Novel and highly selective molecularly imprinted polymers based on the surface of metal-organic frameworks, NH2-MIL-101(Cr) (MIL@MIPS), were successfully fabricated to capture neuronal nitric oxide synthase-postsynaptic density protein-95 (nNOS-PSD-95) uncouplers from Sanhuang Xiexin Decoction (SXD) for stroke treatment. The resultant polymers were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, and X-ray diffraction. The performance tests revealed that MIL@MIPs had a large binding capacity, fast kinetics, and excellent selectivity. Then the obtained polymers were satisfactorily applied to solid-phase extraction coupled with high-performance liquid chromatography to selectively capture nNOS-PSD-95 uncouplers from SXD. Furthermore, the biological activities of components obtained from SXD were evaluated in vivo and in vitro. As a consequence, the components showed a potent neuroprotective effect from the MTS assay and uncoupling activity from the co-immunoprecipitation experiment. In addition, the anti-ischemic stroke assay in vivo was further investigated to determine the effect of reducing infarct size and ameliorating neurological deficit by the active components. Therefore, this present study contributes a valuable new method and new tendency to selectively capture active components for stroke treatment from SXD and other natural medicines.

Bacterial infection often induces chronic repair of wound healing owing to aggravated inflammation. Hydrogel dressing exhibiting intrinsic antibacterial activity may substantially reduce the use of antibiotics for infected wound management. Hence, a versatile hydrogel dressing (rGB/QCS/PDA-PAM) exhibiting skin adaptiveness on dynamic wounds and  mild photothermal antibacterial activity is developed for safe and efficient infected wound treatment. Phenylboronic acid-functionalized graphene (rGB) and oxadiazole-decorated quaternary carboxymethyl chitosan (QCS) are incorporated into a polydopamine-polyacrylamide (PDA-PAM) network with multiple covalent and noncovalent bonds, which conferred the hydrogel with flexible mechanical properties, strong tissue adhesion and excellent self-healing ability on the dynamic wounds. Moreover, the glycocalyx-mimicking phenylboronic acid on the surface of rGB enables the hydrogel to specifically capture bacteria. The enhanced membrane permeability of QCS enhanced bacterial vulnerability to photothermal therapy（PTT）, which is demonstrated by efficient mild PTT antibacteria against methicillin-resistant Staphylococcus aureus in vitro and in vivo at temperatures of <49.6 °C. Consequently, the hydrogel demonstrate accelerated tissue regeneration on MRSA-infected wound in vivo, with an intact epidermis, abundant collagen deposition and prominent angiogenesis. Therefore, rGB/QCS/PDA-PAM is a versatile hydrogel dressing exhibiting inherent antibacterial activity and has considerable potential in treating wounds infected with drug-resistant bacteria.

As the most common malignancy in women, breast cancer causes >40,000 deaths annually. Ribonuclease A (RNase), a new anti-cancer agent, has attracted intense interest due to its high efficacy and specificity. However, RNase suffers from instability, a short half-life in the circulation and poor membrane penetration. To overcome these challenges, we designed a supramolecular nanogel for the cytosolic delivery of RNase. The nanogels were fabricated using host-guest interactions between azobenzene (Azo) and β-cyclodextrin (βCD) conjugated to poly (L-glutamic acid)-graft-poly (ethylene glycol) methyl ether (PLG-g-mPEG). RNase could be loaded inside the nanogels in mild aqueous conditions. Following optimization, the RNase-loading content and efficiency of the nanogel were 23.5 wt% and 50.4%, respectively. In the presence of nitroreductase (NTR), the cross-linking point between Azo and βCD was destroyed due to the conformation transition of Azo, ensuring the hypoxia-sensitive release of cargo from the nanogels in tumors in which NTR is overexpressed. In vitro release profiles revealed that 75.0% of the RNase was released under hypoxic conditions in 72 h, whilst only 19.7% was released under normoxic conditions. Cytotoxicity assays showed that the RNase-loaded nanogels (nano-RNase) were more efficient in inhibiting the proliferation of 4T1 cells than free RNase. In vivo studies showed 68.7% tumor suppression rates (TSR %) in the nano-RNase treated group, whilst free RNase treatment led to a lack of tumor inhibition. To further enhance the hypoxia status of tumors, we combined nano-RNase with a nanoformulation of vascular disrupting agents PLG-g-mPEG/combretastatinA4 (nano-CA4) and obtained a TSR of 91.7%. The hypoxia-sensitive supramolecular nanogels provided a versatile platform for the delivery of RNase, highlighting its applicability for cancer therapy.

