The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.

Abstract: Objective　The study aims to investigate the diagnosis quality and accuracy of syphilis cases reported by medical facilities in Inner Mongolia, understand possible problems and influencing factors in reporting and diagnosis, providing evidence for the better formulation of syphilis control and prevention. Methods　Cross-sectional survey was conducted with 2 counties sampled randomly from 12 municipals of Inner Mongolia, different medical facilities were covered. Syphilis cases reported from July 2019 to June 2020 in medical institutions of different categories were sampled and checked. The quality and accuracy of syphilis case reporting were evaluated according to the identifiers in "Syphilis Diagnosis (WS 273-2018)" and "National STD Case Reporting Quality Management Scheme (2018)". In addition, the basic information of medical institutions and the implementation of syphilis detection in laboratories were investigated, and the physicians who reported the case first were interviewed to understand their mastery of syphilis diagnosis and reporting, thus analyzing the major factors influencing the accuracy of reports. Results　The reporting rate of syphilis in medical institutions in Inner Mongolia was 99.04% (311/314), the missing-report rate was 0.96% (3/314), the timely reporting rate was 98.05% (1 659/1 692), the completeness rate was 99.64% (1 686/1 692), the correct rate was 99.35% (1 681/1 692), the accuracy rate of internet-based input was 84.63% (1 432/1 692). There were statistical differences in the quality (χ2=13.95, P<0.05; χ2=11.40, P<0.05) and accuracy （χ2=30.06, P<0.05; χ2=44.93, P<0.05) of reports among different municipals and different types of medical facilities. The accuracy rate of syphilis reporting by medical institutions was 86.17% (1 458/1 692), the correct rate for classifying diagnosis was 87.06% (1 473/1 692), and the accuracy rate of staging was 90.25% (1 527/1 692). Multivariate logistic regression results showed that whether the first-clinic physician attended training in the past three years [OR=6.26, 95%CI: (2.12-18.46)] and whether they grasped the key points of syphilis classification standard [OR=2.79, 95%CI: (1.21-6.46)] influenced report accuracy. Conclusions　The quality of reports in Inner Mongolia medical institutions is generally high, but the accuracy rate of reporting and correctness of network input have not yet reached the target requirement of 95%. There is still room for improvement in reporting and diagnostic capabilities. It is suggested to further strengthen the training frequency and coverage for physicians on syphilis diagnosis standard.

Objective To evaluate the efficacy of patients with stage T2 bladder cancer who underwent combined treatment of bladder-preserving surgery and adjuvant intra-arterial chemotherapy.Methods The survival data of bladder cancer paients from January 2000 to December 2014 with stage T2N0M0 were retrospectively analyzed.Thirty-five patients of cT2N0M0 receive combined treatment of bladder-preserving surgery and adjuvant intra-arterial chemotherapy(group A),and 80 patients of pT2N0M0 underwent radical cystectomy (RC) (group B).The pathological diagnosis of all patients was urothelial carcinoma.In group A,there were 33(94.2％) males and 2 (5.8％) females;20 (57.1％) tumor size less than 3 cm and 15 (42.9％) larger than 3 cm;24 (68.6％) with single tumor and 11 (31.4％) with multiple tumors;11 (31.4％) patients with primary tumors and 24 (68.6％) recurrent tumors.In group B,there were 71 (88.7％) males and 9 (11.3％) females;35 (43.8％) tumor size less than 3 cm and 45(56.2％) larger than 3 cm;44 (55.0％) with single tumors and 36 (45.0％) with multiple tumors;22(27.5％) patients with primary tumors and 58 (72.5％) recurrent tumors.Results Groups A and B consisted of 35 and 80 patients and median follow-up time was 68 (13-157)and 67 (4-198)months,respectively.There was no significantly statistical difference in disease-specific survival (DSS) between the two groups(P =0.888),76.5％ for group A and 60.6％ for group B respectively.In group A,26 (74.3％) patients achieved complete response (CR) to intra-arterial chemotherapy.Additionally,amounts of 21 (60.0％) patients preserve their functional bladder successfully and their median follow-up time was 69 (13-134)months.8 patients receive delayed radical cystectomy when suffered tumor recurrence and none of them had lymph node metastases.Of those pathological stage was presented as stage T2 5 cases,T3 2 cases and T4 1 case.Importantly,the 8 patients who receive delayed RC did not confer worse DSS when compared with those underwent immediate RC in group B (P =0.809).Cox proportional hazards model showed that tumor number and CR to intra-arterial chemotherapy was independent prognostic factor for disease-free survival (HR =0.238,P =0.007) and DSS(HR =0.085,P =0.004) respectively.During the period of intra-arterial chemotherapy,we did not observe hematological toxicity of grade Ⅳ and the hematological toxicity of grade Ⅰ-Ⅲ was 9 (25.7％),6 (17.1％) and 4 (11.4％).Conclusions For patients with T2N0M0,combined treatment of bladder-preserving surgery and adjuvant intra-arterial chemotherapy could be a therapy with long-term survival outcome and safety.The therapy could be offered as alternative treatment option for patients who were unsuitable for receiving RC.

