Search Results (74)

Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology. Full article

The escalating global burden of Ulcerative Colitis (UC) underscores the urgent need for novel therapeutic strategies. Although dietary modulation is known to influence UC progression, the specific molecular mediators remain largely undefined. Recently, the G protein coupled receptor 35 (GPR35) has emerged as a promising target for maintaining gut homeostasis and promoting intestinal epithelium repair. Yet, whether the therapeutic benefits of dietary polyphenols are mediated through the direct activation of GPR35 remains unexplored. Here, the NanoLuc Binary Technology (NanoBiT) assay was first used to identify the potential GPR35 agonist from a library of 30 natural polyphenolic compounds. We discovered Ellagic acid (EA), a natural polyphenol abundant in fruits and nuts, as the potent GPR35 agonist owing to its most potent agonistic effect. The dose-dependent effect was further confirmed by both NanoBiT and Bret assay. Then, the binding site of the ligand-receptor complex was predicted via molecular docking, and key interactions were validated by site-directed mutagenesis. The results indicated the key binding site of the complex was Gln93, Arg100, Arg151, Phe163 and Ser262. And the conformation of the complex was verified stable by the molecular dynamics simulation. The bioactivity of EA was then evaluated in vivo. And the in vivo experiment indicated that EA alleviated the symptoms of UC. In addition, complementary in vitro assays, including a wound healing (scratch) assay and an SRB proliferation assay, were employed to investigate its effect on intestinal epithelial repair. The in vitro experiment demonstrated that EA enhanced the migration and proliferation of human colonic epithelial cells, an effect that was specifically abolished by the GPR35 antagonist CID2745687, indicating the key role GPR35 played in the intestinal repair. Collectively, our study demonstrates that the natural polyphenolic compound EA promotes epithelial healing and ameliorates colitis by acting as a GPR35 agonist. Full article

Respiratory syncytial virus (RSV) poses a significant global health challenge, particularly affecting infants, the elderly, and immunocompromised individuals. Despite recent progress in the development of vaccines and monoclonal antibodies, effective antiviral therapies remain limited. To advance the discovery of antiviral drugs, we have developed a dual-reporter RSV minigenome system, providing a safe and robust platform for antiviral evaluation. This system incorporates NanoLuc luciferase and superfolder GFP (sfGFP) linked by a self-cleaving P2A peptide, allowing for the simultaneous detection of orthogonal signals. Validation with L polymerase inhibitors confirmed the system’s reliability for screening small-molecule inhibitors. The linear correlation observed between the reporter signals enhances the assay’s reliability for antiviral assessment. This dual-reporter minigenome system advances targeted therapeutic strategies against RSV. Full article

Hepatitis B virus (HBV) remains a major global health threat because covalently closed circular DNA (cccDNA) persists in hepatocytes and limits the efficacy of current antiviral therapies. Effective HBV research and drug screening require culture models that recapitulate the complete viral life cycle and allow for quantitative monitoring of replication. In this study, an 11-amino acid luminescent reporter, HiBiT, was inserted at multiple sites within the preS1 region of a genotype D HBV genome, and the C terminus of preS1 was identified as optimal for maintaining robust replication. We then established HepG2-B4 cells stably replicating HiBiT-HBV with HiBiT at the preS1 C terminus. Extracellular HiBiT activity and supernatant levels of HBV-DNA, HBsAg, and HBcAg increased continuously until day 42 and were reduced by nucleos(t)ide analog treatment, and cccDNA was confirmed by Southern blot analysis. Supernatants from HepG2-B4 cells infected naïve HepG2-NTCP cells and primary human hepatocytes, as shown by extracellular HiBiT activity. Transcriptome analysis revealed distinct gene expression changes in HepG2-B4 cells compared with parental HepG2 cells. These findings indicate that the HepG2-B4 system provides a rapid, quantitative, and scalable platform for HBV replication and infection studies and is suitable for mechanistic investigations and high-throughput antiviral screening. Full article

