Search Results (2,193)

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

Leydig cells (LCs) originate from stem Leydig cells (SLCs) and synthesize testosterone, a hormone indispensable for the development, sustenance, and functionality of the male reproductive system. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) play pivotal roles in animal reproductive processes, yet the functional contributions of lncRNAs in pig LCs remain largely uncharacterized. The aim of this study was to examine how lncRNAs influence the function of LCs and their underlying molecular regulatory mechanisms. To achieve this, RNA-seq was conducted on cells before ethane dimethane sulfonate (EDS) treatment (SLCs and LCs) and after EDS treatment (SLCs), identifying 887 significantly downregulated lncRNAs and 30 upregulated lncRNAs after EDS treatment. Bioinformatics analysis identified lncXIRP1 for further investigation. The effects of lncXIRP1 on LCs proliferation, apoptosis, and expression of genes related to testosterone synthesis were investigated by using RT-qPCR, Western blot, CCK-8 and other methods. Bioinformatics predictions have unveiled the existence of a binding site between lncXIRP1 and IGFBP3. Through RT-qPCR experiments and a dual-luciferase reporter system, it was conclusively demonstrated that lncXIRP1 has the capacity to repress the expression of IGFBP3 mRNA, thereby inhibiting the proliferation and transcription activity of genes associated with testosterone synthesis in LCs and promoting their apoptosis. These results provide a theoretical foundation for further exploration of the impact of lncRNAs on LCs function and improving pig reproductive performance. Full article

Saline-alkali stress and cold damage significantly impact the yield of Brassica napus. G proteins play a crucial role in plant resistance to abiotic stresses, and research on G proteins in Brassica napus (rapeseed) is still in its early stages. In this study, we employed bioinformatics tools to systematically investigate the basic physicochemical properties, phylogenetic relationships, distribution, gene structure, cis-regulatory elements, and expansion patterns of the GS3 gene family in Brassica napus. Additionally, reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the response of the BnGS3-3 gene to salt and low-temperature stresses. Natural variations were found in the promoter region of BnGS3-3. By conducting a promoter-driven luciferase (LUC) assay, the relationship between natural variations in the BnGS3-3 promoter and salt and cold tolerance was analyzed. Furthermore, the impact of these natural variations on flowering time, root length, and yield was explored using phenotypic data from a population. Our research results aim to provide insights into the function and molecular mechanisms of BnGS3-3 in Brassica napus, and to offer valuable genetic resources for molecular breeding to improve salt and low-temperature tolerance in Brassica napus. Full article

Functional U6 promoters are widely utilized in CRISPR gene editing systems for crops. The identification of endogenous U6 promoter activity and the establishment of CRISPR/Cas9 gene editing systems in various crops can enhance the efficiency and accuracy of gene editing in molecular breeding. In this study, four U6 snRNAs were identified in the genome of the oil flax (Linum usitatissimum L.) cultivar Longya 10, which exhibit high homology with the promoter regions of Arabidopsis thaliana U6 snRNA. We cloned and constructed fusion expression vectors with U6 promoter-driven dual-luciferase reporter genes. Transient transformation of flax and Nicotiana benthamiana was performed to measure the relative activity of dual luciferase. The U6-4 on chromosome 14 showed the highest transcriptional activity. Truncations of varying lengths from the 5′ end of this promoter were tested, revealing that a 342 bp U6 promoter fragment possesses high transcriptional activity and an optimal length. Subsequently, we constructed a CRISPR/Cas9 gene editing vector with LuU6-5P/AtU6-P driving LusPDS sgRNA. Agrobacterium-mediated infection of flax hypocotyls yielded transgenic albino flax shoots. DNA from these shoots was used as a template to amplify LusPDS fragments, which were then sequenced. Sequencing analysis revealed that CRISPR/Cas9 vectors using Lu14U6-4-5P achieved higher editing frequencies at LusPDS compared to AtU6-P-driven systems. Full article

