Search Results (85)

Dysferlin direct protein–protein interactions (PPI) previously have been elucidated with surface plasmon resonance (SPR) and predicted to underlie membrane repair in mechanotransducing myofibrils. In mechanotransducing inner ear hair cells, dysferlin is detected with Z-stack confocal immunofluorescence in the stereocilia and their inserts in the tectorial membrane (TM) co-localizing with FKBP8, consistent with the SPR determination of tight, positively Ca²⁺-dependent interaction. FKBP8, a direct binding partner of mechanotransducing TMC1, when overexpressed, evokes an elevation in anti-apoptotic BCL2, inhibition of ryanodine receptor (RYR) activity, and a consequent reduction in Ca²⁺ release. RYR3 has now been immunolocalized to the tip of the TM in close association with a third-row outer hair cell (OHC) stereociliary BCL2-positive insertion. Dysferlin, annexin A2, and Alzheimer’s proteins BACE1 and amyloid precursor protein (APP) are also accumulated in these stereociliary insertions. RYR2 and RYR1 have been immunolocalized to the TM core, in position to influence TM Ca²⁺. Dysferlin PPI pathways also intersect with AD protein pathways in the spiral ganglion (SG). Dysferlin segregates with FKBP8, BACE1, and RYR3 in the interiors of SG type I cell bodies. RYR1, RYR2, PSEN1, BCL2, and caspase 3 are primarily confined to plasma membrane sites. RYR3 pathways traverse the plasma membrane to the cell body interior. Western analysis of dysferlinopathy proteins links FKBP8 and BCL2 overexpression with RYR inhibition, indicative of dysferlin targets that are ameliorative in AD. Full article

Botulinum toxin type A (BTXA) is widely used for the treatment of sialorrhea; however, its mechanism remains unclear. Tight junctions (TJs) are limiting factors for salivary secretion through the paracellular pathway in the salivary gland, among which claudin-1 (Cldn1) is a TJ protein that mainly plays a barrier role. This study observed that Cldn1 was upregulated in BTXA-treated rats’ submandibular glands and SMG-C6 cells. Knockdown of Cldn1 reversed the BTXA-induced reduction in paracellular permeability. The transcription factor specificity protein-1 (Sp1), which binds to the Cldn1 promoter, was also upregulated by BTXA, and its expression was linked to the ERK1/2 pathway. Inhibition of ERK1/2 by U0126 reversed the BTXA-induced upregulation of Sp1 and Cldn1, as well as the reduction in paracellular permeability. MiR-124-3p, which directly targets Sp1, was downregulated by BTXA, but its overexpression counteracted Sp1 and Cldn1 upregulation. Although miR-124-3p did not affect ERK1/2 phosphorylation, ERK1/2 inhibition reversed the BTXA-induced decrease in miR-124-3p expression. These findings reveal a regulatory pathway through which BTXA reduces paracellular permeability in SMG-C6 cells via the ERK1/2/miR-124-3p/Sp1/Cldn1 axis. Full article

Adenosine deaminase (ADA) is one of the most important enzymes in nucleoside metabolism, regulating the levels of adenosine and deoxyadenosine triphosphate (ADT/dATP) on either side of the cell membrane. This small protein (weighing approximately 40 kDa) exhibits deamination properties towards other pharmaceuticals built on adenine as the leading structure, which requires co-administration of ADA inhibitors. 3′-deoxyadenosine (Cordycepin, Cord) is an active compound isolated from the fungus Cordyceps, which has been used in traditional Chinese medicine for over 2000 years. Its anticancer activity is likely related to the inhibition of primer elongation of lagging strands during genetic information replication. Unfortunately, Cord is rapidly deaminated by ADA into inactive 3′-deoxyinosine, necessitating its co-administration with ADA inhibitors. Here, for the first time, the synthesis and discussion of the oxidised form of Cord are presented. The 7,8-dihydro-8-oxo-3′-deoxyadenosine (Cord^OXO) exhibits high resistance to ADA because of its syn conformation, as shown experimentally by UV spectroscopy and RP-HPLC monitoring. Theoretical Density Functional based Tight Binding (DFTB) studies of the Michaelis complex ADA-Cord^OXO have revealed significant distance increases between the “active” H₂O molecule and C6 of the 8-oxo-adenine moiety of Cord^OXO, i.e., 4 Å as opposed to 2.7 Å in the cases of ADA-dAdo and Cord. In conclusion, it can be postulated that the conversion of Cord to Cord^OXO enhances its therapeutic potential; however, this needs to be verified in vitro and in vivo. It should be emphasised that the therapeutic effect, if any, can be achieved theoretically without ADA inhibitors, e.g., pentostatin, thus reducing adverse effects. These promising preliminary results, presented here, warrant further investigations. Full article

