-associated Quality Control complex

Errors during translation can cause ribosome stalling, leaving incomplete nascent chains attached to large ribosomal subunits1-5. Cells rely on the Ribosome-associated Quality Control (RQC) complex to recognize, process, and remove these partially synthesized proteins to maintain proteostasis. Despite its central role, the mechanisms by which the RQC complex orchestrates nascent chain processing and extraction have remained unclear. Here, we present a cryo-EM structure of the RQC complex from budding yeast, revealing how its core eukaryotic components function in nascent chain removal. We show that the Cdc48 protein extracting enzyme, along with its Ufd1-Npl4 adaptor, is recruited by the Ltn1 E3 ubiquitin ligase to extract ubiquitylated stalled peptides from the 60S ribosome. Additionally, we find that the Rqc1 component bridges the 60S ribosome with ubiquitin and Ltn1, facilitating the specific formation of K48-linked polyubiquitin chains on stalled peptides. These findings provide a structural and mechanistic framework for understanding how components of the RQC complex work together to remove faulty translation products from the ribosome, advancing our understanding of cellular protein quality control.

molecular biology↗

bioRxiv2024

Spatiotemporal Regulatory Logics of Mouse Gastrulation

Spatiotemporal coordination of cellular and molecular events is crucial for cell fate commitment during mouse gastrulation. However, the high-precision mechanisms governing the timing and spatial dynamics remain poorly understood. Here, we present a time-series single-cell multi-omic dataset from the mouse gastrulating embryos and construct a hierarchical gene regulatory landscape. Integrating this with real three-dimensional transcriptomic coordinate, we created ST-MAGIC and ST-MAGIC (+) atlas, dissecting the spatiotemporal logics of regulatory networks and signaling responsiveness underpinning the lineage commitment at gastrulation. Specifically, we delineated the multi-omic basis for left-right symmetry breaking events in the gastrula and also revealed the spatiotemporal molecular relay for axial mesendoderm lineage, where early and intermediate transcription factors first open the chromatin regions and setup the responsiveness to signaling, followed by terminal factors to consolidate the transcriptomic architecture. In summary, our study presents a spatiotemporal regulatory logic framework of mouse gastrulation, that advances our understanding of mammalian embryogenesis.

developmental biology↗

bioRxiv2024

Exploring methanogenic archaea and their thermal responses in the glacier-fed stream sediments of Rongbuk River Basin, Mt. Everest

Glacier-fed streams (GFS) are emergent sources of greenhouse gas methane, and methanogenic archaea in sediments contribute largely to stream methane emissions. However, little is known about the methanogenic communities in GFS sediments and their key environmental driving factors. This study analyzed stream sediments from the Rongbuk River basin on Mount Everest for methanogenic communities and their temperature responses through anaerobic microcosm incubations at 5{degrees}C and 15{degrees}C. Diverse methanogens were identified, including acetoclastic, hydrogenotrophic, and methylotrophic types. Substantial methane and CO2 production were detected across altitudes and increased significantly at 15{degrees}C, with both methane and CO2 production rate negatively correlated with altitude. While temperature sensitivity of CO2 production but not CH4 showed a negative altitude correlation. Methanogens increased substantially over long-term incubation, dominating the archaeal community. At 15{degrees}C, the relative abundance of several methanogenic groups was strongly correlated with altitude, with positive correlations observed for Methanomassiliicoccaceae and Methanoregulaceae, and negative correlations for Methanocellaceae and Methanotrichaceae, respectively. Besides altitude, factors like phosphorus, C-to-N ratio, and pH also affected methanogenic structure, methane and CO2 production, and temperature sensitivities. This study offers new insights into methanogens and methane production in GFS sediments, improving our understanding of GFS carbon cycling and its potential responses to climate change.

ecology↗

bioRxiv2024

Structure of Geobacillus stearothermophilus Cas9: insights into the catalytic process and thermostability of CRISPR-Cas9

CRISPR-Cas9 has been developed as a powerful gene editing tool, but the mechanism governing the intricate catalytic process remains incompletely resolved. Here, the cryo-electron microscopy structures of thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) in complex with sgRNA and target DNA are reported. The structure of GeoCas9 in complex with sgRNA reveals a slit termed L1-crevice comprising HNH, RuvC, and L1 helix as a transient storage site of 5 spacer of sgRNA. When 5 spacer is extracted to pair with the target DNA, L1-crevice collapses to trigger the subsequent HNH domain translocation. In addition, structural and biochemical analyses suggest that the resilience of GeoCas9 at elevated temperature is related to the unique PI domain conformation. These results advance our understanding into the catalytic process of Cas9 and unveil the molecular mechanism that accounts for the superior thermal profile of GeoCas9.

molecular biology↗

bioRxiv2024

The transcripts of a gastrula-premarked enhancer prime posterior tissue development through cross-talk with morphogen effector