Benzo[a]pyrene, as the primary component of air pollutants, has been implicated in the pathogenesis of non-small-cell lung cancer (NSCLC). As an m6A reader that facilitates mRNA translation, YTHDF1 serves as a crucial regulator in tumor progression. Therefore, we established Benzo[a]pyrene(B[a]P)-induced bronchial epithelial malignant transformed cells (HBE-P35) to simulate the precancerous lesions of NSCLC and investigated the regulatory axis of YTHDF1 in both HBE-P35 and A549 lung cancer cells. A high level of m6A expression was detected in both HBE-P35 and A549 cells. Over-expression of YTHDF1 was observed in NSCLC tissues and correlated with poor overall survival in NSCLC patients. TMT labeling-based proteomic analysis and clinical lung tissue microarray assays demonstrated that CDK6 and MAP3K6 were positively correlated with YTHDF1 expression. MeRIP and RIP analyses revealed that YTHDF1 mediates the m6A-dependent regulation of CDK6 and MAP3K6 protein expression. The acquisition and deletion of miR-139/145-5p, along with luciferase reporter gene assays, demonstrated that miR-139-5p can target YTHDF1. Therefore, we conclude that YTHDF1 regulates CDK6 and MAP3K6 through m6A in B[a]P-induced HBE-P35 and A549 cells, providing a potential target for lung cancer treatment. Full article

Caigua (Cyclanthera pedata (L.) Schrad.) is a traditional herbal remedy traditionally used in Latin America for its health benefits and to treat metabolic disorders, including diabetes. Despite interest in its herbal use, the phytochemical properties of caigua’s secondary metabolites are poorly known. This study aimed to isolate the main flavone glycosides from the leaves of caigua landrace cultivated in the Camonica Valley (Italy) using flash chromatography and evaluate their potential activity toward peroxisome proliferator-activated receptors (PPARs) and transient receptor potential (TRP) ion channels through luciferase and intracellular calcium assays. We found that the caigua species-specific flavone glycoside, chrysin-6-C-fucopyranoside, showed potent and selective activity toward PPARγ, with no effects on other PPAR subtypes or TRP channels. These findings indicate that the caigua plant could offer a safer alternative to conventional PPARγ agonists, whose use as antidiabetic drugs is limited by severe side effects that currently restrict the clinical use of conventional PPAR agonists. 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

Mastitis poses a severe threat to the global cattle industry, causing huge economic losses. Environmental mastitis is mainly induced by Escherichia coli (E. coli), and the current treatment is still using antibiotics, with problems such as drug resistance and food safety. ALKBH5 is an RNA m⁶A demethylase that plays an important role in various biological processes, while p65 is a key regulator of inflammatory responses. Therefore, studying the interaction between ALKBH5 and p65 in protecting the mammary epithelial barrier provides new insights into the pathogenesis of mastitis. This study revealed that E. coli-induced acute inflammation activated the NF-κB/p65 signaling pathway and disrupted mammary epithelial cell tight junctions. Knockdown of ALKBH5 promoted p65 phosphorylation and inhibited the expressions of the tight junction proteins TJP1, CDH1, and OCLN. Furthermore, motif analysis, CHIP-PCR, and dual luciferase assay confirmed that phosphorylated p65 inhibited TJP1 promoter activity, thereby inhibiting TJP1 expression. In addition, the mouse experiment further demonstrated that knockdown of ALKBH5 aggravated E. coli-induced acute mastitis and epithelial cell tight junction disruption, and promoted E. coli invasion and proliferation. Significantly, this study is the first to demonstrate the details of the interaction between p65 and TJP1 and to declare the molecular mechanism of ALKBH5 in improving the cell tight junction, which lays a potential target and theoretical foundation for the treatment of mastitis and other infectious diseases. Full article

Tea is one of the world’s major non-alcoholic beverages, popular for its health benefits and flavor. Purple-bud tea is particularly rich in anthocyanins, the concentration of which varies depending on the tea cultivar and cultivation conditions. While the genetic regulation of anthocyanin accumulation is well understood, the impact of environmental factors, such as light, on anthocyanin synthesis is less documented. In this study, we analyzed the anthocyanin content and the expression levels of anthocyanin biosynthesis genes (ABGs), CsAN1, and CsHY5, under different light intensities and durations. The expression of both CsAN1 and ABGs was significantly induced by light, with an intensity of 8000 lx particularly effective in promoting anthocyanin accumulation. Furthermore, we explored the effect of shading on anthocyanin content, finding that fifty percent shading reduced anthocyanin content by nearly half. Finally, dual-luciferase reporter assays and yeast one-hybrid assays confirmed the direct regulation of CsHY5 on CsAN1. These findings offer insights into the regulatory mechanisms underlying light-induced anthocyanin biosynthesis in tea plants and suggest a potential method for controlling anthocyanin accumulation in tea production. Full article