The development of functional endothelial monolayers on synthetic vascular grafts remains challenging, particularly for small-diameter vessels (<6 mm) prone to thrombosis. Here, we present a pharmacological strategy combining 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate sodium salt (pCPT-cAMP, a tight junction promoter) with nitric oxide/cGMP pathway agonists 3-morpholinosydnonimine (SIN-1), captopril, and sildenafil) to enhance endothelialization. In human umbilical vein endothelial cells (HUVECs), this four-agent cocktail induced a flat, extended phenotype with a 3-fold increased cell area and 57.5% fewer cells required for surface coverage compared to controls. Immunofluorescence analysis revealed enhanced ZO-1 expression and continuous tight junction formation, while sustained nitric oxide (NO) production (3.9-fold increase) and restored prostacyclin (PGI₂) secretion demonstrated preserved endothelial functionality. Anticoagulation assays confirmed a significant reduction in thrombus formation (p < 0.01) via dual inhibition of platelet activation and thrombin binding. These findings establish a synergistic drug combination that promotes rapid endothelialization while maintaining antithrombogenic activity, offering a promising solution for small-diameter vascular grafts. Further studies should validate long-term stability and translational potential in preclinical models. Full article

We investigated the effect of probiotic Bacillus velezensis strains (LSSA01, 15AP4 and 2084) on pathogens causing post-weaning diarrhea in piglets (Enterotoxigenic Escherichia coli, Clostridium perfringens, Salmonella spp.). We studied the effect of B. velezensis and its cell-free supernatant on (1) pathogen growth; (2) IPEC-J2 cell cytokine and tight junction protein expression; (3) IPEC-J2 cell ‘wound’ recovery; (4) adhesion to IPEC-J2 cells and pathogen exclusion; and (5) Caenorhabditis elegans survival following pathogen exposure. Cell-free supernatant (CFS) from all strains inhibited the growth of ETEC F4 and F18 (by 36.9–53.2%; p < 0.05). One or more strains inhibited C. perfringens and Salmonella spp. (p < 0.05). Strain 2084 CFS increased IL-8 expression (+12.0% vs. control; p < 0.05; 6 h incubation), whereas LSSA01 CFS increased the expression of tight junction proteins (p < 0.05 vs. control; 6 h incubation) and accelerated 96 h ‘wound’ healing. Colony-forming units (CFUs) of all strains displayed a higher binding affinity to IPEC-J2 cells than 12 ETEC isolates, reduced adhesion of ETEC F4 and F18 and extended C. elegans survival over 30 d. The results indicate that probiotic B. velezensis strains have potential for use in the control of PWD pathogens. Full article

Sialyllactose (SL), a bioactive trisaccharide abundant in porcine colostrum, demonstrates multifunctional properties including antimicrobial activity, immune regulation, and apoptosis inhibition. This research uncovers the mechanisms by which SL mitigates enterotoxigenic Escherichia coli (ETEC)-mediated damage to intestinal barrier integrity, employing IPEC-J2 porcine epithelial models. SL pre-treatment effectively blocked pathogen adhesion by competitively binding to cellular receptors, concurrently mitigating inflammation through significant suppression of TNF-α, IL-1β, and IL-6 expression (p < 0.05). Notably, SL exhibited functional parallels to the NF-κB inhibitor BAY11-7082, jointly enhancing tight junction integrity via ZO-1 protein stabilization and inhibiting pro-inflammatory signaling through coordinated suppression of IκB-α/NF-κB phosphorylation cascades. The dual-action mechanism combines molecular interception of microbial attachment with intracellular modulation of the TLR4/MyD88/NF-κB pathway, effectively resolving both pathogenic colonization and inflammatory amplification. These findings position SL as a potential therapeutic application nutraceutical for livestock, with the capacity to address post-weaning porcine enteritis through functional feed formulations that synergistically enhance intestinal barrier resilience while curbing ETEC-mediated inflammatory pathogenesis. Full article

