FUS/TLS Suppresses Enterovirus Replication and Promotes Antiviral Innate Immune Responses.

During viral infection, the dynamic virus-host relationship is constantly in play. Many cellular proteins, such as RNA-binding proteins (RBPs), have been shown to mediate antiviral responses during viral infection. Here, we report that the RBP FUS/TLS (fused in sarcoma/translocated in liposarcoma) acts as a host-restricting factor against infection with coxsackievirus B3 (CVB3). Mechanistically, we found that deletion of FUS leads to increased viral RNA transcription and enhanced internal ribosome entry site (IRES)-driven translation, with no apparent impact on viral RNA stability. We further demonstrated that FUS physically interacts with the viral genome, which may contribute to direct inhibition of viral RNA transcription/translation. Moreover, we identified a novel function for FUS in regulating host innate immune response. We show that in the absence of FUS, gene expression of type I interferons and proinflammatory cytokines elicited by viral or bacterial infection is significantly impaired. Emerging evidence suggests a role for stress granules (SGs) in antiviral innate immunity. We further reveal that knockout of FUS abolishes the ability to form SGs upon CVB3 infection or poly(I·C) treatment. Finally, we show that, to avoid FUS-mediated antiviral response and innate immunity, CVB3 infection results in cytoplasmic mislocalization and cleavage of FUS through the enzymatic activity of viral proteases. Together, our findings in this study identify FUS as a novel host antiviral factor which restricts CVB3 replication through direct inhibition of viral RNA transcription and protein translation and through regulation of host antiviral innate immunity.IMPORTANCE Enteroviruses are common human pathogens, including those that cause myocarditis (coxsackievirus B3 [CVB3]), poliomyelitis (poliovirus), and hand, foot, and mouth disease (enterovirus 71). Understanding the virus-host interaction is crucial for developing means of treating and preventing diseases caused by these pathogens. In this study, we explored the interplay between the host RNA-binding protein FUS/TLS and CVB3 and found that FUS/TLS restricts CVB3 replication through direct inhibition of viral RNA transcription/translation and through regulation of cellular antiviral innate immunity. To impede the antiviral role of FUS, CVB3 targets FUS for mislocalization and cleavage. Findings from this study provide novel insights into interactions between CVB3 and FUS, which may lead to novel therapeutic interventions against enterovirus-induced diseases.

Emerging nanomedicines for effective breast cancer immunotherapy.

Breast cancer continues to be the most frequently diagnosed malignancy among women, putting their life in jeopardy. Cancer immunotherapy is a novel approach with the ability to boost the host immune system to recognize and eradicate cancer cells with high selectivity. As a promising treatment, immunotherapy can not only eliminate the primary tumors, but also be proven to be effective in impeding metastasis and recurrence. However, the clinical application of cancer immunotherapy has faced some limitations including generating weak immune responses due to inadequate delivery of immunostimulants to the immune cells as well as uncontrolled modulation of immune system, which can give rise to autoimmunity and nonspecific inflammation. Growing evidence has suggested that nanotechnology may meet the needs of current cancer immunotherapy. Advanced biomaterials such as nanoparticles afford a unique opportunity to maximize the efficiency of immunotherapy and significantly diminish their toxic side-effects. Here we discuss recent advancements that have been made in nanoparticle-involving breast cancer immunotherapy, varying from direct activation of immune systems through the delivery of tumor antigens and adjuvants to immune cells to altering immunosuppression of tumor environment and combination with other conventional therapies.

One-step in situ biosynthesis of graphene oxide-bacterial cellulose nanocomposite hydrogels.

Graphene oxide-bacterial cellulose (GO/BC) nanocomposite hydrogels with well-dispersed GO in the network of BC are successfully developed using a facile one-step in situ biosynthesis by adding GO suspension into the culture medium of BC. During the biosynthesis process, the crystallinity index of BC decreases and GO is partially reduced. The experimental results indicate that GO nanosheets are uniformly dispersed and well-bound to the BC matrix and that the 3D porous structure of BC is sustained. This is responsible for efficient load transfer between the GO reinforcement and BC matrix. Compared with the pure BC, the tensile strength and Young's modulus of the GO/BC nanocomposite hydrogel containing 0.48 wt% GO are significantly improved by about 38 and 120%, respectively. The GO/BC nanocomposite hydrogels are promising as a new material for tissue engineering scaffolds.

