The Upregulation of a Novel Long Noncoding RNA AK097647 Promotes Enterovirus 71 Replication and Decreases IFN-λ1 Secretion.

BACKGROUND: Enterovirus 71 (EV71) infects millions of children every year in China and has become a challenge to public health. However, there is no effective treatment for EV71 infection. Long noncoding RNAs (lncRNAs) have been found to play various roles in virus replication and infection. OBJECTIVE: We aimed to explore the role of a novel long noncoding RNA AK097647 (lncRNA-AK097647) during EV71 infection. METHODS: To assess the role of lncRNA-AK097647 during EV71 infection, siRNAs were used to silence lncRNA-K097647 expression. RT-qPCR assay and Western blotting were applied to measure the mRNA and protein levels of EV71 VP1 and the phosphorylation of NF-κB. ELISA was used to detect the level of IFN-λ1 expression. RESULTS: The novel lncRNA-AK097647 was upregulated in human rhabdomyosarcoma cells and the blood of hand, foot, and mouth disease patients infected with EV71, as demonstrated by RT-qPCR. Interestingly, RNAi-mediated knockdown of lncRNA-AK097647 dramatically increased the level of IFN-λ1 expression, resulting in the suppression of EV71 replication. In contrast, overexpression of lncRNA-AK097647 decreased the level of IFN-λ1 expression and resulted in increased EV71 replication. In addition, we found that lncRNA-AK097647 could inhibit the phosphorylation of NF-κB. CONCLUSION: These results suggest a novel mechanism by which EV71 evades the IFN-mediated host antiviral response by increasing lncRNA-AK097647 expression.

A biodegradable soy protein isolate-based waterborne polyurethane composite sponge for implantable tissue engineering.

A biodegradable soy protein isolate-based waterborne polyurethane composite sponge (SWPU) was prepared from soy protein isolate (SPI) and polyurethane prepolymer (PUP) by a process involving chemical reaction and freeze-drying. Effects of SPI content (0, 10%, 30%, 50%, 70%) on the micro-structure and physical properties of the composite sponges were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The results showed that the reaction between -NCO of PUP and -NH2 of SPI formed porous SPI-based WPU composite sponges. The results of the water absorption ratio measurement, solvent resistance measurement and compressive testing showed that water absorption, hydrophilicity, and tensile strength in the dry state of the composite sponges increased with the increase of SPI content. Especially, the tensile strength ranged from 0.3 MPa to 5.5 MPa with the increase in SPI content. The cytocompatibility and biodegradability of the composite sponges were evaluated by in vitro cell culture and in vivo implantation experiments. The results indicated that a certain SPI content in the sponges could promote the adhesion, growth, and proliferation of cells, enhance the cytocompatibility and accelerate the degradation speed of composite sponges. During the in vivo implanting period within 9 months, SWPU-50 sponge containing 50% of SPI brought out the lowest activated inflammatory reaction, most newly-regenerated blood capillaries, and best histocompatibility. All results indicated that SWPU-50 composite sponges had greatest potential for tissue engineering.

RASSF4 promotes EV71 replication to accelerate the inhibition of the phosphorylation of AKT.

Enterovirus 71 (EV71) is a neurotropic virus that causes hand, foot and mouth disease (HFMD), occasionally leading to death. As a member of the RAS association domain family (RASSFs), RASSF4 plays important roles in cell death, tumor development and signal transduction. However, little is known about the relationship between RASSF4 and EV71. Our study reveals for the first time that RASSF4 promotes EV71 replication and then accelerates AKT phosphorylation inhibition in EV71-infected 293T cells, suggesting that RASSF4 may be a potential new target for designing therapeutic measures to prevent and control EV71 infection.

Photoguided Shape Deformation of Azobenzene-Containing Polymer Microparticles.

Here we present the generation of uniform microparticles with tunable diameters from azobenzene-based homopolymer by combining the microfluidics technique and emulsion-solvent evaporation route. In addition, the photoinduced deformation behavior of these microspheres, irradiated by a linearly polarized beam with different irradiation time and direction, are systemically studied. The deformation process through real time optical microscope observation can be investigated, benefiting from the uniform and microscaled size of the polymer particles. These results indicate that the deformation degree characterized by relative variation of the long axial for the particles can be controlled by the irradiation time. Moreover, elongated particles with tunable aspect ratio or tilted shape can be generated by manipulating the irradiation direction and/or time. Interestingly, the shape transformation kinetics displays a significant dependence on initial size of the polymer particle. In addition, the shape transformation of the polymer particle can lead to the variation of the orientation and distribution of the encapsulated anisotropic gold nanorods.

miR-27a suppresses EV71 replication by directly targeting EGFR.