The purpose of this study was to investigate the anti-inflammatory effects of Perilla Seed Extract (PSE) and its active ingredient on Inflammatory Bowel Disease (IBD) in vitro and in vivo. Thirty-two C57/BL mice were randomly divided into four groups (n = 8): control group (CON), PBS group, LPS group (LPS 3.5 mg/kg given intraperitoneally [ip] on day 7 of the study only), and PSE group (100 mg/kg orally daily + LPS ip at 3.5 mg/kg on day 7). Mice were euthanized 24 h after LPS administration. MODE-K cells were divided into five groups: control group (CON), LPS group (50 μg/mL LPS for 2 h), and PSE group (low dose, 25 μg/mL PSE + LPS; middle dose, 50 μg/mL PSE + LPS; high dose, 100 μg/mL PSE + LPS). In vivo, compared with the CON group, LPS revealed a significant decrease in the villus length-to-crypt depth ratio (p < 0.01) and goblet cell density per unit area (p < 0.01). Conversely, PSE administration resulted in a significant increase in the villus length-to-crypt depth ratio (p < 0.01) and goblet cell density (p < 0.01). LPS significantly increased the ROS content (p < 0.01), the secretion of inflammatory cytokines of IL-6 (p < 0.01), TNF-α (p < 0.01), and the mRNA expressions of HO-1 (p < 0.01). LPS significantly decreased the mRNA expressions of Occludin (p < 0.01) and Claudin1 (p < 0.01). In contrast, PSE treatment led to a marked decrease in ROS levels (p < 0.01), along with a reduction in the secretion of inflammatory factors IL-6 (p < 0.01) and TNF-α(p < 0.05), as well as the mRNA expressions of HO-1 (p < 0.01). Concurrently, PSE significantly increased the mRNA expressions of Occludin (p < 0.05) and Claudin1 (p < 0.01). In vitro, PSE treatment also significantly reversed LPS-induced inflammation, oxidation and tight junction–related factors. Network pharmacology identified 97 potential targets for PSE in treating IBD, while transcriptomics analysis revealed 342 differentially expressed genes (DEGs). Network pharmacology and transcriptomics analysis indicated that significant pathways included the PI3K-Akt signaling pathway, MAPK signaling pathway, and TNF signaling pathway, of which the PI3K-AKT pathway may represent the primary mechanism. In an in vivo setting, compared with the CON group, LPS led to a significant increase in the protein expression of p-PI3K/PI3K (p < 0.01) and p-AKT1/AKT1 (p < 0.01). Conversely, PSE resulted in a significant decrease in the protein expression of p-PI3K/PI3K (p < 0.01) and p-AKT1/AKT1 (p < 0.01). In vitro, compared with the LPS group, PSE also significantly blocked the protein expression of p-PI3K/PI3K (p < 0.01) and p-AKT1/AKT1 (p < 0.01). The chemical composition of PSE was analyzed using UPLC-MS/MS, which identified six components including luteolin (content 0.41%), rosmarinic acid (content 0.27%), α-linolenic acid (content 1.2%), and oleic acid (content 0.2%). Molecular docking found that luteolin could establish stable binding with eight targets, and luteolin significantly decreased the p-AKT1/AKT1 ratio (p < 0.01) compared to the LPS group in MODE-K cells. In summary, PSE demonstrates efficacy against IBD progression by enhancing intestinal barrier function and inhibiting inflammatory responses and oxidative stress via the PI3K/AKT signaling pathway, and luteolin’s inhibition of AKT1 protein phosphorylation appears to play a particularly crucial role in this therapeutic mechanism. Full article

