Student preparation for laboratory sessions is the first step of conducting laboratory experiments. It makes students maximize use of laboratory time and efficiently perform laboratory exercises in open labs. In view of teaching features and requirements of Microbiology experiment, we designed and developed ‘Online Student Preparation and Management System of Microbiology Experiment’, which integrated func- tions of student preparation for laboratory sessions and teacher management. In the system each experiment consists of six successive parts, viz., learning objectives, principle, materials and equipments, procedure video, manipulation simulation and online quiz. Teaching practices showed that the application of the system enhanced the preparing quality and makes the management of the experiment teaching more normalized and efficient. It was an effective measure in improving experimental teaching of Microbiology.

Objective To investigate the optimal injury time point of cardiac arrest (CA) induced electrically, and establish a reproducible prolonged CA and cardiopulmonary resuscitation (CPR) model in pigs. Methods Forty healthy domestic male pigs were randomly divided into four groups, which were ventricular fibrillation (VF) 8, 10, 11, and 12 minutes groups, each group for 10 animals. In these groups, VF was induced by alternating current delivered to right ventricular endocardium and untreated for 8, 10, 11, and 12 minutes, respectively, followed by 6 minutes of CPR procedure. The resuscitation and survival outcomes were recorded. Hemodynamic parameters and arterial blood gases of animals after successful resuscitation were measured and recorded for 6 hours. Those successful resuscitation animals were regularly evaluated for the neurological deficit score (NDS) and survival outcomes every 24 hours till 96 hours after resuscitation. Results The shortest duration of CPR (minute: 6.9±1.3) and the highest successful ratio of the first defibrillation (7/10) were observed in group VF 8 minutes, and the ratio of successful resuscitation was 100%. The best coronary perfusion pressure (CPP) during the CPR, less neurological impairment, longer survival time, more stable hemodynamics, and shorter time for arterial pH and lactate level restoring to the original state after CPR were also observed in group VF 8 minutes, and no severe damage was found in those animals. The longest duration of CPR (minute:10.3±2.9) and the lowest successful ratio of the first defibrillation (1/10) were observed in group VF 12 minutes, and only 4 animals achieved restoration of spontaneous circulation (ROSC), and no animal survived to CPR 96 hours. The worst CPP during CPR and the highest NDS after resuscitation were also found in VF 12 minutes animals compared to those animals in the other groups. The injuries caused by ischemia and hypoxia in groups VF 10 minutes and VF 11 minutes were in between those of the groups VF 8 minutes and VF 12 minutes, and the duration of CPR were (7.0±2.1) minutes and (8.2±2.6) minutes. There were 9 and 7 animals achieved ROSC in groups VF 10 minutes and VF 11 minutes correspondingly, and 6 and 4 animals survived to 96 hours respectively. Obviously unstable hemodynamics was observed during the period of CPR 2 hours in the two groups. At CPR 1 hour, the heart rates (HR, beats/min) in groups VF 10 minutes and VF 11 minutes increased to 172 (155, 201) and 168 (136, 196) respectively, and the mean arterial pressures (MAP, mmHg, 1 mmHg = 0.133 kPa) declined to 97 (92, 100) and 81 (77, 100), the cardiac output (CO, L/min) decreased to 5.0 (4.0, 5.8), 3.7 (3.0, 5.4) correspondingly. Distinct injuries were found in the two groups [CPR 24-96 hours NDS in groups VF 10 minutes and VF 11 minutes: 180 (110, 255)-20 (0, 400) and 275 (223, 350)-240 (110, 400)], and the arterial pH of the two group decreased to 7.26±0.09 and 7.23±0.09 respectively, and the level of lactate (mmol/L) increased to 9.17±1.48 and 12.80±2.71 correspondingly at CPR 0.5 hour. Significantly lower pH was observed in group VF 11 minutes compared to group VF 8 minutes at CPR 0.5 hour (7.23±0.09 vs. 7.33±0.04, P < 0.05). The highest level of lactate (mmol/L) was also found at the same time point in group VF 11 minutes, which recovered to normal slowly, and was still significantly higher than groups VF 8, 10, 12 minutes (7.58±3.99 vs. 2.55±1.53, 2.13±2.00, 3.40±2.30, all P < 0.05) at CPR 4 hours. Conclusions The longer duration of CA was, the more severe damage would be, the longer CPR time would be required, and the harder of the animals to achieve ROSC. In this prolonged CA and CPR porcine model, 10-11 minutes for untreated VF, was an optimal time point with appropriate successful rate of resuscitation, survival outcomes, and post-resuscitation injuries. Therefore, we recommended 10-11 minutes might be the rational length of no-flow time in this model.

