Search Results (99)

Numerous modern scientific studies have demonstrated that animal venoms harbor a wealth of diverse anticancer active components, serving as a valuable resource for the development of natural antitumor drugs. AI-based computation and prediction models enable rapid screening of extensive active peptides. In this study, the anticancer activity of seven peptides was predicted using our previous deep learning model. Further verification experiments confirmed that Lpep3 can selectively and efficiently inhibit the growth of leukemia cells. Electron microscopy observations revealed cell shrinkage in morphology and honeycomb-like perforations on the cell membrane in the treated group. It is hypothesized that high-concentration peptides disrupt the cell membrane and increase cell permeability, which was confirmed by trypan blue staining and Calcein-AM/PI double-staining assays. Lpep3 induces the release of lactate dehydrogenase (LDH) and ATP in a concentration-dependent manner, further suggesting that this peptide disrupts the cell membrane. In addition, although Lpep3 does not affect the cell cycle of MV-4-11, it can induce cell apoptosis. Western blotting and RT-qPCR results showed that compared with the control group, the expression levels of Bax were upregulated, while the expression level of Bcl-2 protein was downregulated in the Lpep3 group. In vivo experiments demonstrated that Lpep3 has good biological safety, and compared with the control group, the Lpep3 group could inhibit the growth of tumor cells in mice. Collectively, Lpep3 is characterized by high potency and specificity and may serve as a promising lead compound for the development of anti-leukemia drugs. Full article

Background/Objectives: Melanoma is a highly malignant skin tumor with poor response to conventional chemotherapeutic regimens. Melanoma cells induce cytotoxic T cell-mediated immune responses, and immunotherapy has significantly improved survival rates for patients with advanced disease. Methods: Here, we investigate NK cell-mediated melanoma cell killing and its regulation by PD-L1/PD-1 blockade and IFNβ. Four melanoma cell lines were used in this study. To evaluate NK cell cytotoxicity, cells were exposed to NK cells with or without IFNβ. The calcein release assay was used to measure cell death, while specific inhibitors and siRNA silencing were applied to determine the contribution of individual effector pathways. Results: NK cells were able to kill melanoma cells with sensitivity to killing varying between different cell lines. Cytotoxic effects were mainly mediated through activation of the TRAIL signaling cascade. In cell lines with low sensitivity to NK cell killing, expression of PD-L1 was noted and killing by NK cells could be significantly increased by inhibition of the PD-L1/PD-1 checkpoint. Killing of melanoma cells could be further increased by incubation of NK cells with IFNβ. Conclusions: Our results point to a role of NK cells in the killing of melanoma cells and a potential clinical benefit of a combination therapy of IFNβ and anti-PD-1 antibody. Full article

Brassica yellows virus (BrYV) mainly infects cruciferous crops and has been widely prevalent across China. To develop a rapid and highly sensitive method for detecting BrYV in oilseed rape, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established. Four specific primers were designed to target the conserved gene of BrYV, with total RNA extracted from BrYV-infected oilseed rape leaves used as the template for the RT-LAMP assay. The optimal reaction conditions were determined, including a primer concentration ratio of 1:8, MgSO₄ concentration of 4 mM, reaction temperature of 64 °C, and a suitable reaction time of 60 min. Sensitivity analysis demonstrated that the RT-LAMP assay could detect total RNA at a concentration of 0.091 × 10⁻³ μg/μL, which was 100-fold more sensitive than conventional RT-PCR for BrYV detection. In addition to visualizing results by electrophoresis, the RT-LAMP assay could also be easily visualized using calcein-MnCl₂. These results indicate the potential of the established RT-LAMP assay for rapid BrYV detection in oilseed rape plants, which can provide better technical support for field diagnosis, disease forecasting, and the implementation of effective control strategies against the virus. Full article

