Search Results (6,639)

Among all stroke types, ischemic stroke (IS) occurs most frequently, resulting in neuronal death and tissue injury within both the central infarct region and surrounding areas. This study explored the neuroprotective mechanisms of scutellarin, a flavonoid compound, through an integrated strategy that merged in silico analyses (including network pharmacology and molecular docking simulations) with both in vitro and in vivo experimental verification. We identified 1887 IS-related targets and 129 scutellarin targets, with 23 overlapping targets. PPI network analysis revealed five core targets, and molecular docking demonstrated strong binding affinities between scutellarin and these targets. Bioinformatic analyses, including GO functional annotation and KEGG pathway mapping, indicated that the PI3K/AKT cascade represents the primary signaling mechanism. An in vitro experimental system was developed using PC12 cells under oxygen-glucose deprivation conditions to investigate how scutellarin regulates neuronal cell death via the PI3K/AKT pathway. Western blot quantification demonstrated that treatment with scutellarin enhanced the expression of p-PI3K, p-AKT, and Bcl-2 proteins, while simultaneously reducing levels of apoptotic markers Bax and cleaved caspase-3. Furthermore, pharmacological intervention with the selective PI3K inhibitor LY294002 attenuated these molecular alterations, resulting in diminished expression of p-PI3K, p-AKT, and Bcl-2, accompanied by elevated levels of Bax and cleaved caspase-3. In a rat model of middle cerebral artery occlusion, scutellarin administration demonstrated comparable neuroprotective effects, maintaining neuronal survival and modulating apoptotic protein expression via PI3K/AKT pathway activation. Collectively, this study demonstrates the therapeutic potential of scutellarin in cerebral ischemia through PI3K/AKT pathway modulation, suggesting its possible application in treating ischemic disorders. Full article

Two new Ru(II) complexes, mononuclear [RuCl₂(η⁶-p-cymene)(3,4-dmph-κN)] (1) and the binuclear complex [{RuCl(η⁶-p-cymene)}₂(μ-Cl)(μ-3,4-dmph-κ²N,N′)]Cl (2; 3,4-dmph = 3,4-dimethylphenylhydrazine), are synthesized and experimentally and theoretically structurally characterized utilizing ¹H and ¹³C NMR and FTIR spectroscopy, as well as DFT calculations. Degradation product of 2, thus ([{RuCl(η⁶-p-cymene)}₂(μ-Cl)(μ-3,4-dmph-κ²N,N′)][RuCl₃(η⁶-p-cymene)] (2b) was characterized with SC-XRD. In the crystals of 2b, the cationic and anionic parts interact through N-H^...Cl hydrogen bridges. The spectrofluorimetric measurements proved the spontaneity of the binding processes of both complexes and HSA. Spin probing EPR measurements implied that 1 and 2 decreased the amount of bound 16-doxylstearate and implicated their potential to bind to HSA more strongly than the spin probe. The cytotoxicity assessment of both complexes against the MDA-MB-231 and MIA PaCa-2 cancer cell lines demonstrated a clear dose-dependent decrease in cell viability and no effect on healthy HS-5 cells. Determination of the malondialdehyde and protein carbonyl concentrations indicated that new complexes could offer protective antioxidant benefits in specific cancer contexts. Gel electrophoresis measurements showed the reduction in MMP9 activity and indicated the potential of 1 in limiting the cancer cells’ invasion. The annexin V/PI apoptotic assay results showed that 1 and 2 exhibit different selectivity towards MIA PaCa-2 and MDA-MB-231 cancer cells. A comparative molecular docking analysis of protein binding, specifically targeting acetylcholinesterase (ACHE), matrix metalloproteinase-9 (MMP-9), and human serum albumin (HSA), demonstrated distinct binding interactions for each complex. Full article

Perfluorooctanoic acid (PFOA) is one of the most persistent and bioaccumulative water contaminants. Sensitive, rapid, and in-field analysis is needed to ensure safe water supplies. Here, we present a single step (one shot) and rapid sensor capable of measuring PFOA at the sub-quadrillion (ppq) level, 4.5 × 10⁻⁴ ppq, within 10 s. This innovative sensor employs a synergistic combination of a molecularly imprinted polymer (MIP)-modified gold interdigitated microelectrode chip and AC electrothermal effects (ACETs), which enhance detection sensitivity by facilitating the accelerated movement of PFOA molecules towards specific recognition sites on the sensing surface. The application of a predetermined AC signal induces microfluidic enrichment and results in concentration-dependent changes in interfacial capacitance during the binding process. This enables real-time, rapid quantification with exceptional sensitivity. We achieved a linear dynamic range spanning from 0.4 to 40 fg/L (4 × 10⁻⁷–4 × 10⁻⁵ ppt) and demonstrated good selectivity (~1:100) against other PFAS compounds, including perfluorooctanoic acid (PFOS), in PBS buffer. The sensor’s straightforward operation, cost-effectiveness, elimination of the need for external redox probes, compact design, and functionality in relatively resistant environmental matrices position it as an outstanding candidate for deployment in practical applications. Full article