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has broken out several times and was accompanied by neurological disease. microRNAs, a class of small non-coding RNAs that are approximately 20 nucleotides long, play important roles in the regulation of various biological processes, including antiviral defense. However, the roles of miRNAs in EV71 replication and pathogenesis are not well understood. In this study, we found that the expression of miR-27a was significantly decreased in EV71-infected cells. Interestingly, the over-expression of miR-27a could inhibit EV71 replication, as measured by virus titration, qPCR, and Western blotting. We identified EGFR mRNA is a bona fide target of miR-27a by computational analysis and luciferase reporter assays. Furthermore, miR-27a could decrease EGFR expression, as measured by qPCR and Western blotting. Moreover, the inhibition of EGFR expression by miR-27a decreased the phosphorylation of Akt and ERK, which facilitate EV71 replication. These results suggest that miR-27a may have antiviral activity against EV71 by inhibiting EGFR.

Camrelizumab-induced anaphylactic reaction: a case report and literature review.

Camrelizumab is an immune checkpoint inhibitor clinically used to treat various types of tumours. In this study, the authors provided the first report of a case of an anaphylactic reaction induced by camrelizumab in the treatment of a patient with squamous cell carcinoma of the floor of the mouth. The patient, a 58-year-old man, was diagnosed with advanced squamous cell carcinoma of the floor of the mouth, with cancer infiltration and multiple metastases. He underwent treatment for nine cycles, in which cycles 1-5 he received camrelizumab, albumin-bound paclitaxel, and cisplatin (200 mg of camrelizumab each time, every 3 weeks), with no adverse reactions; in cycle 6, he received albumin-bound paclitaxel and cisplatin, with no adverse reactions; and in cycles 7-9, he received camrelizumab and albumin-bound paclitaxel. However, 30 min after 8th administration of camrelizumab (cycle 9), he suddenly developed sweating, a pale complexion, clamminess and cyanosis of the limbs (percutaneous arterial oxygen saturation [SpO2] = 82%, blood pressure [BP] = 79/49 mmHg, heart rate [HR] = 83 beats/min [bpm] and respiratory rate [RR) = 12 bpm). The patient underwent intravenous infusion of methylprednisolone (80 mg) combined with dopamine to boost the BP; he regained consciousness 20 min later, and many parts of his skin appeared smooth, with no desquamation and accompanied by itching erythema, especially on the upper limbs. Approximately 2 h after treatment, the patient's skin erythema subsided (vital sign monitoring results: SpO2 = 100%, BP = 122/84 mmHg, HR = 91 bpm and RR = 17 bpm); the patient did not complain about his obvious discomfort. Despite the rarity of acute anaphylactic reactions among immune-related adverse reactions, great importance should be given to anaphylactic reactions of camrelizumab due to its extensive clinical application.

Coxsackievirus A16 infection triggers apoptosis in RD cells by inducing ER stress.

Coxsackievirus A16 (CA16) infection, which is responsible for hand, foot and mouth disease (HFMD), has become a common health problem in Asia due to the prevalence of the virus. Thus, it is important to understand the pathogenesis of CA16 infection. Viruses that induce endoplasmic reticulum (ER) stress are confronted with the unfolded protein response (UPR), which may lead to apoptotic cell death and influence viral replication. In this study, we found that CA16 infection could induce apoptosis and ER stress in RD cells. Interestingly, apoptosis via the activation of caspase-3, -8 and -9 in the extrinsic or intrinsic apoptotic pathways in RD cells was inhibited by 4-phenyl butyric acid (4PBA), a chemical chaperone that reduces ER stress. These results suggest that CA16 infection leads to ER stress, which in turn results in prolonged ER stress-induced apoptosis. This study provides a new basis for understanding CA16 infection and host responses.

Functionalization of magnetic nanoparticles with dendritic-linear-brush-like triblock copolymers and their drug release properties.

