Objective:To construct a full-length infectious clone of enterovirus D68（EV-D68）expressing enhanced green fluorescent protein（EGFP）by reverse genetics in order to provide an efficient tool for studying the biological characteristics and screening antiviral drugs for EV-D68.Methods:Gene synthesis and overlap PCR techniques were used to construct the full-length clone plasmid pUC57-EV-D68 of EV-D68. The full-length viral sequence was then transferred into the pCAGGS plasmid to obtain the pCAGGS-EV-D68 plasmid. The EGFP gene was amplified and inserted into the pCAGGS-EV-D68 plasmid to construct the pCAGGS-EGFP-EV-D68 plasmid. Then，the two constructed plasmids were transfected into human rhabdomyosarcoma（RD）cells to rescue recombinant viruses RV-EV-D68 and RV-EGFP-EV-D68. The rescued viruses were identified using PCR，Western blot，and immunofluorescence techniques. The antiviral effect of doxycycline was evaluated using the rescued RV-EGFP-EV-D68. Statistical analysis was performed using the two independent samples t-test. Results:The recombinant virus RV-EGFP-EV-D68 capable of expressing EGFP was successfully rescued. Even after 15 serial passages，the virus retained EGFP expression with no significant difference in viral titers compared to the parental virus，indicating its stable passage in RD cells. Besides，the rescued strains exhibited similar replication characteristics to the parental virus. While at 24 and 36 h after infection，the titers of the rescued strains were significantly lower than that of the parental strain（both P<0.05）. This study demonstrated that doxycycline significantly reduced the fluorescence intensity of RV-EGFP-EV-D68-infected RD cells（ P<0.01）. Meanwhile，a negative correlation was observed between the doxycycline concentration and the fluorescence intensity，indicating that the rescued virus could be used for antiviral drug evaluation. Conclusions:This study successfully constructs an infectious clone of EV-D68 expressing EGFP. The rescued recombinant virus RV-EGFP-EV-D68 has been verified to be applicable for the evaluation of antiviral drugs.

Objective:To construct a full-length infectious clone of enterovirus D68（EV-D68）expressing enhanced green fluorescent protein（EGFP）by reverse genetics in order to provide an efficient tool for studying the biological characteristics and screening antiviral drugs for EV-D68.Methods:Gene synthesis and overlap PCR techniques were used to construct the full-length clone plasmid pUC57-EV-D68 of EV-D68. The full-length viral sequence was then transferred into the pCAGGS plasmid to obtain the pCAGGS-EV-D68 plasmid. The EGFP gene was amplified and inserted into the pCAGGS-EV-D68 plasmid to construct the pCAGGS-EGFP-EV-D68 plasmid. Then，the two constructed plasmids were transfected into human rhabdomyosarcoma（RD）cells to rescue recombinant viruses RV-EV-D68 and RV-EGFP-EV-D68. The rescued viruses were identified using PCR，Western blot，and immunofluorescence techniques. The antiviral effect of doxycycline was evaluated using the rescued RV-EGFP-EV-D68. Statistical analysis was performed using the two independent samples t-test. Results:The recombinant virus RV-EGFP-EV-D68 capable of expressing EGFP was successfully rescued. Even after 15 serial passages，the virus retained EGFP expression with no significant difference in viral titers compared to the parental virus，indicating its stable passage in RD cells. Besides，the rescued strains exhibited similar replication characteristics to the parental virus. While at 24 and 36 h after infection，the titers of the rescued strains were significantly lower than that of the parental strain（both P<0.05）. This study demonstrated that doxycycline significantly reduced the fluorescence intensity of RV-EGFP-EV-D68-infected RD cells（ P<0.01）. Meanwhile，a negative correlation was observed between the doxycycline concentration and the fluorescence intensity，indicating that the rescued virus could be used for antiviral drug evaluation. Conclusions:This study successfully constructs an infectious clone of EV-D68 expressing EGFP. The rescued recombinant virus RV-EGFP-EV-D68 has been verified to be applicable for the evaluation of antiviral drugs.