The inhibition of lamellar hydroxyapatite and lamellar magnetic hydroxyapatite on the migration and adhesion of breast cancer cells.

Hydroxyapatite nanoparticles have been reported to exhibit potent anti-tumor effects in some cancer cells. In our previous study, we have successfully synthesized two types of hydroxyapatite nanoparticles, laminated hydroxyapatite (L-HAp) and laminated magnetic hydroxyapatite (LM-HAp). In this study, we wanted to investigate the effects of L-HAp and LM-HAp with various concentrations on human breast cancer MDA-MB-231 cells. Cell proliferation was assessed with a MTT colorimetric assay. Scratch and adhesion assays were used to detect the effects of these two materials on migration and adhesion. The expressions of integrin ß1 and Akt were measured by Western blotting. Our results showed that L-HAp and LM-HAp had little cell cytotoxicity and significantly reduced cell mobility and adhesion. LM-HAp showed greater inhibitor ability on migration and adhesion of MDA-MB-231 cells. Moreover, results from western blotting showed that L-HAp and LM-HAp impacted the phosphorylation of integrin ß1, but showed no regular impact on Akt. This study suggests that L-HAp and LM-HAp may be potential anti-tumor and delivery system for breast cancer therapy.

Water-Induced Expanded Bilayer Vascular Graft with Good Hemocompatibility and Biocompatibility.

Shape memory polymer (SMP) vascular grafts are promising interventional vascular grafts for cardiovascular disease (CAD) treatment; However, hemocompatibility and biocompatibility, which are the critical issues for the SMP vascular grafts, are not systematically concerned. Furthermore, the water-induced SMP grafts are more convenient and safer than the thermally induced ones in case of the bioapplication. Herein, in this work, the new water-induced expanded bilayer vascular graft with the inner layer of crosslinked poly(ε-caprolactone) (cPCL) and the outer layer of water-induced SMP of regenerated chitosan/polyvinyl alcohol (RCS/PVA) are prepared by hot pressing and programming approaches. The results show that the inner and outer layer surfaces of the prepared grafts are smooth, and they exhibit good interfacial interaction properties. The bilayer grafts show good mechanical properties and can be expanded in water with a diameter expansion of ≈30%. When compared with commercial expanded polytetrafluoroethylene (ePTFE), the bilayer graft shows better hemocompatibility (platelet adhesion, hemolysis rate, various clotting times, and plasma recalcification time (PRT)) and in vitro and in vivo biocompatibility, which thus is a promising material for the vascular graft.

Strong, tough, and elastic poly(vinyl alcohol)/polyacrylamide DN hydrogels based on the Hofmeister effect for articular cartilage replacement.

Traditional hydrogels are usually weak and brittle, which limit their application in articular cartilage replacement because cartilage is generally strong, tough, and elastic in nature. Therefore, it is highly desirable to construct hydrogels to mimic the mechanical properties of the native articular cartilage. Herein, in this work, poly(vinyl alcohol)/polyacrylamide (PVA/PAM) DN hydrogels were prepared by in situ polymerization, which were then treated with Hofmeister series ions (Cit3-, SO42-, and Cl-) to achieve H-PVA/PAM DN hydrogels. Among the three Hofmeister ions, the DN hydrogel treated with Cit3- (named PVA/PAM-Cit) showed the densest microstructure and the highest crystallinity degree. In this context, PVA/PAM-Cit exhibited a tensile strength of 18.9 ± 1.6 MPa, a compressive strength of 102.3 ± 7.9 MPa, a tensile modulus of 10.6 ± 2.1 MPa, a compressive modulus of 8.9 ± 0.8 MPa, and a roughness of 66.2 ± 4.2 MJ m-3, respectively, which were the highest strength and modulus, and the second highest toughness when compared with those of the reported PVA and PVA based DN hydrogels so far. It also showed an extreme high elasticity, which could maintain a stress of 99.2% after 500 cycles of fatigue testing. Additionally, PVA/PAM-Cit can promote the adhesion, spreading and proliferation of chondrocytes. These results verify that such a strong, tough, and elastic hydrogel could be a novel candidate material for articular cartilage replacement.