G protein-coupled receptors (GPCRs) constitute the largest and most frequently used family of molecular drug targets. The simplicity of GPCR drug design results from their common seven-transmembrane-helix topology and well-understood signaling pathways. GPCRs are extremely sensitive to slight changes in the chemical structure of compounds, which allows for the reliable design of highly selective and specific drugs. Only recently has the number of GPCR structures, both in their active and inactive conformations, together with their active ligands, become sufficient to comprehensively apply machine learning in decision support systems to predict compound activity in drug design. Here, we describe GPCRVS, an efficient machine learning system for the online assessment of the compound activity against several GPCR targets, including peptide- and protein-binding GPCRs, which are the most difficult for virtual screening tasks. As a decision support system, GPCRVS evaluates compounds in terms of their activity range, the pharmacological effect they exert on the receptor, and the binding mode they could demonstrate for different types and subtypes of GPCRs. GPCRVS allows for the evaluation of compounds ranging from small molecules to short peptides provided in common chemical file formats. The results of the activity class assignment and the binding affinity prediction are provided in comparison with predictions for known active ligands of each included GPCR. Multiclass classification in GPCRVS, handling incomplete and fuzzy biological data, was validated on ChEMBL and Google Patents-retrieved data sets for class B GPCRs and chemokine CC and CXC receptors. Full article

The selection of solvent is pivotal in liquid-phase exfoliation (LPE), as an ideal solvent minimizes the energy required to disrupt the interlayer attractive interactions while stabilizing exfoliated layers to prevent re-agglomeration. This study theoretically analyzed the LPE of Mg(OH)₂ in different solvents, including water, dimethyl sulfoxide (DMSO), dimethylformamide, N-methyl-2-pyrrolidone (NMP), isopropyl alcohol, and 2-butanone, through first-principles calculations combined with experimental validation. DMSO was identified as the most effective solvent for reducing the interlayer attraction, based on exfoliation energy calculations, while NMP was the most efficient for stabilizing exfoliated layers, based on binding energy assessments. Principal component analysis of the solvents’ physicochemical properties reduced the original dataset of seven variables to two dominant factors. The binding energy correlates with planarity and polarity, whereas the exfoliation energy is governed by dipole moment and polarity. The biaxial straining theory successfully clarified the underlying mechanisms behind the established criteria for selecting the optimal solvent. Experimental results confirmed that DMSO outperforms water in the LPE of Mg(OH)₂. These results establish a quantitative framework for solvent selection, enhancing the efficiency of the LPE processes. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by a gradual decline in cognitive function. Currently, there are no effective treatments for this condition. Medicine food homology plants have gained increasing attention as potential natural treatments for AD because of their nutritional value and therapeutic benefits. In this work, we aimed to provide a deeper understanding of how medicine food homology plants may help alleviate or potentially treat AD by identifying key targets, pathways, and small molecule compounds from 10 medicine food homology plants that play an important role in this process. Using network pharmacology, we identified 623 common targets between AD and the compounds from the selected 10 plants, including crucial proteins such as STAT3, IL6, TNF, and IL1B. Additionally, the small molecules from the selected plants were grouped into four clusters using hierarchical clustering. The ConPlex algorithm was then applied to predict the binding capabilities of these small molecules to the key protein targets. Cluster 3 showed superior predicted binding capabilities to STAT3, TNF, and IL1B, which was further validated by molecular docking. Scaffold analysis of small molecules in Cluster 3 revealed that those with a steroid-like core—comprising three fused six-membered rings and one five-membered ring with a carbon–carbon double bond—exhibited better predicted binding affinities and were potential triple-target inhibitors. Among them, MOL005439, MOL000953, and MOL005438 were identified as the top-performing compounds. This study highlights the potential of medicine food homology plants as a source of active compounds that could be developed into new drugs for AD treatment. However, further pharmacokinetic studies are essential to assess their efficacy and minimize side effects. Full article