OBJECTIVE To investigate the effects of soybean isoflavones （SI） on the reproductive development of young mice. METHODS C57BL/6 young mice were randomly divided into control group， SI low-dose and high-dose groups （10， 100 mg/kg）， with 10 mice in each group （half male and half female）. The young mice in each group were given corresponding liquid intragastrically， once a day， for 2 consecutive weeks. After the last administration， the percentage of body weight increase was calculated； serum estradiol and testosterone levels， malondialdehyde （MDA） content， total antioxidant capacity （T-AOC）， superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） activities in the reproductive organs of the young mice were determined. The histopathological changes in the reproductive organs were observed. The cell apoptosis of reproductive organs was detected. RESULTS Compared with the control group， the percentage of body weight increase in female mice was increased significantly in the SI high-dose group， while that of male mice was decreased significantly （P＜0.05 or P＜0.01）. Cystic follicles could be seen in the ovarian tissue in SI groups， a loose arrangement of spermatocytes could be seen in the testicular tissue， and partial epithelial cell shedding could be seen in epididymal tissue. The serum level of testosterone in female young mice and the serum levels of testosterone and estradiol in male young mice in SI groups， GSH-Px activity in the ovarian tissue of female young mice in the SI low-dose group， T-AOC activities in the ovarian tissue of female young mice in SI groups as well as the apoptotic rates of cells in testicular and epididymal tissue of male young mice in SI groups were increased significantly （P＜0.05 or P＜ 0.01）； the serum level of estradiol in female young mice in SI groups， SOD activity in the ovarian tissue of female young mice in the SI high-dose group， and MDA contents in the ovarian tissue of female young mice in SI groups as well as the apoptotic rates of cells in ovarian tissue of female mice in SI groups were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS SI can enhance the antioxidant stress capacity of ovarian tissue in female young mice and reduce their oxidative stress damage， but it has certain toxicity to reproductive organs in male mice.

Objective:We aimed to develop a novel visualized model based on nomogram to predict postoperative overall survival.Methods:This was a multicenter, retrospective, observational cohort study, including participants with histologically confirmed gastric and colorectal cancer who underwent radical surgery from 11 medical centers in China from August 1, 2015 to June 30, 2018. Baseline characteristics, histopathological data and nutritional status, as assessed using Nutrition Risk Screening 2002 (NRS 2002) score and the scored Patient-Generated Subjective Global Assessment, were collected. The least absolute shrinkage and selection operator regression and Cox regression were used to identify variables to be included in the predictive model. Internal and external validations were performed.Results:There were 681 and 127 patients in the training and validation cohorts, respectively. A total of 188 deaths were observed over a median follow-up period of 59 (range: 58 to 60) months. Two independent predictors of NRS 2002 and Tumor-Node-Metastasis (TNM) stage were identified and incorporated into the prediction nomogram model together with the factor of age. The model's concordance index for 1-, 3- and 5-year overall survival was 0.696, 0.724, and 0.738 in the training cohort and 0.801, 0.812, and 0.793 in the validation cohort, respectively.Conclusions:In this study, a new nomogram prediction model based on NRS 2002 score was developed and validated for predicting the overall postoperative survival of patients with gastric colorectal cancer. This model has good differentiation, calibration and clinical practicability in predicting the long-term survival rate of patients with gastrointestinal cancer after radical surgery.

Objective: To document the anatomical structure of the area anterior to the anorectum passing through the levator hiatus between the levator ani slings bilaterally. Methods: Three male hemipelvises were examined at the Laboratory of Clinical Applied Anatomy, Fujian Medical University. (1) The anatomical assessment was performed in three ways; namely, by abdominal followed by perineal dissection, by examining serial cross-sections, and by examining median sagittal sections. (2) The series was stained with hematoxylin and eosin to enable identification of nerves, vessels, and smooth and striated muscles. Results: (1) It was found that the rectourethralis muscle is closest to the deep transverse perineal muscle where the longitudinal muscle of the rectum extends into the posteroinferior area of the membranous urethra. The communicating branches of the neurovascular bundle (NVB) were identified at the posterior edge of the rectourethralis muscle on both sides. The rectum was found to be fixed to the membranous urethra through the rectourethral muscle, contributing to the anorectal angle of the anterior rectal wall. (2) Serial cross-sections from the anal to the oral side were examined. At the level of the external anal sphincter, the longitudinal muscle of the rectum was found to extend caudally and divide into two muscle bundles on the oral side of the external anal sphincter. One of these muscle bundles angled dorsally and caudally, forming the conjoined longitudinal muscle, which was found to insert into the intersphincteric space (between the internal and external anal sphincters). The other muscle bundle angled ventrally and caudally, filling the gap between the external anal sphincter and the bulbocavernosus muscle, forming the perineal body. At the level of the superficial transverse perineal muscle, this small muscle bundle headed laterally and intertwined with the longitudinal muscle in the region of the perineal body. At the level of the rectourethralis and deep transverse perineal muscle, the external urethral sphincter was found to occupy an almost completely circular space along the membranous part of the urethra. The dorsal part of the external urethral sphincter was found to be thin at the point of attachment of the rectourethralis muscle, the ventral part of the longitudinal muscle of the rectum. We identified a venous plexus from the NVB located close to the oral and ventral side of the deep transverse perineal muscle. Many vascular branches from the NVB were found to be penetrating the longitudinal muscle and the ventral part of rectourethralis muscle at the level of the apex of the prostate. The rectourethral muscle was wrapped ventrally around the membranous urethra and apex of the prostate. The boundary between the longitudinal muscle and prostate gradually became more distinct, being located at the anterior end of the transabdominal dissection plane. (3) Histological examination showed that the dorsal part of the external urethral sphincter (striated muscle) is thin adjacent to the striated muscle fibers from the deep transverse perineal muscle and the NVB dorsally and close by. The rectourethral muscle was found to fill the space created by the internal anal sphincter, deep transverse perineal muscle, and both levator ani muscles. Many tortuous vessels and tiny nerve fibers from the NVB were identified penetrating the muscle fibers of the deep transverse perineal and rectourethral muscles. The structure of the superficial transverse perineal muscle was typical of striated muscle. These findings were reconstructed three-dimensionally. Conclusions: In intersphincteric resection or abdominoperineal resection for very low rectal cancer, the anterior dissection plane behind Denonvilliers' fascia disappears at the level of the apex of the prostate. The prostate and both NVBs should be used as landmarks during transanal dissection of the non-surgical plane. The rectourethralis muscle should be divided near the rectum side unless tumor involvement is suspected. The superficial and deep transverse perineal muscles, as well as their supplied vessels and nerve fibers from the NVB. In addition, the cutting direction should be adjusted according to the anorectal angle to minimize urethral injury.
Humans ; Male ; Rectum/surgery* ; Anal Canal/anatomy & histology* ; Rectal Neoplasms/surgery* ; Proctectomy ; Urethra/surgery*