Objective To investigate the relationship between plasma oxidative stress factors levels and organ damage parameters as well as prognosis in patients with sepsis. Methods A case-control study was conducted. Twenty-five patients admitted to surgical intensive care unit (ICU) of the First Affiliated Hospital of Sun Yat-sen University from March to December in 2016 and diagnosed as sepsis were enrolled as study subjects. Another 15 patients without sepsis admitted to surgical ICU in the same period were enrolled as controls. General demographic data, main diagnoses, acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score within 24 hours, clinical laboratory indicators [alanine aminotransferase (ALT), aspartate transaminase (AST), serum creatinine (SCr), blood urea nitrogen (BUN), C-reactive protein (CRP), procalcitonin (PCT), white blood count (WBC)] and oxidative stress indicators [superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO)] as well as length of ICU stay, total hospital stay and 28-day mortality were recorded. Spearman or Pearson correlation method was used to analyze the correlation between oxidative stress indicators and organ damage indicators as well as prognosis in patients with sepsis. Receiver operator characteristic (ROC) curve was plotted to evaluate the predictive value of oxidative stress indicators for 28-day mortality in patients with sepsis. Results The length of ICU stay in sepsis group was significantly longer than that in non-sepsis group [days: 7.0 (5.5, 11.0) vs. 4.0 (1.0, 11.0), P < 0.05], and AST, BUN, CRP, PCT, plasma MDA and NO levels were significantly higher than those in non-sepsis group [AST (U/L): 50.76±19.53 vs. 28.53±14.02, BUN (mmol/L): 9.99±5.26 vs. 6.97±4.32, CRP (mg/L): 109.28±42.79 vs. 60.33±46.68, PCT (μg/L): 5.4 (0.3, 24.0) vs. 0.6 (0.1, 1.5), MDA (ng/L): 488.31±76.68 vs. 399.30±50.23, NO (ng/L): 5.08±0.89 vs. 4.42±0.88, all P < 0.05]. There was no significant difference in gender, age, APACHEⅡ score, total hospital stay, 28-day mortality, ALT, SCr, WBC or plasma SOD activity between the two groups. The correlation analysis between oxidative stress parameters and organ damage parameters as well as prognosis in patients with sepsis showed that MDA and NO were positively correlated with SCr (r value was 0.426 and 0.431, respectively, both P < 0.05), and there was a positive correlation between MDA and NO (r = 0.990, P < 0.01); plasma SOD activity was negatively correlated with 28-day mortality (r = -0.468, P < 0.05), while MDA and NO levels were positively correlated with 28-day mortality (r value was 0.598 and 0.611, respectively, both P < 0.01). ROC curve analysis showed that plasma SOD, MDA and NO levels had a good independent predictive effect on 28-day mortality, the area under ROC curve (AUC) was 0.816±0.087, 0.904±0.078 and 0.912±0.071, and the best cut-off value was 40.76% (sensitivity 68.4%, specificity 100%), 487.93 ng/L (sensitivity 83.3%, specificity 89.5%) and 5.31 ng/L (sensitivity 83.3%, specificity 89.5%), respectively. Conclusions The plasma levels of oxidative stress factors in patients with sepsis are significantly increased, which is closely related to organ damage and poor prognosis. The plasma SOD, MDA and NO levels can be used as independent bio-marker to predict the 28-day mortality of patients with sepsis.

