Search Results (236)

In this study, the pharmacokinetic (PK) characteristics of oleuropein present in the olive tree (Olea europaea) were determined. We developed and validated a highly sensitive ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method to quantify oleuropein and its aglycone derivative, thereby establishing their pharmacokinetic properties in vitro and in vivo. Quantification of oleuropein and oleuropein aglycone was performed using selected reaction monitoring (SRM) with heated electrospray ionization in negative ion mode, employing mass transitions of m/z 275.06 and 307.137 for the respective analytes, and methylparaben as the internal standard. The calibration curve for both exhibited a range from 1 ng/mL to 1000 ng/mL, utilizing a total of 10 calibrator standards. The method demonstrated superior sensitivity, precision, and reproducibility, facilitating accurate quantification of analytes over a wide concentration range in biological matrices. To develop a pharmacokinetic profile, C57BL/6 male mice were administered oleuropein at a dose of 100 mg/kg body weight via oral gavage, and plasma levels were examined by LC-MS/MS. Oleuropein pharmacokinetics were evaluated exclusively, as plasma levels of oleuropein aglycone remained below the limit of quantification throughout the 24 h sampling period. Mass analysis of plasma samples identified multiple glucuronidated and sulfated metabolites, establishing Phase II metabolism as the dominant pathway governing the systemic disposition of oleuropein. In addition, the metabolic stability of the compounds was also investigated in mouse liver microsomes and S9 fractions to define the in vivo stability of oleuropein and oleuropein aglycone. Full article

The increasing prevalence of multidrug-resistant (MDR) pathogens, particularly avian pathogenic Escherichia coli (APEC) and methicillin-resistant Staphylococcus aureus (MRSA), poses a severe threat to the breeding industry and human health. To develop novel antibiotic alternatives, we adopted a “converting virulence into therapy” strategy by leveraging the type VI secretion system (T6SS) of the APEC strain ACN17-20. Guided by the structural analysis of T6SS Protein 00145, we rationally designed a series of amphipathic α-helical polypeptides. Among them, polypeptide A7 emerged as a lead candidate, exhibiting potent broad-spectrum antibacterial activity with negligible cytotoxicity against mammalian cells. Mechanistic studies revealed that A7 exerts a rapid bactericidal effect through a dual mode of action: physical disruption of bacterial membrane integrity leading to cytoplasmic leakage, and induction of lethal oxidative stress via reactive oxygen species (ROS) accumulation. Furthermore, A7 demonstrated excellent efficacy in eradicating pre-formed bacterial biofilms, addressing the challenge of persistent infections in breeding environments. In a mouse sepsis model induced by APEC and MRSA, A7 treatment significantly improved survival rates (60–80%), reduced bacterial loads in vital organs, and attenuated the systemic cytokine storm (TNF-α and IL-1β), thereby alleviating immune-mediated tissue damage. In conclusion, this study identifies polypeptide A7 as a safe therapeutic agent with a dual mechanism of action, providing a promising strategy to combat MDR infections and reduce antibiotic dependence. Full article

Type 2 diabetes mellitus (T2DM) has become a global metabolic disorder, and sprouted wheat (SW) exhibits potential for alleviating metabolic syndromes, although its mechanism remains unclear. This study aimed to investigate the effects and underlying mechanisms of SW on T2DM using a high−fat diet−induced T2DM mouse model. SW intervention significantly improved glycolipid metabolism disorders (p < 0.05), attenuated hepatic mitochondrial injury (p < 0.05) and maintained hepatic homeostasis. SW also reshaped the gut microbiota structure and inhibited the TLR4/NF−κB inflammatory pathway (p < 0.05). Untargeted metabolomics combined with network pharmacology identified five key functional metabolites and four core targets involved in the protective effects of SW. Germination optimized the nutritional composition of wheat, and SW regulated the microbe–liver axis through a multi−component, multi−target and multi-pathway mode. These results reveal the mechanism of SW in improving T2DM−related metabolic disorders and provide experimental support for its application. In the future, SW can be further developed as a dietary nutritional supplement for the prevention and adjuvant treatment of metabolic diseases. Full article

