To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6xHis tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS 115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 °C and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.
Bacterial Proteins ; biosynthesis ; genetics ; Carboxypeptidases ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; Hydrolysis ; Open Reading Frames ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Thermus ; enzymology

Aiming at the contradiction between the current oncology teaching model and the rapid development of modern oncology, this study proposes to use precision medicine principles, integrated medicine (MDT treatment) and problem-based, evidence-based CSCO guide learning in clinical oncology teaching. Through specific cases, students are instructed to study the guidelines, and cultivate students' concepts of evidence-based medicine, students' interest in participating in oncology teaching, and they can initially form treatment strategies. The prospects have been put forward from the establishment of a clinical oncology curriculum system, the construction of a single-disease diagnosis and treatment teaching environment and a team of teaching staff, the reinforcement of students' basic experiments and evidence-based clinical data cross-application learning, and the enhancement of humanistic quality education.

Objective:To detect the expression of has_circ_0001459 in peripheral blood mononuclear cells (PBMCs) of Epstein-Barr virus (EBV)-infected individuals and evaluate its diagnostic value for EBV infection.Methods:The expression profiles of circRNAs in PBMCs of patients with EBV infection were obtained by whole-transcriptome sequencing. Differentially expressed circRNAs with statistical significance were selected based on the criteria of ∣log 2Fold Change∣≥1 and P<0.05. Based on the composition, length and primer specificity, hsa_circ_0001459 with high expression was selected for further research. The relative expression of hsa_circ_0001459 in PBMCs of 60 patients with EBV infection and 45 healthy people was detected by real-time fluorescent quantitative PCR. Receiver operating characteristic (ROC) curve and Kappa analysis were used to analyze the diagnostic value of hsa_circ_0001459 expression for EBV infection. Results:The expression of hsa_circ_0001459 showed an up-regulation trend in patients with EBV infection as compared with that in the healthy people, and the results were consistent with the sequencing results. The area under the ROC curve for screening EBV-infected individuals was 0.83(95%CI: 0.75-0.91) with the sensitivity of 0.80(95%CI: 0.66-0.89) and the specificity of 0.77(95%CI: 0.65-0.86). Kappa analysis indicated that hsa_circ_0001459 was moderately consistent with EBV DNA in the diagnosis of EBV infection (κ=0.51).Conclusions:Hsa_circ_0001459 tended to be highly expressed in PBMCs of EBV-infected individuals, which might be uses as a diagnostic marker for EBV infection.

Objective:To compare the detection rate of genital Chlamydia trachomatis (CT) DNA between urine and urethral/cervical swab samples. Methods:From December 2018 to December 2019, a total of 1 475 outpatients were collected from sexually transmitted disease clinics in 7 medical institutions, such as Department of Venereology, Guangzhou Institute of Dermatology, including 1 118 males and 357 females. One urethral/cervical swab sample and one urine sample were collected successively from each patient. Real-time fluorescence-based PCR was performed to detect CT DNA in urine and urethral/cervical swab samples, and paired chi-square test was used to compare the positive rate of CT DNA between the 2 kinds of samples. Random- or fixed-effect meta-analysis was conducted for the test of heterogeneity and merging of positive rates of CT DNA in the urine and urethral/cervical swabs among 7 medical institutions.Results:The positive rate of CT DNA in the urine samples was significantly higher than that in the swab samples from 4 medical institutions (all P < 0.05) , while there was no significant difference in the positive rate of CT DNA between the 2 kinds of samples from 3 medical institutions (all P > 0.05) . The heterogeneity ( I2) estimates of the CT-DNA positive rate in urine and swab samples among different medical institutions were 78.6% (95% CI: 55.9% - 89.6%) and 73.7% (95% CI: 43.7% - 87.7%) , respectively; meta-analysis showed that the total merged positive rate of CT DNA in the urine samples was 10.8% (95% CI: 7.2% - 15.9%) , which was significantly higher than that in the swab samples (7.8%, 95% CI: 4.9% - 12.1%; χ2 = 39.2, P < 0.05) . Compared with the swab sample-based CT-DNA detection method, the sensitivity, specificity, positive predictive value, negative predictive value and consistency rate of the urine sample-based CT-DNA detection method were 97.0% (128/132) , 96.3% (1 293/1 343) , 71.9% (128/178) , 99.7% (1 293/1 297) , and 96.3% (1 421/1 475) , respectively. The positive rate of CT DNA in the urine samples from 1 118 male patients was 11.0% (95% CI: 7.2% - 16.5%) , which was significantly higher than that in the swab samples (7.6%, 95% CI: 4.9% - 11.8%; χ2 = 34.3, P < 0.05) . There was no significant difference in the positive rate of CT DNA between the urine (11.9%, 95% CI: 7.7% - 17.9%) and cervical swab samples from 357 female patients (10.4%, 95% CI: 7.6% - 14.0%; χ2 = 3.2, P > 0.05) . Conclusions:The positive rate of CT DNA in urine samples is higher than or similar to that in urethral/cervical swab samples. The urine sample-based CT-DNA detection method has characteristics of convenience, non-invasiveness, painlessness and low cost, and is worthy of clinical promotion.

Objective:To investigate the clinical value of shear wave elastography (SWE) in predicting pathological responses to neoadjuvant chemotherapy in breast cancer.Methods:According to the postoperative pathological responses, 56 patients who received neoadjuvant chemotherapy followed by surgical excision in the Fujian Cancer Hospital from August 2019 to September 2020 were divided into responders and non-responders. The relative change rates of tumor maximum diameter(ΔD2, ΔD4) and SWE stiffness (ΔEmax2, ΔEmax4, ΔEmean2, ΔEmean4) were assessed before NAC and after different NAC cycles (t2, t4). Clinical information, including age, T, N stages, ER, PR, HER2, Ki67, and molecular subtype were also considered as the variables. The independent influencing factors of pathological responses after neoadjuvant chemotherapy were obtained by logistic regression analysis and diagnostic test was carried out.Results:There were 23 cases as responders (41.0%, 23/56), and 33 cases as non-responders (58.9%, 33/56). Results of multivariate analysis showed ΔEmax4 and HER2 index were independent influencing factors of pathological responses ( OR=1.11, P<0.001; OR=31.81, P=0.002). Area under curve of the ΔEmax4 (AUC: 0.869, 95% CI: 0.746-0.941) was higher than that of HER2 (AUC: 0.690, 95% CI: 0.545-0.834). The combination of ΔEmax4 and HER2 gave the best prediction of pathological responses (AUC 0.930, 95% CI: 0.829-0.981). the sensitivity, specificity, diagnostic accuracy, postive predictive value, and negative predictive value were 78.26%, 96.97%, 75.23%, 94.73%, and 86.49%, respectively. Conclusions:ΔEmax4 and HER2 are independent predictors of pathological responses after neoadjuvant chemotherapy for breast cancer. Combined ΔEmax4 and HER2 can improve the predictive diagnostic efficacy of pathological responses to chemotherapy for breast cancer.