Search Results (38)

Hydrogen sulfide (H₂S) is a vital gasotransmitter essential for maintaining redox homeostasis and modulating inflammatory responses. Herein, we examined a collection of Drosophila melanogaster knockout (KO) lines generated in our laboratory, lacking key genes involved in transsulfuration (cbs, cse) and sulfide metabolism (tst1), to elucidate the role of this adaptive system in immunity. Genetic ablation of these pathways results in profound H₂S deficiency and hyperhomocysteinemia, leading to chronic oxidative stress. This leads to constitutive activation of major immune pathways—including IMD, Toll, and JAK-STAT—even in the absence of infection, a hallmark of chronic inflammation. Indeed, transcriptomic analysis and qRT-PCR studies revealed significant upregulation of these pathways in double (cbs; cse) and triple (cbs; cse; tst1) KO flies under control conditions. Following septic injury with Bacillus subtilis, double and triple KO flies exhibited increased expression of antimicrobial peptides (AMPs) and pattern recognition receptors compared to control and TST1-/- single KO lines. Notably, double and triple KO lines showed more prolonged immune activation. The triple KO flies exhibited the worst survival rate following septic injury. These results demonstrate that H₂S deficiency causes chronic inflammation through the sustained activation of immune pathways, highlighting sulfur metabolism as a crucial regulator of homeostasis and innate immunity in Drosophila. Full article

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a fatal and highly contagious disease, resulting in enormous losses to the global swine industry. No licensed vaccines or effective therapeutics are currently available to control ASFV infection. Interferons (IFNs) serve as key mediators of host antiviral immunity by inducing interferon-stimulated genes (ISGs), but the specific mechanisms by which individual ISGs restrict ASFV replication remain unclear. Interferon-induced protein with tetratricopeptide repeats 3 (IFIT3, also called ISG60) has been shown to exhibit antiviral activity against various viruses, but its role in ASFV infection has not been previously studied. Here, we used porcine alveolar macrophages (PAMs), the primary target cells of ASFV, to investigate IFIT3’s function in ASFV replication. We found that overexpression of IFIT3 inhibited ASFV replication, while its knockdown enhanced viral propagation. Mechanistically, IFIT3 directly blocked ASFV adsorption to host cells, thereby suppressing all subsequent stages of the viral cycle. IFIT3 also specifically interacted with ASFV F334L, an early viral gene product that encodes the small subunit of ribonucleotide reductase, a key enzyme for viral DNA synthesis. Additionally, IFIT3 positively regulated the STAT1/TBK1/IRF3 signaling axis: its overexpression increased phosphorylation of TBK1 and IRF3, as well as the protein level of STAT1, while IFIT3 knockdown attenuated activation of these molecules. Transcriptomic analysis of IFIT3-knockout PAMs revealed significant suppression of innate immune pathways, including type I interferon, JAK-STAT, and RIG-I-like receptor pathways, along with downregulated expression of core antiviral molecules such as ISG15, MX1, and STAT1. Conversely, pathways related to viral adsorption, endocytosis, and cytoskeleton were activated, and pathways involved in protein translation initiation, endoplasmic reticulum stress, and autophagy were dysregulated, creating a favorable intracellular environment for ASFV replication. In conclusion, IFIT3 restricts ASFV replication possibly by inhibiting viral adsorption and promoting innate immune signaling, identifying it as a potential therapeutic target against ASFV. This study’s limitation is its in vitro PAM model; future work will validate IFIT3’s role in vivo and develop targeted inhibitors. Full article