Avian influenza viruses (AIV) cause severe economic losses in poultry production and pose zoonotic threats, necessitating rapid, field-deployable diagnostics. While real-time PCR is the gold standard, its use is limited in resource-constrained settings. This study aimed to develop and validate optimized loop-mediated isothermal amplification (LAMP) protocols for AIV detection directly at sample collection sites. We optimized Real-Time RT-LAMP and colorimetric LAMP assays targeting the conserved M gene, using primers described in the literature. Analytical sensitivity was assessed with a plasmid standard (10⁶–10⁰ copies/μL); specificity was evaluated against 27 AIV strains (H1–H12) and heterologous avian viruses (Newcastle disease, infectious bronchitis, Gumboro, and laryngotracheitis viruses). Reverse transcription was integrated into the LAMP reaction. Real-Time LAMP with SYBR Green achieved 100% analytical sensitivity (95% CI: 80–100; detection limit: Ct = 38), while colorimetric LAMP (cresol red, malachite green, calcein) detected 10² plasmid copies (Ct = 32) with 91.67% sensitivity (95% CI: 76.1–100). No cross-reactivity occurred. These optimized LAMP protocols offer sensitivity and specificity comparable to PCR, require minimal equipment, and enable rapid AIV screening, significantly enhancing early detection and epidemiological surveillance in field conditions. Full article

Lung cancer remains a leading cause of cancer-related mortality worldwide, where conventional chemotherapy is often limited by severe side effects and drug resistance. Ginsenosides, the primary bioactive triterpenoid saponins isolated from the root of Panax ginseng C. A. Mey, have demonstrated potential in combating non-small-cell lung cancer (NSCLC). However, their efficacy under nutrient-deficient conditions remains unclear. This study aimed to investigate the effects of ginsenosides on the growth and death of lung cancer cells under low-nutrient conditions and to explore the underlying mechanisms. A549 cells were divided into two groups: one cultured in 10% serum and another under serum-free conditions, followed by treatment with ginsenosides CK, Rh2(S), and Rg3(S) for 24 h. Cell proliferation and apoptosis were evaluated using a CCK-8 assay, Calcein/PI fluorescence staining, Hoechst 33258 staining, and flow cytometry. Potential targets and signaling pathways of ginsenosides were predicted using network pharmacology and bioinformatics analyses. The mRNA expression of key genes was measured by qRT-PCR, and mitochondrial membrane potential was assessed using JC-1 staining. The results showed that ginsenosides induced dose-dependent apoptosis in serum-starved A549 cells. Bioinformatics analysis suggested the involvement of the PI3K/Akt/FoxO signaling pathway, which was supported by decreased Akt mRNA levels and increased FoxO mRNA expression. Furthermore, mRNA levels of Bim, Caspase-3, Caspase-8, and Caspase-9 were significantly upregulated, accompanied by a loss of mitochondrial membrane potential. These findings indicate that under serum deprivation, ginsenosides enhance apoptosis in A549 cells, likely through the regulation of the PI3K/Akt/FoxO pathway. Full article

This study investigates the scavenger activity of Polynucleotide High Purification Technology (PN HPT^TM), alone or in combination with hyaluronic acid (PN HPT^TM + HA) against oxidative stress induced by hydrogen peroxide (H₂O₂). Since oxidative stress is implicated in numerous pathological conditions, identifying effective antioxidants is crucial for therapeutic development. We employed a cell-free fluorometric assay based on Calcein-AM, a fluorescence probe whose signal increases proportionally to the generation of reactive oxygen species (ROS), to evaluate the ability to neutralize ROS under varying oxidative stress conditions and determine the dose- and time-dependent effects of these compounds. PN HPT^TM, HA, and PN HPT^TM + HA were tested at various concentrations over multiple time points. Our results demonstrated that all tested treatments significantly lowered ROS levels compared to the untreated control. Notably, the PN HPT^TM -based compounds exhibited robust scavenging activity, with PN HPT^TM + HA displaying the strongest and most consistent ROS-neutralizing effect across all concentrations and time points. This enhanced performance suggests a synergistic interaction between PN HPT^TM and HA, potentially due to complementary mechanisms of free radical scavenging and structural stabilization. These findings highlight the potential of PN HPT^TM and PN HPT^TM + HA as effective antioxidative agents, offering potential for therapeutic applications where oxidative stress is central, including wound healing and tissue regeneration. Full article

