Objective To prepare virus-like particles (VLPs) for yellow fever virus (YFV) using the pACYCDuet-1 single-plasmid dual expression system. Methods Gene fragments including NS1, NS5, and 5' untranslated region (UTR) and 3' UTR fragment of YFV were cloned into the expression plasmid pACYCDuet-1-MS2-TMpac (carrying MS2 coat protein gene) and transformed into E. coli BL21(DE3) for expression. VLPs were observed by TEM. After DNase I/RNase A digestion, RNA was extracted and transcribed into cDNA, which was subjected to fluorescence quantitative PCR detection. Results According to SN/T 3560-2013, double digestion and colony PCR of pACYCDuet-1-MS2-TMpac-YFV both produced expected bands. SDS-PAGE showed a target band at 25-35 kDa. RT-PCR amplified VLPs after enzyme digestion, and TEM revealed VLPs with clear edges and translucent interiors. Thus, the plasmid was successfully constructed, and the phage coat protein packaged YFV genes with nuclease resistance. Conclusion VLPs containing specific gene fragments of yellow fever virus have been successfully prepared. These nuclease-resistant VLPs can serve as safe and stable quality control materials to ensure the reliability of laboratory detection for YFV.

Objective To build an automatic segmentation model of temporomandibular joint(TMJ)based on magnetic resonance im-aging(MRI)using deep learning method.Methods The MRI data of TMJ of 104 subjects were collected,with the articular disc,con-dyle and glenoid fossa marked.The adaptive U-Net framework(nnU-Net)was used to construct a segmentation model,which was sub-jected to both quantitative and qualitative assessments.Results The segmentation model demonstrated excellent accuracy in segmenta-tion.In the segmentation of different joint structures,the model achieved Dice of 0.77 for the articular disc,0.85 for the condyle,and 0.66 for the glenoid fossa.The model showed similar segmentation performance when processing MRI images in both open-mouth and closed-mouth states.Conclusion This study developed an automatic segmentation model for TMJ MRI based on deep learning,which can assist clinicians in diagnosing anterior displacement of the TMJ disc.

Objective To build an automatic segmentation model of temporomandibular joint(TMJ)based on magnetic resonance im-aging(MRI)using deep learning method.Methods The MRI data of TMJ of 104 subjects were collected,with the articular disc,con-dyle and glenoid fossa marked.The adaptive U-Net framework(nnU-Net)was used to construct a segmentation model,which was sub-jected to both quantitative and qualitative assessments.Results The segmentation model demonstrated excellent accuracy in segmenta-tion.In the segmentation of different joint structures,the model achieved Dice of 0.77 for the articular disc,0.85 for the condyle,and 0.66 for the glenoid fossa.The model showed similar segmentation performance when processing MRI images in both open-mouth and closed-mouth states.Conclusion This study developed an automatic segmentation model for TMJ MRI based on deep learning,which can assist clinicians in diagnosing anterior displacement of the TMJ disc.

T-cell exhaustion is characterized by the stepwise and progressive loss of T cell functions under conditions of antigen-persistence, which occurs following chronic infections and tumor outgrowth. Exhausted T cells present functional defects, express multiple inhibitory receptors and show reprogrammed transcriptional regulation. As T cell exhaustion is correlated to its dysfunction to control infections and tumors, exploring new strategies to target exhausted T cell may reverse this dysfunctional state and reinvigorate immune response. This study takes CD8+ T cell as an example, which acts as an important subset involved in exhaustion state, discuss current understanding of the properties of exhausted T cell and the mechanisms that promote and maintain this state, and reveal new therapeutic targets for chronic infection and cancer.

Objective To investigate the safety of thyroid gland resection and primary resection in the treatment of thyroid nodule and its influence to thyroid function.Methods 86 patients with thyroid nodules were selected as the research subjects.The patients were randomly divided into two groups.43 cases in the observation group implemented the thyroid gland resection treatment.43 cases in the control group received thyroid gland subtotal resection.The curative effect,safety,thyroid function and other indicators were compared between the two groups.Results The operation time and hospitalization time in the observation group after operation[(105.65 ± 12.54)min;(6.35 ± 2.01)d]were shorter than those in the control group[(149.41 ± 13.68)min;(9.62 ± 2.45)d].The amount of bleeding during operation in the control group [(134.51 ± 9.64) mL] was significantly higher than the observation group [(84.62 ± 6.35) mL],there was significant difference between the two groups (P ＜ 0.05).The total effective rate of the observation group was 95.55％,which was significantly higher than 72.09％ in the control group,the difference was statistically significant (P ＜ 0.05).Before operation,the between the two groups had no significant difference (P ＞ 0.05).After surgical treatment,serum FT3 and serum FT4 levels in two groups were decreased,but those in the observation group[(11.62 ± 3.02),(51.24 ± 7.25)pmol/L] were significantly lower than the control group [(14.14 ± 5.11) pmoL/L;(60.52 ± 6.35) pmol/L],there were obvious differences (P ＜ 0.05).The incidence rate of complications such as throat edema,hemorrhage,postoperative hoarseness in the observation group was 9.30％,which in the control group was 23.26％,the difference was statistically significant (P ＜ 0.05).Conclusion The thyroid gland resection therapy for thyroid nodules has high safety and significant curative effect,it can quickly improve the thyroid function,reduce relapse rate,has higher application value in benign and malignant tumors indistinguishable after surgery.It can reduce the length of stay in hospital,is conducive to the recovery of patients,it is worthy of clinical promotion.