Aflatoxin B1 (AFB1) is a prevalent and highly toxic mycotoxin in the food and feed chain and can directly injure the intestinal epithelium. Yet, its upstream determinants linking epithelial stress to cytotoxicity remain insufficiently defined. Here, we used porcine intestinal epithelial IPEC-J2 cells to characterize AFB1-induced cytotoxic and transcriptomic responses and to determine the role of the tight-junction scaffold, Cingulin-like 1 (CGNL1), a candidate gene identified through genome-scale CRISPR knockout library screening. The results showed that AFB1 exposure reduced cell viability in a dose-dependent manner and induced oxidative stress. RNA-seq profiling analysis revealed broad transcriptional remodeling, with activation of inflammatory pathways (including NF-κB and JAK–STAT signaling). Based on our constructed CGNL1-knockout IPEC-J2 cell line (CGNL1-KO IPEC-J2) using CRISPR/Cas9, it was found that CGNL1 deficiency markedly alleviated AFB1-induced cytotoxicity and oxidative stress. Comparative transcriptomics analysis showed that CGNL1 knockout attenuated AFB1-triggered aberrant expression of some CGNL1-dependent AFB1-responsive genes related to immune response under AFB1 challenge. Together, these findings identify CGNL1 as a potential modulator of epithelial susceptibility to AFB1 and support its involvement in the regulation of toxin-induced oxidative response. Full article

Signal transducer and activator of transcription 2 (STAT2) is a key component of the type I interferon (IFN-I/III) signaling pathway, which is pivotal in host defense against cancer and viral infections and in shaping immune responses. Building on our previously reported conditional Stat2 knockout (KO) mouse, we expand its utility by validating additional tissue-specific models and exploring novel functional contexts. Mice carrying loxP-flanked Stat2 alleles were crossed with CMV-Cre, Cdx2-Cre or CD11c-Cre mice. Deletion of STAT2 was validated by PCR genotyping and western blotting in the relevant tissues. To confirm defective IFN-I signaling with STAT2 deletion, IFN-β stimulation of splenocytes from CMV-Cre Stat2 KO mice showed a lack of induction of canonical IFN-I target genes, confirming functional disruption of the pathway. In vivo, global Stat2 deletion significantly impaired the antitumor efficacy of IFN-β treatment. Similarly, lung fibroblasts isolated from globally deleted Stat2 KO mice showed defective antiviral responses to IFN-β. Tissue-specific Cre models demonstrated selective ablation of STAT2 in target compartments without affecting its expression in non-target tissues. Together, these studies expand our published conditional Stat2 KO findings and highlight the value of this model as a versatile platform for dissecting STAT2-dependent signaling pathways in a tissue- and disease-specific manner. Full article

The role of Signal Transducer and Activator of Transcription 2 (STAT2) in cancer remains poorly understood. STAT2 is a key mediator of type I interferon (IFN) signaling, activating the expression of IFN-stimulated genes with antiviral and antiproliferative effects. However, emerging evidence suggests that STAT2 can also promote tumor growth. Here, we show that high STAT2 mRNA expression in colon cancer tumors correlates with reduced overall survival in patients. In preclinical models, deletion of STAT2 in tumor cells suppressed tumor growth, whereas STAT2 overexpression enhanced tumor growth, supporting its pro-tumorigenic role. To determine whether this function depends on type I IFN receptor (IFNAR1) signaling, we generated IFNAR1 knockout (IFNAR1 KO) colon carcinoma cells and compared their growth with parental and STAT2-deficient (STAT2 KO) tumor cells. Loss of type I IFN signaling was confirmed by western blot and qPCR analyses. In vitro, IFNAR1 KO and STAT2 KO tumor cells proliferated at similar rates. However, in xenograft tumor transplantation models, IFNAR1 KO cells formed larger tumors while STAT2 KO tumor cells formed smaller ones compared to parental tumor cells. These findings indicate that STAT2 promotes colorectal cancer growth through mechanisms independent of IFNAR1 signaling. Full article

