Search Results (577)

Enhancer-promoter interactions occur in the chromatin loci delineated by the CCCTC-binding zinc-finger protein CTCF. CTCF binding is frequently perturbed in genetic disorders and cancer, allowing for misregulation of genes. Here, we developed a panel of chimeric proteins consisting of either full-length or truncated CTCF fused with programmable DNA-binding module dCas9 and fluorescent tracker EGFP. We found that the recruitment of a chimeric protein based on the CTCF N-terminal domain and two zinc-finger domains to the human HOXD locus leads to the de novo formation of a spatial contact with a nearby cohesin/CTCF-bound region, anchoring several chromatin loops. This chimeric protein did not show binding to CTCF motifs and did not affect the epigenetic and transcription profile of the locus. Recruitment of this chimeric protein is also able to restore chromatin loops, lost after deletion of an endogenous CTCF-binding site. Together, our data indicate that the ectopic recruitment of the CTCF N-terminal part could be an appropriate tool for 3D genome engineering. Full article

Passion fruit (Passiflora edulis Sims), belonging to the Passifloraceae family, is an economically important plant in tropical and subtropical regions. The advances in functional genomics research of passion fruit have been significantly hindered by its recalcitrance to regeneration and stable transformation. This study establishes the first efficient Agrobacterium rhizogenes-mediated hairy root transformation system for passion fruit. Utilizing the eGFP marker gene, transformation efficiencies of 11.3% were initially achieved with strains K599, MSU440, and C58C1, with K599 proving most effective. Key transformation parameters were systematically optimized to achieve the following: OD₆₀₀ = 0.6, infection duration 30 min, acetosyringone concentration 100 μM, and a dark co-cultivation period of 2 days. The system’s utility was further enhanced by incorporating the red visual marker RUBY, enabling direct, instrument-free identification of transgenic roots via betaxanthin accumulation. Finally, this system was applied for functional analysis using PeMYB123, which may be involved in proanthocyanidin accumulation. Overexpression of PeMYB123 produced a higher content of proanthocyanidin in hairy roots. Additionally, the PeANR gene involved in the proanthocyanidin pathway was strongly activated in the transgenic hairy roots. This rapid and efficient visually simplified hairy root transformation system provides a powerful tool for functional gene studies in passion fruit. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article

Natural products, characterized by their structural novelty, multi-target capabilities, and favorable toxicity profiles, represent a prominent reservoir for the discovery of innovative anticancer therapeutics. In the current investigation, we identified ajuforrestin A, a diterpenoid compound extracted from Ajuga lupulina Maxim, as a potent agent against lung cancer. In vitro, this compound markedly curtailed the proliferation of A549 cells. Mechanistic explorations revealed that ajuforrestin A could arrest A549 cells in the G0/G1 phase of the cell cycle, provoke apoptosis in cancer cells, and impede their migration by modulating the STAT3 and FAK signaling cascades. Angiogenesis is indispensable for tumor formation, progression, and metastatic dissemination. Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 are established as crucial mediators in tumor neovascularization, a process fundamental to both the expansion of tumor cells and the development of new blood vessels within the tumor milieu. Through the combined application of a Tg(fli1:EGFP) zebrafish model and SPR experimentation, we furnished strong evidence for the ability of ajuforrestin A to obstruct tumor angiogenesis via selective engagement with VEGFR-2. Finally, a zebrafish xenograft tumor model demonstrated that ajuforrestin A could effectively restrain tumor growth and metastasis in vivo. Ajuforrestin A therefore shows considerable promise as a lead compound for the future development of therapies against non-small cell lung cancer (NSCLC). Full article

Snail and Zeb1 have been suggested as markers for the hybrid/mixed epithelial (E)/mesenchymal (M) state of cancer cells. Such cancer cells co-express E- and M-specific transcripts and possess cancer stem cell properties. M13HS-2/-8 tumor hybrid clones derived from human M13SV1-EGFP-Neo breast epithelial cells and human HS578T-Hyg breast cancer cells exhibited co-expression of Snail and Zeb1. To explore the impact of Snail on stemness/epithelial-to-mesenchymal transition (EMT)-related properties in M13HS-2/-8 tumor hybrid clones, Snail was knocked out (KO) using CRISPR/Cas9. Mammosphere formation, colony formation, Western blot analyses, cell migration, and invasion assays were conducted for the characterization of Snail knockout cells. Interestingly, Snail-KO in M13SV1-EGFP-Neo cells resulted in the up-regulation of vimentin and N-cadherin, suggesting EMT induction, which was associated with a significantly enhanced colony formation capacity. In contrast, EMT marker pattern and colony formation capacities of M13HS-2/-8 Snail-KO tumor hybrid clones remained unchanged. Notably, the mammosphere formation capacities of M13HS-2/-8 Snail-KO tumor hybrid clones were significantly reduced. The migratory behavior of all Snail-KO cells was not altered compared with their wild-type counterparts. In contrast, M13HS-2 hybrids and their M13HS-2 Snail-KO variant exhibited a markedly enhanced invasive capacity. Therefore, Snail plays a role as a mediator of stemness properties rather than mediating EMT. Full article

