Search Results (632)

Conessine is a steroidal alkaloid found in many plants. The pharmacological efficacies of conessine on various ailments, including antiviral effects against Zika, Herpes, and Coronavirus, were reported. However, the effect of conessine on the influenza virus was still unknown. In this study, conessine exhibited a strong inhibitory effect against influenza A virus (IAV) infection. We examined the effect of conessine on IAV using green fluorescent protein (GFP)-expressing Influenza A/PR8/34 and wild-type A/PR8/34. The fluorescence-activated cell sorting, fluorescence microscopy, cytopathic effect analysis, and plaque assay demonstrated that conessine significantly inhibits IAV infection. Consistently, immunofluorescence results showed that conessine strongly reduces the expression of IAV proteins. The time-of-drug-addition assay revealed that conessine could affect the viral attachment and entry into the cells upon IAV infection. Further, conessine eradicated the virus before binding to the cells in the early stage of viral infection. Our results suggest that conessine has strong anti-viral efficacy against IAV infection and could be developed as an anti-influenza viral agent. Full article

Genetic transformation is an essential tool for investigating gene function and editing genomes. Kiwifruit, recognized as a significant global fresh fruit crop, holds considerable economic and nutritional importance. However, current genetic transformation techniques for kiwifruit are impeded by low efficiency, lengthy culture durations (a minimum of six months), and substantial labor requirements. In this research, we established an efficient system for shoot regeneration and the stable genetic transformation of the ‘Hayward’ cultivar, utilizing leaf explants in conjunction with two strains of Agrobacterium that harbor the expression vector pBI121-35S::GFP, which contains the green fluorescent protein (GFP) gene as a visible marker within the T-DNA region. Our results show that 93.3% of leaf explants responded positively to the regeneration medium, producing multiple independent adventitious shoots around the explants within a six-week period. Furthermore, over 71% of kanamycin-resistant plantlets exhibited robust GFP expression, and the entire transformation process was completed within four months of culture. Southern blot analysis confirmed the stable integration of GFP into the genome, while RT-PCR and fluorescence microscopy validated the sustained expression of GFP in mature plants. This efficient protocol for regeneration and transformation provides a solid foundation for micropropagation and the enhancement of desirable traits in kiwifruit through overexpression and gene silencing techniques. Full article

Methods such as AlphaFold have revolutionized protein structure prediction, making quantitative prediction of the thermodynamic stability of individual proteins and their complexes one of the next frontiers in computational protein modeling. Here, we develop methods for using protein language models (PLMs) with protein mutational datasets related to protein tertiary and quaternary stability. First, we demonstrate that fine-tuning of a ProtT5 PLM enables accurate prediction of the largest protein mutant stability dataset available. Next, we show that mutational impacts on protein function can be captured by fine-tuning PLMs, using green fluorescent protein (GFP) brightness as a readout of folding and stability. In our final case study, we observe that PLMs can also be extended to protein complexes by identifying mutations that are stabilizing or destabilizing. Finally, we confirmed that state-of-the-art simulation methods (free energy perturbation) can refine the accuracy of predictions made by PLMs. This study highlights the versatility of PLMs and demonstrates their application towards the prediction of protein and complex stability. Full article

Background/Objectives: Antibiotic resistance presents an urgent public health threat. By developing a streamlined and effective method for studying bacteriophage induction, this research marks a step further in understanding how antibiotic-resistant genes might spread across different environments. This knowledge is essential for creating strategies to reduce the spread of antimicrobial resistance (AMR), particularly from a One Health perspective. In this study, we develop and validate a Green Fluorescent Protein (GFP)-based method as a proxy for bacteriophage induction. This method screens compounds for their potential to promote bacteriophage induction. Methods: This study utilized a recA-GFP construct in Escherichia coli to measure fluorescence as an indicator of SOS response activation. The experiments involved treating E. coli cultures with varying concentrations of the DNA-damaging chemical mitomycin C and measuring fluorescence over time. Additionally, droplet digital PCR (ddPCR) quantified bacteriophage induction in a lambda phage-carrying E. coli strain, allowing for correlation analysis between the two methods. Results: The recA-driven SOS response depended on both dose and time, with increasing concentrations of mitomycin C leading to higher fluorescence. ddPCR analysis confirmed that mitomycin C induced prophage activation, with gene ratios increasing at higher drug concentrations over time. A strong Spearman correlation (>0.7) was noted between fluorescence and ddPCR results at elevated concentrations and relevant time points, indicating the validity of the GFP-based model as a proxy for bacteriophage induction. Conclusions: The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based E. coli model is a reliable, cost-effective, and efficient tool for studying phage induction and its potential role in AMR spread. This method could facilitate the screening of environmental samples and specific drugs to evaluate their impact on bacteriophage induction, which opens the door for applications such as screening for antibiotic resistance dissemination. Full article