Hierarchical porous bacterial cellulose scaffolds with natural biomimetic nanofibrous structure and a cartilage tissue-specific microenvironment for cartilage regeneration and repair.

The limited three-dimensional (3D) nano-scale pore structure and lack of biological function hamper the application of bacterial cellulose (BC) in cartilage tissue engineering. To address this challenge, 3D hierarchical porous BC/decellularized cartilage extracellular matrix (DCECM) scaffolds with structurally and biochemically biomimetic cartilage regeneration microenvironment were fabricated by freeze-drying technique after EDC/NHS chemical crosslinking. The BC/DCECM scaffolds exhibited excellent mechanical properties, water superabsorbency and shape-memory properties. Compared with the BC control, the BC/DCECM scaffolds exhibited enhanced cell adhesion and proliferation. Cartilage regeneration in vitro and in vivo indicated that the BC/DCECM scaffolds achieved satisfactory neocartilage tissue regeneration with superior original shape fidelity, exterior natural cartilage-like appearance and histologically cartilage-specific lacuna formation and ECM deposition. Furthermore, the BC/DCECM scaffolds achieved superior repair outcomes, as hyaline cartilage-like tissue formed within the defect sites. The present study constitutes a strong step toward the further application of BC in cartilage tissue engineering.

Simultaneous engineering of nanofillers and patterned surface macropores of graphene/hydroxyapatite/polyetheretherketone ternary composites for potential bone implants.

Incorporating bioactive nanofillers and creating porous surfaces are two common strategies used to improve the tissue integration of polyetheretherketone (PEEK) material. However, few studies have reported the combined use of both strategies to modify PEEK. Herein, for the first time, dual nanoparticles of graphene oxide (GO) and hydroxyapatite (HAp) were incorporated into PEEK matrix to obtain ternary composites that were laser machined to create macropores with diameters ranging from 200 µm to 600 µm on the surfaces. The surface morphology and chemistry, mechanical properties, and cellular responses of the composites were investigated. The results show that micropatterned pores with a depth of 50 µm were created on the surfaces of the composites, which do not significantly affect the mechanical properties of the resultant composites. More importantly, the incorporation of GO and HAp significantly improves the cell adhesion and proliferation on the surface of PEEK. Compared to the smooth surface composite, the composites with macroporous surface exhibit markedly enhanced cell viability. The combined use of nanofillers and surface macropores may be a promising way of improving tissue integration of PEEK for bone implants.

Enwrapping Polydopamine on Doxorubicin-Loaded Lamellar Hydroxyapatite/Poly(lactic-co-glycolic acid) Composite Fibers for Inhibiting Bone Tumor Recurrence and Enhancing Bone Regeneration.

Simultaneous prevention of bone tumor recurrence and promotion of repairing bone defects resulting from tumorectomy remain a challenge. Herein, we report a polydopamine (PDA)-coated composite scaffold consisting of doxorubicin (DOX)-loaded lamellar hydroxyapatite (LHAp) and poly(lactic-co-glycolic acid) (PLGA) in an attempt to reach dual functions of tumor inhibition and bone repair. The DOX was intercalated into LHAp, and the DOX-loaded LHAp was incorporated into PLGA solution to prepare a DOX-intercalated LHAp/PLGA (labeled as DH/PLGA) scaffold that was coated with PDA to obtain a PDA@DH/PLGA scaffold. The morphology, structure, wettability, mechanical properties, drug release, biocompatibility, and in vitro and in vivo bioactivities of the PDA@DH/PLGA scaffold were evaluated. It is found that PDA coating not only improves hydrophilicity and mechanical properties, but also leads to more sustainable drug release. More importantly, the PDA@DH/PLGA scaffold shows significantly inhibited growth of tumor cells initially and subsequent improved adhesion and proliferation of osteoblasts. In addition, the PDA coating improves the bioactivity of the DH/PLGA scaffold as suggested by the in vitro biomineralization. Further in vivo study demonstrates the improved bone growth around PDA@DH/PLGA over DH/PLGA after 20 days of drug release. The dual functional PDA@DH/PLGA scaffold shows great promise in the treatment of bone tumor.