Objective: To explore the clinical outcome of advanced testicular nonseminomatous germ cell cancer patients undergoing post chemotherapy retroperitoneal lymph node dissection (PC-RPLND), and to analyze the relevant prognostic factors of lymph node pathological. Methods: A total of 43 consecutive testicular nonseminomatous germ cell cancer patients underwent PC-RPLND between March 2001 and December 2014 in Department of Urology at Sun Yat-sen University Cancer Center were retrospectively reviewed. The average age of the patients was (29.0±11.5) years (ranging from 12 to 58 years). Before PC-RPLND, 22 patients were classified as phase Ⅱ, while 21 were phase Ⅲ. Primary tumor histology revealed seminomatous elements in 19 cases, embryonal cell carcinoma in 22 cases, yolk sac tumor in 13 cases, chorionic carcinoma in 3 cases, mature teratomatous elements in 11 and immature teratomatous elements in 2 cases. Patients were treated with cisplatin-based chemotherapy after orchectomy and then underwent surgical resection of retroperitoneal lymph nodes.After PC-RPLND, all patients underwent a periodic review including the blood routine, biochemistry routine and computed tomography or ultrasonograph of the chest, the abdomen and the pelvis. The association of pathological data with patient′s clinic features and the correlations between molecular features detected with each other were assessed by the t test, χ² and Fisher′s exact test. Multivariate logistic regression were used to assess prognostic factors. Results: The median operative time was 278 minutes (ranging from 50 to 715 minutes). Median blood loss was 425 ml (ranging from 50 to 5 000 ml). Eight patients received blood transfusion intra-operatively, 2 patients underwent adjunctive surgical procedures, 4 patients developed ileus and 4 had an ascites chylosus following PC-RPLND, 1 patient had a postoperative hyperthermia and retrograde ejaculation was present in 10 patients. The transverse diameter of the residual tumor in patients ranged from 0.8 to 18.2 cm. Necrosis, teratoma and viable germ cell tumors were found in 15, 17 and 11 of all patients. The median follow-up time was 46 months (ranging from 6 to 169 months). There were 39 patients had no tumor recurrence, 7 patients were found recurrence after PC-RPLND, 5 died of malignant germ cell tumor. The normal serum lactate dehydrogenase (LDH) level before chemotherapy (HR=25.811, 95%CI: 0.678 to 982.624, P=0.017) and relative changes more than 50% in retroperitoneal lymph node size (HR=0.016, 95%CI: 0 to 0.698, P=0.032) were statistically significant prognostic factors of the presence of necrosis. Conclusions Since most residual masses are not sensitive to chemotherapy, PC-RPLND is still an essential part of the treatment of metastatic testicular nonseminomatous germ cell cancer. Patients with the normal serum LDH level before chemotherapy and a shrinkage of 50% or more in retroperitoneal mass have a considerably chance of having necrosis in the retroperitoneum resection. This may help to refine the selection of candidates for PC-RPLND.