Bio-based corrosion inhibitor formulations are incredibly promising for mitigating corrosion, offering an environmentally sustainable approach while providing effective protection against material degradation. This study explores the corrosion inhibition potential of Ammi visnaga essential oil (AVEO) on carbon steel (CS) in a 1 mol/L hydrochloric acid (HCl) medium, combining electrochemical impedance spectroscopy (EIS), potentiodynamic polarization (PDP), linear polarization resistance (LPR), weight loss (WL) analysis, density functional theory (DFT), density-functional tight-binding (DFTB) modeling, and molecular dynamics (MD) simulation. The AVEO was extracted through hydrodistillation, and its chemical profile was characterized to identify key active compounds. EIS and PDP results revealed that the AVEO effectively inhibited corrosion through the formation of a protective layer on the steel surface, exhibiting inhibition efficiencies of up to 84% at 3 g/L, with a mixed-type corrosion inhibition action. Nyquist plots displayed an increased polarization resistance with the AVEO concentration, indicating an enhanced surface coverage and reduction in active corrosion sites. WL studies further supported these findings, showing decreased corrosion rates proportional to the AVEO concentration, while temperature variation studies showed a decreased performance at higher temperatures. Scanning electron microscope (SEM) analysis supported the formation of an effective protective layer on the CS surface upon the addition of AVEO to the HCl medium. DFTB modeling and MD simulations were employed to evaluate the interaction between major AVEO constituents and the steel surface, providing insight into the adsorption behavior and the electronic contributions at the molecule–metal interface. The combined experimental and theoretical findings indicate that AVEO holds promise as a natural, eco-friendly corrosion inhibitor, with implications for sustainable metal protection in acidic environments. Full article

Gene regulation depends on the interaction between chromatin-associated factors, such as transcription factors (TFs), which promote chromatin loops to ensure tight contact between enhancer and promoter regions. So far, positive interactions that lead to gene activation have been the main focus of research, but regulations related to blocking or inhibiting factor binding are also essential to maintaining a defined cellular status. To understand these interactions in greater detail, I investigated the possibility of the muscle differentiation factor Mef2 to prevent early Hox factor binding, leading to the proper timing of regulatory processes and the activation of differentiation events. My investigations relied on a collection of publicly available genome-wide binding data sets of Mef2 and Ubx (as the Hox factor), Capture-C interactions, and ATAC-seq analysis in Mef2 mutant cells. The analysis indicated that Mef2 can form possible chromatin loops to Ubx-bound regions. These regions contain low-affinity Ubx binding sites, and the chromatin architecture is independent of Mef2’s function. High levels of Ubx may disrupt the loops and allow specific Ubx bindings to regulate defined targets. In summary, my investigations highlight that the use of many publicly available data sets enables computational approaches to make robust predictions and, for the first time, suggest a molecular function of Mef2 as a preventer of Hox binding, indicating that it may act as a timer for muscle differentiation. Full article

As a zoonotic pathogen, S. suis serotype 2 (SS2) can cause severe diseases in both pigs and humans, and develop resistance to antibiotics. Plant natural compounds are regarded as promising alternatives to conventional antibiotics. Phillyrin is the major bioactive components of Chinese herbal medicine Forsythia suspensa. In this study, we explored the activity and action mechanism of phillyrin against SS2. The results showed that phillyrin could disrupt membrane integrity, destroy intracellular structures, and increase the exosmosis of DNA. Results of PCR revealed that phillyrin affected bacterial-virulence-related genes’ expression levels. Meanwhile, phillyrin significantly decreased the adhesion activity, inhibited lactate dehydrogenase (LDH) secretion, and reduced biofilm formation of SS2 in Newborn pig trachea epithelial (NPTr) cells. Furthermore, phillyrin protected tight junction protein of NPTr cells from SS2. We reported that phillyrin (0.1 mg/kg) treatment after bacterial challenge significantly improved the survival rate, ameliorated pulmonary inflammation, and inhibited the accumulation of multiple cytokines (IL-1, IL-6, IL-8, and TNF-α). Molecular docking showed that phillyrin had a good binding activity with the Ala88 and Asp111 of suilysin (SLY), one of the most important virulence factors of SS2. Collectively, phillyrin possesses antibacterial and anti-inflammatory activities, and is a promising candidate for preventing SS2 infection. Full article