Fabrication of Robust, Shape Recoverable, Macroporous Bacterial Cellulose Scaffolds for Cartilage Tissue Engineering.

Recently, the fabricating of three-dimensional (3D) macroporous bacterial cellulose (MP-BC) scaffolds with mechanically disintegrated BC fragments has attracted considerable attention. However, the successful implementation of these materials depends mainly on their mechanical stability and robustness. Here, a non-toxic crosslinker, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), is employed to induce crosslinking reactions between BC fragments. In addition to their large pore sizes, the EDC/NHS-crosslinked MP-BC scaffolds exhibit excellent compression properties and shape recovery ability, owing to the EDC/NHS-induced crosslinking on the BC nanofibers. The results of in vitro studies reveal that the biocompatibility of MP-BC scaffolds is better than that of pristine BC scaffolds because the former provided more space for cell proliferation. The results of in vivo studies show that the neocartilage tissue with native cartilage appearance and abundant cartilage-specific extracellular matrix deposition is successfully regenerated in nude mice. The findings reveal the immense application potential of mechanically robust BC scaffolds with controllable pore sizes and shape-recoverable properties in tissue engineering.

Ultrathin, Strong, and Highly Flexible Ti3C2Tx MXene/Bacterial Cellulose Composite Films for High-Performance Electromagnetic Interference Shielding.

The fabrication of ultrathin films that are electrically conductive and mechanically strong for electromagnetic interference (EMI) shielding applications is challenging. Herein, ultrathin, strong, and highly flexible Ti3C2Tx MXene/bacterial cellulose (BC) composite films are fabricated by a scalable in situ biosynthesis method. The Ti3C2Tx MXene nanosheets are uniformly dispersed in the three-dimensional BC network to form a mechanically entangled structure that endows the MXene/BC composite films with excellent mechanical properties (tensile strength of 297.5 MPa at 25.7 wt % Ti3C2Tx) and flexibility. Importantly, a 4 µm thick Ti3C2Tx/BC composite film with 76.9 wt % Ti3C2Tx content demonstrates a specific EMI shielding efficiency of 29141 dB cm2 g-1, which surpasses those of most previously reported MXene-based polymer composites with similar MXene contents and carbon-based polymer composites. Our findings show that the facile, environmentally friendly, and scalable fabrication method is a promising strategy for producing ultrathin, strong, and highly flexible EMI shielding materials such as the freestanding Ti3C2Tx/BC composite films for efficient EMI shielding to address EMI problems of a fast-developing modern society.

Improved Removal of Toxic Metal Ions by Incorporating Graphene Oxide into Bacterial Cellulose.

The efficient removal of toxic metal ions from waste water is of critical importance in environmental protection. In this study, we report the incorporation of graphene oxide (GO) into bacterial cellulose (BC) and the effect on the removal of metal ions from waste water. The as-prepared BC/GO adsorbents have a three-dimensional (3D) network structure with interconnected pores and high porosity. The adsorption capacities and efficiencies of the BC/GO adsorbents with varying GO contents were compared by using Cu2+, Cd2+, and Pb2+ as model heavy metal ions. The incorporated GO into the BC/GO adsorbents plays a critical role in removing metal ions through strong electrostatic interactions between the positive metal ions and the negative functional groups on GO. In addition, the effects of pH, contact time, adsorbent dose, and ion concentration on the adsorption behavior of the BC/GO adsorbents were investigated. The data from adsorption kinetics indicate that the adsorption of Cu2+, Cd2+, and Pb2+ on BC/GO obeys a pseudo-second-order model, while the adsorption isotherms vary with the type of metal ions. The desorption and readsorption experiments of the BC/GO adsorbents demonstrate good recyclability. It has been demonstrated that incorporating GO into BC is an effective way to improve the adsorption behavior of BC.

Interpenetrated nano- and submicro-fibrous biomimetic scaffolds towards enhanced mechanical and biological performances.