Novel water-soluble dendritic-linear-brush-like triblock copolymer polyamidoamine-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) (PAMAM-b-PDMAEMA-b-PPEGMA)-grafted superparamagnetic iron oxide nanoparticles (SPIONs) were successfully prepared via a two-step copper-mediated atom transfer radical polymerization (ATRP) method. The macroinitiators were immobilized on the surface of Fe(3)O(4) nanoparticles via effective ligand exchange of oleic acid with the propargyl focal point PAMAM-typed dendron (generation 2.0, denoted as propargyl-D(2.0)) containing four carboxyl acid end groups, following a click reaction with 2'-azidoethyl-2-bromoisobutylate (AEBIB). PDMAEMA and PPEGMA were grown gradually from nanoparticle surfaces using the "grafting from" approach, which rendered the SPIONs soluble in water and reversed aggregation. To the best of our knowledge, this is the first report that describes the functionalization of magnetic nanoparticles with dendritic-linear-brush-like triblock copolymers. The modified nanoparticles were systematically studied via TEM, FT-IR, DLS, XRD, NMR, TGA, and magnetization measurements. DLS measurement confirmed that the obtained dendritic-linear-brush-like triblock copolymer-grafted SPIONs had a uniform hydrodynamic particle size of average diameter less than 30 nm. The dendritic-linear-brush-like triblock copolymer-grafted SPIONs possessed excellent biocompatibility by methyl tetrazolium (MTT) assays against NIH3T3 cells and hemolysis assays with rabbit erythrocytes. Furthermore, an anticancer drug, doxorubicin (Dox), was used as a model drug and loaded into the dendritic-linear-brush-like triblock copolymer-grafted SPIONs, and subsequently, the drug releases were performed in phosphoric acid buffer solution pH = 4.7, 7.4, or 11.0 at 37 °C. The results verify that the dendritic-linear-brush-like triblock copolymer-grafted SPIONs possess pH-responsive drug release behavior. The Dox dose of the loaded and free drug required for 50% cellular growth inhibition was 2.72 and 0.72 µm/mL, respectively, according to MTT assay against a Hella cell line in vitro. Therefore, on the basis of its biocompatibility and drug release effect, the modified SPION could provide a charming opportunity to design some excellent drug delivery systems for therapeutic applications.

Combination of Anti-PD-1 Antibody, Anlotinib and Pegaspargase "Sandwich" With Radiotherapy in Localized Natural Killer/T Cell Lymphoma.

Asparaginase/pegaspargase containing regimens combined with radiotherapy are highly effective and considered the cornerstone of localized Natural killer/T-cell lymphoma (NKTL) treatment. However, these chemotherapy regimens inevitably cause relatively high incidence of treatment-related adverse events (TRAEs). Herein we retrospectively evaluated the efficacy and safety of the combined regimen of anti-PD-1 antibody, anlotinib and pegaspargase "sandwich" with radiotherapy in localized NKTL. Anti-PD-1 antibody and pegaspargase at 2500 U/m2 were administered on day 1, while anlotinib (12 mg once a day) was orally administered on days 1-14. The treatment was repeated every 3 weeks. All the eight patients included received 3 cycles of the regimen followed by radiotherapy and an additional 3 cycles. The overall response rate was 100%, and the complete response rate was 87.5%. With a median follow-up time of 35.5 months (range, 34.03-40.90 months), median PFS and OS times were not reached. The 3-year PFS and OS rates were 100% and 100%, respectively. All patients were alive at the last follow-up. No treatment-related death and no grade 4 TRAE was reported. No grade 3/4 hematological toxicity was detected, and half of the patients didn't report any hematological toxicity. This study indicates that anti-PD-1 antibody combined with anlotinib and pegaspargase is a promising chemoradiotherapy regimen for localized NTKL, with mild toxicity and good tolerance.

Green fabrication of seedbed-like Flammulina velutipes polysaccharides-derived scaffolds accelerating full-thickness skin wound healing accompanied by hair follicle regeneration.

A novel seedbed-like scaffold was firstly fabricated by the "frozen sectioning" processing method using Flammulina velutipes as a raw material. The Flammulina velutipes polysaccharides scaffold is composed of a natural structure imitating the "ground" (connected and aligned hollow tubes with porous walls). Meanwhile, its biologically active components include polysaccharides and proteins, mimicking the "plant nutrition" in the seedbed. To further optimize the ground and nutrition components, Flammulina velutipes polysaccharides-derived scaffolds (FPDSs) were fabricated via the treatment of original Flammulina velutipes polysaccharides scaffold (labeled FPS) by NaOH, cysteine (labeled as FPS/NaOH, FPS/Cys, respectively). FPDSs were characterized by SEM, FTIR, XRD, water absorption and retention, and mechanical evaluations. From the results, FPS/NaOH and FPS/Cys lost the characteristic big tubes of original strips and had higher water absorption capacities comparing to FPS. Simultaneously, FPS/NaOH had better ductility, FPS/Cys had showed increased stiffness. Biological activities of FPDSs were tested against different types of bacteria exhibiting excellent anti-bacterial activity, and FPS/NaOH and FPS/Cys had dramatically higher anti-bacterial activity than FPS. The cytocompatibility of FPDSs was evaluated utilizing mouse fibroblast cell line (L929), and all FPDSs showed good cytocompatibility. The FPDSs were further applied to a rat full-thickness skin wound model, and they all exhibited obviously accelerated re-epithelialization, among which FPS/NaOH showed the greatest efficiency. FPS/NaOH could shorten the wound-healing process as evidenced by dynamic alterations of the expression levels of specific stagewise markers in the healing areas. Similarly, FPS/NaOH can efficiently induce hair follicle regeneration in the healing skin tissues. In summary, FPDSs exhibit potential functions as seedbeds to promote the regeneration of the "seed" including hair follicles and injured skin, opening a new avenue for wound healing.