Zearalenone (ZEN), a toxic estrogenic mycotoxin in cereals, threatens human and animal health through reproductive, immune, and cytotoxic effects, necessitating sensitive detection methods. While nanobodies offer advantages over conventional antibodies for on-site ZEN detection, their application remains unexplored. This study aimed to develop an anti-ZEN nanobody derived from an anti-ZEN phage display nanobody library. An alpaca was immunized with a ZEN-bovine serum albumin (ZEN-BSA) antigen, achieving peak serum antibody titers (1:25,600) following four immunizations. A high-capacity phage display nanobody library (1.0 × 10¹¹ plaque-forming units/mL) was constructed. Following four rounds of biopanning, an enrichment factor of 479 was achieved. Phage ELISA screening identified six phage display nanobodies with specific ZEN-binding activity, and multiple sequence alignment revealed four unique nanobody sequences. The selected phage display nanobody, designated phage-V44, was expressed and purified, and its presence was validated by SDS-PAGE and western blotting, which detected a single approximately 17 kDa band consistent with the expected nanobody size. We established a working curve for an indirect competitive enzyme-linked immunoassay (ELISA) for ZEN, which showed an IC50 value of 7.55 ng/mL. The specificity and affinity of the V44 were also verified. Collectively, the study successfully constructed an anti-ZEN phage display nanobody library, screened four specific ZEN-binding phage display nanobodies, and prepared the anti-ZEN nanobody V44. Thereby establishing a foundation for the nanobody’s future integration into rapid on-site detection methods for ZEN in both animal feed and human food products. Full article

Natural killer (NK) cell-derived exosomes (NK-Exos) are emerging as a promising avenue in cancer immunotherapy due to their inherent tumor-targeting properties and their capacity to deliver therapeutic agents directly to malignant cells. This research delves into the boosted anti-tumor potency of NK-Exos that has been genetically enhanced to overexpress NKG2D, a vital activating receptor, along with interleukin-24 (IL24), a cytokine renowned for its selective suppressive impact on tumor cells. NKG2D facilitates the recognition of tumor cells by binding to stress-induced ligands, while IL24 induces apoptosis and modulates immune responses to enhance tumor destruction. The NK-Exos engineered to express both NKG2D and IL24 significantly enhanced tumor targeting and increased the apoptosis rate of tumor cells by 30% in A549 and by 20% in HELA at 48 h compared with non-modified NK-Exos, respectively. Furthermore, this enhancement also impacted cell proliferation, with inhibition rates increasing by 30%, 15%, and 15% in A549, HELA, and MCF-7 cells, respectively, and it reduced A549 cell migration by 10%. The integration of NKG2D and IL24 within NK-Exos confers a dual therapeutic mechanism, synergistically amplifying their efficacy in cancer treatment. The utility of NK-Exos co-expressing NKG2D and IL24 offers a novel approach to overcome the limitations of current therapies, providing prolonged tumor suppression and precise targeting of malignant cells and holding great promise for clinical application. Full article

Background/Objectives: Porcine reproductive and respiratory syndrome virus (PRRSV) is classified into various lineages based on the phylogenetic variation of orf5, which encodes a major surface glycoprotein GP5 containing both neutralizing and non-neutralizing linear epitopes. Several positively selected sites have been identified on the GP5 ectodomain, indicating host immune pressure on these sites. This present study aimed to investigate the kinetics of antibody responses to GP5 and to map the epitope-specific response to the GP5 ectodomain from different PRRSV lineages after vaccination with commercially available modified live virus (MLV) vaccines. Methods: Post-weaning pigs were vaccinated with MLV vaccines derived from either lineage 1D (Prevacent PRRS^®) or lineage 5 (Ingelvac PRRS^®). Animals were challenged with a heterologous (lineage 1A) strain at 64 days post-vaccination (dpv). Blood samples were collected at various times post-vaccination and challenge. Kinetics of antibody response to different PRRSV antigens were monitored and virus neutralization against archetypal and contemporary strains belonging to lineage 5 and 1A were evaluated. In addition, antibody responses to peptides derived from the GP5 ectodomain of different viral lineages were assessed. Results: Our results showed that the GP5-specific antibody response observed between 18 and 35 dpv was delayed compared to responses to the viral nucleocapsid protein. The polyclonal antibody response in both vaccinated groups showed similar levels of binding to variant GP5 peptides from different sub-lineages. Notably, in both vaccinated groups, the antibody directed to a peptide representing the GP5 ectodomain of a lineage 1C strain (variant 1C.5) displayed a rise in titer at 64 dpv, which was further increased by the challenge with the lineage 1A strain. Less than 50% of animals developed heterologous neutralizing antibodies post-vaccination with both MLV vaccines. However, higher neutralization titers were observed in all vaccinated animal post-challenge. Conclusions: Together, these data provide insights into the antibody responses to the GP5 ectodomain in MLV-vaccinated swine herds. Full article