Objective To explore the effectiveness and adverse effect of high frequency oscillation ventilation (HFOV) combined with Sildenafil (SIL) treatment on newborns with persistent pulmonary hypertension (PPHN).Methods A total of 89 cases of PPHN infants collected from Chengdu Women and Children's Central Hospital from Sep.2010 to Sep.2012 were randomly divided into HFOV group,constant mechanical ventilation (CMV) group,HFOV combined SIL group (HFOV + SIL group) and CMV combined with SIL group (CMV + SIL group).The arterial blood gas,pulmonary artery pressure (PAP) and adverse reactions were monitored before and 3 days after treatment.SNK multiple comparison method andx2 test were performed for data before and after treatment among groups for continuous variables and categorical variables,respectively.Results The levels of pa (O2) [(79.1 ± 13.7) mmHg (1 mmHg =0.133 kPa),(77.9 ±14.6) mmHg,(85.4 ±15.2) mmHg],Sa(O2) [(87.8 ±13.4)％,(88.4±15.6)％,(96.1±15.9)％],pa(CO2)[(42.5±11.3) mmHg,(40.2 ±10.5) mmHg,(35.6 ±8.7) mmHg] and PAP [(31.1 ± 8.1) mmHg,(30.4 ± 9.5) mmHg,(25.8 ± 7.3) mmHg] were all improved significantly in CMV + SIL group,HFOV group and HFOV + SIL group compared with those in CMV group[(69.9 ± 12.3) mmHg,(81.1 ± 14.9)％,(48.1 ±9.5) mmHg,(35.6 ±8.9) mmHg] (F =4.629 3,3.673 2,5.865 3,4.849 5,P ＜0.05),especially for HFOV + SIL group(P ＜ 0.05).No significant difference in such indicators was observed between CMV + SIL group and HFOV group (P ＞ 0.05).The effective rate in HFOV + SIL group (90％) was the highest among the 4 groups (x2 =7.938,P ＜ 0.05).During the treatment,all neonates have no adverse reaction.Conclusion The combined use of SIL and HFOV might be a more effective and safer method in the treatment of PPHN of neonate.

Objective To obtain highly purified astrocytes and identify the cells in each stage to support further studies.Methods The cerebral cortex of a neonatal SD rat was isolated and prepared into single cell suspension.The obtained cells were purified by differential adherence and shook at a constant temperature.By inverted phase contrast microscopy and HE staining,cell morphology was observed.The immunofluorescence staining with anti-mouse GFAP was used to identify the cells.Results The primary cortical cells developed rapidly at 3 d after culture and covered the flasks at 9-12 d.At this time,the cells showed stratification and the astrocytes lay at the lower layer.GFAP positive rate was only about (67.2 ±7.1)%.After the first passage,GFAP positive rate increased obviously (84.0±6.0)%. However, oligodendrocytes and microglias could not be removed completely,and the cells also showed stratification.Through 3 times of passages,we obtained many single species of astrocytes showing satellite shape with 2 or 3 processes,big cell body and round or oval-shaped nuclei leaned to one side.Immunofluorescence staining showed that nearly all of the cells were strong positive and the positive rate reached as high as (97.6 ± 2.4 )%.Conclusion Through differential adherence and shaking at a constant temperature,more astrocytes of high purity and in good state can be obtained.

ObjectiveTo evaluate the long-term therapeutic efficacy of muscle-region alignment needling plus dermalneedle therapy in treating post-stroke upper-limb spasticity.MethodTotally 488 patients with post-stroke upper-limb spasticity were randomized into a treatment group and a control group, 244 in each group. Besides theessentialrehabilitation treatment, the treatment group was intervened by muscle-region alignment needling plus dermal needle therapy, while the control group was given regular Western medication.Thetwo groups were intervened for 3 weeks and were followed up for 6 months. The neurologic function, Functional Comprehensive Assessment (FCA), and Stroke Speciality-Quality of Life (SS-QOL) were observed for the follow-up study.ResultThe total effective rate was 93.4% in the treatment group versus 61.5% in the control group, and the difference was statistically significant (P<0.05); the neurologic function, FCA score, and SS-QOL score in the treatment group were significantly superior to that in the control group at the end of the follow-up study (P<0.05).ConclusionMuscle-region alignment needling plus dermal needle therapycan produce a content long-term therapeutic efficacy in treating post-stroke upper-limb spasticity.

Objective: To observe the clinical efficacy of acupuncture treatment for migraine. Methods: Forty cases were randomly allocated to a treatment group and a control group, 20 cases in each group. Cases in the treatment group were treated with acupuncture, while cases in the control group were treated with oral Sibelium. After that, the changes of cerebral blood flow were observed before and after treatment. Results: There was significant difference in clinical efficacies between two groups (P<0.05). There were also significant differences in arterial blood flow velocities of before and after treatment. Acupuncture can produce substantial differences (P<0.05) in blood flow velocities of vertebral artery (VA), middle cerebral artery (MCA) and anterior cerebral artery (ACA) during an increased flow rate. It can also produce statistical differences in blood flow velocities of VA during a decreased flow rate (P<0.05). Conclusion: Acupuncture can effectively alleviate the pain of migraine sufferers and exert a two-way regulation on the cerebral blood flow.

Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template, double stranded cDNA of HNG with FLAG in its C-terminal was obtained, which was cloned into the plasmid pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time, the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG, 25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h, then cell morphology, MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h, cell viability decreased to (65.8±5.3)%, and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG, Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast, pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.