Interactions between chemical drugs and their target proteins are fundamental to drug screening and precision therapy in modern clinical medicine. However, elucidating these interactions within living cells remains challenging due to the limited availability of efficient detection methods. Despite substantial efforts, technical limitations still impede the identification of direct interactors. In this study, we present a simple method to detect the binding between a chemical drug and its target proteins in live cells. This approach utilizes a self-labeling protein (SLP) tag system, specifically HaloTag which is a modified haloalkane dehalogenase, combined with spatially localized expression of the SLP. To implement this system, dasatinib was conjugated to a HaloTag ligand, and the HaloTag protein was expressed in specific intracellular compartments, such as endosomes or F-actin structures. Upon treatment of cells with the HaloTag ligand-conjugated dasatinib, green fluorescent protein (GFP)-fused cytoplasmic dasatinib target proteins were observed to co-localize with the HaloTag at these subcellular structures, thereby indicating direct drug–target binding. This method provides a good spatial resolution with a high signal-to-noise ratio and low false-positive signals across a high background and false-positive/false-negative signals from endogenous proteins and/or non-specific binding. In this context, we believe that our method is a useful platform for visualizing the binding between chemical drugs and their cytoplasmic targets within living systems. Full article

Background/Objectives: While adenovirus (Ad) therapies have been proven to be effective in local administration, systemic Ad treatments have shown limited success due to pre-existing antibodies in the human blood that neutralize the virus. We developed a liposome coating procedure that protects the Ad from pre-existing neutralizing antibodies in human blood. To assess the in vivo stability of the liposomes, the present study used a novel in vivo method to quantitatively assess the protective capabilities of liposome-encapsulated Ad (DfAd) from neutralizing antibodies. Methods: The assay systemically administers DfAd with a green fluorescent protein transgene (DfAd-GFP) into pre-immunized mice and allows it to circulate in the presence of neutralizing antibodies; the infected blood is extracted and used to transduce HEK293 cells, which emits fluorescence in the presence of protected, un-neutralized Ad. Results: The PEGylated liposome formulation provides 12× protection in vivo relative to unencapsulated Ads. In vitro optimization of the liposome coating reveals a strong correlation between the structural stability of liposomes and protection against anti-Ad neutralizing antibodies, where DSPE-PEG2000-carboxylic acid (DSPE-PEG2000-CA) is a critical component for liposome stability and increasing protection against antibody neutralization of the encapsulated Ad. Conclusions: The findings in the present study confirm that the DfAd liposome can protect against neutralizing antibodies in blood circulation. The novel in vivo assay for liposome protection against neutralizing antibodies and in vitro experiments in the present study provide new tools and insights toward designing liposome–Ad complexes for the systemic treatment of cancer. Full article

Our previous study demonstrated that an Escherichia coli heterologous secretion expression system, mediated by superfolder green fluorescent protein (sfGFP) mutants, significantly enhances recombinant lipase yield and reduces large-scale production costs. In this study, we identified mScarlet3, a fast-folding fluorescent protein, as another effective mediator of secretion expression in E. coli. A novel lipolytic enzyme, named LipHu6, was identified through sequence alignment. Secretion expression of LipHu6 was achieved by fusing mScarlet3 to either its N- or C-terminus. The specific activity of mScarlet3-LipHu6 reached 669,151.75 U/mmol, slightly surpassing that of LipHu6 alone (646,682.69 U/mmol) and markedly exceeding that of sfGFP_(-15)-LipHu6 (492,432.39 U/mmol). Notably, N-terminal mScarlet3 fusion had no impact on LipHu6 hydrolytic activity toward short-chain p-nitrophenyl fatty acyl esters (C2–C8). In contrast, mScarlet3-LipHu6 exhibited approximately 1.5- and 1.7-fold increases in hydrolytic activity toward p-nitrophenyl palmitate (p-NPP, C16) and p-nitrophenyl stearate (p-NPS, C18), respectively. In conclusion, this study establishes a novel E. coli heterologous secretion expression system mediated by mScarlet3, offering a highly efficient and cost-effective strategy for the large-scale production of lipolytic enzymes. Full article