Cancer remains a leading cause of death worldwide, highlighting the urgent need for novel and more effective treatments. Natural products, with their structural diversity, represent a valuable source for the discovery of anticancer compounds. In this study, we screened 750 Antarctic extracts to identify potential inhibitors of human topoisomerase 1 (hTOP1), a key enzyme in DNA replication and repair, and a target of cancer therapies. Bioassay-guided fractionation led to the identification of palmitic acid (PA) as the active compound from the Antarctic sponge Artemisina plumosa, selectively inhibiting hTOP1. Our results demonstrate that PA irreversibly blocks hTOP1-mediated DNA relaxation and specifically inhibits the DNA religation step of the enzyme’s catalytic cycle. Unlike other fatty acids, PA exhibited unique specificity, which we confirmed through comparisons with linoleic acid. Molecular dynamics simulations and binding assays further suggest that PA interacts with hTOP1-DNA complexes, enhancing the inhibitory effect in the presence of camptothecin (CPT). These findings identify PA as a hTOP1 inhibitor with potential therapeutic implications, offering a distinct mechanism of action that could complement existing cancer therapies. Full article

Although peptides have been shown to have biological functions in neurodegenerative diseases, their role in Parkinson’s disease has been understudied. A previous study by our group, which used a 6-hydroxydopamine zebrafish model, suggested that nine intracellular peptides may play a part in this condition. In this context, our aim is to better understand the role of five of these nine peptides. The selection of peptides was made based on their precursor proteins, which are fatty acid binding protein 7, mitochondrial ribosomal protein S36, MARCKS-related protein 1-B, excitatory amino acid transporter 2 and thymosin beta-4. The peptides were chemically synthesized in solid phase and characterized by high-performance liquid chromatography and mass spectrometry. Circular dichroism was performed to determine the secondary structure of each peptide, which showed that all five peptides maintain a random structure in the aqueous solutions that were studied. Two molecules show a helical profile in trifluoroethanol, a known structuring agent. Cell viability by the MTT assay indicates that all five peptides are not cytotoxic in all concentrations tested in both mouse and human cell lines. Behavioral assay using a 6-OHDA zebrafish larvae model suggest that all peptides help in the recovery of motor function with 24 h treatment at two concentrations. Three peptides showed a complete recovery from the 6-OHDA-induced motor impairment. Further studies are needed to better understand the mechanism of action of these peptides and whether they are truly a potential ally against Parkinson’s disease. Full article

Background/Objectives: Fragile X syndrome (FXS) is a disease of pathologic epigenetic silencing induced by RNA. In FXS, an expanded CGG repeat tract in the FMR1 gene induces epigenetic silencing during embryogenesis. FMR1 silencing can be reversed with 5-aza-deoxyctidine (5-aza-dC), a nonspecific epigenetic reactivator; however, continuous administration of 5-aza-dC is problematic due to its toxicity. We describe an approach to restore FMR1 expression in FXS neurons by transient treatment with 5-aza-dC, followed by treatment with 2HE-5NMe, which binds the CGG repeat expansion in the FMR1 mRNA and could block the resilencing of the FMR1 gene after withdrawal of 5-aza-dC. Methods: This study uses immunofluorescence and fluorescent in situ hybridization (FISH) to measure whether FMR1 expression is maintained in FXS post-mitotic neurons treated with 2HE-5NMe. Genome-wide profiling of histone marks was used to monitor epigenetic changes and drug selectivity in response to 5-aza-dC followed by 2HE-5NMe treatment. Changes to dendritic morphology were visualized using confocal microscopy. Results: In this study, we find that 2HE-5Nme maintains FMR1 in a reactivated state after reactivation using 5-aza-dC in post-mitotic neurons. FMR1 reactivation in neurons results in the re-expression of FMRP and reversal of FXS-associated dendritic spine defects. Conclusions: These results demonstrate that an RNA-binding small molecule can achieve gene-specific epigenetic control and provide an approach for the restoration of FMRP in FXS neurons. Full article