Influenza A virus (IAV) infection constitutes a major public health threat. Severe influenza virus infection can induce intense inflammatory responses and lung injury, leading to serious clinical symptoms or even death. The utility of current anti-influenza drugs is often limited by side effects and the emergence of drug-resistant strains. Based on the critical role of L-type voltage-gated calcium channels (L-VGCCs) in influenza virus replication, this study investigates the antiviral activity and mechanism of verapamil, a classic L-type calcium channel antagonist, against H1N1-UI182 virus. Verapamil, an L-type calcium channel blocker, is widely used in the treatment of cardiovascular diseases and has a well-established safety profile. Through molecular dynamics (MD) simulation and network pharmacology analysis, we predicted the stable binding mode of verapamil to the target protein (PDB id: 6JPA) and its potential multi-target network. In vitro, verapamil exhibited antiviral activity against H1N1-UI182 in MDCK cells, enhancing the survival rate of infected cells and reducing viral nucleoprotein (NP) expression. In a lethal H1N1-UI182 infection mouse model, verapamil treatment markedly improved survival rates, alleviated weight loss and lung pathological damage, exhibiting a dose-dependent protective effect. Lung tissue analysis showed that verapamil effectively reduced the lung index and viral load, suppressed the activation of the Nuclear factor kappa B (NF-κB) signaling pathway, and decreased the expression of key inflammatory factors, thereby mitigating the cytokine storm. A comparison of administration regimens indicated that pre-treatment yielded optimal efficacy, suggesting verapamil acts primarily during the early stage of the viral life cycle. This study systematically elucidates that verapamil exerts antiviral and immunomodulatory effects by regulating the NF-κB pathway. Network pharmacology analysis suggested the potential involvement of multiple targets and pathways, including EGFR, SRC, and phospholipase D signaling, providing hypotheses for future mechanistic investigation. This paper supports a drug repurposing strategy against drug-resistant influenza viruses and highlights its significant potential for clinical translation. Full article

Photobiomodulation (PBM) has shown promising potential to enhance bone regeneration; however, its optimal delivery parameters and interactions with osteoconductive scaffolds remain insufficiently defined. This preclinical study is the first to incorporate a pilot dosimetry evaluation to standardize 980-nm PBM delivery and ensure that effective irradiance reached the target surface of critical-size calvarial defects in mice. The primary aim was to evaluate the effectiveness of this novel 980-nm PBM protocol delivered using either flat-top (FT) or standard Gaussian (ST) handpieces in enhancing bone regeneration in critical-size defects (CSDs), both with and without Bio-Oss^® grafting. A total of 120 adult mice were allocated into twelve experimental groups (n = 10 per group): untreated (control), Bio-Oss^® alone, PBM alone, and PBM combined with Bio-Oss^®, using either FT or ST handpieces, and evaluated at 30 and 60 days. Animals received 980 nm irradiation at 0.6 W (nominal power output–set on laser interface) in continuous-wave mode for 60 s, three times per week, for two consecutive weeks. Pilot dosimetry included power meter measurements to determine the therapeutic power reaching the defect surface area and temperature monitoring to ensure safe energy delivery. The dosimetry study demonstrated that, after accounting for the optical properties of mouse shaved skin and the Bio-Oss^® graft covered with Bio-Gide^® membrane, the effective irradiance reaching the base of the defect surface area was 1.131 W/cm² for the FT handpiece and 0.413 W/cm² for the ST handpiece. This dose was sufficient to induce significant regenerative effects. Histological, Masson’s trichrome, and immunohistochemical analyses for Runx2, OCN, GLI1, CD34, and CTSK were performed to characterize early and late osteogenic events. The combination of PBM and Bio-Oss^® significantly accelerated bone regeneration compared with PBM alone, with the FT handpiece producing the most uniform and advanced osteogenesis. PBM enhanced progenitor activation, osteoblast differentiation, angiogenesis, matrix deposition, and late-stage remodeling, demonstrating a synergistic effect with the scaffold, whereas Bio-Oss^® alone or defect alone showed limited early regenerative potential. These findings highlight the effectiveness of this novel standardized PBM dosimetry and uniform beam profile (FT), supporting their use as a foundation for future randomized controlled trials in craniofacial bone repair. Full article