The regulatory mechanisms governing cell fate determination, particularly lineage diversification during mammalian embryonic development, remain poorly understood with in-depth regulatory paradigms yet to be fully elucidated. Here, leveraging the epigenetic landscape of mouse gastrula, we identified p-Enh, a pre-marked enhancer in primitive streak region, as pivotal regulator for posterior tissue development in mouse embryos. Morphological and single-cell transcriptomic analyses confirmed embryonic lethality phenotype with disrupted posterior tissue development trajectories in p-Enh-KO embryos. Molecularly, apart from regulating the neighboring coding-gene Cdx2 in cis, our findings suggest that p-Enh also modulate the global transcriptome and epigenomic landscape, which might through the transient production of eRNA in trans. Further investigation revealed p-Enh participate in the regulatory cascades of TGF-{beta} signaling. Chemical modulation of TGF-{beta} signaling can largely rescue the posterior development deficiency in in vitro gastruloids through a Cdx2-independent mechanism. Thus, we propose a potential model in which the broadly distributed p-Enh transcripts within the nucleus could serve as essential cross-modular coordinators, priming the posterior development of mouse embryo.

developmental biology↗

bioRxiv2024

Proteome analysis provides insights into sex differences in Holothuria Scabra

Sex-determining mechanism is still ambiguous for sea cucumber Holothuria scabra which only manifests gonochorism in gonad. In this study, proteomic analysis was employed to delineate sex-related proteins and genes in gonads of H. scabra, subsequently validated through Quantitative real-time polymerase chain reaction (qRT-PCR). A total of 5,313 proteins were identified via proteome sequencing. Among these, 817 proteins exhibited expression in both the ovary and testis, with 445 proteins displaying up-regulation and 372 proteins showing down-regulation. Furthermore, 136 and 69 proteins were identified as ovary-specific and testis-specific Differentially Abundant Proteins (DAPs), respectively. For the validation of 75 DAP coding genes, 9 genes were considered to be reliable. Notably, 25 ovary-bias proteins enriched in ribosome pathway strongly indicated the crucial role of ribosome in ovary. And 5S/18S rRNA ratio in H. Scabra may have potencial to establish a nondestructive method to distinguish sexes unambiguously. This study serves to furnish novel evidence pertaining to sex differences in H. scabra.

molecular biology↗

bioRxiv2023

Antibodies Expand the Scope of Angiotensin Receptor Pharmacology

G protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue, and cellular level. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays, and structural studies, we develop maternally selective heavy chain-only antibody ("nanobody") antagonists against the angiotensin II type I receptor (AT1R) and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to AT1R with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

pharmacology and toxicology↗

bioRxiv2023

A reverse-central-dogma pathway underlies ribosome-antibiotic efficacy and accelerates resistance evolution

In bacteria, the precise coordination of DNA replication, transcription and translation is mediated by dynamic interactions among the corresponding macromolecular machineries, playing a pivotal role in maintaining cellular homeostasis. Here we show that such coordination is hijacked by ribosome antibiotics to trigger secondary damage that predominantly contributes to their efficacy. Specifically, through developing a transcription dynamics profiling method, complemented by genetic and biochemical approaches, we unveil that translation inhibition disrupts transcription-translation coupling, leading to premature stalling of RNA polymerase (RNAP) at genome scale, which subsequently triggers extensive genomic instability. Moreover, mutagenic repairment of genomic lesions is mediated by heterogenous hyperactivation of SOS response, facilitating an inducible evolutionary path towards genetic resistance characterized by a unique mutation spectrum. Our findings thereby reveal a yet overlooked reverse-central-dogma pathway underlying actions of ribosome inhibitors, and establish a framework to understand antibiotic efficacy and induced mutagenesis through network disorder.

microbiology↗

bioRxiv2023

A novel bacterial signal transduction system specifically senses and responds to peptidoglycan damage

The peptidoglycan (PG) layer is a mesh-like structure within the cell envelope essential for maintenance of cell shape and resistance to osmotic stress, and therefore is a primary target of many important and widely used antibiotics, such as {beta}-lactams. In Gram-negative bacteria, while signal transduction systems that monitor the state of the inner- and outer-membranes have been extensively studied and well understood, much less is known about how cells sense and respond to PG-targeting stresses. Here we show that a novel bacterial two-component system (PghKR) from Shewanella oneidensis is capable of sensing and responding to PG damage. This system is specifically activated in cells exposed to various PG-targeting antibiotics or carrying a defect in PG synthesis, resulting in induced expression of blaA and relV, which encode a {beta}-lactamase conferring resistance to {beta}-lactams and a small ppGpp synthetase responsible for antibiotic tolerance, respectively. Intriguingly, the PghKR homologs are widespread among several classes of Proteobacteria and the periplasmic domain of sensor kinase PghK contains a family 9 carbohydrate-binding module that is required for signal perception, implying that the signals could be the glycan fragments of PG. Overall, our results provide critical insights into the regulation of PG homeostasis in Gram-negative bacteria.

microbiology↗