Carbon dots have emerged as promising nanocarriers for drug delivery due to their unique physicochemical properties and biocompatibilities. Here, the potential of leaf-derived carbon dots (named as SBL_CD), derived from Seabuckthorn (Hippophae rhamnoides L.), was illustrated as a novel nano-formulation for bioactive compound delivery. Seabuckthorn leaves, rich in flavonoids, are the waste product during the production of Seabuckthorn fruits. The wasted leaves were utilized to synthesize carbon dots via a hydrothermal method. The resulting SBL_CD, characterized by TEM, FT-IR and Raman spectroscopy, exhibited a diameter of ~5 nm in both amorphous and quasi-crystalline forms. Applications of SBL_CD in cultures demonstrated robust properties of anti-inflammation and inducing neuronal cell differentiation. Furthermore, SBL_CD was able to encapsulate luteolin, a bioactive flavonoid. The enhanced delivery efficiency translated to superior biological activity, with SBL_CD-luteolin requiring only 1.50 μg/mL in achieving the EC₅₀ efficacy, as compared to 6.82 μg/mL for free luteolin in pNF200-Luc expression assays. This approach not only valorizes Seabuckthorn leaf by-products but also potentially improves the efficacy of encapsulated flavonoids. The development of SBL_CD as a multifunctional platform for flavonoid delivery represents a promising strategy in enhancing the efficacy of neuroactive compounds, combining anti-inflammatory effects (>70% cytokine suppression) with enhanced cellular uptake (4.5-fold increase). Full article

Neutralizing antibodies (nAbs) are key indicators of protection against SARS-CoV-2, and their measurement remains essential for monitoring vaccine responses and population immunity. While the plaque reduction neutralization test (PRNT) is the gold standard, it relies on replicative viruses and is not suited for high-throughput applications. Here, both an in-house and a commercial pseudovirus-based neutralization (PBN) assay were standardized and compared with PRNT to assess performance and concordance. The in-house PBN employed a VSV-ΔG pseudovirus encoding NanoLuc and displaying the SARS-CoV-2 Spike from the Wuhan or Omicron BA.1 variants in HEK293T-hACE2 cells, whereas the commercial assay (Integral Molecular, Philadelphia, PA, USA) used a lentiviral backbone with Renilla or GFP reporters and Wuhan or Omicron XBB.1.5/XBB.1.9 Spikes in Vero E6-ACE2-TMPRSS2 cells. Both assays showed strong correlations with PRNT, the commercial assay; moreover, they offered superior reproducibility and scalability, while the in-house version provided a cost-effective alternative suitable for BSL-2 settings. A total of 600 serum samples from vaccinated individuals were analyzed by commercial PBN at collection time points, from pre-vaccination to twelve months post–second dose, enabling large-scale screening, revealing marked differences in neutralization between Wuhan and Omicron XBB.1.5/1.9, and allowing unbiased classification of low, medium, and high responders using k-means clustering. The geometric mean titers (log₁₀ GMT) highlighted a ~1.5 log₁₀ (eightfold) reduction in neutralizing activity against Omicron, reflecting antibody waning and antigenic drift. Altogether, this study integrates assay standardization, PRNT comparison, and large-scale immune profiling, establishing a robust framework for harmonized pseudovirus-based neutralization testing. Full article

Bluetongue virus (BTV) is the causative agent of the significant livestock disease Bluetongue (BT), which causes severe economic losses associated with its considerable impact on the health and trade of ruminants. Background/Objectives: BTV infection and vaccination against the virus typically result in the induction of antibodies with the capacity to neutralize viral infection. Classic neutralization approaches resemble the methodology applied for neutralizing antibodies (NAbs) quantification. To improve long-standing and new-generation methodologies for the quantification of NAbs or evaluation of antivirals, we offer here the development of a new luciferase-based microneutralization approach as a proof-of-concept. Methods: Central to this innovative approach is the recently generated set of replication-competent reporter-expressing recombinant BTV, where the NanoLuc luciferase protein expression serves as a quantifiable readout for viral replication. After evaluating a set of heat-inactivated serum samples with neutralizing activity (measured via SNTs), these were incubated with 100 PFU of NLuc-expressing rBTV of serotype 1, 4 or 8 and Vero cells were infected with the serum–virus mixture. Then, the luminescent signal was measured at 48 h post-infection. Results: Using the proposed NLuc-based assay and the luminescent signal in the supernatant, we could detect neutralizing activity as soon as 48 h post-infection. Importantly, we were able to observe a strong correlation between NAbs titers measured by classic microneutralization assay and by our bioluminescent approach (BTV-1 Spearman r = 0.932901; p-value < 0.0001; BTV-4 Spearman r = 0.8070192; p-value < 0.0001; BTV-8 Spearman r = 0.9983; p-value < 0.0001). In addition, the NLuc-based assay displayed a serotype-specific character potentially equivalent to classic SNT methods. Conclusions: In summary, our reporter-based microneutralization assay provides a rapid and suitable method to quantify BTV-neutralizing antibodies in serum samples of natural hosts after vaccination or infection, with a serotype-specificity equivalent to classic SNT methods. Full article