Near-infrared (NIR) imaging, including NIR-I (800–1000 nm) and NIR-II (1000–1700 nm), has been primarily evaluated using separate cameras with different detectors, limiting comparison. We investigated whether using a single-camera system capable of both NIR-I and NIR-II acquisition, NIR-II improves spatial resolution and contrast-to-noise ratio (CNR) for nanoparticle-based imaging. Dual-mode, dual-Gd ICG (DMDG-ICG) nanoparticles were characterized for absorption and fluorescence. A custom NIR imaging system using a single InGaAs camera enabled visualizing both NIR-I and -II windows. In vitro, capillary tubes containing nanoparticles in water, in tissue-mimicking Intralipid, or covered with mouse skin were imaged, and full-width-half maximum (FWHM) and CNR were measured. In vivo, the mouse femoral artery was imaged after IV nanoparticle delivery. DMDG-ICG showed strong fluorescence at both NIR-I and NIR-II. Scatter greater at NIR-I than NIR-II increased with depth and tissue layers. FWHM was lower and CNR higher at NIR-II versus NIR-I for up to 10 mm depth (p < 0.05, n = 3) in Intralipid. In vivo, femoral artery CNR was also higher at NIR-II (p < 0.05, n = 3). Using a single-camera system allowing direct comparison, NIR-II imaging provided greater penetration, spatial resolution, and CNR compared to NIR-I. The findings support the utility of NIR-II for vascular and molecular imaging applications. Full article

The all-d-enantiomeric-peptide RD2 was developed for the treatment of Alzheimer’s disease. This study aimed to develop a specific and highly sensitive liquid chromatography-mass-spectrometric (UHPLC-ESI-QTOF) method for quantifying RD2 in the mouse brain and to validate it according to the ICH M10 guideline to investigate the pharmacokinetic profile of RD2 in its target organ. Sample preparation, chromatographic separation and quantification were very challenging due to RD2’s highly hydrophilic properties, the complex matrix and the required lower limit of quantification (LLOQ). Chromatographic separation was performed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm particle size) within 5 min at 50 °C with a flow rate of 0.5 mL·min⁻¹. Mobile phases consisted of water and acetonitrile with 0.2% formic acid and 0.015% heptafluorobutyric acid. Ions were generated by electrospray ionization in the positive mode, and RD2 was quantified by QTOF-MS. The developed extraction method revealed complete recovery. The linearity of the calibration curve was in the range of 2 ng·mL⁻¹ to 500 ng·mL⁻¹ (R² > 0.99) with a LLOQ of 5 ng·mL⁻¹. The intraday and interday accuracy and precision ranged from 0.4% to 12.2% and from 1.0% to 12.0%. RD2 remained stable in the freshly homogenized brain even after several freeze–thaw cycles, but stability decreased over time during long-term storage at −80 °C. Using this validated method, RD2-spiked brain homogenate samples and samples of a pharmacokinetic study with RD2 in mice were analyzed. Full article

