Objective To explore the possibility of mutual regulation between TRiC/CCT complex(TCP-1 ring complex/chaperonin containing TCP-1)and mTOR complex 1(mechanistic target of rapamycin complex 1).Methods co-immunoprecipi-tation and Western blot were adopted to explore the interaction between TRiC/CCT complex and mTORC1.Rapamycin was used to inhibit the activity of mTORC 1.Soluble and insoluble cell protein samples were prepared,and the changes in protein ex-pression levels of TRiC/CCT complex-assisted folding were compared.The changes were observed via non-denaturing gel elec-trophoresis to explore the influences of mTORC1 on the integrity of TRiC/CCT complex.The CCT2 or CCT5 gene in HEK-293T cells was silenced by lentivirus-mediated shRNA to explore the influences of TRiC/CCT complex on the expression of key components of mTORC1 and the phosphorylation level of downstream substrates.Results Interaction between each subunit of TRiC/CCT complex and mTORC1 was proved by co-immunoprecipitation experiment.The activity of mTORC1 was inhibited by rapamycin.Expression levels of β-actin,β-Tubulin,and STAT3 insoluble proteins were not significantly increased,and the molecular size of the complex did not change significantly in the non-denaturing electrophoresis gel,indicating that mTORC1 had no regulative effect on formation of TRiC/CCT complex.CCT2 or CCT5 was silenced in HEK-293T.The protein expres-sion levels of mTOR,Raptor,and mLST8 were decreased,and the phosphorylation levels of p70S6K and Akt were decreased,re-vealing that the TRiC/CCT complex had a regulative effect on mTORC1 and downstream molecules.Conclusion The protein expression level of each component of mTORC1 was regulated by TRiC/CCT complex,thus affecting the phosphorylation level and activity status of the downstream substrates.

Objective To explore the possibility of mutual regulation between TRiC/CCT complex(TCP-1 ring complex/chaperonin containing TCP-1)and mTOR complex 1(mechanistic target of rapamycin complex 1).Methods co-immunoprecipi-tation and Western blot were adopted to explore the interaction between TRiC/CCT complex and mTORC1.Rapamycin was used to inhibit the activity of mTORC 1.Soluble and insoluble cell protein samples were prepared,and the changes in protein ex-pression levels of TRiC/CCT complex-assisted folding were compared.The changes were observed via non-denaturing gel elec-trophoresis to explore the influences of mTORC1 on the integrity of TRiC/CCT complex.The CCT2 or CCT5 gene in HEK-293T cells was silenced by lentivirus-mediated shRNA to explore the influences of TRiC/CCT complex on the expression of key components of mTORC1 and the phosphorylation level of downstream substrates.Results Interaction between each subunit of TRiC/CCT complex and mTORC1 was proved by co-immunoprecipitation experiment.The activity of mTORC1 was inhibited by rapamycin.Expression levels of β-actin,β-Tubulin,and STAT3 insoluble proteins were not significantly increased,and the molecular size of the complex did not change significantly in the non-denaturing electrophoresis gel,indicating that mTORC1 had no regulative effect on formation of TRiC/CCT complex.CCT2 or CCT5 was silenced in HEK-293T.The protein expres-sion levels of mTOR,Raptor,and mLST8 were decreased,and the phosphorylation levels of p70S6K and Akt were decreased,re-vealing that the TRiC/CCT complex had a regulative effect on mTORC1 and downstream molecules.Conclusion The protein expression level of each component of mTORC1 was regulated by TRiC/CCT complex,thus affecting the phosphorylation level and activity status of the downstream substrates.

Objective In order to explore the influence and effect of case teaching method based on real patients in oto-rhinolaryngology on clinical practice ability of general practice.Methods 96 trainees of general practice in otolaryngology de-partment from January 2018 to January 2021 were randomly divided into two groups:a CBL group and a control group.In the CBL group,CBL teaching method was adopted based on real patients in Otorhinolaryngology.Conventional teaching method was used in the control group.Results The theoretical scores of written test and the examination of clinical skill operation scores of the CBL group and the control group were analyzed.There was significant difference between the two groups(P＜0.05).The students in CBL group were more satisfied with clinical thinking ability,analysis and problem solving ability,active learning abil-ity,learning efficiency,teacher-student interaction,doctor-patient communication ability and consultation skills than the control group.There was significant difference between the two groups(P＜0.05).Conclusion The CBL teaching method can obvi-ously improve the learning enthusiasm and clinical practice ability of general resident training doctors.The teaching quality has been significantly improved.It is of great value to cultivate excellent general practitioners.