Background/Objectives: RAF pathway aberrations are one of the hallmarks of lung cancer. Sorafenib is a multi-kinase inhibitor targeting the RAF pathway and is FDA-approved for several cancers, yet its efficacy in lung cancer is controversial. Previous clinical research showed that a ARAF p.S214C mutation exhibited exceptional responsiveness to sorafenib in lung adenocarcinoma. Methods: Considering this promising clinical potential, the oncogenic potential and sorafenib response of the ARAF p.S214C mutation were investigated using lung cancer models. ARAF p.S214C mutant, ARAF wild-type (WT), and EGFP control genes were ectopically expressed in lung adenocarcinoma cell lines retroviral transduction. In vitro and in vivo sorafenib sensitivity studies were performed, followed by transcriptomics and proteomics analyses. Results: Compared to the ARAF-WT and EGFP-engineered cells, the ARAF p.S214C-engineered cells activated Raf-MEK-ERK signaling and exhibited enhanced oncogenic potential in terms of in vitro cell proliferation, colony and spheroid formation, migration, and invasion abilities, as well as in vivo tumorigenicity. The ARAF p.S214C-engineered cells also displayed heightened sensitivity to sorafenib in vitro and in vivo. RNA sequencing and reverse-phase protein array analyses demonstrated elevated expression of genes and proteins associated with tumor aggressiveness in the ARAF p.S214C mutants, and its sorafenib sensitivity was likely moderated through inhibition of the cell cycle and DNA replication. The ERK and PI3K signaling pathways were also significantly deregulated in the ARAF p.S214C mutants regardless of sorafenib treatment. Conclusions: This study demonstrates the oncogenicity and sorafenib sensitivity of the ARAF p.S214C mutation in lung cancer cells, which may serve as a biomarker for predicting the sorafenib response in lung cancer patients. Importantly, investigating the gene–drug sensitivity pairs in clinically exceptional responders may guide and accelerate personalized cancer therapies based on specific tumor mutations. Full article

E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G₁-S phase transition and DNA synthesis with its heterodimeric partners (DP1 or DP2). The dimerization domain is the E2Fs and DP1 protein interaction interface and is believed to function in protein dimerization. In this study, eight E2F transcription factors (PcE2F1–8) of large yellow croaker Pseudosciaena crocea and one dimerization partner (PcDP1) are identified in the genome of large yellow croakers. The prediction of E2Fs conserved domains revealed that PcE2F1–6 has one DNA-binding domain (DBD) and one dimerization-binding domain (DD), while PcE2F7–8 only possess two duplicate DBDs but not DD, indicating that E2F7–8 cannot form the E2F/DP1 heterodimer. To explore whether PcDP1 is a partner of PcE2F1–6, the ORF of PcE2F1–6 was cloned. Subsequently, its sequence characteristics, the expression pattern in healthy fish, and subcellular co-localization were analyzed, and an interaction between PcDP1 and PcE2F1–6 were detected directly by yeast two-hybrid and BiFC. The PcE2F1, PcE2F2, PcE2F3, PcE2F4, PcE2F5, and PcE2F6 genes encode a protein of 454, 448, 444, 392, 362, and 396 amino acids, respectively, with accession numbers QFZ93593.1, QFZ93594.1, QFZ93595.1, QFZ93596.1, QFZ93597.1, and QFZ93598.1, respectively. Sequence characteristics analysis found that PcE2F1–5 but not PcE2F6 proteins share the pocket protein-binding domain sequestering in dimerization domains and transactivation domains. The PcE2F1,2,4 proteins possess one nuclear localization signal (NLS), and PcE2F3 protein possess two NLSs, but there is no NLS in PcE2F5 and 6 protein. Moreover, PcE2F4 also contains one NES. However, PcE2F1–6 proteins were all located in nucleus by using Euk-mPloc 2.0 programs and were confirmed by performing the Cherry and EGFP reporter assay. Regarding co-expression of DP1, only E2F4 can transfer DP1’s subcellular location from cytoplasm to the nucleus. RT-qPCR analysis indicated that PcE2F1–6 are constitutively and tissue specifically expressed in all of the tissues tested of a healthy large yellow croaker. The PcE2F1–6, except for PcE2F3, mRNA levels were all detected higher in the liver. PcE2F1–4 were also highly specifically expressed in the kidney, PcE2F4,6 in the brain, and PcE2F5 in the spleen of a healthy large yellow croaker, respectively. Using a yeast two-hybrid system, PcE2F4 interacting with PcDP1 was identified. The interaction between PcE2F4 and PcDP1 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Collectively, these results indicate that an interaction between PcE2F4 and PcDP1 was detected, which may form heterodimer E2F4/DP1 to regulate cell cycles and immune-related pathways in large yellow croakers. Full article

