技术资料

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

Efficient generation of antibodies by B cells is one of the prerequisites of protective immunity. B cell activation by cognate antigens via B cell receptors (BCRs),or pathogen-associated molecules through pattern-recognition receptors,such as Toll-like receptors (TLRs),leads to transcriptional and metabolic changes that ultimately transform B cells into antibody-producing plasma cells or memory cells. BCR signaling and a number of steps downstream of it rely on coordinated action of cellular membranes and the actin cytoskeleton,tightly controlled by concerted action of multiple regulatory proteins,some of them exclusive to B cells. Here,we dissect the role of Missing-In-Metastasis (MIM),or Metastasis suppressor 1 (MTSS1),a cancer-associated membrane and actin cytoskeleton regulating protein,in B cell-mediated immunity by taking advantage of MIM knockout mouse strain. We show undisturbed B cell development and largely normal composition of B cell compartments in the periphery. Interestingly,we found that MIM-/- B cells are defected in BCR signaling in response to surface-bound antigens but,on the other hand,show increased metabolic activity after stimulation with LPS or CpG. In vivo,MIM knockout animals exhibit impaired IgM antibody responses to immunization with T cell-independent antigen. This study provides the first comprehensive characterization of MIM in B cells,demonstrates its regulatory role for B cell-mediated immunity,as well as proposes new functions for MIM in tuning receptor signaling and cellular metabolism,processes,which may also contribute to the poorly understood functions of MIM in cancer. View Publication

Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells,macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR),while decreasing at higher doses over a broad physiological range,AID and Blimp1 expression,CSR,somatic hypermutation and plasma cell differentiation. In human and mouse B cells,butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3'UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors,not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes,these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models. View Publication

Microelectrode material and cell culture medium have significant roles in the signal-to-noise ratio and cell well-being in in vitro electrophysiological studies. Here,we report an ion beam assisted e-beam deposition (IBAD) based process as an alternative titanium nitride (TiN) deposition method for sputtering in the fabrication of state-of-the-art TiN microelectrode arrays (MEAs). The effects of evaporation and nitrogen flow rates were evaluated while developing the IBAD TiN deposition process. Moreover,the produced IBAD TiN microelectrodes were characterized by impedance,charge transfer capacity (CTC) and noise measurements for electrical properties,AFM and SEM for topological imaging,and EDS for material composition. The impedance (at 1 kHz) of brand new 30 $\mu$m IBAD TiN microelectrodes was found to be double but still below 100 k$\Omega$ compared with commercial reference MEAs with sputtered TiN microelectrodes of the same size. On the contrary,the noise level of IBAD TiN MEAs was lower compared with that of commercial sputtered TiN MEAs in equal conditions. In CTC IBAD TiN electrodes (3.3 mC/cm2) also outperformed the sputtered counterparts (2.0 mC/cm2). To verify the suitability of IBAD TiN microelectrodes for cell measurements,human pluripotent stem cell (hPSC)-derived neuronal networks were cultured on IBAD TiN MEAs and commercial sputtered TiN MEAs in two different media: neural differentiation medium (NDM) and BrainPhys (BPH). The effect of cell culture media to hPSC derived neuronal networks was evaluated to gain more stable and more active networks. Higher spontaneous activity levels were measured from the neuronal networks cultured in BPH compared with those in NDM in both MEA types. However,BPH caused more problems in cell survival in long-term cultures by inducing neuronal network retraction and clump formation after 1-2 weeks. In addition,BPH was found to corrode the Si3N4 insulator layer more than NDM medium. The developed IBAD TiN process gives MEA manufacturers more choices to choose which method to use to deposit TiN electrodes and the medium evaluation results remind that not only electrode material but also insulator layer and cell culturing medium have crucial role in successful long term MEA measurements. View Publication

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However,whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis,we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic,autochthonous tumorigenesis. Mechanistically,single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program,driven by the Hippo pathway effector Yap. In vivo,Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments,we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation,nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice,thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis. View Publication

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections,this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies,permeable membrane based monolayer approaches are needed. In this paper,we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures,which were characterized with respect to epithelial cell types,polarization,barrier function,and gene expression. In addition,viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated,with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research. View Publication

We have designed and validated a set of robust and non-toxic protocols for directly evaluating the properties of engineered neural tissue. These protocols characterize the mechanical properties of engineered neural tissues and measure their electrophysical activity. The protocols obtain elastic moduli of very soft fibrin hydrogel scaffolds and voltage readings from motor neuron cultures. Neurons require soft substrates to differentiate and mature,however measuring the elastic moduli of soft substrates remains difficult to accurately measure using standard protocols such as atomic force microscopy or shear rheology. Here we validate a direct method for acquiring elastic modulus of fibrin using a modified Hertz model for thin films. In this method,spherical indenters are positioned on top of the fibrin samples,generating an indentation depth that is then correlated with elastic modulus. Neurons function by transmitting electrical signals to one another and being able to assess the development of electrical signaling serves is an important verification step when engineering neural tissues. We then validated a protocol wherein the electrical activity of motor neural cultures is measured directly by a voltage sensitive dye and a microplate reader without causing damage to the cells. These protocols provide a non-destructive method for characterizing the mechanical and electrical properties of living spinal cord tissues using novel biosensing methods. View Publication

CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity,the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here,we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore,we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system. View Publication

The enteric pathogen Lawsonia intracellularis is one of the main causes of diarrhea and compromised weight gain in pigs worldwide. Traditional cell-line cultures have been used to study L. intracellularis pathogenesis. However,these systems fail to reproduce the epithelial changes observed in the intestines of L. intracellularis-infected pigs,specifically,the changes in intestinal cell constitution and gene expression. A more physiologically accurate and state-of-the-art model is provided by swine enteroids derived from stem cell-containing crypts from healthy pigs. The objective of this study was to verify the feasibility of two-dimensional swine enteroids as in vitro models for L. intracellularis infection. We established both three- and two-dimensional swine enteroid cultures derived from intestinal crypts. The two-dimensional swine enteroids were infected by L. intracellularis in four independent experiments. Enteroid-infected samples were collected 3 and 7 d postinfection for analysis using real-time quantitative PCR and L. intracellularis immunohistochemistry. In this study,we show that L. intracellularis is capable of infecting and replicating intracellularly in two-dimensional swine enteroids derived from ileum. View Publication

Hepatocellular carcinoma (HCC) is among the most common causes of cancer death in men. Whether or not a longitudinal follow-up of circulating tumor cells (CTCs) before and at different time points during systemic/targeted therapy is useful for monitoring the treatment response of patients with locally advanced or metastatic HCC has been evaluated in this study. Blood samples (n = 104) were obtained from patients with locally advanced or metastatic HCC (n = 30) for the enrichment of CTCs by a negative selection method. Analysis of the blood samples from patients with defined disease status (n = 81) revealed that those with progressive disease (PD,n = 37) had significantly higher CTC counts compared to those with a partial response (PR) or stable disease (SD; n = 44 for PR + SD,p = 0.0002). The median CTC count for patients with PD and for patients with PR and SD was 50 (interquartile range 21-139) and 15 (interquartile range 4-41) cells/mL of blood,respectively. A longitudinal analysis of patients (n = 17) after a series of blood collections demonstrated that a change in the CTC count correlated with the patient treatment response in most of the cases and was particularly useful for monitoring patients without elevated serum alpha-fetoprotein (AFP) levels. Sequential CTC enumeration during treatment can supplement standard medical tests and benefit the management of patients with locally advanced or metastatic HCC,in particular for the AFP-low cases. View Publication

Hepatocyte death during acetaminophen (APAP) intoxication elicits a reactive inflammatory response,with hepatic recruitment of neutrophils and monocytes,which further aggravates liver injury. Neutrophil elastase (NE),secreted by activated neutrophils,carries degradative and cytotoxic functions and maintains a proinflammatory state. We investigated NE as a therapeutic target in acetaminophen-induced liver injury (AILI). C57BL/6 mice were administered a toxic dose of APAP,2 h prior to receiving the NE inhibitor sivelestat,N-acetylcysteine (NAC),or a combination therapy,and were euthanized after 24 and 48 h. Upon APAP overdose,neutrophils and monocytes infiltrate the injured liver,accompanied by increased levels of NE. Combination therapy of NAC and sivelestat significantly limits liver damage,as evidenced by lower serum transaminase levels and less hepatic necrosis compared to mice that received APAP only,and this to a greater extent than NAC monotherapy. Lower hepatic expression of proinflammatory markers was observed in the combination treatment group,and flow cytometry revealed significantly less monocyte influx in livers from mice treated with the combination therapy,compared to untreated mice and mice treated with NAC only. The potential of NE to induce leukocyte migration was confirmed in vitro. Importantly,sivelestat did not impair hepatic repair. In conclusion,combination of NE inhibition with sivelestat and NAC dampens the inflammatory response and reduces liver damage following APAP overdose. This strategy exceeds the standard of care and might represent a novel therapeutic option for AILI. View Publication

PURPOSE To assess the potential for CUE-101,a novel therapeutic fusion protein,to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors,including head and neck squamous cell carcinoma (HNSCC),cervical,and anal cancers. EXPERIMENTAL DESIGN CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex,an HPV16 E7 peptide epitope,reduced affinity human IL2 molecules,and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101,whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS CUE-101 demonstrates selective binding,activation,and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice,and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS Consistent with its design,CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells,a favorable safety profile,and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689). View Publication