Although acetaminophen (APAP) overdose represents the predominant cause of drug-induced acute liver failure (ALF) worldwide and has been extensively studied, the modes of cell death remain debatable and the treatment approach for APAP-induced acute liver failure is still limited. This study investigated the mechanisms of APAP hepatotoxicity in primary mouse hepatocytes (PMHs) by using integrated methods (MTT assay, HPLC analysis for glutathione (GSH), Calcein-AM for labile iron pool detection, confocal microscopy for lipid peroxidation and mitochondrial superoxide measurements, electron microscopy observation, and Western blot analysis for ferritin), focusing on the role of iron dysregulation under oxidative stress. Our results showed that 20 mM APAP treatment induced characteristic features of ferroptosis, including GSH depletion, mitochondrial dysfunction, and iron-dependent lipid peroxidation. Further results showed significant ferritin degradation and subsequent iron releasing. Iron chelator deferoxamine (DFO) and N-acetylcysteine (NAC) could alleviate APAP-induced hepatotoxicity, while autophagy inhibitors did not provide a protective effect. In vitro experiments confirmed that hydrogen peroxide directly damaged ferritin structure, leading to iron releasing, which may aggravate iron-dependent lipid peroxidation. These findings provide evidence that APAP hepatotoxicity involves a self-amplifying cycle of oxidative stress and iron-mediated oxidative damaging, with ferritin destruction playing a key role as a free iron source. This study offers new insights into APAP-induced liver injury beyond conventional cell death classifications, and highlights iron chelation as a potential therapeutic strategy alongside traditional antioxidative treatment with NAC. Full article

Background/Objectives: P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, plays a key role in multidrug resistance by actively exporting chemotherapeutic agents and xenobiotics from cells. Overexpression of P-gp significantly reduces intracellular drug accumulation and compromises treatment efficacy. Despite extensive research, clinically approved P-gp inhibitors remain elusive due to toxicity, poor specificity, and limited efficacy. This study investigates DMH1, a selective type I BMP receptor inhibitor, as a novel P-gp inhibitor. Methods: DMH1 cytotoxicity was assessed in P-gp-overexpressing (PC3-TxR, K562/Dox) and P-gp-deficient (PC3) cell lines using MTT assays. P-gp inhibition was evaluated using calcein AM retention and daunorubicin (DNR) accumulation assays. Kinetic analysis determined DMH1’s effect on P-gp-mediated transport (Vmax and Km). ATPase activity assays were performed to assess DMH1’s impact on ATP hydrolysis. Preliminary molecular docking (CB-Dock2) was used to predict DMH1’s binding site on the human P-gp structure (PDB ID: 6QEX). Results: DMH1 showed no cytotoxicity in P-gp-overexpressing or deficient cells. It significantly enhanced intracellular accumulation of Calcein AM and DNR, indicating effective inhibition of P-gp function. Kinetic data revealed that DMH1 reduced Vmax without affecting Km, consistent with noncompetitive, allosteric inhibition. DMH1 also inhibited ATPase activity in a dose-dependent manner. Docking analysis suggested DMH1 may bind to an allosteric site in the transmembrane domain, potentially stabilizing the inward-facing conformation. Conclusions: DMH1 is a promising noncompetitive, allosteric P-gp inhibitor that enhances intracellular drug retention without cytotoxicity, supporting its potential as a lead compound to overcome multidrug resistance and improve chemotherapeutic efficacy. Full article