Breast cancer remains a leading cause of mortality among women despite advances in early detection and targeted therapies, underscoring the need for safer and more effective treatment options. Drug repurposing offers a promising strategy by leveraging existing pharmacological agents with established safety profiles. Desloratadine, a second-generation H1-histamine receptor antagonist widely prescribed for allergic conditions, has attracted interest in oncology because histamine signaling influences proliferation, angiogenesis, and immune responses, yet its anticancer potential remains poorly understood. In this study, we investigated its effects in MCF-7 breast cancer cells, which harbor wild-type TP53. Desloratadine inhibited cell viability in a dose-dependent manner, with an IC₅₀ of 14.2 µg/mL. Mechanistic analyses revealed that growth inhibition was primarily mediated through apoptosis, confirmed by Hoechst 33342 staining, ROS generation, annexin V/PI staining, and caspase-dependent pathways. Gene expression profiling demonstrated upregulation of TP53, FAS, and BAX, alongside reduced PARP-1 and NF-κB expression, with no detectable STAT3 or BCL2 expression. Flow cytometry indicated accumulation of cells in the sub-G₁ phase and G₂/M arrest, consistent with apoptosis induction. Molecular docking further supported these findings, showing that Desloratadine binds with high affinity to p53 (−7.0 kcal/mol), FAS (−6.8 kcal/mol), and NF-κB (−6.5 kcal/mol), forming stabilizing hydrogen bonds and hydrophobic interactions aligned with the observed gene expression changes. To confirm the functional role of TP53, we generated CRISPR-Cas9 knockout MCF-7 cells. Compared with wild-type cells, these knockout cells displayed markedly reduced sensitivity to Desloratadine, with the IC₅₀ shifting from 14.2 µg/mL to 36.4 µg/mL, demonstrating that p53 is a key mediator of the drug’s cytotoxic effect. Collectively, these findings identify Desloratadine as a potential repurposed drug candidate for breast cancer therapy, acting at least in part through a p53-dependent apoptotic pathway. Full article

In eutherian mammals, embryo implantation is a critical process for a successful pregnancy. In mice, the activation of the leukemia inhibitory factor (LIF) receptor–STAT3 signaling axis induces embryo adhesion and decidualization. The LIF receptor is believed to function as a heterodimer composed of LIFR (encoded by Lifr) and GP130 (encoded by Il6st); however, their distinct expression patterns in the uterine epithelium immediately prior to implantation suggest divergent functional roles. In this study, we generated uterine epithelium-specific Lifr knockout (Lifr eKO) mice and conducted a comprehensive gene expression analysis of the endometrium before implantation. We compared these results with those from uterine epithelium-specific Gp130 knockout (Gp130 eKO) mice. Similarly to Gp130 eKO mice, Lifr eKO mice were completely infertile. We identified 299 genes with expression changes greater than twofold following gene deletion; among these, 31 genes were downregulated and 57 genes were upregulated in both eKO models. Many of the downregulated genes were previously implicated in uterine function. Hub gene analysis identified Erbb2 and c-Fos as key regulators in both models. Further experiments using an ERBB2 inhibitor suggested that LIFR–ERBB2-mediated signaling plays a crucial role in embryo implantation. Full article

Ischemic stroke is a leading cause of death and disability worldwide. Currently, there is an unmet clinical need for pharmacological treatments that can improve ischemic stroke outcomes. In this study, we investigated the role of brain peroxisome proliferator-activated receptor alpha (PPARα) in ischemic stroke pathophysiology. We used a well-established model of cerebral ischemia in PPARα transgenic mice and conducted the RNA sequencing (RNA-seq) of mouse stroke brains harvested 48 h post-middle cerebral artery occlusion (MCAO). PPARα knockout (KO) increased brain infarct size following stroke, indicating a protective role of PPARα in brain ischemia. Our RNA-seq analysis showed that PPARα KO altered the expression of genes in mouse brains with known roles in ischemic stroke pathophysiology. We also identified many other differentially expressed genes (DEGs) upon the loss of PPARα that correlated with increased infarct size in our stroke model. Gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis revealed the upregulation of gene signatures for the positive regulation of leukocyte proliferation, apoptotic processes, acute-phase response, and cellular component disassembly in mouse stroke brains with PPARα KO. In addition, pathway analysis of our RNA-seq data revealed that TNFα signaling, IL6/STAT3 signaling, and epithelial–mesenchymal transition (EMT) gene signatures were increased in PPARα KO stroke brains. Our study highlights PPARα as an attractive drug target for ischemic stroke due to its transcriptional regulation of inflammation-, apoptosis-, and EMT-related genes in brain tissue following ischemia. Full article