Atrial fibrillation (AF) is closely linked to atrial remodeling, while its underlying immune mechanisms remain elusive. This study sought to investigate the role of SPP1⁺ macrophages in the development and progression of AF and further elucidate the underlying mechanisms. Single-nucleus RNA sequencing was performed on right atrial tissues from 3 patients with persistent AF and 3 with sinus rhythm (all with rheumatic valvular heart disease). The results revealed significant immune cell infiltration in AF atrial tissues, with a marked increase in the proportion of SPP1⁺ macrophages, which exhibited the strongest intercellular communication with cardiomyocytes. Phenotypic scoring indicated that apoptosis was the dominant mode of cardiomyocyte death in AF. Immunohistochemical and Western blot analyses confirmed elevated levels of pro-apoptotic proteins (Bax, Cleaved-Caspase3) and reduced levels of the anti-apoptotic protein Bcl2 in AF tissues. In a mouse model with macrophage-specific SPP1 overexpression, increased AF inducibility and duration were observed, accompanied by enhanced cardiomyocyte apoptosis. In vitro co-culture experiments using SPP1-overexpressing RAW264.7 macrophages and HL-1 cardiomyocytes confirmed that SPP1⁺ macrophages could induce cardiomyocyte apoptosis. Mechanistically, KEGG and GSEA analyses identified downregulation of the PI3K/AKT pathway in AF. Treatment with the PI3K/AKT activator Recilisib reversed apoptosis and restored p-PI3K/p-AKT levels in HL-1 cells co-cultured with SPP1-overexpressing RAW264.7 macrophages. These findings demonstrate that SPP1⁺ macrophages accumulate in atrial tissues of AF patients and induce cardiomyocyte apoptosis by downregulating the PI3K/AKT pathway, thereby increasing AF susceptibility. Full article

Background/Objectives: Impaired intestinal barrier function is a hallmark of inflammatory bowel disease (IBD). Akkermansia muciniphila and its outer membrane protein Amuc_1100 can enhance this barrier, but the clinical application of Amuc_1100 is limited by the fastidious growth of its native host. This study aimed to overcome this by utilizing the robust probiotic Lacticaseibacillus paracasei L9 for targeted Amuc_1100 delivery. Methods: We engineered Lc. paracasei L9 to express Amuc_1100 via intracellular (pA-L9), secretory (pUA-L9), and surface-display (pUPA-L9) strategies. Their efficacy was assessed in Lipopolysaccharide (LPS)-induced macrophages and a dextran sulfate sodium (DSS)-induced colitis mouse model, evaluating inflammation, barrier integrity, and mucosal repair. Results: The secretory (pUA-L9) and surface-display (pUPA-L9) strains most effectively suppressed pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in macrophages. In mice, both strains alleviated colitis and outperformed native A. muciniphila in improving disease activity. Crucially, they exhibited distinct, specialized functions: pUA-L9 acted as a systemic immunomodulator, reducing pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α), elevating anti-inflammatory mediators (IL-4 and IL-10), and promoting goblet cell differentiation; notably, the inhibitory effect of pUA-L9 on IL-6 expression was approximately 2-fold greater than that of pUPA-L9. In contrast, pUPA-L9 excelled in local barrier repair, uniquely restoring mucus layer integrity (Muc1, Muc2, and Tff3) and reinforcing tight junctions (ZO-1, Occludin, Claudin1, Claudin3, and Claudin4). In particular, pUPA-L9 increased Muc2 expression by approximately 3.6-fold compared with pUA-L9. Conclusions: We demonstrate that the subcellular localization of Amuc_1100 within an engineered probiotic dictates its therapeutic mode of action. The complementary effects of secretory and surface-displayed Amuc_1100 offer a novel, spatially targeted strategy for precision microbiome therapy in IBD. Full article

Glucagon is an endogenous peptide that is produced in the pancreas. Via glucagon receptors, glucagon increases the beating rate in cultured rat neonatal cardiomyocytes and also in isolated right atrial preparations from adult rats. Moreover, in living adult mice, injections of glucagon can elevate the heart rate. It is unknown whether these effects of glucagon in living adult mice are mediated via central glucagon receptors or via a direct effect on cardiac glucagon receptors. Thus, we tested the hypothesis that glucagon can exert a direct positive chronotropic effect in the adult mouse heart. We measured the contractile effects of cumulatively increasing concentrations of glucagon (0.1–100 nM) in isolated paced (1 Hz) left atrial preparations, in isolated spontaneously beating right atrial preparations and in isolated spontaneously beating retrogradely perfused whole hearts. We detected in isolated right atrial preparations time- and concentration-dependent positive chronotropic effects of glucagon that were reversed by the glucagon receptor antagonists SC203972 and desglucagon. The positive chronotropic effects of glucagon were also attenuated by 1 µM of ivabradine, an inhibitor of the hyperpolarization-activated cation channels (HCN), but not by 100 nM rolipram, a phosphodiesterase 4 inhibitor, nor by 10 µM of propranolol, a β-adrenoceptor antagonist. Moreover, the positive chronotropic effects of glucagon were also attenuated by stimulation of the A₁-adenosine receptor or muscarinic receptors. Glucagon decreased the force of contraction in right atrial preparations. In left atrial preparations, glucagon failed to alter the force of contraction. In isolated adult mouse hearts perfused in the Langendorff mode, 10 nM of glucagon increased the beating rate and reduced left ventricular force of contraction. The gene expression of the glucagon receptors was lowest in the left atrium, higher in the ventricle and highest in the right atrium of adult mice. In summary, glucagon exerted a positive chronotropic effect in the mouse heart via glucagon receptors, mediated, at least in part, via HCN channels in the sinus node. Full article