Objective:To explore the advantages of the novel individualized 3D printing artificial vertebral body in spine reconstruction and to evaluate its clinical effect.Methods:From January 2017 to December 2018, the 15 patients who underwent total vertebrectomy and spine reconstruction with individualized 3D printing artificial vertebral body were analyzed retrospectively. There were 8 males and 7 females, with the mean age 39.5 years (range: 20-57), including 12 primary tumors and 3 metastatic tumors. According to tumor location and surrounding soft tissue invasion range, simple posterior or combined anterior and posterior approach were used for total vertebral resection, and the defection was reconstructed by 3D printing artificial vertebral body. The operation time, intraoperative bleeding volume, postoperative stability of artificial vertebral body and bone ingrowth of adjacent vertebral body, preoperative and postoperative neurological changes, preoperative and postoperative VAS score, local control and survival of patients were analyzed.Results:The mean operation time was 412.0 min (range: 135-740 min), and the mean blood loss was 4 140.0ml (range: 100-14 000 ml). The mean follow-up time was 23.2 months (range: 12-35 months), and no one loss to follow-up. One case had pleural rupture, one case had cerebrospinal fluid leakage and one case had L5 nerve root palsy. All patients recovered after active symptomatic treatment. Compare with the preoperative VAS score (4.7±1.1), the differences of VAS score at 7 d postoperative and last follow-up (1.6±0.6 and 1.0±0.5) were significantly reduced ( P<0.001). Three patients with Frankel grade C gradually recovered to grade D, and no change were found in grade D and Grade E patients, there was no significant improved at last follow-up. Preliminary bone growth was found between the artificial vertebral body and the adjacent vertebral body 3 months after operation. The bone growth was more obvious at 12 months post-operation, and the artificial vertebral body fused with the adjacent vertebral bodies to form bone integration. At 24 months post-operation, the integration of the artificial vertebral body was more accurate. During the follow-up period, there was no loosening or displacement of the artificial vertebral body and no failure of internal fixation. A case of hemangioendothelioma and a case of epithelioid angiosarcoma died at 33 months and 35 months postoperatively. One patient with chondrosarcoma had local recurrence at16 months post-operation. After treated with arotinib, the tumor did not progress. The other 12 patients had no tumor recurrence or distant metastasis. Conclusion:After spinal tumor resection, individualized 3D printing artificial vertebral body can be used to accurate restoration of spinal continuity, and provide nice interface matching and bone growth between artificial vertebral body and the adjacent vertebral endplates. Moreover, the immediate and long-term stability of the artificial vertebral body can meet the needs of spinal reconstruction.

OBJECTIVETo observe the effect of electroacupuncture (EA) combined with general anesthesia for the immune function of patients treated with laparoscopic radical rectectomy for rectal cancer.

METHODSFifty patients who would receive selective laparoscopic radical rectectomy for rectal cancer with general anesthesia were randomly divided into an observation group and a control group,25 cases in each one. Fifteen minutes before anesthesia induction,patients in the observation group were treated with EA at Zusanli (ST 36) and Sanyinjiao (SP 6) until the end of operation. Sham acupuncture without piercing the skin was applied at the same acupoints in the control group, and electrodes were connected without stimulation. Interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-6 (IL-6) were quantitatively tested before anesthesia (T), at the time of abdomen closing (T) and one hour after anesthesia anabiosis (T). And serum procalcitonin (PCT) level, leucocyte count and the number of cases with increasing leucocyte (the standard number>10×10/L) were measured on the first day after operation.

RESULTSThe levels of IL-4 and IL-6 were increased apparently and the ratio of IFN-γ/IL-4 was decreased at Tcompared with those before treatment in the control group (all<0.05), but obvious change did not appear in the observation group (all>0.05). The ratio of IFN-γ/IL-4 was enhanced (<0.05),and the levels of IL-4 and IL-6 were reduced (both<0.05) at Tin the observation group compared with those in the control group. The level of PCT of the observation group was markedly lower than that of the control group on the first day after operation (<0.05). There was no statistical significance about leucocyte count and the number of cases with increasing leucocyte between the two groups (both>0.05).