Developing fibrous scaffolds with hierarchical structures that closely mimic natural extracellular matrix (ECM) is highly desirable. However, fabricating scaffolds with true nanofibers (<100 nm) and submicrofibers (<1 µm) remains a big challenge. In this work, to mimic the fibrillar structure of natural ECM, bacterial cellulose (BC) nanofibers were hybridized with cellulose acetate (CA) submicrofibers for the first time. The interpenetrated nano-submicron fibrous BC/CA scaffold was fabricated using the combined electrospinning and modified in situ biosynthesis method. The BC/CA scaffold has an integrated symmetrical nanostructure in which BC nanofibers (42 nm in diameter) penetrate into the submicrofibrous CA (820 nm in diameter) scaffold. The BC/CA scaffold shows an interconnected porous structure with a high porosity of >90%. Additionally, the combination of CA submicrofibers with BC nanofibers leads to significantly improved mechanical properties over nanofibrous BC and submicrofibrous CA scaffolds and enlarged pores over nanofibrous BC scaffold. In addition, the biological behaviors of prepared BC/CA on MC3T3-E1 cells were investigated. Results suggested that BC/CA scaffold is beneficial for cell migration and proliferation. Moreover, the BC/CA scaffold shows higher alkaline phosphatase (ALP) activity, and calcium depositions. In addition, the hierarchical structures can effectively improve the expression of osteogenic gene (ALP mRNA and Runx2 mRNA) and protein (ALP). We believe that the methodology might provide biomimetic morphological microenvironments for enhanced tissue regeneration.

Controllable synthesis of biomimetic nano/submicro-fibrous tubes for potential small-diameter vascular grafts.

Mimicking the morphological structure of native blood vessels is critical for the development of vascular grafts. Herein, small-diameter composite vascular grafts that integrate the nanofibrous bacterial cellulose (BC) and submicrofibrous cellulose acetate (CA) were fabricated via a combined electrospinning and step-by-step in situ biosynthesis. Scanning electron microscopy (SEM) observation shows the nano/submicro-fibrous morphology and well-interconnected porous structure of the BC/CA grafts. It is found that the BC/CA graft with a suitable BC content demonstrates lower potential of thrombus formation and enhanced endothelialization as compared to the BC and CA counterparts. Western blotting and RT-qPCR results suggest that the BC/CA-2 graft promotes endothelialization by improving expressions of genes vWF-1 and CD31 and protein CD31. The in vivo tests demonstrate much lower inflammatory response to the BC/CA graft. These results suggest that the BC/CA graft shows a great potential as an artificial graft for rapid formation of an endothelial cell monolayer.

Liposome-mediated transient transfection reduces cholesterol-dependent coxsackievirus infectivity.

Liposome-mediated gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Coxsackievirus B3 is an important human pathogen associated with various diseases. Here we reported that liposome-mediated transient transfection of plasmid cDNA inhibited coxsackieviral replication at the levels of RNA, protein and viral progeny release. These inhibitory effects were observed in various cell types and by using different liposome reagents. We further showed that the inhibition was likely due to the lack of virus attachment. Moreover, we showed that addition of cholesterol restored, at least in part, the viral infectivity. Interestingly, we found that membrane cholesterol levels were unchanged during transfection, indicating that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Our data suggest that liposome-mediated cDNA transient transfection inhibits coxsackievirus infectivity via inhibition of viral attachment, which is likely occurring through the changes of membrane cholesterol integrity.

Surface controlled calcium phosphate formation on three-dimensional bacterial cellulose-based nanofibers.

Studies on the early calcium phosphate (Ca-P) formation on nanosized substrates may allow us to understand the biomineralization mechanisms at the molecular level. In this work, in situ formation of Ca-P minerals on bacterial cellulose (BC)-based nanofibers was investigated, for the first time, using the X-ray absorption near-edge structure (XANES) spectroscopy. In addition, the influence of the surface coating of nanofibers on the formation of Ca-P minerals was determined. Combined with XRD analysis, XANES results revealed that the nascent precursor was ACP (amorphous calcium phosphate) which was converted to TCP (ß-tricalcium phosphate), then OCP (octacalcium phosphate), and finally to HAP (hydroxyapatite) when phosphorylated BC nanofibers were the templates. However, the formation of nascent precursor and its transformation process varied depending on the nature of the coating material on nanofibrous templates. These results provide new insights into basic mechanisms of mineralization and can lead to the development of novel bioinspired nanostructured materials.