In animals, a trade-off exists between reproduction and growth, which are the most fundamental traits. Males and females exhibit profound differences in reproduction and growth in fish species. However, the precise molecular mechanism governing this phenomenon is still not clear. Here, we uncovered that chr23-miR-200s and dmrt1 knockout specifically caused an impairment in reproduction and an increase in body growth in female and male zebrafish, respectively. Chr23-miR-200s and Dmrt1 directly regulate the stat5b gene by targeting its 3′UTR and promoter. The loss of stat5b completely abolished the elevated growth performance in chr23-miR-200s-KO or dmrt1^−/− zebrafish. Moreover, the dmrt1 transgenic zebrafish had significantly lower body length and body weight than the control males, accompanied by a significant reduction in stat5b expression in the liver of transgenic fish. In summary, our study proposes a regulatory model elucidating the roles of chr23-miR-200s and Dmrt1 in controlling the sexually dimorphic trade-off between reproduction and growth. Full article

The healthy gut masks a dynamic balance between pro- and anti-inflammatory activities, largely due to microbial factors in the lumen. IL-10 is vital among the anti-inflammatory mediators, yet confirming constitutive versus stimulated secretion in any cell type is difficult due to the cellular complexity in the gut. Seeking to determine whether intestinal epithelial cells are programmed to constitutively make IL-10, we confirmed that IL-10 mRNA was present in enteroids from C57BL/6 mice and IL-10 protein was co-localized with a Paneth cell marker but not with markers for goblet or tuft cells. Paneth cells positive for IL-10 also possessed apical and basal IL-10RA, while cells negative for IL-10 had only basal IL-10RA, suggesting a possible autocrine role for IL-10. Indeed, Paneth cells in IL-10 gene knockout (IL-10KO) enteroids possessed lower levels of anti-microbial protein mRNAs, which could not be restored by adding IL-10. Enteroids passaged onto Transwell^® filters to form monolayers were treated with IL-10 and STAT3 phosphorylation was measured. Apically applied IL-10 resulted in a stronger STAT3 signal than basally applied cytokine. Our results indicate that a subpopulation of Paneth cells constitutively secrete IL-10 apically, which binds apical IL-10RA, impacting the expression of anti-microbial proteins unique to Paneth cells. Full article

Background/Objectives: Lung cancer ranks as the leading cause of cancer-related deaths globally and is highly associated with cisplatin resistance due to both intrinsic and extrinsic mechanisms. Proliferating Cell Nuclear Antigen (PCNA) plays a critical role in molecular processes, such as DNA replication and repair, chromatin structure maintenance, and cell cycle progression. PCNA is known as a molecular marker for proliferation and an excellent inhibition target to shut down highly proliferative cells. One of the mechanisms of cisplatin resistance is the increase in DNA repair, and studies have reported an association between PCNA, lung cancer, and cisplatin treatment. The present study aimed to characterize the absence of PCNA in A549 lung adenocarcinoma cells. Methods: Employing a CRISPR/Cas9 gene-editing approach, we generated a monoclonal cell culture, termed PKO (PCNA knockout). Results: PKO cells exhibited a residual PCNA expression, significantly decreased clonogenic potential and ubiquitylation at K164 residue. IC50 assay suggested that PKO cells could not acquire cisplatin resistance when compared to PX. After cisplatin treatment, PKO cells presented impaired ubiquitylation and did not have increased STAT3 phosphorylation (Tyr705), a previously characterized mechanism of cisplatin resistance. Conclusions: We suggest that PCNA participates in cisplatin resistance in A549, partially by DNA damage tolerance through failure on PCNA monoubiquitylation, and its inhibition may be an approach to circumvent cisplatin resistance. Full article