Objective:To investigate the risk factors of lymph node metastasis and the clinical significance of deep submucosal invasion in patients with T1 stage colorectal cancer.Methods:From January 30, 2010 to December 31, 2019, at Shandong Provincial Hospital Affiliated to Shandong First Medical University, among patients with T1 stage colorectal cancer, 41 patients underwent radical surgery for colorectal cancer (surgery group) and 23 patients received endoscopic submucosal dissection (ESD) (ESD group) were enrolled. The tumor gross type, maximum diameter, histologically poorly differentiated components, degree of invasion (the type of mucosal muscle destruction, the width and depth of invasion), the budding grade of tumor, and whether with vascular tumor thrombus were recorded. The additional treatment and prognosis of patients were collected by telephone follow-up. The risk factors of lymph node metastasis in stage T1 colorectal cancer, the correlation between the complete muscularis mucosa destruction and the width and depth of invasion in the ESD group, and the effects of additional treatment after operation on the prognosis of patients were analyzed. Independent sample t test and chi-square test were used for statistical analysis. Results:The rate of lymph node metastasis in patients with poorly differentiated components or vascular tumor thrombus was higher than that in patients without poorly differentiated components or vascular tumor thrombus (3/6 vs. 12.1%, 7/58; 3/4 vs. 11.7%, 7/60), and the differences were statistically significant ( χ2=5.934 and 11.409, both P<0.05). All patients in the surgery group had complete muscularis mucosa destruction. In ESD group, the width of tumor invasion was ≥ 2 mm in 16 cases, including complete destruction of muscularis mucosa in 15 cases and partial destruction in one case; the width of tumor invasion was <2 mm in seven cases, including complete destruction of muscularis mucoa in two cases and partial destruction in five cases; the depth of infiltration was ≥ 2 000 μm in 14 cases, including complete destruction of muscularis mucosa in 13 cases and partial destruction in one case; the depth of infiltration was <2 000 μm in nine cases, including complete destruction of muscularis mucosa in four cases and partial destruction in five cases. The complete muscularis mucosa destruction was related with tumor of invasion width ≥ 2 mm and invasion depth ≥ 2 000 μm (15/16 vs.2/7, 13/14 vs. 4/7), and the differences were statistically significant ( χ2=10.729, 6.659, both P<0.05). Among the 64 patients with T1 stage colorectal cancer in this study, six cases (9.4%) had poor prognosis; five cases (7.8%) died, and three of them (4.7%) were tumor-related deaths. Adjuvant therapy was added in 10 cases in surgery group and 10 cases in ESD group, and there were no poor prognosis in those patients. There were no significant difference in the incidences of poor prognosis of patients without additional treatment and patients with additional treatment of the two groups (9.7% (3/31) vs. 0 (0/10) and 23.1% (3/13) vs. 0 (0/10)) (both P>0.05). Conclusion:When T1 stage colorectal cancer with tumor submucosal invasion, clinicians should comprehensively evaluate the prognostic risk based on various pathological characteristics such as the degree of tumor differentiation, vascular tumor thrombus and mucosal muscle destruction.

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.
Codon/genetics* ; Humans ; Pichia/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales ; Vascular Endothelial Growth Factor A/genetics* ; Vascular Endothelial Growth Factors