Conditional transgenic systems and multi-copy target transgenes were used to produce transient fluorescent protein expression in adult Drosophila melanogaster, with the goal of developing an in vivo assay of protein turnover. Free-moving flies were assayed at multiple time points using video, and decay in fluorescence was used to calculate protein half-life. Additional experiments involved image capture of anesthetized flies. The half-life of eGFP was increased by the proteasome inhibitor bortezomib, both in vivo and in vitro, indicating proteasomal degradation of eGFP. The accumulation of eGFP in vivo was decreased by the protein synthesis inhibitor cycloheximide, without affecting half-life. The half-lives of several fluorescent proteins were determined, using both tissue-general and tissue-specific expression, in flies of both sexes and varying ages. Typical half-life values varied by fluorescent protein. DsRED showed a greater half-life than eGFP, and little if any degradation was detected for mCherry. Half-life also varied by tissue, with greater eGFP half-life observed in muscle relative to other tissues. Increased half-life with age was detected for DsRED but not for eGFP. Limited effects were observed for sex and female mating status. Taken together, the data indicate the in vivo assays are promising tools for the study of protein degradation regulated by protein sequence, subcellular compartment, tissue and small molecules. Full article

Background/Objectives: Biodegradable polymers have emerged as promising platforms for drug delivery. Produced by microbiomes, polyhydroxyalkanoates (PHAs) offer excellent biocompatibility, biodegradability, and environmental sustainability. In this study, we report the surface functionalization of PHA-based nanoparticles (NPs) using the SpyTag–SpyCatcher system to enhance cellular uptake. Methods: Initial conjugation with mEGFP-SpyTag enabled visualization, followed by decoration with HER2-specific Affibody-SpyCatcher and/or TAT-SpyCatcher peptides. The prepared NPs retained a diameter of <200 nm and a negatively charged surface. Results: Affibody-functionalized NPs significantly enhanced internalization and cytotoxicity in HER2-overexpressing SK-BR-3 cells, whereas TAT-functionalized NPs promoted uptake across various cell types, independently of HER2 expression. Dual-functionalized NPs exhibited synergistic or attenuated effects based on the HER2 expression levels, highlighting the critical role of ligand composition in targeted delivery. Conclusions: The results of this study demonstrate that the SpyTag–SpyCatcher-mediated surface engineering of PHA NPs offers a modular and robust strategy for active targeting in nanomedicine. Full article

Pseudorabies virus (PRV) is a highly contagious pathogen in swine that can cross species barriers and infect other mammals, including humans. Given the potential for interspecies transmission and its threat to public health, understanding the molecular biology of PRV strains is essential for developing effective control measures and preparing for future pandemics. In this study, a novel PRV strain, PRV-HL-2021, was isolated from an outbreak in Heilongjiang Province, China. The viral genome was used to establish a reverse genetics system based on a fosmid library of the PRV-HL-2021 genome. This system facilitated the creation of recombinant PRV, including one expressing EGFP and another with deletions in the US9, gI, and gE genes. PRV-HL-2021 was found to be highly lethal to mice in vivo. The recombinant PRV strains, such as rPRV-US9-EGFP and rPRV-delgI/gE/US9, exhibited growth characteristics similar to the parental PRV-HL-2021 strain. The isolation and characterization of PRV-HL-2021 contribute to a better understanding of the genetic diversity of PRV strains. The developed reverse genetics system provides valuable tools for investigating viral functions, creating genetically modified PRV strains, and advancing the development of safer vaccines. These findings will enhance strategies for controlling PRV outbreaks and mitigating its impact on both animal and public health. Full article