STING (stimulator of interferon genes) signaling pathway has attracted considerable attention in cancer immunotherapy due to its capacity to boost vigorous antitumor immunity. However, the shortage of effective STING agonists limits the promotion of STING pathway in cancer treatment. Herein, we present an approach for in situ activation of STING pathway with nanoparticles delivered DNA-targeting chemo agents, based on the understanding that cytosol DNA is a pre-requisite for STING pathway activation. Through in vitro screening among several DNA-targeting chemo agents, we identified 7-ethyl-10-hydroxycamptothecin (SN38) as the most potent drug for stimulating interferon (IFN)-β secretion and proved that this process is mediated by the passage of DNA-containing exosomes from treated tumor cells to bone marrow-derived dendritic cells (BMDCs) and subsequent activation of the STING pathway. Furthermore, we designed a polymeric-SN38 conjugate that could self-assemble into nanoparticles (SN38-NPs) for in vivo application. The SN38-NPs formulation reduced toxicity of free SN38, effectively stimulated the activation of STING pathway in E0771 tumors, and resulted in a tumor suppression rate (TSR%) of 82.6%. Our results revealed a new mechanism of SN38 in cancer treatment and should inspire using more DNA-targeting agents, especially in nanoformulation, for activating STING pathway and cancer chemoimmunotherapy.

Repairing cartilage defects using thermosensitive hydrogels is an attractive treatment strategy, but the poor mechanical properties and limited understanding of the interactions between hydrogels and cells limit their application. Methods: In this study, a thermosensitive hydroxypropyl chitin hydrogel (HPCH) was functionalized with methacrylate groups to synthesize photocrosslinkable glycidyl methacrylate-modified HPCH (GM-HPCH). GM-HPCH could form a gel in situ through a thermosensitive sol-gel transition and its mechanical properties can be improved by UV irradiation. Cell viability, cell adhesion and anti-apoptosis activity of GM-HPCH were evaluated. Transforming growth factor-β1 (TGFβ1) was introduced into the GM-HPCH hydrogel to fabricate the composite hydrogel. The macrophage immunomodulation, MSC recruitment and chondrogenesis of the composite hydrogel were evaluated. Results: With high biocompatibility, GM-HPCH could protect chondrocytes from apoptosis. Both the in vitro and in vivo experiments showed that GM-HPCH + TGFβ1 shifted the recruited macrophages from M1 to M2 and promoted chondrogenic gene expression. Additionally, the composite hydrogel could promote the migration of marrow stromal cells (MSCs) in the Transwell test and increase migrated gene expression. The fluorescent tracking of MSCs confirmed MSC homing in the rat chondral defect with the help of GM-HPCH. The macroscopic evaluation and histological results at 6 weeks and 12 weeks postsurgery showed that GM-HPCH + TGFβ1 can achieve superior cartilage healing. Conclusions: The GM-HPCH + TGFβ1 hydrogel effectively promoted cartilage repair via immunomodulating macrophages, recruiting MSCs and promoting chondrogenesis; thus it is a promising injectable hydrogel for cartilage regeneration.

Biofilms formed on skin wound lead to inflammation and a delay of healing. In the present work, a novel textile pile debridement material was prepared and treated by plasma. Samples before and after plasma treatment were characterized by a series of methods, including scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and water uptake capacity. Besides, mechanical, coagulation, and in vitro biofilm removal performances of the textile pile debridement material were evaluated, with a medical gauze as a control. The results demonstrate that the plasma treatment produced corrosions and oxygen-containing polar groups on the fiber surface, offering an enhanced water uptake capacity of the textile pile debridement material. In addition, compressive tests certify the mechanical performances of the textile pile debridement material in both dry and wet conditions. The results from a kinetic clotting time test suggest a favorable ability to promote blood coagulation. Furthermore, the results of an MTT cell viability assay, SEM, and confocal laser scanning microscopy (CLSM) illustrate that the textile pile debridement material demonstrates a more superior in vitro biofilm removal performance than medical gauze. All of these characterizations suggest that the textile pile debridement material can offer a feasible application for clinical wound debridement.