Purpose: Central lymph node metastasis (CNM) are highly prevalent but hard to detect preoperatively in papillary thyroid carcinoma (PTC) patients, while the significance of prophylactic compartment central lymph node dissection (CLND) remains controversial as a treatment option. We aim to establish a nomogram assessing risks of CNM in PTC patients, and explore whether prophylactic CLND should be recommended. Materials and Methods: One thousand four hundred thirty-eight patients from two clinical centers that underwent thyroidectomy with CLND for PTC within the period 2016–2019 were retrospectively analyzed. Univariate and multivariate analysis were performed to examine risk factors associated with CNM. A nomogram for predicting CNM was established, thereafter internally and externally validated. Results: Seven variables were found to be significantly associated with CNM and were used to construct the model. These were as follows: thyroid capsular invasion, multifocality, creatinine > 70 μmol/L, age < 40, tumor size > 1 cm, body mass index < 22, and carcinoembryonic antigen > 1 ng/mL. The nomogram had good discrimination with a concordance index of 0.854 (95% confidence interval [CI], 0.843 to 0.867), supported by an external validation point estimate of 0.825 (95% CI, 0.793 to 0.857). A decision curve analysis was made to evaluate nomogram and ultrasonography for predicting CNM. Conclusion A validated nomogram utilizing readily available preoperative variables was developed to predict the probability of central lymph node metastases in patients presenting with PTC. This nomogram may help surgeons make appropriate surgical decisions in the management of PTC, especially in terms of whether prophylactic CLND is warranted.

Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting E. coli DH5alpha. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub-cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase.
Amidohydrolases ; genetics ; isolation & purification ; metabolism ; Arthrobacter ; genetics ; Bacteriophage lambda ; genetics ; Chromatography, Thin Layer ; Cloning, Molecular ; Electrophoresis, Agar Gel ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Gene Library ; Promoter Regions, Genetic ; genetics

BACKGROUNDDaxx has been identified as a nuclear protein that involves in apoptosis and transcriptional repression. Daxx co-localizes with the promyelocytic leukemia (PML) protein and regulates transcription. Human Daxx (hDaxx) is a protein that functions as a transcriptional regulation through its interaction with some DNA-associated proteins. The aim of this study was to explore the transcriptional regulatory effect of hDaxx interacting with adenovirus (Ad) 12 E1B (Ad12E1B) 55-kDa oncoprotein.

METHODSThe co-localization of hDaxx-Ad12E1B or hDaxx-PML protein in the nucleus was observed under a confocal microscope. Interaction of hDaxx and Ad12E1B was analyzed by yeast two-hybrid assay. Direct binding of hDaxx and Ad12E1B was analyzed using coimmunoprecipitation and Western blot in vivo and in vitro. The activity of a luciferase reporter gene, which was regulated by an hDaxx modulated thymidine kinase (TK) promoter, was detected in an automat luminometer.

RESULTSAd12E1B, which co-localized with hDaxx in the nuclei of G401-CC3 cells, disrupted the co-localization of hDaxx and PML in the PML oncogenic domains (PODs). hDaxx bound directly to Ad12E1B in vivo and in vitro. hDaxx interacted with Ad12E1B along its full length. Ad12E1B enhanced transcriptional repression activity of hDaxx.

CONCLUSIONAd12E1B disrupts the co-localization of hDaxx with PML in PODs and enhances transcriptional repression activity of hDaxx.