Mango is a vital fruit crop in tropical and subtropical regions, yet pests and diseases cause 30–70% production losses. Developing disease-resistant cultivars through transgenic methods could mitigate these issues. Agrobacterium-mediated callus transformation is a common genetic engineering approach, but successful transgenic mango plants from callus remain unreported due to severe browning and necrosis post-infection. We hypothesized that Agrobacterium-induced immune responses trigger callus death, hindering transformation. To improve efficiency, we engineered an Agrobacterium strain carrying the type III secretion system (T3SS) and effector gene AvrPto. Compared to controls, infected calluses exhibited elevated reactive oxygen species (ROS), along with up-regulated ROS-related, gallic acid biosynthesis, and defense genes. Calluses infected with T3SS-AvrPto-harboring Agrobacterium showed delayed browning and necrosis versus those infected with the empty vector (NV). The transformation rate with Agrobacterium (T3SS-AvrPto-EGFP) reached 1.6%, while Agrobacterium (NV-EGFP) failed entirely. These findings demonstrate that T3SS and AvrPto enhance mango transformation efficiency, offering a promising strategy for breeding multi-resistant varieties. Full article

The liver plays a pivotal role in metabolism, detoxification, and protein synthesis and comprises several cell types, including hepatocytes and cholangiocytes. Primary human hepatocytes in 2D cultures rapidly dedifferentiate and lose their function, making their use as a reliable cell model challenging. Therefore, developing robust three-dimensional cell culture models is crucial, especially for diseases lacking reliable animal models. The aim of this study was to optimize a protocol for the directed differentiation of induced pluripotent stem cells into liver progenitor cells, achieving the high-level expression of specific markers. As a result, we established a 2D culture of liver progenitor cells capable of differentiating into three cell types: a 3D organoid culture containing hepatocyte- and cholangiocyte-like cells and a 2D cell culture comprising stellate-like cells. To evaluate gene delivery efficiency, liver progenitor cells were transduced with various rAAV serotypes carrying an eGFP reporter cassette at different multiplicities of infection (MOIs). Our results revealed that rAAV serotype 2/2 at MOI of 100,000 achieved the highest transduction efficiency of 93.6%, while electroporation demonstrated a plasmid delivery efficiency of 54.3%. These findings suggest that liver progenitor cells are a promising tissue-like cell model for regenerative medicine and demonstrate high amenability to genetic manipulation, underscoring their potential in gene therapy and genome editing studies. Full article

Bone marrow-derived macrophages (BMMs) exhibit dynamic behavior and functional capabilities in response to specific microenvironmental stimuli. Recent investigations have proved that BMMs play crucial roles in promoting necrotic lesion resolution. Despite substantial advancements in understanding their activation and interaction with injured livers, researchers face challenges to develop effective treatments based on manipulating BMMs function. Caveolin-1 (Cav-1) is the major structural protein on the plasma membrane. We previously reported that Cav-1 knockout (KO) mice exhibited less functional damage and necrosis in carbon tetrachloride (CCl₄)-induced liver injury. We hypothesize that the activation and recruitment of BMMs are involved in the resolution of necrotic lesions in Cav-1 KO mice. Wild-type (WT) and Cav-1 KO mice were injected with CCl₄ (10% v/v) to induce acute liver injury model. Blood samples and hepatic tissues were harvested for serum alanine transaminase (ALT) activity assessment, histopathological examination through hematoxylin–eosin (H&E) staining, and BMMs subpopulation analysis via flow cytometry. Then, primary BMMs were isolated and cultured to investigate the effect of Cav-1 on BMMs polarization, migration, and activation of STAT3 signal pathway. Validation of hepatic macrophage depletion was induced by administrating clodronate liposomes (CLs), and BMMs reconstitution was evaluated by EGFP labelled BMMs. Following this, hepatic macrophages were depleted by CLs, BMMs were isolated from Cav-1 KO, and WT mice were cultured and administrated to evaluate the protective role of Cav-1-deleted BMMs on the resolution of hepatocellular necrosis and apoptosis in acute liver injury. The BMMs ratio significantly increased from 2.12% (1D), 4.38% (1W), and 5.38% (2W) in oil control mice to 7.17%, 14.90%, and 19.30% in CCl₄-treated mice (p < 0.01 or p < 0.001). Concurrently, Cav-1 positive BMMs exhibited a marked elevation from 6.41% at 1D to 24.90% by 2W (p = 0.0228). Cav-1 KO exerted protective effects by reducing serum ALT by 26% (p = 0.0265) and necrotic areas by 28% (p = 0.0220) and enhancing BMMs infiltration by 60% (p = 0.0059). In vitro, Cav-1 KO BMMs showed a decrease in CD206 fluorescence intensity (p < 0.001), a time-dependent upregulation of arginase-1 mRNA (p < 0.05 or p < 0.01), a 1.22-fold increase in phosphorylated STAT3 (p = 0.0036), and impaired wound healing from 12 to 24 h (p < 0.001). The macrophage-depleting action in livers by CL injection persists for a minimum of 48 h. Administrated EGFP⁺ BMMs emerged as the predominant population following CL injection for a duration of 48 h. Following clodronate liposome-mediated hepatic macrophage depletion, the adoptive transfer of Cav-1 KO BMMs demonstrated therapeutic efficacy in CCl₄-induced acute liver injury. In CCl₄-induced acute liver injury, the adoptive transfer of Cav-1 KO BMMs reduced necrosis by 12.8% (p = 0.0105), apoptosis by 25.2% (p = 0.0127), doubled macrophages infiltration (p = 0.0269), and suppressed CXCL9/10 mRNA expression (p = 0.0044 or p = 0.0385). BMMs play a key role in the resolution of liver necrotic lesions in CCl₄-induced acute liver injury. Cav-1 depletion attenuates hepatocellular necrosis and apoptosis by accelerating BMMs recruitment and M2 polarization. Cav-1 in macrophages may represent a potential therapeutic target for acute liver injury. Full article