Chitin-derived hydrogels are commonly used in bone regeneration because of their high cell compatibility; however, their poor mechanical properties and little knowledge of the interaction between the materials and host cells have limited their practical application. Methods: To evaluate osteoinductivity and enhance the mechanical properties of a newly synthesized thermosensitive hydroxypropyl chitin hydrogel (HPCH), a mesenchymal stem cell (MSC)-encapsulated HPCH was infused into a three-dimensional-printed poly (ε-caprolactone) (PCL)/ nano-hydroxyapatite (nHA) scaffold to form a hybrid scaffold. The mechanical properties and cell compatibility of the scaffold were tested. The interaction between macrophages and scaffold for angiogenesis and osteogenesis were explored in vitro and in vivo. Results: The hybrid scaffold showed improved mechanical properties and high cell viability. When MSCs were encapsulated in HPCH, osteo-differentiation was promoted properly via endochondral ossification. The co-culture experiments showed that the hybrid scaffold facilitated growth factor secretion from macrophages, thus promoting vascularization and osteoinduction. The Transwell culture proved that MSCs modulated the inflammatory response of HPCH. Additionally, subcutaneous implantation of MSC-encapsulated HPCH confirmed M2 activation. In situ evaluation of calvarial defects confirmed that the repair was optimal in the MSC-loaded HPCH + PCL/nHA group. Conclusions: PCL/nHA + HPCH hybrid scaffolds effectively promoted vascularization and osteoinduction via osteogenesis promotion and immunomodulation, which suggests promising applications for bone regeneration.

Photodynamic therapy (PDT) as a non-aggressive therapy with fewer side effects has unique advantages over traditional treatments. However, PDT still has certain limitations in clinical applications, mainly because most photosensitizers utilized in PDT are hydrophobic compounds, which will self-aggregate in the aqueous phase and cause undesirable effects. In order to resolve this, we utilized the self-polymerization of dopamine molecules under alkaline conditions to coat cerium oxide nanorods (CeONR) with a dense polydopamine (PDA) film. Thereafter, thiolated galactose (Gal-SH) and hypericin (Hyp) were modified and loaded onto the surface to construct CeONR@PDA-Gal/Hyp, respectively, which can be used for targeted photodynamic therapy of human hepatoma HepG2 cells. CeONR@PDA-Gal/Hyp was characterized by transmission electron microscope (TEM), Zeta potential, Ultraviolet-visible (UV-Vis), and fluorescence spectroscopy, respectively. This hypericin delivery system possesses good biocompatibility and specific targeting ability, where the galactose units on the surface of CeONR@PDA-Gal/Hyp can specifically recognize the asialo-glycoprotein receptors (ASGP-R), which overexpress on HepG2 cell membrane. Furthermore, Hyp will detach from the surface of CeONR@PDA-Gal/Hyp after the nanorods enter cancer cells, and shows excellent PDT effect under the irradiation of light with a wavelength of 590 nm. Our work exemplifies a novel targeted delivery of hydrophobic photosensitizers for cancer treatment.

The mucilage from Brasenia schreberi (BS) exhibits various biological activities, including antialgal, antibacterial, soluble-fiber properties, and excellent lubricating behavior. Thus, the extraction and wide use of mucilage in the food industry are crucial. In this study, the high-speed shear-assisted extraction of mucilage from BS was optimized by using response surface methodology (RSM). The optimal extraction conditions were as follows: Extraction temperature of 82 °C, extraction time of 113 min, liquid-solid ratio of 47 mL/g, and shear speed of 10,000 rpm. Under these conditions, the actual yield of BS mucilage was 71.67%, which highly matched the yield (73.44%) predicted by the regression model. Then, the BS mucilage extract was powdered to prepare the capsule, and the excipients of the capsule were screened using a single-factor test to improve the disintegration property and flowability. The final capsule formulation, which consisted of: 39% BS mucilage powder (60 meshes); 50% microcrystalline cellulose (60 meshes) as the filler; both 10% sodium starch glycolate and PVPP XL-10 (3:1, 60 meshes) as the disintegrant; both 1% colloidal silicon dioxide and sodium stearyl fumarate (1:1, 100 meshes) as the glidant by weight; were used for preparing the weights of a 320 mg/grain of capsule with 154.7 ± 0.95 mg/g polysaccharide content. Overall, the optimized extraction process had a high extraction rate for BS mucilage and the capsule formulation was designed reasonably.