Given their role in viral polyprotein processing, viral proteases (PRs) are excellent targets for antiviral therapy. Most assays developed for screening PR inhibitors are in vitro assays, and therefore have several limitations, including the inability to account for cell permeability, toxicity and the need for compounds activation within cells. The development of cellular reporters overcoming these limitations is therefore highly desirable. In this study, we developed two different Bioluminescence Resonance Energy Transfer (BRET)-based reporters for Human Immunodeficiency virus-1 (HIV-1) PR, allowing the simultaneous monitoring of cell viability and HIV-1 PR activity. The reporters employ two different BRET pairs as donor and acceptor moieties: Renilla luciferase (RLuc) with Yellow Fluorescent Protein (YFP), and Nano luciferase (NLuc) with mNeonGreen (mNG), both linked by the HIV-1 p2/p7 cleavage site. While both reporters specifically detected HIV-1 protease activity, mNG-p2/p7-NLuc exhibited higher sensitivity, increased energy transfer and better spectral separation between donor and acceptor emissions, resulting in a significantly higher BRET ratio. mNG-p2/p7-NLuc was used to quantify the effect of a panel of protease inhibitors in living cells, assessing simultaneously cell viability and HIV-1 PR activity. Additionally, it was employed to measure the potency of well-known HIV-1 PR inhibitors. Together, these findings demonstrate the utility of the mNG-p2/p7-NLuc reporter as a cell-based tool for the evaluation of HIV-1 PR activity and inhibitor efficacy. Its dual-readout capability provides a valuable platform for antiviral drug screening in physiologically relevant conditions. Full article

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by limited treatment options and poor prognosis. Although immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown clinical promise, many TNBC patients exhibit resistance or limited response, underscoring the need to understand regulatory mechanisms of PD-L1 expression. Here, we performed a kinome-wide inhibitor screen using a HEK293A cell line stably expressing a NanoLuc-tagged PD-L1 construct lacking its endogenous promoter, to identify post-translational regulators of PD-L1 stability. We identified focal adhesion kinase (FAK) as a novel modulator of PD-L1. FAK inhibition significantly decreased PD-L1 levels in HEK293A cells but paradoxically increased PD-L1 expression in TNBC cell lines. Mechanistically, FAK directly interacts with PD-L1 to modulate its stability independently of its kinase activity. Functionally, FAK inhibition enhanced membrane PD-L1 expression and reduced T-cell-mediated cancer cell killing, suggesting increased immune evasion. These findings reveal a novel role for FAK in immune modulation and suggest that combining FAK inhibitors with PD-L1 blockade may offer a promising strategy for TNBC treatment. Full article

Reporter viruses are valuable tools for studying infections at the cellular level and in living animals. They also enable rapid, high-throughput antiviral drug screening and serological studies. We previously developed a bioluminescence-based reporter virus, rTN09-PA-Nluc, derived from influenza A/Tennessee/1-560/2009 (TN09, pH1N1) in which a NanoLuc (Nluc) reporter protein was fused to the PA protein. Reduced growth of rTN09-PA-Nluc in MDCK cells and mice was restored by mutations arising from mouse adaptation. Here, to test the hypothesis that the growth defect resulted from the PA-Nluc protein fusion, we generated the luciferase reporter virus rTN09-PA-Nluc/SG, which undergoes StopGo translation to yield separate PA and NLuc proteins along with a proportion of the PA-Nluc fusion. The rTN09-PA-Nluc/SG virus had greater protein expression and increased replication in MDCK cells compared to rTN09-PA-Nluc. The reporter virus encoding StopGo translation was superior to the virus without it in bioluminescence-based virus neutralization assays in vitro, providing results in 24 h as opposed to 3 days using unmodified influenza virus and standard neutralization assay protocols. However, the reporter virus encoding StopGo translation remained attenuated in mice. Mouse-adaptive mutations were needed for full virulence and efficient non-invasive imaging in mice. Overall, these findings demonstrate the benefit of incorporating StopGo translation into influenza reporter viruses for in vitro assays, yet mouse-adapted mutations appeared superior in mice. Full article