Detection of Salmonella, a highly diverse foodborne pathogen, is paramount to ensure safety and protection of the animal industry and its consumers. Salmonella enterica serovar Typhimurium is among the most important non-typhoidal serovars causing gastroenteritis worldwide. However, traditional serovar identification is labor- and resource-intensive, while typical molecular tools require expensive reagents and equipment. Hence, this study developed and optimized a calcein-based and closed-tube loop-mediated isothermal amplification (LAMP)-based assay to detect S. Typhimurium following enrichment steps compared with an optimized PCR assay. The PCR assay showed 100% specificity in silico confirmed through DNA sequencing. For actual specificity testing, both PCR and LAMP showed 100% specificity to S. Typhimurium. For DNA sensitivity, while PCR showed a limit of detection of 22 pg/μL, LAMP showed a 100-fold higher sensitivity at 220 fg/μL. Meanwhile, for pure culture sensitivity, both assays detected at least 4.98 × 10⁴ CFU/mL. Parallel testing of 208 raw meat samples from wet markets in Metro Manila, Philippines, showed corroboration and statistical association of the optimized PCR and LAMP with 89.42% and 90.87% positivity rates for S. Typhimurium, respectively. Hence, the developed closed-tube and calcein-based LAMP assay is potentially a powerful yet simple, sensitive, and fast method for S. Typhimurium detection. Full article

Background: Multidrug resistance (MDR) in triple-negative breast cancer (TNBC) leads to treatment failure and tumor recurrence. Dysregulation of the MYC oncogene is associated with the pathogenesis of TNBC and the development of chemoresistance via overexpression of ATP-binding cassette (ABC) transporters. Therefore, in the present study, we aimed to identify molecules from a natural product origin that prevent the development of MDR in TNBC by targeting the MYC signaling. Methods: The cell viability of TNBC was evaluated using sulforhodamine assay. Protein levels were detected by western blots or enzyme-linked immunosorbent assays. Intracellular calcein and hoechst33342 accumulation assay aimed to evaluate the inhibitory ability of phytocompounds on drug-efflux functions of ABCB1 and ABCG2 transporters. The Cancer Genome Atlas (TCGA) database was used to explore clinical genomic data. Furthermore, the zebrafish xenotransplantation model bearing Dil-labeled TNBC cells was applied to testify the in vivo effects of phyto-sesquiterpene lactones. Results: The results of the present study demonstrated that the phyto-sesquiterpene lactones exhibited an MDR prevention effect by repressing efflux activities of ABCB1 and ABCG2 transporters. Mechanistic studies showed that phyto-sesquiterpene lactones inducted TNBC cell apoptosis and cell cycle G2/M arrested by blocking the STAT3/MYC pathway. Clinical genomic data demonstrated that the percentages of MYC amplification and mRNA were upregulated approximately two-fold higher in the TNBC patients than the non-TNBC breast cancer patients. The survival of patients with an alteration in MYC was significantly lower in TNBC as compared to other subtypes. Moreover, the results of the zebrafish xenograft model confirmed that phyto-sesquiterpene lactones exerted stronger inhibitory effects on TNBC tumor growth in vivo. Conclusions: In conclusion, these three phyto-sesquiterpene lactones were promising candidates for TNBC treatment and shed light on the prevention of developing MDR TNBC. Full article