Osteosarcoma is the most common primary malignant bone tumor in adolescents and young adults; yet survival outcomes have remained stagnated for decades, underscoring the urgent need for new therapeutic strategies. Creatine kinase (CK)—comprising cytosolic CKB and mitochondrial CK—maintains malignant behaviors by supporting high-energy phosphate transfer through the phosphocreatine (pCr) shuttle. Here, we pharmacologically inhibited CK activity in osteosarcoma models and evaluated proliferation, cell death modalities, mitochondrial function, stemness, motility, and tumor behavior. CK blockade consistently suppressed growth and clonogenicity and induced apoptosis as the predominant mode of death. It impaired ATP buffering capacity and disturbed mitochondrial homeostasis, accompanied by reduced expression of stemness-associated markers and diminished migration and invasion. In mouse models, CK inhibition significantly restrained tumor progression and dissemination. These results indicate that disabling the CK-pCr energy-buffering system reprograms cellular energetics toward apoptosis and less aggressive phenotypes. Our findings support targeting the CK pathway as a tractable metabolic vulnerability and a rational partner for cytotoxic regimens, with pathway-specific signaling alterations representing downstream consequences of central energetic collapse. Full article

To identify novel peptides with potential antiviral activities, a database search was performed based on the primary sequence of the peptide I24 (CLAFYACFC), the effective part of the antiviral peptide TAT-I24 consisting of peptide I24 and the cell penetrating TAT-peptide (amino-acids 48–60; GRKKRRQRRRPPQ). A Protein BLAST search identified several sequences with high similarity to I24 in diverse proteins, some of which are known to be involved in the interaction with nucleic acids. Selected sequences and newly designed variants of I24 were synthesized as TAT fusion peptides and tested for antiviral activity in two well-established models: baculovirus transduction of HEK293 cells and mouse cytomegalovirus (MCMV) infection of NIH/3T3 cells. Several of the TAT-fusion peptides exhibited antiviral activities with a potency comparable to TAT-I24. The ability of these peptides to bind double-stranded DNA suggested the same mode of action. Several peptides caused swelling of red blood cells (RBC) but with only one peptide clearly inducing haemolysis. With two exceptions, RBC swelling was observed with antivirally active peptides but not with less active peptides, indicating that antiviral activities are linked to an effect on membrane integrity of target cells. Structural prediction of the TAT-fusion peptides indicated formation of two α-helical elements, with several of these peptides showing remarkable similarity when subjected to structural alignment. Full article