CONCLUSIONSEA at Zusanli (ST 36) and Sanyinjiao (SP 6) could alleviate the depressing immune function and inflammatory reaction of patients after laparoscopic radical rectectomy for rectal cancer.

AIM: To explore the intervention effect of Dahuangtang pellets (DHT) on diabetic nephropathy (DN) based on the AMP-activated protein kinase/mammalian target of rapamycin/unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway. METHODS: Eight mice were randomly assigned to the model group, the dapagliflozin group, and the DHT (high, medium, and low dosage) group out of a total of 40 C57BL/KSJ-db/db (hereafter referred to as db/db) mice; another 10 C57BL/KSJ-db/dm mice were used as the normal group, saline was provided to the normal and model groups, and the mice in the treatment group received the appropriate medications. The medications were given for 10 consecutive weeks, once per day, to the mice in the treatment group. At weeks 0, 4, 8, and 10 of administration, fasting blood glucose (FBG) was assessed by drawing blood at a predetermined time from the tail vein; Urine samples were taken at 0, 5, and 10 weeks after treatment to evaluate the levels of albumin and creatinine, and the urinary albumin-creatinine ratio (ACR) was computed. After 10 weeks, mice in each group were assayed for 24 h total urine protein, serum creatinine (Scr), urea nitrogen (BUN) levels; Western blotting analysis was conducted to detect the expression of p-AMPK, p-mTOR, and p-ULK1, as well as the expression of autophagy related proteins homolog of yeast Atg6 (Beclin-1), autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3), P62 in renal tissue; Immunohistochemistry was used to measure the expression of podocyte lacunar membrane proteins (Nephrin, Podocin) in renal tissues; The pathological morphology of renal tissue was observed by light microscopy and transmission electron microscopy. RESULTS: Compared with the model group, FBG, ACR, and 24 h total urine protein were reduced in the dapagliflozin group and DHT groups of mice, and there was no statistically significant difference in Scr and BUN; In renal tissues, there is increased expression of p-AMPK and p-ULK1, decreased expression of p-mTOR, increased expression of LC3II / LC3I and Beclin-1, and decreased expression of P62 (P<0.01, P< 0.05); differentially upregulated in glomeruli are the podocyte lacunar membrane proteins Nephrin and Podocin (P<0.01, P<0.05); renal pathologic damage was reduced to varying degrees; transmission electron microscopy showed an increase in the number of autophagic vesicles and autophagic lysosomes. CONCLUSION: DHT can delay the development of DN by regulating the AMPK / mTOR / ULK1 signaling pathway, enhancing podocyte autophagy, and protecting glomeruli.