Linear DNA constructs are used in gene delivery and therapy application due to their capacity of integration into the mammalian genome, offering stable transgene expression. Compared to circular plasmids, linear DNA also has the advantage that its dimension and steric hindrance are directly correlated to the length of the nucleotide chain. These considerations make linear DNA an effective choice for gene delivery pilot studies, where formulations and transfection efficiency calculations are studied considering the nucleic acid dimensions. Meanwhile, the development of DNA–chitosan nanoparticles (NPs) has gained significant interest for their potential in nucleic acid delivery, especially as non-viral gene delivery systems and for embedding linear DNA fragments, as well as gene delivery to the lung. This study explored an easy polyelectrolyte complexing preparation of linear DNA-loaded chitosan nanoparticles. Among the different formulations of nanoparticles prepared, the optimal one exhibited a size of approximately 290 nm, an encapsulation efficiency of 86% and a zeta potential of 25 mV. Additionally, this study examined how the concentration of DNA in solution influenced nanoparticle formation, encapsulation efficiency and particle size. In particular, transient transfection of the chitosan–linear DNA fragment complex, encoding for green fluorescent protein (GFP), was conducted in human pulmonary distal lung cells (NCI-H441 cells), demonstrating successful cellular internalization and protein expression. These studies highlight the potential of DNA–chitosan NPs in nucleic acid delivery, particularly for pulmonary applications. Future works will focus on formulating the achieved carrier into an inhalable dosage form to improve its translational application. Full article

Viral replicons are efficient tools to understand the mechanisms of viral replication and screen antiviral drugs. In this study, a viral-cDNA-based replicon of Japanese encephalitis virus (JEV), which is the causative agent of Japanese encephalitis, was constructed by replacing the viral structural proteins with a green fluorescent protein (JEV-GFP replicon). The resulting JEV-GFP replicon was used as a tool to screen antiviral drugs targeting JEV nonstructural proteins, and the five compounds JNJ-A07, HZ-1157, NITD-2, quinine, and NITD008 were obtained, which significantly inhibited the replication of the JEV-GFP replicon and JEV in vitro, and the properties of these five compounds were also analyzed. The CC₅₀, EC₅₀, and SI indices of these five compounds were analyzed. In addition, the JEV-GFP replicon was used as a tool to identify the residues of viral nonstructural proteins involved in RNA replication, and the cysteine residue at position 4 of nonstructural protein 1 was found to be essential for JEV RNA replication. These data suggested that the noninfectious JEV-GFP replicon could be used as tool for different purposes, such as antiviral drug screening and gene function studies. Full article

For steady transgenic expression, lentiviral vector-mediated gene delivery is a commonly used technique. One question that needs to be explored is how external lentiviral vectors and overexpressed genes perturb cellular homeostasis, potentially altering transcriptional networks. In this study, two Human Embryonic Kidney 293T (HEK293T)-derived cell lines were established via lentiviral transduction, one overexpressing green fluorescent protein (GFP) and the other co-overexpressing GFP and ADORA3 following puromycin selection to ensure stable genomic integration. Genes with differentially transcript utilization (gDTUs) and differentially expressed genes (DEGs) across cell lines were identified after short-read and long-read RNA-seq. Only 31 genes were discovered to have changed in expression when GFP was expressed, although hundreds of genes showed variations in transcript use. In contrast, even when co-overexpression of GFP and ADORA3 alters the expression of more than 1000 genes, there are still less than 1000 gDTUs. Moreover, DEGs linked to ADORA3 overexpression play a major role in RNA splicing, whereas gDTUs are highly linked to a number of malignancies and the molecular mechanisms that underlie them. For the analysis of gene expression data from stable cell lines derived from HEK293T, our findings provide important insights into changes in gene expression and alternative splicing. Full article

Microalgae such as Phaeodactylum tricornutum are promising cell biofactories for the production of high-value molecules, including monoclonal antibodies (mAbs). However, to date, the production of mAbs in P. tricornutum using the inducible nitrate reductase (NR) promoter has yielded only a limited amount of mAbs. Therefore, the identification of a robust promoter that produces high yields of mAbs is crucial for the development of a cost-effective expression system. To date, only a few endogenous promoters have been characterized in P. tricornutum. In this study, we identified thirty-three potential “strong” endogenous promoters based on our previously published transcriptomic data from the P. tricornutum Pt3 strain. These putative promoter sequences were cloned into an episomal vector and fused to the gene encoding enhanced green fluorescent protein (eGFP). Their strength was assessed by measuring eGFP fluorescence, which reflects the level of eGFP protein expression. Of the thirty-three promoters, thirteen were able to successfully drive eGFP protein expression. Among them, the best results were obtained with the VOC promoter, which allowed a significant increase in eGFP expression compared to that induced by the NR promoter. These results contribute to the identification of new genetic tools that can be used in future studies to increase the yield of production of recombinant proteins in P. tricornutum at an industrial scale. Full article