Background/Objectives: Lactoferrin (Lf) is an iron-binding glycoprotein with multiple bioactivities, including promotion of cell proliferation and differentiation, immunomodulation, and antimicrobial activity. Lf, a basic glycoprotein, can bind to α-lactalbumin (α-Lac), an acidic whey protein. The current study aimed to evaluate whether Lf forms protein complexes with α-Lac and proteins/peptides from whey protein hydrolysate (WPH) and nonfat bovine milk powder (MP) and whether forming protein complexes influences resistance to gastrointestinal digestion and affects the bioactivities of Lf in human intestinal epithelial cells (HIECs and differentiated Caco-2 cells). Methods: Lf was blended with α-Lac, WPH, or MP. Assays were conducted to evaluate the bioactivities of proteins (Lf, α-Lac, WPH, or MP) and Lf–protein blends on HIECs and Caco-2 cells. Results: (1) Lf forms complexes with α-Lac and proteins/peptides from WPH and MP; (2) compared with Lf alone, complexed Lf shows greater resistance to in vitro digestion; (3) forming protein complexes does not affect Lf’s binding to the Lf receptor or its uptake by HIECs; and (4) forming protein complexes does not impact Lf’s bioactivities, including the promotion of cell proliferation and differentiation, reduction of cell permeability by upregulating tight-junction proteins, immune modulation through the regulation of IL-18, inhibition of enteropathogenic Escherichia coli growth, and modulation of immune responses to EPEC infection. Conclusions: Lf forms complexes with α-Lac and other milk proteins/peptides from WPH and MP in protein blends, and forming complexes does not affect the functionalities of Lf. Full article

Cadmium is a contributing factor to cardiovascular diseases and highly toxic to vascular endothelial cells. It has a distinct mode of injury, causing the de-endothelialization of regions in the monolayer structure of endothelial cells in a concentration-dependent manner. However, the specific molecules involved in the cadmium toxicity of endothelial cells remain unclear. The purpose of this study was to identify the specific molecular mechanisms through which cadmium affects endothelial detachment. Cadmium inhibited the expression of claudin-5 and zonula occludens (ZO)-1, which are components of tight junctions (strongest contributors to intercellular adhesion), in a concentration- and time-dependent manner. Compared to arsenite, zinc, and manganese, only cadmium suppressed the expression of both claudin-5 and ZO-1 molecules. Moreover, the knockdown of claudin-5 and ZO-1 exacerbated cadmium-induced endothelial cell injury and expansion of the detachment area, whereas their overexpression reversed these effects. CRE-binding protein inhibition reduced cadmium toxicity, suggesting that CRE-binding protein activation is involved in the cadmium-induced inhibition of claudin-5 and ZO-1 expression and endothelial detachment. These findings provide new insights into the toxicological mechanisms of cadmium-induced endothelial injury and risk of cardiovascular disease. Full article

The enzyme ROCK1 plays a pivotal role in the disruption of the tight junction protein CLDN1, a downstream effector influencing various cellular functions such as cell migration, adhesion, and polarity. Elevated levels of ROCK1 pose challenges in HCV, where CLDN1 serves as a crucial entry factor for viral infections. This study integrates a drug screening protocol, employing a combination of quantitative structure–activity relationship machine learning (QSAR-ML) techniques; absorption, distribution, metabolism, and excretion (ADME) predictions; and molecular docking. This integrated approach allows for the effective screening of specific compounds, using their calculated features and properties as guidelines for selecting drug-like candidates targeting ROCK1 inhibition in HCV treatment. The QSAR-ML model, validated with scores of 0.54 (R²), 0.15 (RMSE), and 0.71 (CCC), demonstrates its predictive capabilities. The ADME-Docking study’s final results highlight notable compounds from ZINC15, specifically ZINC000071318464, ZINC000073170040, ZINC000058568630, ZINC000058591055, and ZINC000058574949. These compounds exhibit the best ranking Vina scores for protein–ligand binding with the crystal structure of ROCK1 at the C2 pocket site. The generated features and calculated pIC₅₀ bioactivity of these compounds provide valuable insights, facilitating the identification of structurally similar candidates in the ongoing exploration of drugs for ROCK1 inhibition. Full article