Adaptor Proteins, Signal Transducing ; Adenovirus E1B Proteins ; physiology ; Carrier Proteins ; analysis ; genetics ; Cell Line, Tumor ; Humans ; Intracellular Signaling Peptides and Proteins ; Neoplasm Proteins ; analysis ; Nuclear Proteins ; analysis ; genetics ; Promyelocytic Leukemia Protein ; Repressor Proteins ; physiology ; Transcription Factors ; analysis ; Transcription, Genetic ; Tumor Suppressor Proteins

Objective To assess the efficacy and adverse-event profile of monotherapy oradd-on therapy with topiramate in elderly patients with epilepsy. Methods A multicenter, prospective,open-label, observational study of topiramate, for either monotherapy or add-on therapy, was performedamong 119 elderly patients with epilepsy in the Outpatient Department of Neurology of 52 generalhospitals in China. After the baseline evaluation, topiramate was given at the target dose of 200 mg/dayover an 8-week titration period. In the subsequent 12-week maintenance period, the topiramate dose wasadjusted (200-300 mg) according to the clinical results. The patients were followed up for 6 months, withmonthly visits and regular physical, neurological and laboratory examinations. Results After themaintenance period, 106 patients (89.1%) showed a seizure frequency reduction by 50% or greater fromthe baseline, among whom 65 patients(93.8%)received the monotherapy and 54(83.8%) had the add-ontherapy. Topiramate monotherapy and add-on therapy resulted in a complete seizure control in over 50%of the patients with various types of seizure. In patients with disease course less than 1 year, between 1and 3 years, between 4 and 6 years, and over 6 years, topiramate monotherapy resulted in seizurereduction by 92.86%, 91.67%, 100%, and 94.44%, and the add-on therapy reduced the seizure by80.00%, 85.71%, 70.00%, and 86.36%, respectively. When combined with carbamazepine, valproatesodium, phenytoin, phenol barbital, and diazepam, the total effective rate oftopiramate was 79.41%,87.50%, 85.71%, 0%, and 80.00%, respectively. Topiramate, especially in monotherapy, caused onlymild or moderate adverse effects. Conclusion Topiramate is effective and safe for either monotherapyor add-on therapy of epilepsy in elderly patients. The types of epilepsy, disease course, or the drugs usedin the add-on therapy do no obviously affect the efficacy of topiramate for seizure control.

OBJECTIVEThis study aims to establish and evaluate the methodology of isolated rabbit eye (IRE) test.

METHODSIRE test was performed according to modifications of the in vitro toxicology (INVITTOX) Protocol No.85: Rabbit enucleated eye test by European Centre for the Validation of Alternative Methods (ECVAM), and then 26 chemicals and 26 cosmetic products were tested in both in vitro IRE and in vivo Draize tests. A statistical analysis was conducted to determine the relevance of the IRE test to the data generated in the Draize test.

RESULTSIRE test was established successfully in our laboratory. It was shown that ranking correlation and class concordance were fairly well between the IRE test and the Draize test for 26 reference chemicals (Fisher's Exact Test χ(2)=51.314, P<0.001; McNemar P=0.261; Gamma=0.960, P<0.001; Kappa=0.843, P<0.001) and 26 cosmetic products (Fisher's Exact Test χ(2)=15.522, P<0.001; McNemar P=0.311; Gamma=0.967, P<0.001; Kappa=0.611, P<0.001).

CONCLUSIONIRE test was established successfully for in vitro testing of eye irritation as an alternative to Draize test.

Animal Testing Alternatives ; Animals ; Cosmetics ; toxicity ; Eye ; drug effects ; Irritants ; toxicity ; Rabbits ; Toxicity Tests ; methods

OBJECTIVETo establish a stable recipient mouse model for stem cell transplantation into seminiferous tubules and improve the traditional techniques for transplantation.

METHODSSixty male ICR mice were equally divided into Groups A, B, C and D, and injected with Busulfan at 15 mg/kg, 30 mg/kg, 40 mg/kg and 0 mg/kg, respectively. The survival rate was recorded every day, and the testis weight and spermatogenesis of testicular tubules were determined at 4, 8 and 12 weeks after the injection. We improved the stem cell transplantation technique and designed a new transplantation device, which connected the nozzle end, syringe and puncture needle by a three-way joint. The nozzle end was used for tentative injection, and the syringe for drawing and then injecting the cell suspension.