To facilitate intracellular growth and replication, the virulent human malaria parasite P. falciparum remodels its host erythrocyte by exporting many proteins into the host cell cytosol. Along with a few other exported proteins, the parasite CLAG3 protein is then inserted in the host erythrocyte membrane, exposing a small variant loop to host plasma and contributing to essential nutrient acquisition via the plasmodial surface anion channel (PSAC). To explore trafficking mechanisms and develop therapies that block host cell remodeling, we have now used a split NanoLuc reporter and performed a high-throughput screen for inhibitors of parasite CLAG3 trafficking and insertion at the host membrane. We screened ~52,000 small molecules and uncovered 65 chemically diverse hits. Hits that inhibit the NanoLuc reporter without blocking protein export were filtered out by a secondary screen whose signal does not depend on protein export. Because chemicals that interfere with parasite maturation were found to compromise CLAG3 export indirectly, a third screen using a NanoLuc reporter-tagged intracellular protein was used to evaluate nonspecific toxicity. Although our relatively small chemical screen did not identify bona fide inhibitors of CLAG3 host membrane insertion, these studies establish a framework for larger screens to identify novel export inhibitors. Such novel inhibitors will provide important insights into how Plasmodia remodel their host cells and may seed the development of therapies that block the export and membrane insertion of proteins needed for intracellular parasite survival. Full article

Background: Influenza A virus (IAV) is the major pathogen responsible for influenza pandemics and can cause seasonal influenza outbreaks. In general, viral infection of host cells increases reactive oxygen species (ROS) levels, a process that triggers cell death, lung injury (LI), and other damage mechanisms. Methods: In our previous study, we revealed that selenoproteins may inhibit IAV replication at the cellular level. In this study, we determined the effect of selenoprotein M (SelM) on Nanoluc-IAV-PR8 replication through Nanoluc analysis. The mechanism through which selenoprotein inhibits the replication of the influenza virus was investigated using the SelM knockout cell line, nano-luciferase reporter assays, RNAi, qPCR, Western blot, and confocal microscopy. Results: Our experimental results show that SelM can effectively inhibit the replication of influenza A viruses and could potentially be used as a broad-spectrum inhibitor for IAV therapy in future clinical treatments. The increase in ROS levels induced by IAV infection was found to be inhibited by SelM, which possesses an important Sec functional site, thus weakening the ability of IAV to replicate in cells. Conclusions: The results of this study highlight SelM as a selenoprotein that can effectively inhibit IAV replication. Full article

Flavonoids, like Hesperetin, have been shown to be an ACE2 receptor agonists with antioxidant and pro-apoptotic activity and can induce apoptosis in cancer cells. ACE2 receptors are abundant in lung cancer cells. Here, we explored the application of Hesperetin bound to PegPLGA-coated nanoparticles (Hesperetin nanoparticles, HNPs) and anti-CD40 antibody as an aerosol treatment for lung tumor-bearing mice. The Hesperetin nanoparticles (HNPs) were engineered using a nano-formulation microfluidic technique and polymeric nanoparticles. The in vitro studies were performed in human A549 (ATCC) and murine LL/2-Luc2 (ATCC) lung cancer cell lines. A syngeneic orthotopic murine model of lung cancer was generated in wild (+/+) C57/BL6 background mice with luciferase-positive cell line LL/2-Luc2 cells. Lung tumor-bearing mice were treated via aerosol inhalation with HNP, anti-CD40 antibody, or both. Survival was used to analyze the efficacy of the aerosol treatment. The cohorts were also analyzed for body condition score, weight, and liver and kidney function. Analysis of an orthotopic murine lung cancer model demonstrated a differential uptake of the HNPs and anti-CD40 by the cancer cells. A higher survival rate was observed when the combination of aerosol treatment with HNPs was added with the treatment with anti-CD40 (p < 0.001), as compared to anti-CD40 alone (p < 0.01). Moreover, two tumor-bearing mice survived long-term with the combination treatment, and their tumors were diminished. Subsequently, these two mice were shown to be refractory to the development of subcutaneous tumors, indicating systemic resilience to developing new tumors. Using an inhalation-based administration, we successfully established a treatment model of increased therapeutic efficacy with HNPs and anti-CD40 in an orthotopic murine lung cancer model. Our findings open the possibility of improved lung cancer treatment using nanoparticles like flavonoids and immunoadjuvants. Full article

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic. Full article

Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin–luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies. Full article