Background/Objectives: Potency testing of clinical-grade lentiviral vectors (LVVs) is critical to support a drug’s commercial approval. Careful consideration should be paid to the development of a suitable potency test during the drug’s clinical development. We aimed to develop an affordable, quantitative test for our CAR19-LVV, based on a measure of transgene’s functional activity. Methods: Several indicators of functional activity of CAR19-LVV were explored in a co-culture setting of CAR-transduced Jurkat cells and CD19-expressing target cells. The selected assay was further developed and subjected to validation. Assay’s adaptability to other CAR-encoding LVV and autologous CAR-T cell products was also investigated. Results: Measure of CD69 expression on the membrane of Jurkat-CAR-expressing cells is a specific indicator of CAR functionality. Quantification of CD69 in terms of mean fluorescence intensity (MFI), coupled with an intra-assay standard curve calibration, allows for a quantitative assay with high precision, specificity, robustness, linearity and accuracy. The assay has also shown optimal performance for a CARBCMA-LVV product. Importantly, we show that in primary T cells, CD69 expression reflects CAR-T cell cytotoxicity. After adaptation, we have applied a CD69-based potency test, with simultaneous measurement of CAR-T cell cytotoxicity, to autologous CAR-T cell products, demonstrating the assay’s specificity also in this context. Conclusions: We developed a validated, in vitro cell-based potency test, using a quantitative flow-cytometry method, for our CAR19-LVV. The assay is based on the detection of T-cell activation upon CAR binding to antigen, which is a measure of transgene functionality. The assay was easily adapted to another CAR-encoding LVV, targeting a different molecule. Furthermore, the same assay principle can be applied in the context of autologous CAR-T cell products. The quantitative CD69 potency assay shows reduced variability among autologous products compared to the IFNγ assay and allows for simultaneous evaluation of traditional semi-quantitative cytotoxicity, thereby directly evaluating the drug’s mechanism of action (MoA) in the same assay. Full article

G-protein coupled receptors (GPCRs) are the largest family of membrane proteins engaged in transducing signals from the extracellular environment into the cell. GPCR-biased signaling occurs when two different ligands, sharing the same binding site, induce distinct signaling pathways. This selective signaling offers significant potential for the design of safer and more effective drugs. Although its molecular mechanism remains elusive, big efforts are made to try to explain this mechanism using a wide range of methods. Recent advances in computational techniques and AI technology have introduced a variety of simulations and machine learning tools that facilitate the modeling of GPCR signal transmission and the analysis of ligand-induced biased signaling. In this review, we present the current state of in silico approaches to elucidate the structural mechanism of GPCR-biased signaling. This includes molecular dynamics simulations that capture the main interactions causing the bias. We also highlight the major contributions and impacts of transmembrane domains, loops, and mutations in mediating biased signaling. Moreover, we discuss the impact of machine learning models on bias prediction and diffusion-based generative AI to design biased ligands. Ultimately, this review addresses the future directions for studying the biased signaling problem through AI approaches. Full article

A biosensor was designed for detecting Pseudomonas aeruginosa (P. aeruginosa) bacteria in whole milk samples. The sensing layer involved the antifouling linking molecule 3-(2-mercaptoethanoxy)propanoic acid (HS-MEG-COOH), which was covalently linked to an aptamer for binding P. aeruginosa. The aptasensor uses the thickness shear mode (TSM) system for mass-sensitive acoustic sensing of the bacterium. High concentrations (10⁵ CFU mL⁻¹) of nonspecific bacteria, E. coli, S. aureus, and L. acidophilus, were tested with the aptasensor and caused negligible frequency shifts compared to P. aeruginosa. The aptasensor has high selectivity for P. aeruginosa, with an extrapolated limit of detection (LOD) of 86 CFU mL⁻¹ in phosphate-buffered saline (PBS) and 157 CFU mL⁻¹ in milk. To improve the sensitivity of the sensor, gold nanoparticles (AuNPs) were functionalized with the same aptamer for P. aeruginosa and flowed through the sensor following bacteria, reducing the extrapolated LOD to 68 CFU mL⁻¹ in PBS and 46 CFU mL⁻¹ in milk. The frequency variations in the aptasensor are proportional to various concentrations of P. aeruginosa (10²–10⁵ CFU mL⁻¹) with and without AuNPs, respectively. The low and rapid mass-sensitive detection demonstrates the ability of the aptasensor to quantitatively identify bacterial contamination in buffer and milk. Full article