Bergamot [Citrus × limon (L.) Osbeck, syn. C. × bergamia (Risso) Risso & Poit.] is primarily cultivated in the Calabria region of Italy and exploited in the food and perfumery industry. The epicarp of its fruit is a rich source of essential oil (BEO) containing mainly monoterpenes, which are known for their diverse biological activities, including antimicrobial, anti-inflammatory, antiproliferative, and neuromodulatory effects. Emerging evidence suggests that oxidative stress plays a central role in the pathogenesis of neurodegenerative diseases, particularly Alzheimer’s disease (AD), where it contributes to neuronal dysfunction and cell death. Moreover, heavy metal exposure has been identified as a key environmental factor exacerbating oxidative stress and neurodegeneration in AD. This study aimed to explore whether BEO could mitigate heavy metal (Cd²⁺, Hg²⁺, and Pb²⁺)-induced neurotoxicity in SH-SY5Y cells, a model system for brain cells. MTT and calcein-AM assays were performed to examine the viability of the SH-SY5Y cells after exposure to each heavy metal itself, or in combination with BEO, whereas the LDH assay was carried out to determine the effects of BEO towards necrotic cell death induced by heavy metals. Furthermore, DCFH-DA was performed to determine whether BEO could protect SH-SY5Y from heavy metal-induced oxidative stress. This study also investigated the antibacterial properties of BEO on different Gram-positive and Gram-negative bacterial strains belonging to the ATCC collection. These results suggest that BEO may help counteract heavy metal-induced neuronal damage, particularly Cd²⁺ toxicity, potentially reducing one of the environmental risk factors associated with AD. Additionally, its antimicrobial properties reinforce its relevance in preventing infections that may contribute to neuroinflammation in AD. Full article

Chemotherapy resistance is a significant barrier to effective cancer treatment. A key mechanism of resistance at the single-cell level is the overexpression of drug transporters in the ABC family, particularly P-glycoprotein (P-gp), which leads to multidrug resistance (MDR). Inhibitors of these transporters can help re-sensitize cancer cells to chemotherapeutics. This study evaluated elacridar (GG918 and GF120918), a potent third-generation P-gp inhibitor, for its ability to reverse MDR in paclitaxel (PAC)-resistant ovarian cancer cell lines. Sensitive and PAC-resistant cells were cultured in two-dimensional (2D) and three-dimensional (3D) models. MDR1 gene expression was analyzed using Q-PCR, and P-gp protein expression was examined via Western blot and immunofluorescence. Drug sensitivity was evaluated with MTT assays, and P-gp activity was analyzed by flow cytometry and fluorescence microscopy. Elacridar effectively inhibited P-gp activity and increased sensitivity to PAC and doxorubicin (DOX) in 2D cultures but not cisplatin (CIS). In 3D spheroids, P-gp activity inhibition was observed via Calcein-AM staining. However, no re-sensitization to PAC occurred and limited improvement was observed for DOX. These findings suggest that elacridar effectively inhibits P-gp in both 2D and 3D conditions. However, its ability to overcome drug resistance in 3D models is limited, highlighting the complexity of tissue-specific resistance mechanisms. Full article

Background/Objectives: This study investigates the structural and biophysical properties of the wild-type antimicrobial peptide LyeTx I, isolated from the venom of the spider Lycosa erythrognatha, and its analog LyeTx I-b, designed to enhance antibacterial activity, selectivity, and membrane interactions by the acetylation and increased amphipathicty. Methods: To understand the mechanisms behind these enhanced properties, comparative analyses of the structural, topological, biophysical, and thermodynamic aspects of the interactions between each peptide and phospholipid bilayers were evaluated. Both peptides were isotopically labeled with ²H₃-Ala and ¹⁵N-Leu to facilitate structural studies via NMR spectroscopy. Results: Circular dichroism and solid-state NMR analyses revealed that, while both peptides adopt α-helical conformations in membrane mimetic environments, LyeTx I-b exhibits a more amphipathic and extended helical structure, which correlates with its enhanced membrane interaction. The thermodynamic properties of the peptide–membrane interactions were quantitatively evaluated in the presence of phospholipid bilayers using ITC and DSC, highlighting a greater propensity of LyeTx I-b to disrupt lipid vesicles. Calcein release studies reveal that both peptides cause vesicle disruption, although DLS measurements and TEM imaging indicate distinct effects on phospholipid vesicle organization. While LyeTx I-b permeabilizes anionic membrane retaining the vesicle integrity, LyeTx I promotes significant vesicle agglutination. Furthermore, DSC and calcein release assays indicate that LyeTx I-b exhibits significantly lower cytotoxicity toward eukaryotic membranes compared to LyeTx I, suggesting greater selectivity for bacterial membranes. Conclusions: Our findings provide insights into the structural and functional modifications that enhance the antimicrobial and therapeutic potential of LyeTx I-b, offering valuable guidance for the design of novel peptides targeting resistant bacterial infections and cancer. Full article