In this study, a facile and effective method is adopted to prepare mechanochemically robust super antifouling membrane surfaces. During the process, vinyl trimethoxy silane (VTMS) was used as the reactive intermediate for coupling the hydrophilic inorganic SiO2 nanoparticle layer on to the organic ultra-high-molecular-weight polyethylene (UHMWPE) membrane surface, which created hierarchical nanostructures and lower surface energy simultaneously. The physical and chemical properties of the modified UHMWPE composite membrane surface were investigated. FTIR and XPS showed the successful chemical grafting of VTMS and SiO2 immobilization, and this modification could effectively enhance the membrane's surface hydrophilicity and filtration property with obviously decreased surface contact angle, the pure water flux and bovine serum albumin (BSA) rejection were 805 L·m-2·h-1 and 93%, respectively. The construction of the hydrophilic nano-SiO2 layer on the composite membrane surface for the improvement of membrane antifouling performance was universal, water flux recovery ratio values of BSA, humic acid (HA), and sodium alginate (SA) were all up to 90%. The aim of this paper is to provide an effective approach for the enhancement of membrane antifouling performance by the construction of a hydrophilic inorganic layer on an organic membrane surface.

This research demonstrates the use of fill density as an effective tool for controlling the drug release without changing the formulation composition. The merger of hot-melt extrusion (HME) with fused deposition modeling (FDM)-based 3-dimensional (3-D) printing processes over the last decade has directed pharmaceutical research towards the possibility of printing personalized medication. One key aspect of printing patient-specific dosage forms is controlling the release dynamics based on the patient's needs. The purpose of this research was to understand the impact of fill density and interrelate it with the release of a poorly water-soluble, weakly acidic, active pharmaceutical ingredient (API) from a hydroxypropyl methylcellulose acetate succinate (HPMC-AS) matrix, both mathematically and experimentally. Amorphous solid dispersions (ASDs) of ibuprofen with three grades of AquaSolveTM HPMC-AS (HG, MG, and LG) were developed using an HME process and evaluated using solid-state characterization techniques. Differential scanning calorimetry (DSC), powder X-ray diffraction (pXRD), and polarized light microscopy (PLM) confirmed the amorphous state of the drug in both polymeric filaments and 3D printed tablets. The suitability of the manufactured filaments for FDM processes was investigated using texture analysis (TA) which showed robust mechanical properties of the developed filament compositions. Using FDM, tablets with different fill densities (20-80%) and identical dimensions were printed for each polymer. In vitro pH shift dissolution studies revealed that the fill density has a significant impact (F(11, 24) = 15,271.147, p < 0.0001) and a strong negative correlation (r > -0.99; p < 0.0001) with the release performance, where 20% infill demonstrated the fastest and most complete release, whereas 80% infill depicted a more controlled release. The results obtained from this research can be used to develop a robust formulation strategy to control the drug release from 3D printed dosage forms as a function of fill density.

This paper presents an easy and low-cost flame treatment method to improve the bonding performance of GF/EP (Glass Fiber-Reinforced Epoxy) pultrusion plates, which are using widely for large size wind blades. In order to explore the effect of flame treatment on the bonding performance of the precast GF/EP pultruded sheet vs. the infusion plate, the GF/EP pultruded sheets were treated with different flame treatment cycles and were embedded in the fiber fabrics during the vacuum-assisted resin infusion process (VARI). The bonding shear strengths were measured by tensile shear tests. It is found that after 1, 3, 5, and 7 flame treatments, the tensile shear strength between the GF/EP pultrusion plate and infusion plate increased by 8.0%, 13.3%, 22.44%, and -2.1%, respectively. This indicates that the maximum tensile shear strength can be obtained after five times of flame treatment. In addition, DCB and ENF tests were also adopted to characterize the fracture toughness of the bonding interface with the optimal flame treatment. It is found that the optimal treatment gives increments of 21.84% and 78.36% for G I C and G II C, respectively. Finally, the surficial topography of the flame-treated GF/EP pultruded sheets were characterized by optical microscopy, SEM, contact angle test, FTIR, and XPS. The results show that flame treatment plays an impact on the interfacial performance through the combination of physical meshing locking and chemical bonding mechanism. Proper flame treatment would remove the weak boundary layer and mold release agent on the surface of the GF/EP pultruded sheet, etch the bonding surface and improve the oxygen-containing polar groups, such as C-O and O-C=O, to improve the surface roughness and surface tension coefficient of pultruded sheet to enhance the bonding performance. Excessive flame treatment destroys the integrity of epoxy matrix on bonding surface which results into the exposure of the glass fiber, and the carbonization of release agent and resin on the surface loosen the surficial structure, which reduces the bonding properties.