Heat shock transcription factor (Hsf) plays a crucial role in the signal transduction pathways of plants in response to drought stress. However, studies exploring the specific functions and mechanisms of action of the Hsf family in tea plants (Camellia sinensis L.) remain limited. In this study, we identified 31 members of the CsHsf family from the C. sinensis genome. CsHsf10 was determined to be a potential drought-resistant candidate gene by screening 10 highly expressed genes in mature leaves and confirming results through RT-qPCR. Correlation analysis indicates that CsHsf10 may enhance the drought resistance of tea plants by participating in the tea polyphenol synthesis pathway and regulating the expression of antioxidant enzyme genes. Furthermore, overexpression experiments in Arabidopsis and antisense oligonucleotide experiments in tea plants corroborated that CsHsf10 exerts a significant positive regulatory effect on drought resistance in tea plants. Yeast one-hybrid assays and dual luciferase reporter gene experiments demonstrated that CsHsf10 can directly target CsPOD17, significantly promoting its transcriptional expression. Additionally, we found that the expression of CsHsf10 contributes to the increased accumulation of catechin components in tea plants under drought stress. These findings suggest that, during the response of tea plants to drought stress, CsHsf10 not only enhances antioxidant capacity by regulating the activity of antioxidant enzymes but also optimizes the physiological state of tea plants by influencing the accumulation of secondary metabolites, thereby significantly improving their drought resistance. Full article

Background/Objectives: Murine models play a key role in guiding formulation and immunogenicity studies across various vaccine platforms, including mRNA-based vaccines. Typically, a narrow age range (6 to 8 weeks) is used in these studies. Here, we investigated whether widening this age range could provide greater flexibility in experimental design without impacting pre-clinical outcomes. Methods: To achieve this, we evaluated two commonly used lipid nanoparticle (LNP) formulations (based on SM102 and ALC-0315 ionizable lipids) containing either firefly luciferase or ovalbumin mRNA in female BALB/c mice aged 4, 8, and 16 weeks. LNPs were prepared and purified via microfluidics, and their size, polydispersity, zeta potential, and encapsulation efficiency were measured. Mice were injected intramuscularly, and the in vivo bioluminescence and antibody titers were measured to evaluate mRNA expression profiles and immunogenicity across the three age groups. Results: Our findings show that the 4-week-old mice exhibited higher protein expression following mRNA administration compared to the older groups; however, no significant differences were observed between the 8- and 16-week-old mice. Despite the initial higher protein expression, the antibody responses after the prime dose were lower in the 4-week-old mice compared to the other two groups. However, following the booster dose, antibody levels were comparable across all three age groups. Conclusions: By identifying a broader age range window, we provide greater flexibility in study design, enhance data comparability across studies, and promote more efficient use of animal resources, all while maintaining reliable and representative results in these murine models. Full article

Puccinia polysora Underw, the pathogen that causes southern corn rust (SCR), delivers effectors to manipulate host immune responses. However, the mechanisms by which these effectors modulate host defenses are not well characterized. In this study, we found that the P. polysora effector PpEX is highly upregulated during infection. PpEX suppresses plant immune responses that are initiated by chitin, including the activation of mitogen-activated protein kinases (MAPKs) and the expression of pathogenesis-related (PR) genes. Maize plants transiently expressing PpEX exhibited higher pathogen infection rates, larger colony areas, and greater fungal biomass on their leaves compared to the control group. By employing TurboID proximity labeling technology coupled with mass spectrometry analysis, we discovered potential target proteins of PpEX in maize. The split-luciferase system enabled us to identify ZmMPK3, a component of the MAPK signaling pathway, as an interacting partner of PpEX among the candidate proteins. This interaction was subsequently confirmed by co-immunoprecipitation (Co-IP) experiments. Additionally, we verified that ZmMPK3 plays a positive role in regulating maize resistance to SCR. Thus, PpEX may function as a virulence effector that dampens plant PTI immunity by interacting with ZmMPK3 and impeding the MAPK signaling pathway. Full article