ObjectiveTo investigate the effect and mechanism of Shenqi Tangluo pill （SQTLP） on oxidative stress injury of skeletal muscle of type 2 diabetes mellitus （T2DM） mice based on nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1）/NAD（P）H quinone oxidoreductase 1 （NQO1） pathway. MethodA total of 60 7-week-old male db/db mice ［specific pathogen-free （SPF） grade］ were selected and fed for one week for adaption. They were divided into the model control group， SQTLP low-， medium- and high-dose （19， 38， and 76 g·kg^-1） groups and metformin group （0.26 g·kg^-1） by gavage. Each group consisted of 12 mice. Twelve male db/m mice of the same age were selected as the blank group. The intervention was implemented continuously for 8 weeks. Fasting blood glucose （FBG） was detected. Fasting serum insulin （FINS） levels were detected by enzyme-linked immunosorbent assay （ELISA）， and the homeostasis model assessment-insulin resistance （HOMA-IR） index and the homeostasis model assessment-insulin sensitivity index （HOMA-ISI） were calculated. Oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were conducted. The activities of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） and the contents of malondialdehyde （MDA） and reduced nicotinamide adenine dinucleotide phosphate （NADPH） in skeletal muscle tissues were detected by biochemical kits. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in skeletal muscle tissues. The levels of reactive oxygen species （ROS） and 4-hydroxynonenal （4-HNE） in skeletal muscle tissue were detected by immunofluorescence （IF）. The expression levels of Nrf2， HO-1， NQO1 and glutamate-cysteine ligase catalytic subunit （GCLC） proteins in skeletal muscle tissues were detected by Western blot. ResultCompared with those in the blank group， FBG， FINS and HOMA-IR in the model group were significantly increased （P<0.05）， while HOMA-ISI was decreased （P<0.05）. The results of OGTT and ITT showed that blood glucose was significantly increased at all time points （P<0.05）， and glucose tolerance and insulin tolerance were significantly impaired. SOD and GSH-Px activities in skeletal muscle tissues were significantly decreased （P<0.05）， and MDA and NADPH contents were significantly increased （P<0.05）. In skeletal muscle tissues， the arrangement of muscle fibers was loose， the nucleus was disordered， and inflammatory cells were infiltrated. The expression levels of ROS and 4-HNE in skeletal muscle tissues were significantly increased （P<0.05）. The protein expression levels of Nrf2， HO-1， NQO1 and GCLC in skeletal muscle tissues were significantly decreased （P<0.05）. Compared with those in the model group， FBG， FINS and HOMA-IR in the metformin group were significantly decreased （P<0.05）， while HOMA-ISI was increased （P<0.05）. The results of OGTT and ITT showed that blood glucose in the metformin group was significantly decreased at all time points （P<0.05）. The activities of SOD and GSH-Px in skeletal muscle tissues were significantly increased （P<0.05）， while the contents of MDA and NADPH were significantly decreased （P<0.05）. No obvious abnormality was found in the skeletal muscle tissue of the metformin group. The expressions of ROS and 4-HNE in skeletal muscle tissues were decreased （P<0.05）. The protein expression levels of Nrf2， HO-1， NQO1 and GCLC in skeletal muscle tissues were significantly increased （P<0.05）. Compared with those in the model group， FBG， FINS and HOMA-IR in the SQTLP medium- and high-dose groups were significantly decreased （P<0.05）， while HOMA-ISI was increased （P<0.05）. The results of OGTT and ITT showed that the glucose tolerance and insulin tolerance of mice were improved in each dose group of SQTLP. The GSH-Px activity in the SQTLP low-dose group was significantly increased （P<0.05）， and the NADPH content was decreased （P<0.05）. The activities of SOD and GSH-Px in the SQTLP medium- and high-dose groups were significantly increased （P<0.05）， while the contents of MDA and NADPH were significantly decreased （P<0.05）. The skeletal muscle tissue injury of mice in each dose group of SQTLP was ameliorated to different degrees. In the SQTLP medium- and high-dose groups， the expressions of ROS and 4-HNE were decreased （P<0.05）， and the protein expression levels of Nrf2， HO-1， NQO1 and GCLC were significantly increased （P<0.05）. Compared with those in the SQTLP low-dose group， FBG and HOMA-IR in the SQTLP high-dose group were significantly decreased （P<0.05）， while HOMA-ISI was increased （P<0.05）. The results of OGTT and ITT showed that the SQTLP high-dose group significantly improved the glucose tolerance and insulin tolerance of mice. The activities of SOD and GSH-Px in skeletal muscle tissues were significantly increased （P<0.05）， while the contents of MDA and NADPH were significantly decreased （P<0.05）. No obvious abnormality was found in the skeletal muscle tissue， the expressions of ROS and 4-HNE were decreased （P<0.05）， and the protein expression levels of Nrf2， HO-1， NQO1 and GCLC were significantly increased （P<0.05） in the skeletal muscle tissue of the SQTLP high-dose group. ConclusionSQTLP can significantly improve IR in T2DM mice， and the mechanism is related to SQTLP activating the Nrf2/HO-1/NQO1 signaling pathway， promoting the expression of antioxidant enzymes， and thus improving the oxidative stress injury in the skeletal muscle.