Trametes trogii is a member of the white-rot fungi family, which has a unique ability to break down recalcitrant lignin polymers to CO2 and water, and they have enormous potential to biodegrade a wide range of toxic environmental pollutants. Because of its industrial potential, the identification of lignin-degrading enzyme systems in Trametes is an important area of research. Development and utilization of industrial value genes are suffering due to deficiency knowledge of genome available for their manipulation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the global pandemic of COVID-19. SARS-CoV-2 genome encodes a main protease (nsp5, also called Mpro) and a papain-like protease (nsp3, also called PLpro), which are responsible for processing viral polyproteins to assemble a functional replicase complex. In this study, we found that Mpro of SARS-CoV-2 can cleave human MAGED2 and other mammalian orthologs at Gln-263. Moreover, SARS-CoV and MERS-CoV Mpro can also cleave human MAGED2, suggesting MAGED2 cleavage by Mpro is an evolutionarily conserved mechanism of coronavirus infection in mammals. Intriguingly, Mpro from Beta variant cleaves MAGED2 more efficiently than wild type, but Omicron Mpro is opposite. Further studies show that MAGED2 inhibits SARS-CoV-2 infection at viral replication step. Mechanistically, MAGED2 is associated with SARS-CoV-2 nucleocapsid protein through its N-terminal region in an RNA-dependent manner, and this disrupts the interaction between SARS-CoV-2 nucleocapsid protein and viral genome, thus inhibiting viral replication. When MAGED2 is cleaved by Mpro, the N-terminal of MAGED2 will translocate into the nucleus, and the truncated MAGED2 is unable to suppress SARS-CoV-2 replication. This work not only discovers the antiviral function of MAGED2 but also provides new insights into how SARS-CoV-2 Mpro antagonizes host antiviral response. IMPORTANCE Host factors that restrict severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain elusive. Here, we found that MAGED2 can be cleaved by SARS-CoV-2 main protease (Mpro) at Gln-263. SARS-CoV and MERS-CoV Mpro can also cleave MAGED2, and MAGED2 from multiple species can be cleaved by SARS-CoV-2 Mpro. Mpro from Beta variant cleaves MAGED2 more efficiently efficiently than wild type, but Omicron is the opposite. MAGED2 depletion enhances SARS-CoV-2 infection, suggesting its inhibitory role in SARS-CoV-2 infection. Mechanistically, MAGED2 restricts SARS-CoV-2 replication by disrupting the interaction between nucleocapsid and viral genomes. When MAGED2 is cleaved, its N-terminal will translocate into the nucleus. In this way, Mpro relieves MAGED2' inhibition on viral replication. This study improves our understanding of complex viral-host interaction and provides novel targets to treat SARS-CoV-2 infection.

α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the "stem" region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the "stem" region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.

The development of energy storage devices that can endure large and complex deformations is central to emerging wearable electronics. Hydrogels made from conducting polymers give rise to a promising integration of high conductivity and versatility in processing. However, the emergence of conducting polymer hydrogels with a desirable network structure cannot be readily achieved using conventional polymerization methods. Here we present a cryopolymerization strategy for preparing an intrinsically stretchable, compressible and bendable anisotropic polyvinyl alcohol/polyaniline hydrogel with a complete recovery of 100% stretching strain, 50% compressing strain and fully bending. Due to its high mechanical strength, superelastic properties and bi-continuous phase structure, the as-obtained anisotropic polyvinyl alcohol/polyaniline hydrogel can work as a stretching/compressing/bending electrode, maintaining its stable output under complex deformations for an all-solid-state supercapacitor. In particular, it achieves an extremely high energy density of 27.5 W h kg-1, which is among that of state-of-the-art stretchable supercapacitors.