Multitarget drug discovery, including host-directed therapy, is particularly promising for tuberculosis (TB) due to the resilience of Mycobacterium tuberculosis (Mtb) as well as the complexity of the host’s immune response. In this proof-of-concept study, we used high-content imaging to test a novel panel of dual glycogen synthase kinase 3 beta (GSK-3β) and histone deacetylase (HDAC) 1 and 6 inhibitor candidates for their efficacy in reducing the growth of green fluorescent protein (GFP)-expressing mycobacteria in human primary macrophages. We demonstrate that all ten test compounds, also including the GSK-3β inhibitor SB415286, exhibit an antimycobacterial effect of 20–60% at low micromolar doses and are non-toxic to host cells. Mtb growth showed a positive correlation with the respective 50% inhibitory concentration (IC50) values of GSK-3β, HDAC1, and HDAC6 in each compound, indicating that compounds with a potent IC50 value for HDAC1, in particular, corresponded to higher antimycobacterial activity. Furthermore, the results from multiparametric flow cytometry and a customized multiplex RNA array demonstrated that SB415286 and selected compounds, C02 and C06, could modulate immune polarization and inflammation in Mtb-infected macrophages involving an enhanced expression of CCL2, IL-10 and S100A9, but a decrease in inflammatory mediators including COX-2, TNF-α, and NFκB. These data suggest that GSK-3β inhibition alone can decrease the intracellular growth of mycobacteria and regulate macrophage inflammation, while dual GSK-3β/HDAC inhibitors enhance this efficacy. Accordingly, the tailored design of dual GSK-3β/HDAC inhibitors could represent an innovative approach to host-directed therapy in TB. Full article

Background: Lipid nanoparticles (LNPs) and polyethyleneimine (PEI) have independently been used for DNA complexation and delivery. However, non-ideal gene delivery efficiency and toxicity have hindered their clinical translation. We developed DNA-PEI-LNPs as a strategy to overcome these limitations and enhance DNA delivery and transgene expression. Methods: Three microfluidic mixing protocols were evaluated: (i) LNPs without PEI, (ii) a single-step process incorporating PEI in the organic phase, and (iii) a two-step process with DNA pre-complexed with PEI before LNP incorporation. The influence of DNA/PEI ratios (1:1, 1:2, 1:3) and DNA/lipid ratios (1:10, 1:40) on particle properties and delivery efficiency was examined. Results: In luciferase formulations, higher DNA/lipid ratios (1:40) produced smaller particles (136 nm vs. 188 nm) with improved cellular uptake (77% vs. 50%). The two-step method with higher DNA/PEI ratios improved transfection efficiency, with LNP-Luc/PEI 1:3 (40) achieving ~1.9 × 10⁶ relative light units (RLU) in luciferase expression. In green fluorescent protein (GFP) studies, LNP-GFP/PEI 1:3 (40) showed ~23.8% GFP-positive cells, nearly twofold higher than LNP-GFP (40) at ~12.6%. Conclusions: These results demonstrate the capability of microfluidic-prepared DNA-PEI-LNPs to improve DNA delivery and transgene expression through optimized formulation strategies and selection of appropriate preparation methods. Full article

Berbamine (BBM) is a bibenzyl isoquinoline present in the traditional Chinese herbal medicine Berberis amurensisis Rupr. The present study demonstrates that BBM exerts strong antiviral efficacy against influenza A virus (IAV) infection. We examined the anti-IAV effect of BBM using green fluorescent protein (GFP)-expressing influenza A and H1N1 IAV. The fluorescence microscopy, fluorescence-activated cell sorting analysis, and plaque assay showed that BBM significantly hinders IAV infection. The immunofluorescence analysis confirmed the anti-influenza activity of BBM. From the time-of-addition and hemagglutination inhibition results, it is elucidated that the antiviral effect of BBM is closely related to its inhibitory effect against viral binding and entry at an early infection stage. Our findings imply that BBM has the potential to be developed as a potent antiviral drug against influenza viral infection. Full article

Nano-luciferase binary technology (NanoBiT)-based pseudoviral sensors are innovative tools for monitoring viral infection dynamics. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells via its trimeric surface spike protein, which binds to the human angiotensin-converting enzyme II (hACE2) receptor. This interaction is crucial for viral entry and serves as a key target for therapeutic interventions against coronavirus disease 2019 (COVID-19). Aptamers, short single-stranded DNA (ssDNA) or RNA molecules, are highly specific, high-affinity biorecognition elements for detecting infective pathogens. Despite their potential, optimizing viral infection assays using traditional protein–protein interaction (PPI) methods often face challenges in optimizing viral infection assays. In this study, we selected and evaluated aptamers for their ability to interact with viral proteins, enabling the dynamic visualization of infection progression. The NanoBiT-based pseudoviral sensor demonstrated a rapid increase in luminescence within 3 h, offering a real-time measure of viral infection. A comparison of detection technologies, including green fluorescent protein (GFP), luciferase, and NanoBiT technologies for detecting PPI between the pseudoviral spike protein and hACE2, highlighted NanoBiT’s superior sensitivity and performance, particularly in aptamer selection. This bioluminescent system provides a robust, sensitive, and early-stage quantitative approach to studying viral infection dynamics. Full article