RESULTSOnly one mouse in Group C died after the injection. At 4 weeks after Busulfan treatment, the testis weight decreased apparently in Groups A, B and C, with significant differences from D (P < 0.05). The differences remained significant at 8 weeks (P < 0.05), except between Groups A and D (P > 0.05), but at 12 weeks none of the first three groups showed any significant difference from Group D (P > 0.05). At 4 and 8 weeks, the rate of hollow seminiferous tubules was < 50% in Group A and > 50% in Groups B and C, and almost returned to normal at 12 weeks, with no significant differences among the three groups (P > 0.05), but it remained unchanged in Group D. The improved transplantation device increased the success rate (> 90%), lowered the donor cell loss (< 50 microl cell suspension needed for both testes) and shortened the process ( < 10 min for one testis).

CONCLUSIONIntraperitoneal injection of Busulfan at 30 mg/kg is suitable for the establishment of the recipient mouse model of stem cell transplantation. The improved transplantation device and methods help promote the efficiency and success rate of the transplantation operation.

Animals ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred ICR ; Seminiferous Tubules ; cytology ; Stem Cell Transplantation ; methods ; Testis

OBJECTIVETo study the effect of Platycarya strobilacea Sieb. et Zucc (PSZ) extract on methuosis of human nasopharyngeal carcinoma CNE1 and CNE2 cells and explore the underlying mechanism.

METHODSCNE1 and CNE2 cells were treated with 1 mg/mL PSZ extract and the expressions of Rac1 mRNA and Rac1 protein were detected using RT-qPCR and Western blotting, respectively. Results CNE1 and CNE2 cells showed obvious morphological changes typical of methuosis following treatment with PSZ extract characterized by cell merging, accumulation of large cytoplasmic vacuoles, and membrane rupture without obvious changes in the nuclei. PSZ treatment resulted in up-regulated Rac1 mRNA and Rac1 protein expressions in the cells. Application of EHT 1864 obviously blocked the effect of PSZ extract in inducing methuosis in CNE1 and CNE2 cells.

CONCLUSIONPSZ extract can induce methuosis in CNE1 and CNE2 cells by inducing the overexpression of Rac1.

Objective: To explore changing trend in prognosis of primary hypopharyngeal carcinoma and to analyze the reasons at the Eye, Ear, Nose and Throat Hospital of Fudan University. Methods: We retrospectively analyzed the clinical data of 461 patients with primary hypopharyngeal carcinoma treated at the Eye, Ear, Nose and Throat Hospital of Fudan University from 2003 to 2007 (Group 1) and 2010 to 2014 (Group 2) according to the inclusion criteria. 142 from Group 1, including 133 males and 9 females, rangedfrom 38 to 82 years old and 319 from Group 2, including 313 males and 6 females, ranged from 39 to 81 years old, were included in this work. The laryngeal function preservation rate, survival outcome, application and effect of pre-and post-operative adjuvant therapy were compared. SPSS 24.0 was used for statistical analyses. Results: There were 62 patients with early disease (T1-2N0) including 18 in Group 1 and 44 in Group 2, in whom 3 (16.7%) underwent surgical procedures with laryngeal function preservation in Group 1, while, 30 (68.2%) underwent laryngeal function preservation surgery in Group 2. The laryngeal function preservation rate showed an obviously upward trend in recent years (χ²=13.617, P<0.001), whereas, the recurrence-free survival rate (RFS) and overall survival rate (OS) showed no significant differences between two groups (P=0.469 and 0.808, respectively). Among the 399 patients with advanced disease, 124 were in Group 1 and 275 in Group 2. After propensity score matching (PSM) was used, the OS rate was significantly higher for Group 2 than Group 1 (P=0.017), while the application of laryngeal function preservation surgery was significantly higher in Group 2 (χ²=4.686, P=0.030). The application rates of preoperative adjuvant therapy and postoperative adjuvant chemotherapy were significantly higher in group 2 than in group 1 (χ²=5.687, P=0.017; χ²=19.407, P<0.001). Conclusion The application of laryngeal function preserving surgery significantly increases the retention rate of laryngeal functions in patients with early-stage hypopharyngeal carcinoma, with similar long-term survival outcomes. The application of comprehensive treatment including preoperative adjuvant therapy and postoperative adjuvant treatments, especially postoperative chemoradiotherapy, shows an obvious increase in the function preservation rate and long-term survival in patients with advanced hypopharyngeal carcinoma.