To further increase the quantity and density of functional groups on adsorbent, terminal triethylenetetramine hyperbranched dendrimer-like polymer modified silica-gel (SG-TETA and SG-TETA2) was synthesized. The hyperbranched dendrimer-like polymer was successfully introduced onto silica gel and new cavities were formed, which was demonstrated by FTIR, SEM, and BET. The highest adsorption capacities of SG-TETA and SG-TETA2 obtained from Langmuir model toward Au(III) were 2.11, and 2.27 mmol g-1, respectively, indicating that SG-TETA2 possessing more functional groups had a better adsorption ability. Moreover, the adsorbents combined with Au(III) ion through chelation and electrostatic attraction mechanism, after which reduction reactions for Au(III) ion loaded on adsorbents proceeded. SG-TETA2 had better adsorption selectivity than SG-TETA in removing Au(III) in Au-existed ion solution systems. SG-TETA2 had higher overall adsorption capacities compared to silica-gel-based hyperbranched polymers functionalized by diethylenetriamine. Therefore, the effective recovery makes SG-TETA2 a practical adsorbent in removing Au(III) ion from e-wastes and industrial effluents with much prospect.

Osteosarcoma (OS) tissue resection with distinctive bactericidal activity, followed by regeneration of bone defects, is a highly demanded clinical treatment. Biodegradable Mg-based implants with desirable osteopromotive and superior mechanical properties to polymers and ceramics are promising new platforms for treating bone-related diseases. Integration of biodegradation control, osteosarcoma destruction, anti-bacteria, and bone defect regeneration abilities on Mg-based implants by applying biosafe and facile strategy is a promising and challenging topic. Here, a black Mn-containing layered double hydroxide (LDH) nanosheet-modified Mg-based implants was developed. Benefiting from the distinctive capabilities of the constructed black LDH film, including near-infrared optical absorption and reactive oxygen species (ROS) generation in a tumor-specific microenvironment, the tumor cells and tissue could be effectively eliminated. Concomitant bacteria could be killed by localized hyperthermia. Furthermore, the enhanced corrosion resistance and synergistic biofunctions of Mn and Mg ions of the constructed black LDH-modified Mg implants significantly facilitated cell adhesion, spreading and proliferation and osteogenic differentiation in vitro, and accelerated bone regeneration in vivo. This work offers a new platform and feasible strategy for OS therapeutics and bone defect regeneration, which broadens the biomedical application of Mg-based alloys.

The protein α-hemolysin (α-HL) is a self-assembling exotoxin that binds to the membrane of a susceptible host cell. In this paper, experimental studies show that cyrtominetin (CTM) can inhibit the hemolytic activity of α-HL. To understand how CTM can affect hemolytic activity, molecular dynamics simulations were carried out for α-HL-CTM complex and these results were compared with the crystal structure of monomeric α-HL. With this approach, the analysis revealed that the inhibition of CTM involves CTM directly binding to α-HL. Due to the binding of CTM, the conformation of the critical "Loop" region was restrained. This mechanism was confirmed by the experimental data. These findings indicate that CTM hinders the lysis activity of α-HL through a novel mechanism.

Alpha-hemolysin (α-HL) is a self-assembling, channel-forming toxin produced by most Staphylococcus aureus strains as a 33.2-kDa soluble monomer. Upon binding to a susceptible cell membrane, the monomer self-assembles to form a 232.4-kDa heptamer that ultimately causes host cell lysis and death. Consequently, α-HL plays a significant role in the pathogenesis of S. aureus infections, such as pneumonia, mastitis, keratitis and arthritis. In this paper, experimental studies show that oroxylin A (ORO), a natural compound without anti-S. aureus activity, can inhibit the hemolytic activity of α-HL. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were performed to understand the formation of the α-HL-ORO complex. This combined approach revealed that the catalytic mechanism of inhibition involves the direct binding of ORO to α-HL, which blocks the conformational transition of the critical "Loop" region of the α-HL protein thereby inhibiting its hemolytic activity. This mechanism was confirmed by experimental data obtained from a deoxycholate-induced oligomerization assay. It was also found that, in a co-culture system with S. aureus and human alveolar epithelial (A549) cells, ORO could protect against α-HL-mediated injury. These findings indicate that ORO hinders the lytic activity of α-HL through a novel mechanism, which should facilitate the design of new and more effective antibacterial agents against S. aureus.