技术资料

Expansion microscopy (ExM) has enabled nanoscale imaging of tissues by physically enlarging biological samples in a swellable hydrogel. However,the increased sample size and water-based environment pose challenges for deep imaging using conventional inverted confocal microscopes,particularly due to the limited working distance of high-numerical-aperture (NA) water immersion objectives. Here,we introduce a practical imaging alternative that utilizes an inverted water-dipping objective and a refractive-index-matched optical path using fluorinated ethylene propylene (FEP) film. Through point spread function (PSF) measurements and simulations,we show that the FEP film introduces predominantly defocus-like wavefront profiles characteristic of high NA systems,which result in an easily correctable axial shift of the focal plane. To ensure stable immersion and refractive index continuity,we use an arrangement relying on an FEP film,Immersol W,water and a FEP-based imaging dish. This configuration achieves sub-micron lateral and axial resolution,supports large tile-scan acquisitions,and maintains image quality across depths exceeding 800 µm. We validate the system by imaging 4×-expanded U2OS cells and human cerebral organoids. Our approach provides a low-cost,plug-and-play solution for high-resolution volumetric imaging of expanded samples using standard inverted microscopes. View Publication

Baculovirus,an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems,is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering,we developed SHOT 2.0,an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56,with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow,enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform,we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types,including iPSCs and liver cancer cells,with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox,providing a flexible platform for genome editing and future gene delivery. View Publication

Understanding how chemical stress perturbs human lung physiology requires models that capture dynamic molecular responses in real time. Here,we established a CRISPR/Cas9-engineered human induced pluripotent stem cell (hiPSC)-derived lung organoid expressing endogenous G3BP1–mCherry,enabling live,non-destructive visualization of stress granule (SG) formation under toxicant exposure. The organoids recapitulated airway and alveolar epithelial diversity and displayed lamellar body-like ultrastructures,indicating advanced maturation. Time-lapse imaging revealed rapid and reversible SG dynamics across chemically distinct stressors,while cytotoxicity assays showed that these organoids are significantly more sensitive than conventional 2D or cancer-derived lung models. Importantly,SG dynamics were linked to exposure duration–dependent changes in epithelial barrier integrity,indicating that SG formation precedes overt epithelial injury and serves as an early indicator of toxicant-induced cellular stress. Integration with high-content screening enabled quantitative,image-based analysis of cellular stress phenotypes,greatly enhancing throughput and mechanistic insight,thereby provided next-generation New Approach Methodologies for lung toxicity assessment. Together,this hiPSC-derived lung organoid SG reporter platform links early molecular stress adaptation to tissue-level responses,offering a predictive and mechanistically informative framework for human-relevant lung toxicity evaluation. View Publication

Background: Lenalidomide is an immunomodulatory drug approved in the treatment of autoimmune disease,inflammation,and cancer. Its impact continues to grow due to its diverse spectrum of effects hampered only by toxicities and reduced efficacy. Therefore,development of strategies that enhance function while reducing drawbacks remains a prime goal. Objective and Hypothesis: The mechanisms of action of lenalidomide on the activity of natural killer cells (NK cells) remains understudied yet could be critical for the development of strategies to enhance its efficacy. These cells are critical drivers of anti-tumor immune responses which are often functionally suppressed in malignancies. NK cell and T cell survival and function is driven by the IL-2 family of cytokines (IL-2 or IL-15) and work has shown that lenalidomide potentially works by increasing the secretion of IL-2 by other lymphocytes,such as CD4+ T helper cells. Thus,we hypothesized that improving NK activity with IL-2 family of cytokines could lead to enhanced lenalidomide-induced responses of these cells. Results: We show that lenalidomide does not affect NK cell viability but reduces their proliferation through cell cycle arrest which could be overcome by exogenous addition of IL-2 family of cytokines. Moreover,lenalidomide induced the secretion of IL-2 on isolated NK cells although it also modulated NK receptor expression,such as NKp46,trough downregulation of PI3K/AKT pathway reduction. This was overcome by exogeneous addition of IL-2 family of cytokines increasing natural cytotoxicity,through higher perforin and granzyme expression. Mechanistically,this increased gene and protein expression occurred through the activation of STAT5 by lenalidomide which was also enhanced through the exogenous addition of IL-2 family of cytokines and modulation of IL-2R subunit changes. Conclusions: These data provide a rationale for the combination of lenalidomide with IL-2 family of cytokines to enhance the effectiveness of NK cells. View Publication

Drug resistance and relapse remain key challenges in pancreatic cancer. Here,we have used RNA sequencing (RNA-seq),chromatin immunoprecipitation (ChIP)-seq,and genome-wide CRISPR analysis to map the molecular dependencies of pancreatic cancer stem cells,highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular,the nuclear hormone receptor retinoic-acid-receptor-related orphan receptor gamma (ROR$\gamma$),known to drive inflammation and T cell differentiation,was upregulated during pancreatic cancer progression,and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth and a marked improvement in survival. Further,a large-scale retrospective analysis in patients revealed that ROR$\gamma$ expression may predict pancreatic cancer aggressiveness,as it positively correlated with advanced disease and metastasis. Collectively,these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells,suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients. View Publication