Nipah virus (NiV), a highly lethal zoonotic pathogen causing encephalitis and respiratory diseases with mortality rates up to 40–70%, faces research limitations due to its strict biosafety level 4 (BSL-4) containment requirements, hindering antiviral development. To address this, we generated two NiV minigenome replicons (Fluc- and EGFP-based) expressed via helper plasmids encoding viral N, P, and L proteins, enabling replication studies under BSL-2 conditions. The minigenome replicon recapitulated the cytoplasmic inclusion body (IB) formation observed in live NiV infections. We further demonstrated that IB assembly is driven by liquid–liquid phase separation (LLPS), with biochemical analyses identifying the C-terminal N core domain of the N protein, as well as N₀ and XD domains and the intrinsically disordered region (IDR) of the P protein, as essential structural determinants for LLPS-mediated IB biogenesis. The targeted siRNA silencing of the 5′ and 3′ untranslated regions (UTRs) significantly reduced replicon-derived mRNA levels, validating the regulatory roles of these regions. Importantly, the minigenome replicon demonstrated sensitivity to type I/II/III interferons and antivirals (remdesivir, azvudine, molnupiravir), establishing its utility for drug screening. This study provides a safe and efficient platform for investigating NiV replication mechanisms and accelerating therapeutic development, circumventing the constraints of BSL-4 facilities while preserving key virological features. Full article

Buckwheat is a promising crop with grains that are rich in nutrients and bioactive compounds. Genome sequence data for common and Tartary buckwheat have recently become available. Currently, there is a critical need for the development of a simple and reliable transient gene expression protocol, as well as a stable genetic transformation method, to facilitate metabolic engineering of bioactive compounds, functional analysis of genes, targeted editing, and, in a long-term perspective, to accelerate the breeding process in buckwheat. In this paper, we report optimized methods for Agrobacterium-mediated transient and stable transformation of Fagopyrum esculentum and F. tartaricum. Leaf and cotyledon tissues were infiltrated with an A. tumefaciens-bearing construct containing eGFP and GUS reporter genes. Histochemical staining and Western blotting were used to confirm the expression of reporter proteins. We also demonstrate the usefulness of the developed method for engineering the gramine biosynthetic pathway in buckwheat. HvAMIS and HvNMT genes were transiently expressed in buckwheat leaves, and the de novo production of gramine was confirmed by LC-MS. Moreover, in planta genetic transformation of common and Tartary buckwheat with a reporter gene (eGFP) and selectable marker gene (NptII) was achieved by Agrobacterium-mediated vacuum infiltration. Genomic integration of the construct was confirmed by polymerase chain reaction (PCR), whereas the production of eGFP was confirmed by fluorescence microscopy. Full article