Background: Epigenetic alterations, including DNA methylation, play a crucial role in the development of oral squamous cell carcinoma (OSCC) by regulating the expression of tumor suppressor genes and oncogenes. This study investigated the methylation status of miR-124-3 and its role in OSCC progression. Methods: This study applied the Illumina Infinium MethylationEPIC BeadChip assay to profile >850,000 CpG sites in paired OSCC and normal tissues. The methylation data were validated by further analyzing the methylation level of miR-124-3 by using a bisulfite pyrosequencing assay. We investigated whether miR-124-3 acts as a tumor suppressor by establishing miR-124-3-overexpressing OSCC cells and subjecting them to cell proliferation, colony formation, and migration assays. Dual-luciferase reporter assay was used to validate the target genes of miR-124-3 in OSCC cells. Results: The Infinium MethylationEPIC BeadChip and bisulfite pyrosequencing assays consistently identified hypermethylation of miR-124-3 in OSCC tissues relative to normal oral tissues. It was especially notable that miR-124-3 methylation levels were markedly higher in late-stage tumors than in early-stage, and differed significantly between early-stage tumor and normal tissues, indicating that miR-124-3 methylation is an early event in OSCC development. Methylation of miR-124-3 contributes markedly to the downregulation of the gene, leading to the increased expression of its target gene, leucine-rich repeat-containing 1 (LRRC1), which is considered to be positively associated with cancer progression. Moreover, overexpression of miR-124-3 suppressed the proliferation and migration of OSCC cells, while silencing the expression of LRRC1 produced similar tumor-suppressive effects. Luciferase reporter assays confirmed that miR-124-3 directly targets the 3′ untranslated region of LRRC1 to downregulate LRRC1 expression. Conclusions: Hypermethylation-mediated downregulation of miR-124-3 results in increased LRRC1 expression, which drives OSCC progression. These findings highlight DNA methylation of miR-124-3 as a potential biomarker for the early detection of OSCC and a therapeutic target for OSCC treatments. Full article

Cell lines and their corresponding expression plasmids are extensively utilized in the study of insect physiology and pathology. In this research, four single-cell cultured lines (Px4-1 to Px4-4) of Plutella xylostella were established from eggs. The promoter for the P. xylostella elongation factor 1α (PxEF1α), known for its high driving activity in cells, was cloned and used to construct expression plasmids. Dual-luciferase activity assays and EGFP expression analyses demonstrated that the PxEF1α promoter exhibited the strongest driving activity in Px4-2 cells, comparable to that of the immediate-early 1 promoter associated with the homologous region 5 enhancer (AcIE1^hr5) from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). In contrast, the driving activity of PxEF1α in cells derived from Spodoptera frugiperda, Trichoplusia ni, and Helicoverpa armigera was lower. Furthermore, the PxEF1α promoter was successfully employed to drive inward rectifier potassium 2A (Kir2A) expression in Px4-2 cells. The electrophysiological properties of the insect Kir2A channel were successfully characterized for the first time. It was observed that the PxKir2A channel possesses typical inward rectifier potassium channel properties and can be inhibited by nanomolar concentrations of VU625 and VU590. This study offers a novel approach for the expression and investigation of foreign gene function in insect cells and provides a valuable tool for the in-depth study of key biomolecules in P. xylostella. Full article

Thiamine, crucial for energy metabolism, is associated with various human diseases when deficient. We studied how variations in the SLC19A3 gene, encoding THTR2, a thiamine transporter, may influence type 2 diabetes (T2DM) and gout (arthritis urica, AU). We characterized the SLC19A3 gene variants using bioinformatics and analyzed DNA samples from controls, T2DM, and gout patients to explore associations with physical/laboratory parameters. In human cells, we used a luciferase reporter assay to assess how these variants affect gene expression. We examined four large haplotypes (H1–4) in this gene, identified lead SNPs for the minor variants (MV), and explored potential transcription factor binding sites. We found that in T2DM patients, H3-MV correlated significantly with impaired glucose metabolism (pHOMA = 0.0189, pHbA1c% = 0.0102), while H4-MV correlated with altered uric acid (p = 0.0008) and white blood cell levels (p = 0.0272). In AU patients, H3-MV correlated with increased basophil granulocyte levels (p = 0.0273). In model cell lines, H3-MV presence increased gene expression (p = 0.0351), influencing responses to thiamine depletion and metformin (p = 0.0016). Although H4-MV did not directly affect luciferase expression, thiamine and fedratinib co-treatment significantly enhanced gene expression in thiamine-depleted cells (p = 0.04854). Our results suggest a connection between selected SLC19A3 variants and the severity of metabolic diseases or their response to treatment. Full article