Periodontitis is an irreversible,bacteria-induced,chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria,neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However,periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation,which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod,Filifactor alocis. With the discovery of these oral bacterial species,there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently,few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus,this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis,cytokine and chemokine signaling pathways,and apoptosis. Moreover,F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient,dampened p38 MAPK activation by secondary stimuli TNF$\alpha$ but not by fMLF. Functionally,the F. alocis-mediated inhibition of p38 activation by TNF$\alpha$ resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNF$\alpha$. Since the modulatory effect was characteristic of viable F. alocis only,we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses. View Publication

Integrating nutrient sensing with the synthesis of complex molecules is a central feature of metabolism. Yet the regulatory mechanisms underlying such integration are often unknown. Here,we establish that the transcription regulators Rtg1/3 are key determinants of sphingolipid homeostasis in the human fungal pathogen Candida albicans. Quantitative analysis of the C. albicans lipidome reveals Rtg1/3-dependent alterations in all complex sphingolipids and their precursors,ceramides. Mutations in the regulators render the fungus susceptible to myriocin,a sphingolipid synthesis inhibitor. Rtg1/3 exert control on the expression of several enzymes involved in the synthesis of sphingolipids' building blocks,and the regulators are activated upon engulfment of C. albicans cells by human neutrophils. We demonstrate that Rtg1p and Rtg3p are regulated at two levels,one in response to sphingolipids and the other by the nutrient sensor TOR. Our findings,therefore,indicate that the Rtg1/3 system integrates nutrient sensing into the synthesis of complex lipids. View Publication

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation,here,we compared,in multiple cell and in vivo models,mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1m$\Psi$),synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1m$\Psi$ made by the modified process,while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA. View Publication

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis,regeneration and cancer,little is known about the post-translational regulation of LGR5. Here,we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation,increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically,we find that both NEDD4 and NEDD4L negatively regulate Wnt/$\beta$-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers,which subsequently enhances tumour predisposition and progression. View Publication

Autoantibodies to transglutaminase 2 (TG2) are hallmarks of celiac disease. To address B cell tolerance and autoantibody formation to TG2,we generated immunoglobulin knock-in (Ig KI) mice that express a prototypical celiac patient-derived anti-TG2 B cell receptor equally reactive to human and mouse TG2. We studied B cell development in the presence/absence of autoantigen by crossing the Ig KI mice to Tgm2-/- mice. Autoreactive B cells in Tgm2+/+ mice were indistinguishable from their naive counterparts in Tgm2-/- mice with no signs of clonal deletion,receptor editing,or B cell anergy. The autoreactive B cells appeared ignorant to their antigen,and they produced autoantibodies when provided T cell help. The findings lend credence to a model of celiac disease where gluten-reactive T cells provide help to autoreactive TG2-specific B cells by involvement of gluten-TG2 complexes,and they outline a general mechanism of autoimmunity with autoantibodies being produced by ignorant B cells on provision of T cell help. View Publication

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically,it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose,a large set of proteins representing diverse structures and functions,some of which are known or potential drug targets,were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86{\%} of the targets,as determined by luminescence-based plate assays,blotting,and imaging. In order to determine whether endogenously tagged proteins yield more representative models,cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases,only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches. View Publication

This study traced intravenously administered induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSC) and assessed the impact of iPSC-MSC on preserving renal function in SD rat after 5/6 nephrectomy. The results of in vitro study showed that FeraTrack™Direct contrast particles (ie intracellular magnetic labelling) in the iPSC-MSC (ie iPS-MSCSPIONs ) were clearly identified by Prussian blue stain. Adult-male SD rats (n = 40) were categorized into group 1 (SC),group 2 [SC + iPS-MSCSPIONs (1.0 × 106 cells)/intravenous administration post-day-14 CKD procedure],group 3 (CKD),group 4 [CKD + iPS-MSCSPIONs (0.5 × 106 cells)] and group 5 [CKD + iPS-MSCSPIONs (1.0 × 106 cells)]. By day-15 after CKD induction,abdominal MRI demonstrated that iPS-MSCSPIONs were only in the CKD parenchyma of groups 4 and 5. By day 60,the creatinine level/ratio of urine protein to urine creatinine/kidney injury score (by haematoxylin and eosin stain)/fibrotic area (Masson's trichrome stain)/IF microscopic finding of kidney injury molecule-1 expression was lowest in groups 1 and 2,highest in group 3,and significantly higher in group 4 than in group 5,whereas IF microscopic findings of podocyte components (ZO-1/synaptopodin) and protein levels of anti-apoptosis ((Bad/Bcl-xL/Bcl-2) exhibited an opposite pattern to creatinine level among the five groups (all P {\textless} .0001). The protein expressions of cell-proliferation signals (PI3K/p-Akt/m-TOR,p-ERK1/2,FOXO1/GSK3$\beta$/p90RSK),apoptotic/DNA-damage (Bax/caspases8-10/cytosolic-mitochondria) and inflammatory (TNF-$\alpha$/TNFR1/TRAF2/NF-$\kappa$B) biomarkers displayed an identical pattern to creatinine level among the five groups (all P {\textless} .0001). The iPS-MSCSPIONs that were identified only in CKD parenchyma effectively protected the kidney against CKD injury. View Publication