Natural product honokiol exhibits antiviral effects against Micropterus salmoides rhabdovirus (MSRV) both in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is a formidable pathogen, presenting a grave menace to juvenile largemouth bass. This viral infection frequently leads to epidemic outbreaks, resulting in substantial economic losses within the aquaculture industry. Unfortunately, at present, there are no commercially available vaccines or pharmaceutical treatments to combat this threat. In order to address the urgent need for therapeutic strategy to resist MSRV infection, the antiviral activity of natural product honokiol against MSRV was explored in this study. Firstly, cellular morphology was directly observed in an inverted microscope when treated with honokiol after MSRV infection. The results clarified that honokiol significantly lessened cytopathic effect (CPE) induced by MSRV and protected the integrity of GCO cells. Furthermore, the viral nucleic acid expression (G gene) was detected by reverse transcription real-time quantitative PCR (RT-qPCR) and the results indicated that honokiol significantly decreased the viral loads of MSRV in a concentration-dependent manner, and honokiol showed a high antiviral activity with IC50 of 2.92 µM. Besides, honokiol significantly decreased the viral titre and suppressed apoptosis caused by MSRV. Mechanistically, honokiol primarily inhibited the initial replication of MSRV and discharge of progeny virus to exert anti-MSRV activity. More importantly, in vivo experiments suggested that honokiol (40 mg/kg) expressed a fine antiviral activity against MSRV when administrated with intraperitoneal injection, which led to a notable 40% improvement in the survival rate among infected largemouth bass. In addition, it also resulted in significant reduction in the viral nucleic acid expression within liver, spleen and kidney at 2, 4 and 6 days following infection. What is more, 100 mg/kg honokiol with oral administration also showed certain antiviral efficacy in MSRV-infected largemouth bass via improving the survival rate by 10.0%, and decreasing significantly the viral nucleic acid expression in liver, spleen and kidney of largemouth bass on day 2. In summary, natural product honokiol is a good candidate to resist MSRV infection and has promising application prospects in aquaculture.

Fatigue Effect on Minimal Toe Clearance and Toe Activity during Walking.

This study investigates the effects of fatigue on the process of walking in young adults using the developed clog-integrated sensor system. The developed sensor can simultaneously measure the forefoot activity (FA) and minimum toe clearance (MTC). The FA was evaluated through the change in the contact area captured by a camera using a method based on a light conductive plate. The MTC was derived from the distance between the bottom surface of the clog and ground obtained using a time of flight (TOF) sensor, and the clog posture was obtained using an acceleration sensor. The induced fatigue was achieved by walking on a treadmill at the fastest walking speed. We evaluated the FA and MTC before and after fatigue in both feet for 14 participants. The effects of fatigue manifested in either the FA or MTC of either foot when the results were evaluated by considering the participants individually, although individual variances in the effects of fatigue were observed. In the dominant foot, a significant increase in either the FA or MTC was observed in 13 of the 14 participants. The mean MTC in the dominant foot increased significantly (p = 0.038) when the results were evaluated by considering the participants as a group.

Synthesized Magnolol Derivatives Improve Anti-Micropterus salmoides Rhabdovirus (MSRV) Activity In Vivo.

Micropterus salmoides rhabdovirus (MSRV) is a primary viral pathogen in largemouth bass aquaculture, which leads to tremendous economic losses yearly. Currently, there are no approved drugs for the treatment and control of this virus. Our previous studies screened the herb Magnolia officinalis from many traditional Chinese medicines, and we isolated and identified magnolol as its main active compound against multiple rhabdoviruses, including MSRV. On the basis of the structure-activity relationship and pharmacophore model of magnolol, two new magnolol derivatives, namely, hydrogenated magnolol and 2,2'-dimethoxy-magnolol, were designed and synthesized. Their anti-MSRV activities were systematically investigated both in vitro and in vivo. By comparing the half-maximal inhibitory concentration (IC50), it was found that hydrogenated magnolol possessed a higher anti-MSRV activity than magnolol and 2,2'-dimethoxy-magnolol, with an IC50 of 13.37 µM. Furthermore, hydrogenated magnolol exhibited a protective effect on the grass carp ovary (GCO) cell line by reducing the cytopathic effect induced by MSRV. Further studies revealed that hydrogenated magnolol did not directly impact virions or interfere with MSRV adsorption. It worked within the 6-8 h of the phase of virus replication. In vivo treatment of MSRV infection with magnolol and hydrogenated magnolol showed that they significantly improved the survival rate by 44.6% and 62.7%, respectively, compared to MSRV-infected groups. The viral load measured by the expression of viral glycoprotein in the organs including the liver, spleen, and kidney also significantly decreased when fish were intraperitoneally injected at a dose of 20 mg/kg. Altogether, the structural optimization of magnolol via hydrogenation of the propylene groups increased its anti-MSRV activity both in vitro and in vivo. These results may provide a valuable reference for anti-MSRV drug discovery and development in aquaculture.