Over the past few decades, Fc fragment-conjugated proteins, such as Protein A, have been extensively utilized across a range of applications, including antibody purification, site-specific immobilization of antibodies, and the development of biosensing platforms. In this study, building upon our group prior research, we designed and screened an affinity DNA functional ligand (A-DNAFL) and experimentally validated its binding affinity (K_D = 6.59 × 10⁻⁸) toward mouse IgG antibodies, whose binding performance was comparable to that of protein A. Systematic evaluations were performed to assess the binding efficiency under varying pH levels and ionic strength conditions. Optimal antibody immobilization was achieved in PBST-B buffer under physiological pH 7.2–7.4 and containing approximately 154 mM Na⁺ and 4 mM K⁺. Two competitive binding assays confirmed that the A-DNAFL binds to the Fc fragment of murine IgG antibody. Furthermore, molecular docking simulations were employed to investigate the interaction mode, revealing key residues involved in binding as well as the contributions of hydrogen bonding and hydrophobic interactions to complex stabilization. Leveraging these insights, A-DNAFL was utilized as a tool for oriented immobilization of antibodies on the sensing interface, enabling the construction of an immunosensor for ricin detection. Following optimization of immobilization parameters, the biosensor exhibited a detection limit of 30.5 ng/mL with the linear regression equation is lg(Response) = 0.329 lg(C_ricin) − 2.027 (N = 9, R = 0.938, p < 0.001)—representing a 64-fold improvement compared to conventional protein A-based methods. The system demonstrated robust resistance to nonspecific interference. Sensing interface reusability was also evaluated, showing only 8.55% signal reduction after two regeneration cycles, indicating that glycine effectively elutes bound antibodies while preserving sensor activity. In summary, the A-DNAFL presented in this study represents a novel antibody-directed immobilization material that serves as a promising alternative to protein A. It offers several advantages, including high modifiability, low production cost, and a relatively small molecular weight. These features collectively contribute to its broad application potential in biosensing, antibody purification, and other areas of life science research. Full article

Gnetol (trans-2,3′,5′,6-tetrahydroxystilbene), a naturally occurring stilbene structurally related to resveratrol (trans-3,5,4′-trihydroxystilbene; RES), has been reported to possess multiple health-promoting activities. In order to support its potential nutraceutical application, a reliable chromatography–tandem mass spectrometry (LC–MS/MS) assay was developed and validated for the quantitative determination of gnetol in mouse plasma and tissue samples, using isotopically labeled RES-¹³C₆ serving as the internal standard (IS). Electrospray ionization (ESI) was performed in negative mode, with multiple reaction monitoring (MRM) transitions m/z 243.2 → 175.0 for gnetol and m/z 233.1 → 191.0 for the IS. Chromatographic separation was achieved on a reversed-phase HPLC column using a 5-min gradient delivery of acetonitrile and 2 mM ammonium acetate at 0.5 mL/min and 40 °C. The linear calibration curve covered the concentration range of 5.0–1500 ng/mL, and the method validation confirmed its selectivity, accuracy, precision, stability, and dilution integrity. The developed method was subsequently applied to a biodistribution study in mice after oral administration of gnetol at 400 µmol/kg (equivalent to 97.7 mg/kg). Gnetol was rapidly absorbed and extensively distributed in key pharmacologically relevant organs. Despite its poor aqueous solubility, oral uptake was not significantly hindered. Collectively, these findings demonstrate that gnetol exhibits favorable absorption and tissue distribution profiles, supporting its promise as a candidate for nutraceutical development. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the most common chronic liver disorders worldwide and can lead to inflammation, fibrosis, and liver cancer. To better understand the impact of an unbalanced hypercaloric diet on liver phenotype in impaired autophagy, the study compared C57BL/6J wild type (WT) and MAPK15-ERK8 knockout (KO) male mice with C57BL/6J background fed for 17 weeks with “Western-type” (WD) or standard diet (SD). Liver features were monitored in vivo by high-frequency ultrasound (HFUS) using a semi-quantitative and parametric assessment of pathological changes in the parenchyma complemented by computer-aided diagnosis (CAD) methods. Liver histology was considered the reference standard. WD induced liver steatosis in both genotypes, although KO mice showed more pronounced dietary effects than WT mice. Overall, HFUS reliably detected steatosis-related parenchymal changes over time in the two mouse genotypes examined, consistent with histology. Furthermore, this study demonstrated the feasibility of extracting quantitative features from conventional B-mode ultrasound images of the liver in murine models at early clinical stages of MASLD using a computationally efficient and vendor-independent CAD method. This approach may contribute to the non-invasive characterization of genetically engineered mouse models of MASLD according to the principles of replacement, reduction, and refinement (3Rs), with interesting translational implications. Full article