Objective:To investigate the difference of lymphocyte subsets between elderly patients with rheumatoid arthritis and non-elderly patients and its clinical significance.Methods:A total of 124 patients with rheumatoid arthritis in Affiliated Hospital of Nantong University from January, 2017 to December, 2019 were enrolled.The patients were divided into elderly group(≥60 years old, 34 cases)and non-elderly group(<60 years old, 90 cases). Rheumatoid arthritis activity(DAS-28)scoring was performed for each patient.Peripheral blood mononuclear cells(PBMCs)were extracted by Ficoll density centrifugation.Lymphocytes were labeled and detected by 18-color flowcytometry with more than 30 fluorescent antibodies.Results:DAS-28 scoring showed that the disease activity score of the elderly group(4.56±1.89)was higher than that of the non-elderly group(3.37±1.49)( t=3.633, P<0.001). Flow cytometry showed that MAIL%T(mucus-associated lymphoid tissue T cell subset)( Z=-2.798, P=0.005), Tn%CD8 T cells(initial CD8 T cells)( Z=-2.179, P=0.029), VD2% T(Vδ2+ T, γδT cell subtype)( Z=-2.806, P=0.005), PD1-CD28-%Th( Z=-2.050, P=0.040)and IGM+ D-%B( Z=-2.376, P=0.017)were lower in the elderly group than in the non-elderly group.While, CD45+ CD27+ %CD8 T cells( Z=-3.069, P=0.002), abT%T cell(αβT cells)( Z=-2.103, P=0.035), CD27-CD28+ %T cells( Z=-2.341, P=0.019), ASC%PBMC( Z=-2.341, P=0.019)and ASC%CD19+ ( Z=-2.000, P=0.046)subgroup expression were higher in the elderly group than in the non-elderly group. Conclusions:The disease activity of elderly patients with rheumatoid arthritis is significantly higher than that of younger patients.The expressions of abT%T and CD4% abT in effector T cells of elderly patients with rheumatoid arthritis are higher than those of younger patients, while the expression of VD2% T is lower.The expression level of CD45RA+ CD27+ %CD8 T with cytotoxic effect is higher; However, the expression level of Tn%CD8 T in naive cells is lower.

Objective:To investigate the overall clinical performance of nursing students and analyze its influencing factors to provide evidence for the improvement.Methods:A questionnaire survey was carried out among 288 nursing students from Qilu Hospital of Shandong University in March to May 2020. The survey included a general data questionnaire, Holistic Clinical Assessment Tool, Clinical Learning Environment, Supervision and Nurse Teacher evaluation scale, Medical Student Safety Attitudes and Professionalism Survey of nursing students. The factors associated with overall clinical performance of nursing students were analyzed by t test, analysis of variance, Pearson correlation analysis and multiple linear regression. Results:The total score of Holistic Clinical Assessment Tool was 114.95 ± 22.91. The total score of Clinical Learning Environment, Supervision and Nurse Teacher evaluation scale was 156.70 ± 26.98. The total score of Medical Student Safety Attitudes and Professionalism Survey was 123.65 ± 31.00. The influencing factors included social practice experience and clinical learning environment, which accounted for 28.0% of the total variation of overall clinical performance of nursing students.Conclusions:Educators and managers of nursing students' schools and internship hospitals should actively explore the educational reform practice to promote the improvement of clinical ability, so as to promote the improvement of the overall clinical ability of nursing students.