Aligned soy protein isolate-modified poly(L-lactic acid) nanofibrous conduits enhanced peripheral nerve regeneration.

OBJECTIVE: Repair and regeneration of peripheral nerve defect by engineered conduits have greatly advanced in the past decades while still facing great challenges. APPROACH: In this work, we fabricated a new highly oriented poly(L-lactic acid) (PLLA)/soy protein isolate (SPI) nanofibrous conduit (HO-PSNC) for nerve regeneration. MAIN RESULTS: Firstly, we observed that SPI could efficiently modify PLLA for the electrospinning of PLLA/SPI nanofibers with enhanced physical and biological properties. Incorporation of SPI decreased the fiber diameter and ductility of PLLA/SPI nanofibrous films (PSNFs), improved the tensile strength and surface wettability of PSNFs and increased the in vivo degradability of the PSNFs. When the hybrid ratio of SPI was 20 and 40%, PSNFs could efficiently promote neural cell extension and differentiation in vitro. Based on these data, 20% SPI (PSNF-20) was chosen for further investigation. Next, PSNF-20 with different fiber orientations (random/low orientation, medium, and high orientation, respectively) were developed and used for evaluating neural cell behaviors on the materials. Results revealed that the PSNF-20 with highly oriented nanofibers (HO-PSNF-20) or mediumly oriented nanofibers (MO-PSNF-20) showed a better performance in directing cell extension and enhancing neurite outgrowth. Finally, the highly oriented nanofibers conduits (HO-PSNC-20) were used to bridge sciatic nerve defect in rats with highly oriented PLLA and autografts as controls. HO-PSNC-20 exhibited a significant promotion in nerve regeneration and functional reconstruction comparing to highly oriented PLLA as proven by the evaluations of walking track, electrophysiology, toluidine blue nerve staining, transmission electron microscopy, neural factors staining and qPCR, and gastrocnemius histology. SIGNIFICANCE: In conclusion, nerve conduit fabricated from aligned electrospinning of SPI-modified PLLA nanofibers is promising for peripheral nerve regeneration.

Preparation of poly(MAA-g-EG) hydrogel nanoparticles by a thermally-initiated free radical dispersion polymerization.

Poly(methacrylic acid-grafted-poly(ethylene glycol)) (P(MAA-g-EG)) hydrogel nanoparticles (HNPs) were prepared by a thermally-initiated free radical dispersion polymerization method. The effects of various reaction parameters on the preparation of HNPs were investigated, including the quantity of monomer, temperature, initiator dosage, crosslinker dosage, and co-stabilizer concentration. The reaction temperature at 75 degrees C was found to be suitable for preparing stable and small P(MAA-g-EG) HNPs. By adding a little amount of polyvinyl alcohol in the reaction media, P(MAA-g-EG) HNPs with narrow size distribution could be obtained. The effects of pH and the crosslinker dosage on the equilibrium swelling behavior of P(MAA-g-EG) HNPs were also studied. The P(MAA-g-EG) HNPs perform pH-responsive swelling behavior, which is strongly influenced by the crosslinker dosage.

Improvement in physical and biological properties of chitosan/soy protein films by surface grafted heparin.