Objectives: The industrial production of influenza vaccines is facing significant challenges, particularly in improving virus production efficiency. Despite advances in cell culture technologies, our understanding of the production characteristics of high-yield suspension cell lines remains limited, thereby impeding the development of efficient vaccine production platforms. This study aims to investigate the key features of STAT1 knockout suspension-adapted MDCK cells (susMDCK-STAT1-KO) in enhancing influenza A virus (IAV) production. Methods: Suspension-adapted susMDCK-STAT1-KO cells were compared to suspension-adapted wild-type MDCK cells (susMDCK) for IAV production. Virus quantification, gene expression analysis, and cholesterol deprivation assays were performed. Metabolite profiles, viral RNA quantification, and lipid and dry weight measurements were also conducted to assess the viral replication and release efficiency. Results: The susMDCK-STAT1-KO cells exhibited significantly improved virus adsorption (64%) and entry efficiency (75%) for the H1N1 virus, as well as accelerated viral transcription and replication for both the H1N1 and H9N2 viruses. Virus release was identified as a limiting factor, with a 100-fold higher intracellular-to-extracellular viral RNA ratio. However, the STAT1-KO cells showed a 2.39-fold higher release rate (750 virions/cell/h) and 3.26-fold greater RNA release for the H1N1 virus compared to wild-type cells. A gene expression analysis revealed enhanced lipid metabolism, particularly cholesterol synthesis, as a key factor in viral replication and release. Cholesterol deprivation resulted in reduced viral titers, confirming the critical role of intracellular cholesterol in IAV production. Conclusions: This study demonstrates the enhanced influenza virus production capacity of susMDCK-STAT1-KO cells, with significant improvements in viral yield, replication, and release efficiency. The findings highlight the importance of STAT1-mediated immune modulation and cholesterol metabolism in optimizing virus production. These insights provide a foundation for the development of more efficient vaccine production platforms, with implications for large-scale industrial applications. Full article

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing in B cells. To assess the functional impact of the proposed causal variant in C-type lectin domain family 16, member A (CLEC16A), we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), the CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus—particularly the risk allele—drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and could underlie the signal at 16p13. Full article

The genomic activity of 1,25(OH)₂D₃ is mediated by vitamin D receptor (VDR), whilst non-genomic is associated with protein disulfide isomerase family A member 3 (PDIA3). Interestingly, our recent studies documented that PDIA3 is also involved, directly or indirectly, in the modulation of genomic response to 1,25(OH)₂D₃. Moreover, PDIA3 was also shown to regulate cellular bioenergetics, possibly through the modulation of STAT signaling. Here, the role of VDR and PDIA3 proteins in membrane response to 1,25(OH)₂D₃ and calcium signaling was investigated in squamous cell carcinoma A431 cell line with or without the deletion of VDR and PDIA3 genes. Calcium influx was assayed by Fura-2AM or Fluo-4AM, while calcium-regulated element (NFAT) activation was measured using a dual luciferase assay. Further, the levels of proteins involved in membrane response to 1,25(OH)₂D₃ in A431 cell lines were analyzed via Western blot analysis. The deletion of either PDIA3 or VDR resulted in the decreased baseline levels of Ca²⁺ and its responsiveness to 1,25(OH)₂D₃; however, the effect was more pronounced in A431∆PDIA3. Furthermore, the knockout of either of these genes disrupted 1,25(OH)₂D₃-elicited membrane signaling. The data presented here indicated that the VDR is essential for the activation of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), while PDIA3 is required for 1,25(OH)₂D₃-induced calcium mobilization in A431 cells. Taken together, those results suggest that both VDR and PDIA3 are essential for non-genomic response to this powerful secosteroid. Full article

Pancreatic cancer is among the cancers with the highest mortality rates. Most of the patients are found to have advanced cancer, losing the chance of surgical treatment, and there is an urgent need to find new treatment methods. Targeted therapy for specific genes that play a key role in cancer is now an important means to improve the survival rate of patients. We determined that CD73 is highly expressed in pancreatic cancer by flow cytometry and qRT-PCR assays combined with bioinformatics techniques. Application of CRISPR/Cas9 technology to knockout CD73 in human and murine cell lines, respectively, revealed that CD73 inactivation inhibited cell growth and migration and induced G1 cell cycle arrest. We also found that CD73 deletion inhibited the ERK/STAT3 pathway and activated the E-cadherin pathway. In addition, a CRISPR/Cas9 protein kinase library screen was performed and identified Pbk, Fastk, Cdk19, Adck5, Trim28, and Pfkp as possible genes regulating CD73. Full article