Morinda officinalis oligosaccharides (MOO) are an oral drug approved in China for the treatment of depression in China. However, MOO is hardly absorbed so that their anti-depressant mechanism has not been elucidated. Here, we show that oral MOO acted on tryptophan → 5-hydroxytryptophan (5-HTP) → serotonin (5-HT) metabolic pathway in the gut microbiota. MOO could increase tryptophan hydroxylase levels in the gut microbiota which accelerated 5-HTP production from tryptophan; meanwhile, MOO inhibited 5-hydroxytryptophan decarboxylase activity, thus reduced 5-HT generation, and accumulated 5-HTP. The raised 5-HTP from the gut microbiota was absorbed to the blood, and then passed across the blood-brain barrier to improve 5-HT levels in the brain. Additionally, pentasaccharide, as one of the main components in MOO, exerted the significant anti-depressant effect through a mechanism identical to that of MOO. This study reveals for the first time that MOO can alleviate depression via increasing 5-HTP in the gut microbiota.

Beh?et′s syndrome (BS) is classified among variable vessel vasculitis with unknown etiology and extensively heterogeneous clinical features. It is easily missed or misdiagnosed due to the lack of a specific laboratory diagnosis index. Based on the evidence and guidelines from China and other countries, the Chinese Rheumatology Association developed the standardization of diagnosis and treatment of BS. The purposes are: (1) To standardize the detection and interpretation of key indicators for BS; (2) To standardize rational management for BS patients with topical and systemic organ involvement to reduce complications and improve outcomes.

Objective:To explore the effect and mechanism of Guizhitang with different proportions of Cinnamomi Ramulus and Paeoniae Alba Radix on overactive cardiac sympathetic nerves in salt-sensitive hypertensive rats. Method:Randomly divide the forty male 6-week-old salt-sensitive hypertensive rats into five groups： the normal control group， the model group， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶2 group，and the Cinnamomi Ramulus and Paeoniae Alba Radix 2∶1 group， each group has 8 animals， the normal control group was fed with low-salt feed， and the remaining four groups were fed with 8% high-salt feed. After 4 weeks of feeding， gastric feeding was started. Give both the normal control group and model group saline and the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group， the 1∶2 group，and the 2∶1 group， were given Guizhitang aqueous solution at 4.0， 5.5 and 5.5 g·kg^-1， respectively. Continuous gavage intervention was held for 4 weeks. IITC multi-channel non-invasive sphygmomanometer was used to detect changes of systolic blood pressure before and after treatment in rats. Left ventricular anterior wall end-diastolic thickness （LVAWd） and interventricular septal diastolic thickness （IVSd） were detected by echocardiography. Hematoxylin-eosin（HE）staining and Masson staining were used to observe the myocardial morphological changes of rats in each group， Western blot was used to detect the expression of nerve growth factor （NGF）， growth-associated protein 43 （GAP43） and tyrosine hydroxylase （TH） protein. Result:After 4 weeks of intervention with Guizhitang， compared with the normal control group， the blood pressure， LVAWd and IVSd of the model group were significantly increased， and the expressions of NGF， TH and GAP43 protein were significantly increased （P<0.01）. HE and Masson staining results showed that the model group had myocardial cell edema， a large number of inflammatory cell infiltration， myocardial fiber hyperplasia and disordered arrangement， and a large amount of collagen deposition could be seen in the intercellular substance. Compared with model group， the systolic blood pressure of rats in each Guizhitang group increased slowly， and the expression of NGF， TH and GAP43 protein decreased （P<0.05，P<0.01），the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group has the best effect. The results of echocardiography shows that the 1∶1 Guishao group could reduce LVAWd and IVSd levels （P<0.05，P<0.01）， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶2 group could reduce IVSd level （P<0.05）， there was no statistical difference in LVAWd， there was no statistical difference in LVAWd and IVSd in 2∶1 group. In terms of myocardial morphology， each group of Guizhitang can reduce cell edema and inflammatory cell infiltration， reduce myocardial fiber hyperplasia and collagen deposition， and improve the disorder of myocardial fiber arrangement. Among them， the 1∶1 group has the best effect. Conclusion:Guizhitang can inhibit the overactive activation of cardiac sympathetic nervous system，reduce the extent of myocardial fibrosis， inflammatory infiltration and myocardial hypertrophy， and protect salt-sensitive hypertension rats， whose mechanism may be related to regulating the expression of heart NGF.Among them， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group is better than the 1∶2 and 2∶1 group in reducing myocardial fibrosis， inflammatory infiltration and myocardial hypertrophy.