PEG-modified subunit vaccine encoding dominant epitope to enhance immune response against spring viraemia of carp virus.

Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) can infect almost all fish of cyprinids, which bring huge economic losses to aquaculture. Glycoprotein (G), as the most important antigenic determinant protein of SVCV, is widely considered as an effective method against SVCV. In our previous study, we found that G3 (131 aa) is the potential dominant antigen epitope that induces strong immune responses similar to G protein (510 aa). Here, in order to further improve the immune effect, we reported a subunit vaccine (PEG-G3) constructed by PEG-modified dominant epitope protein (G3). The results of serum antibody production, enzyme activities and immune-related genes expression showed that PEG-G3 induces significantly stronger immune protective responses against SVCV than G3. PEG modification significantly increased the serum antibody level of the vaccine, which increased significantly after immunization and reached the peak at 21 day post-vaccination. T-AOC and AKP activities in the lowest concentration group (5 µg) of PEG-G3 were significantly higher than those in the highest concentration group (20 µg) of G3. In PEG-G3 group, the expression of almost all genes increased at least 4 times compared with the control group. After 14-day challenge, the RPS (relative percentage survival) of the highest concentration of PEG-G3 group was 53.6%, while that of G3 group is 38.9%. Therefore, this work shows that PEG modification and dominant epitope screening may be effective methods to improve the immune protective effect of vaccines and to resist the infection of aquatic animal viral diseases.

Titanosilicate Derived SiO2/TiO2@C Nanosheets with Highly Distributed TiO2 Nanoparticles in SiO2 Matrix as Robust Lithium Ion Battery Anode.

Carbon-coated SiO2/TiO2 (SiO2/TiO2@C) nanosheets consisting of TiO2 nanoparticles uniformly embedded in SiO2 matrix and a carbon-coating layer are fabricated by using acidified titanosilicate JDF-L1 nanosheets as template and precursor. SiO2/TiO2@C has unique structural features of sheetlike nanostructure, ultrafine TiO2 nanoparticles distributed in SiO2 matrix, and carbon coating, which can expedite ion diffusion and electron transfer and relieve volume expansion efficiently, and thus, the synergetic combination of these advantages significantly enhances its Li storage capability. As anode of lithium-ion batteries (LIBs), SiO2/TiO2@C nanosheets exhibit a high capacity of 998 mAh g-1 at 100 mA g-1 after 100 cycles. Moreover, an ultrahigh capacity of 410 mAh g-1 retains at 2000 mA g-1 after 400 cycles. A mixed reaction mechanism of capacitance and diffusion-controlled intercalation is revealed by qualitative and quantitative analysis.

Protective role of fentanyl in lipopolysaccharide-induced neuroinflammation in BV-2 cells.

Neurosurgery always results in neuroinflammation, which may activate microglial cells. Previous studies have demonstrated that fentanyl could be used for the induction or maintenance of anesthesia prior to surgery. However, it is unknown if fentanyl attenuates neuroinflammation prophylactically. Cell viability in groups that were treated with different concentrations of fentanyl (0.01, 0.1, 1 or 5 µmol/l) was analyzed by an MTT assay. BV-2 microglial cells were treated with lipopolysaccharide (LPS) at a concentration of 1 µg/ml to mimic neuroinflammation in vitro. BV-2 cells were pretreated with 5 µmol/l fentanyl prior to stimulation by LPS. The protein levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-10 in the culture medium were assessed by ELISA. The mRNA level of toll-like receptor (TLR)4 was evaluated by reverse transcription-quantitative polymerase chain reaction analysis. The protein levels of TLR4, glycogen synthase kinase (GSK)-3ß and phosphorylated (p)-GSK-3ß in BV-2 cells were assessed by western blot analysis. The MTT assay demonstrated that low concentrations of fentanyl (0.01, 0.1 or 1 µmol/l) did not affect the cell viability of BV-2 cells, while 5 µmol/l fentanyl significantly reduced BV-2 cell viability. The results of ELISA revealed that LPS significantly upregulated the release of TNF-α, IL-1ß and IL-10, which were repressed by fentanyl pretreatment. Fentanyl pretreatment significantly reduced the LPS-induced elevation of TLR4 at mRNA and protein levels as well as p-GSK-3ß protein levels in BV-2 cells. In conclusion, fentanyl pretreatment protects BV-2 cells from LPS-induced neuroinflammation by inhibiting TLR4 expression and GSK-3ß activation. Neuroinflammation induced by surgery serves an important role in the development of postoperative cognitive dysfunction (POCD) and targeting the TLR4 and GSK-3ß signaling pathway may provide a novel therapeutic approach for the treatment of POCD.