Mechanical properties and cytotoxicity of nanoplate-like hydroxyapatite/polylactide nanocomposites prepared by intercalation technique.

Hydroxyapatite (HAp) in the forms of fiber, needle, and whisker has been employed as fillers in polymer composites. Herein, nanoplate-like HAp synthesized by template-assisted self-assembly was used to reinforce polylactide (PLA) nanocomposites via the solution intercalation method. Dynamic and static mechanical properties and cytotoxicity of the as-prepared HAp/PLA nanocomposites were assessed in addition to characterizations by XRD, FTIR, and TGA. XRD analysis confirms the formation of exfoliated structure in the HAp/PLA nanocomposites. The HAp/PLA nanocomposites exhibit better static and dynamic mechanical properties than unreinforced PLA. Furthermore, the HAp/PLA nanocomposite with an optimum HAp content of 20wt% (20HAp/PLA) demonstrates not only the best mechanical performance but also the highest thermal stability among the nanocomposite samples. Cell studies using a mouse fibroblast cell line (L929) suggest that 20HAp/PLA shows excellent biocompatibility, which makes it a promising material for biomedical applications.

Evolution of morphology of bacterial cellulose scaffolds during early culture.

Morphological characteristics of a fibrous tissue engineering (TE) scaffold are key parameters affecting cell behavior. However, no study regarding the evolution of morphology of bacterial cellulose (BC) scaffolds during the culture process has been reported to date. In this work, BC scaffolds cultured for different times starting from 0.5h were characterized. The results demonstrated that the formation of an integrated scaffold and its 3D network structure, porosity, fiber diameter, light transmittance, and the morphology of hydroxyapatite (HAp)-deposited BC scaffolds changed with culture time. However, the surface and crystal structure of BC fibers did not change with culture time and no difference was found in the crystal structure of HAp deposited on BC templates regardless of BC culture time. The findings presented herein suggest that proper selection of culture time can potentially enhance the biological function of BC TE scaffold by optimizing its morphological characteristics.

Creation of macropores in three-dimensional bacterial cellulose scaffold for potential cancer cell culture.

There is an increasing need for an effective in vitro model that can resemble the 3-D nature of tumor microenvironments. In this work, a 3-D bacterial cellulose (BC) scaffold with macropores was fabricated by a facile freeze drying method for potential culture of cancer cells. This in vitro study reported, for the first time, the role of macropores in the adjustment of cancer cell behavior when compared with previous results cultured in BC scaffolds without macropores. The scaffold was characterized by SEM and mercury intrusion porosimeter. A human breast cancer cell line (MDA-MB-231) cultured in the macroporous BC scaffold was examined via cell proliferation, histological and SEM analyses. The results demonstrated that the macroporous scaffold provided a good environment for cell viability, adhesion, proliferation, and infiltration. These findings suggested that the macroporous BC scaffold might have great potential for use in the in vitro culture of cancer cells.

Preparation of three-dimensional braided carbon fiber-reinforced PEEK composites for potential load-bearing bone fixations. Part I. Mechanical properties and cytocompatibility.

In this study, we focused on fabrication and characterization of three-dimensional carbon fiber-reinforced polyetheretherketone (C3-D/PEEK) composites for orthopedic applications. We found that pre-heating of 3-D fabrics before hot-pressing could eliminate pores in the composites prepared by 3-D co-braiding and hot-pressing techniques. The manufacturing process and the processing variables were studied and optimum parameters were obtained. Moreover, the carbon fibers were surface treated by the anodic oxidization and its effect on mechanical properties of the composites was determined. Preliminary cell studies with mouse osteoblast cells were also performed to examine the cytocompatibility of the composites. Feasibility of the C3-D/PEEK composites as load-bearing bone fixation materials was evaluated. Results suggest that the C3-D/PEEK composites show good promising as load-bearing bone fixations.