A series of chitosan/soy protein isolate (SPI) composite films (CS-n, n=0, 10 and 30, corresponding to SPI content in the composites) were prepared. Heparin was grafted onto the surface of CS-n to fabricate a series of heparinized films (HCS-n). CS-n and HCS-n were characterized by ATR-Fourier transform infrared spectroscopy and water contact angle. The surface heparin density was measured by toluidine blue assay. The results showed that heparin has been successfully grafted onto the surface of CS-n. Heparin evenly distributed on the surface of the films and the heparin content increased with the increase of SPI content, and the hydrophilicity of the films was enhanced due to the grafted heparin. The cytocompatibility and hemocompatibility of CS-n and HCS-n were evaluated by cell culture (MTT assay, live/dead assay, cell morphology and cell density observation), platelet adhesion test, plasma recalcification time (PRT) measurement, hemolysis assay and thrombus formation test. HCS-n showed higher cell adhesion rate and improved cytocompatibility compared to the corresponding CS-n. HCS-n also exhibited lower platelet adhesion, longer PRT, higher blood anticoagulant indexes (BCI) and lower hemolysis rate than the corresponding CS-n. The improved cytocompatibility and hemocompatibility of HCS-n would shed light on the potential applications of chitosan/soy protein-based biomaterials that may come into contact with blood.

The long non-coding RNA expression profile of Coxsackievirus A16 infected RD cells identified by RNA-seq.

Coxsackievirus A16 (CVA16) is one of major pathogens of hand, foot and mouth disease (HFMD) in children. Long non-coding RNAs (IncRNAs) have been implicated in various biological processes, but they have not been associated with CVA16 infection. In this study, we comprehensively characterized the landscape of IncRNAs of normal and CVA16 infected rhabdomyosarcoma (RD) cells using RNA-Seq to investigate the functional relevance of IncRNAs. We showed that a total of 760 IncRNAs were upregulated and 1210 IncRNAs were downregulated. Out of these dysregulated IncRNAs, 43.64% were intergenic, 22.31% were sense, 15.89% were intronic, 8.67% were bidirectional, 5.59% were antisense, 3.85% were sRNA host IncRNAs and 0.05% were enhancer. Six dysregulated IncRNAs were validated by quantitative PCR assays and the secondary structures of these IncRNAs were projected. Moreover, we conducted a bioinformatics analysis of an IncRNAs (ENST00000602478) to elucidate the diversity of modification and functions of IncRNAs. In summary, the current study compared the dysregulated IncRNAs profile upon CVA16 challenge and illustrated the intricate relationship between coding and IncRNAs transcripts. These results may not only provide a complete picture of transcription in CVA16 infected cells but also provide novel molecular targets for treatments of HFMD.

Cellulose/soy protein isolate composite membranes: evaluations of in vitro cytocompatibility with Schwann cells and in vivo toxicity to animals.

OBJECTIVE: To evaluate the in vitro cytocompatibility of cellulose/soy protein isolate composite membranes (CSM) with Schwann cells and in vivo toxicity to animals. METHODS: A series of cellulose/soy protein isolate composite membranes (CSM) were prepared by blending, solution casting and coagulation process. The cytocompatibility of the CSM to Schwann cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by direct cells culture of Schwann cells on the surfaces of the CSM, respectively. The in vivo toxicity of the CSM to animals were also evaluated by acute toxicity testing, skin sensitization testing, pyrogen testing and intracutaneous stimulation testing, respectively, according to the ISO 10993 standard. RESULTS: The MTT assay showed that the cell viability of Schwann cells cultured in extracts from the CSM was higher than that from the neat cellulose membrane without containing SPI component. The direct cells culture indicated that the Schwann cells could attach and grow well on the surface of the CSM and the incorporation of SPI into cellulose contributed to improvement of cell adhesion and proliferation. The evaluations of in vivo biological safety suggested that the CSM showed no acute toxicity, no skin sensitization and no intracutaneous stimulation to the experimental animals. CONCLUSION: The CSM had in vitro cytocompatibility with Schwann cells and biological safety to animals, suggesting potential for the applications as nerve conduit for the repair of nerve defect.

Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways.

Autophagy is an important homeostatic process for the degradation of cytosolic proteins and organelles and has been reported to play an important role in cellular responses to pathogens and virus replication. However, the role of autophagy in Coxsackievirus A16 (CA16) infection and pathogenesis remains unknown. Here, we demonstrated that CA16 infection enhanced autophagosome formation, resulting in increased extracellular virus production. Moreover, expression of CA16 nonstructural proteins 2C and 3C was sufficient to trigger autophagosome accumulation by blocking the fusion of autophagosomes with lysosomes. Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy. Expression of viral protein 2C enhanced IRGM promoter activation, thereby increasing IRGM expression and inducing autophagy. CA16 infection inhibited Akt/mTOR signaling and activated extracellular signal-regulated kinase (ERK) signaling, both of which are necessary for autophagy induction. In summary, CA16 can use autophagy to enhance its own replication. These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).