B7-H3 in combination with regulatory T cell is associated with tumor progression in primary human non-small cell lung cancer.

B7-H3 belongs to the co-inhibitory B7 family and plays an important role in the adaptive immune response in regulating T cells. In human malignancies, B7-H3 is reported to be involved in tumor immune evasion. However, the detailed molecular mechanism of B7-H3 in tumor evasion remains unclear, particularly in non-small cell lung cancer (NSCLC). Regulatory T cells (Tregs) are known as a key player in the inhibition of immune mechanisms. The study demonstrated the correlation between B7-H3 on tumor cells and the number of Tregs in the tumor microenvironment in NSCLC. B7-H3 was examined in tumor tissues from 110 patients with NSCLC by immunohistochemical analysis. Forkhead box P3+ (FOXP3+) Tregs in those spencimens were also detected and numbered. Survival curves were drawn using the Kaplan-Meier method and compared by the log-rank test. High B7-H3 expression in tumor cells significantly correlated with male gender, squamous NSCLC, advanced stage and shorter overall survival (OS) (P = 0.035, P = 0.004, P = 0.037, P = 0.014, respectively). Meanwhile, FOXP3 expression in tumor-infiltrating lymphocytes (TILs) was associated with male gender, regional lymph node involvement, advanced stage and worse OS (P = 0.009, P = 0.015, P = 0.014, P = 0.034, respectively). Significant correlation was identified between the expression of B7-H3 and the number of FOXP3+ TILs (P = 0.013). Patients with B7-H3 high/FOXP3 high had poorer OS (P = 0.006), suggesting that B7-H3 and Tregs may play a cooperatively role in tumor immune evasion, leading to poor outcomes for NSCLC patients.

[Influence of penetrative needling of Shendao (GV 11) on the symptom score and serum IgE content in chronic urticaria patients].

OBJECTIVE: To observe the changes of symptom scores and serum IgE level after treatment with thick-needle subcutaneous penetration of Shendao (GV 11) in chronic urticaria patients. METHODS: A total of 60 chronic urticaria patients were randomly divided into acupuncture group (n = 30) and medication group (n = 30). Subcutaneous penetrative needling was applied to GV 11 with thick acupuncture needle (retained for 4 h/time, once daily, 5 times/week) for patients of acupuncture group and Levocetirizine Hydrochloride tablets (5 mg/time, once daily in the first two weeks, then, once every other day in the 3rd and 41th weeks, and once every 3 days in the last two weeks) were given to patients of medication group. Serum IgE content was assayed before and 2,6, 12 weeks after the treatment by chemiluminescent technique. Symptom scores were obtained by "0-3 four levels assessment" method in the light of the size of the wheal and the itching severity. RESULTS: Self-comparison indicated that the symptom scores and serum IgE levels declined significantly (P < 0.01) 2 and 6 weeks (Wks) after the treatment in both acupuncture and medication groups,and 12 Wks after the treatment in the acupuncture group (P < 0.01). Comparison between two groups showed that the symptom score and serum IgE content of acupuncture group were significant lower than those of medication group 12 Wks after the treatment (P < 0.05). No significant differences were found between two groups in the symptom scores and serum IgE levels before, 2 and 6 Wks after the treatment (P > 0.05). A positive correlation exists between the symptom score and the serum IgE level before and after the treatment in both groups. CONCLUSION: Thick-needle subcutaneous penetration of Shendao (GV 11) can effectively improve clinical symptoms of chronic urticaria patients, which may be closely related to its effect in lowering peripheral blood IgE level.

[Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151].

AIM: To construct and express the single chain antibody (scFv) in E.coli HB2151 by cloning the variable region genes from hybridoma against bFGF. METHODS: Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction (RT-PCR). V(L) and V(H) were fused by a short peptide linker containing 15 amino acids (Gly(4)Ser)(3) using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151. RESULTS: The V(H) gene contained 375 base pairs and encoded 125 amine acid residues. The V(L) gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) gene and the V(L) gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of V(H)-linker-V(L). Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-5E-scFv was constructed correctly. SDS-PAGE and ELISA analysis showed that scFv was successfully expressed in E.coli HB2151 and the expression protein had specific antigen binding activity. CONCLUSION: The variable region genes of anti-bFGF mAbs have been cloned successfully and single chain antibody fragments have been constructed and expressed, which will be a great help to the study of humanized antibodies against bFGF.