This study aimed to investigate the effect of phenylethanol glycoside from Cistanche tubulosa (CPhGs) on the prevention of bovine serum albumin (BSA)-induced hepatic fibrosis in rats. Investigation of the mechanisms of the anti-hepatic fibrosis effect was focused on CPhGs’ influence on the “gut–liver” regulation, including the gut microbiota, intestinal barrier, systemic lipopolysaccharide (LPS) concentration, and LPS-related signaling pathway. The results show that CPhGs restored the diversity of gut microbiota, increased the relative abundance of Bacteroidetes, and decreased the relative abundance of Firmicutes and Proteobacteria in the fibrotic rats. In addition, CPhGs promoted the enrichment of probiotics such as Blautia, Oscillospira, Ruminococcus, Odoribacter, Bacteroides, and Parabacteroides in intestines of these rats. Furthermore, CPhGs reduced histopathological injury in the intestine and restored the tight junctions of the intestine by increasing the expression of ZO-1, occludin, and E-cadherin. CPhGs efficiently reduced serum LPS and liver lipopolysaccharide-binding protein (LBP) levels and inhibited the LPS-TLR4/MyD88/NF-κB pathway, which is related to protein expression in the liver. Correlation analysis confirmed that these beneficial bacteria were negatively associated with pathological damage, while LPS and harmful bacteria were positively associated with liver injury. Our fecal microbiota transplantation (FMT) experiment confirmed that gut microbiota is an important part of disease progression and that CPhGs is useful for the prevention and treatment of hepatic fibrosis. Our data demonstrate that the anti-hepatic fibrosis mechanism of CPhGs was mediated by regulation of the “gut–liver” axis. These results can stimulate consideration for its use in clinical practices. Full article

Tight junctional complexes (TJCs) between cerebral microvascular endothelial cells (CMECs) are essential parts of the blood–brain barrier (BBB), whose regulation closely correlates to the BBB’s integrity and function. hCMEC/D3 is the typical cell line used to imitate and investigate the barrier function of the BBB via the construction of an in vitro model. This study aims to investigate the protective effect of the deep-sea-derived fibrinolytic compound FGFC1 against H₂O₂-induced dysfunction of TJCs and to elucidate the underlying mechanism. The barrier function was shown to decline following exposure to 1 mM H₂O₂ in an in vitro model of hCMEC/D3 cells, with a decreasing temperature-corrected transendothelial electrical resistance (tcTEER) value. The decrease in the tcTEER value was significantly inhibited by 80 or 100 µM FGFC1, which suggested it efficiently protected the barrier integrity, allowing it to maintain its function against the H₂O₂-induced dysfunction. According to immunofluorescence microscopy (IFM) and quantitative real-time polymerase chain reaction (qRT-PCR), compared to the H₂O₂-treated group, 80~100 µM FGFC1 enhanced the expression of claudin-5 (CLDN-5) and VE-cadherin (VE-cad). And this enhancement was indicated to be mainly achieved by both up-regulation of CLDN-5 and inhibition of the down-regulation by H₂O₂ of VE-cad at the transcriptional level. Supported by FGFC1’s molecular docking to these proteins with reasonable binding energy, FGFC1 was proved to exert a positive effect on TJCs’ barrier function in hCMEC/D3 cells via targeting CLDN-5 and VE-cad. This is the first report on the protection against H₂O₂-induced barrier dysfunction by FGFC1 in addition to its thrombolytic effect. With CLDN-5 and VE-cad as the potential target proteins of FGFC1, this study provides evidence at the cellular and molecular levels for FGFC1’s reducing the risk of bleeding transformation following its application in thrombolytic therapy for cerebral thrombosis. Full article