In this study, the cytotoxic effects of brake wear particles (≥250 nm ceramic/ceramic wear particles (CCWPs) and ≤100 nm ceramic/steel wear particles (CSWPs)) and 100 nm iron (III) oxide ultrafine particles (IOUFPs) on human lung carcinoma (A549) and Chinese hamster ovary (CHO) cells were investigated. Cell viability was determined using the MTT and Calcein AM methods. Oxidative stress was assessed by measuring reactive oxygen species (ROS), intracellular reduced glutathione (GSH), and malondialdehyde (MDA) concentrations under exposure to the above particles in the concentration range of 10–80 µg/mL. The initial assessments of CCWPs and CSWPs on the cell viability were performed after a 4-h exposure but later extended to 24 h to investigate the time-dependent of the cell viability and oxidative stress. MTT and Calcein AM assays indicated that the A549 cells are less susceptible to CCWPs and CSWPs than the CHO cells when exposed for both 4 h and 24 h. This study highlights that oxidative stress induced by CCWPs, CSWPs, and IOUFPs is cell-specific. While CCWPs did not affect glutathione (GSH) levels in the CHO cells, it significantly reduced GSH levels in A549 cells, with the exception of 80 µg/mL. Both CCWPs and CSWPs increased the lipid peroxidation in both cell types; however, the A549 cells demonstrated lower sensitivity to these treatments. Full article

Parkinson’s disease (PD) is the second most common neurodegenerative disorder globally that lacks any disease-modifying drug for prevention or treatment. Oxidative stress has been identified as one of the key pathogenic drivers of Parkinson’s disease (PD). Edaravone, an approved free-radical scavenger, has proven to have potential against PD by targeting multiple key pathologies, including oxidative stress, focal mitochondria, and neuroinflammation. However, its bioavailability is potentially restricted due to its poor solubility and short half-life. This study aims to develop a simple and effective drug delivery system for edaravone to enhance its solubility, stability, and bioavailability to improve its neuroprotective efficacy. An MPEG-2000-DSPE-edaravone (MDE) micelle was prepared via solvent evaporation using MPEG-2000-DSPE as a carrier to encapsulate edaravone. The morphology, particle size, zeta potential, chemical structure, and edaravone loading of MDE were evaluated. We then investigated whether such targeted edaravone delivery could provide enhanced neuroprotection. A cell model of PD was established in PC12 cells through exposure to rotenone. The effects of MDE on PC12 cells treated with or without rotenone were evaluated using a cell counting kit-8, calcein acetoxymethyl ester (AM)–propidine iodide (PI) staining, and flow cytometry. Cell migration was evaluated using a wound healing assay. Additionally, the intracellular antioxidant study was performed using an ROS-level-detecting DCFH-DA probe, and the mitochondrial membrane potentials were evaluated using a JC-1 assay. MDE with a drug-loading content of 17.6% and an encapsulation efficiency of 92.8% was successfully prepared. The resultant MDE had a mean particle size of 112.97 ± 5.54 nm with a zeta potential of −42 mV. Cytotoxicity assays confirmed that the MDE (≤200 ug/mL) exhibited promising cytocompatibility with no significant effect on cell viability, cell cycle regulation, or apoptosis levels. Likewise, compared with the free edaravone, no effect on cell migration was noted for MDE. MDE might be able to target edaravone delivery into PC12 cells, increasing the mitochondrial membrane potential and providing a significant local antioxidant effect. The results demonstrated that MPEG-2000-DSPE could be a promising material for enhancing edaravone’s aqueous solubility, stability, and antioxidant effects. MDE could be a potential drug formulation for treating PD and other diseases in which oxidative stress plays a key role in pathogenesis. Full article