Objective To investigate the dynamic changes in early gastric antrum contraction in patients with craniocerebral injury. Methods The patients with craniocerebral injury admitted to neurosurgery intensive care unit (ICU) of the First Affiliated Hospital of Sun Yat-sen University from July to November in 2018 were enrolled. The changes in antral contraction frequency (ACF), antral contraction amplitude (ACA) and antral motility index (MI) were dynamically observed at 1-6 days after injury by ultrasonography. According to Glasgow coma score (GCS), the patients were divided into moderate to severe craniocerebral (GCS ≤ 11) and mild craniocerebral injury groups (GCS > 11). The differences in ACF, ACA and MI between the two groups were compared to observe the effect of craniocerebral injury on gastric antral motility. The patients were divided into simple supratentorial and supratentorial combined infratentorial lesion groups according to the lesion location of craniocerebral injury. The differences in ACF, ACA and MI between the two groups were compared to analyze the influence of lesion location on gastric antrum activity. Results A total of 68 patients with craniocerebral injury were screened during the study period, 50 patients were in accorded with the admission criteria, 17 patients were withdrawn from the observation because they could not tolerate the ultrasonography of gastric antrum or discharged from ICU. Finally, 33 patients were enrolled in the analysis. ① The ACF, ACA and MI at 1 day after injury were lower [ACF (times/min): 1.67 (0.00, 2.00), ACA: 42.06 (0.00, 44.45)%, MI: 0.70 (0.00, 0.87)], and then gradually increased, till 6 days after injury, ACF was 1.83 (1.25, 2.79) times/min, ACA was 56.80 (33.25, 60.77)%, and MI was 0.89 (0.50, 1.70), which showed no differences among all time points (all P > 0.05). ② The contractile function of gastric antrum in two groups of patients with different degrees of craniocerebral injury was decreased, especially ACA in patients with moderate to severe craniocerebral injury (n = 22), which showed significant differences at 3 days and 5 days after injury as compared with mild craniocerebral injury [n = 11; 3 days: 35.05 (0.00, 53.69)% vs. 58.51 (49.90, 65.45)%, 5 days: 39.88 (0.00, 77.01)% vs. 56.94 (41.71, 66.66)%, both P < 0.05], indicating that the degree of craniocerebral injury affected the contractive function of gastric antrum. However, there was no significant difference in ACF or MI between the two groups at different time points after injury. ③ The contractile function of gastric antrum was decreased after craniocerebral injury in both groups of patients with different lesion locations of craniocerebral injury. The ACF, ACA, and MI at 3-4 days in patients with supratentorial combined infratentorial lesion (n = 12) were slightly lower than those in patients with simple supratentorial lesion [n = 21; 3 days: ACF (times/min) was 0.83 (0.00, 2.00) vs. 2.25 (0.00, 3.00), ACA was 35.05 (0.00, 53.60)% vs. 49.93 (0.00, 63.44)%, MI was 0.29 (0.00, 1.07) vs. 1.23 (0.00, 1.61); 4 days: ACF (times/min) was 1.42 (0.50, 2.63) vs. 2.00 (1.63, 2.63), ACA was 30.45 (21.69, 60.61)% vs. 43.29 (38.41, 53.35)%, MI was 0.50 (0.15, 1.45) vs. 0.97 (0.66, 1.28)] without statistical differences (all P > 0.05), indicating that the lesion location might not affect the contractive function of gastric antrum. Conclusion In the early stage of craniocerebral injury, the contractile function of gastric antrum was decreased, and the more severe the craniocerebral injury, the worse contractive function of gastric antrum.[Key words] Craniocerebral injury; Antral contraction; Enteral nutrition; Antral ultrasonography

Objective To investigate the complications of therapeutic endoscopic retrograde cholangiopancreatography(ERCP) in patients with metabolic syndrome and their prevention. Methods Clinical data of 300 patients who underwent therapeutic ERCP in the First Affiliated Hospital of Soochow University from March 2013 to January 2016 were analyzed retrospectively. Among these patients, 100 of them(group A)had metabolic syndrome and 200 others(group B)did not. Age, sex and post-operative hyperamylasemia,acute pancreatitis,infection of biliary tract and upper gastrointestinal bleeding of two groups were compared. In addition,effects of BMI,TG,BP and FPG on the occurrence of complications in group A were analyzed. Results No difference was found in age and sex between group A and B(P>0.05),but group A had higher incidences of post-operative hyperamylasemia[16.0%(16/100)VS 6.0% (12/200)],acute pancreatitis[14.0%(14/100)VS 4.0%(8/200)], infection of biliary tract[12.0% (12/100)VS 4.0%(8/200)]and upper gastrointestinal bleeding[8.0%(8/100)VS 1.0%(2/200)] (P<0.05). Meanwhile, patients had higher incidences of complications(post-operative hyperamylasemia, acute pancreatitis,infection of biliary tract and upper gastrointestinal bleeding)when BMI[56.10%(46/82) VS 22.22%(4/18)],TG[56.41%(44/78)VS 27.27%(6/22)],BP[64.29%(27/42)VS 39.66% (23/58)]or FPG[54.44%(49/90)VS 10.00%(1/10)]increased in group A(P < 0.05).Conclusion Patients with metabolic syndrome have a higher incidence of complications after therapeutic ERCP than patients without. Therefore,careful preparation, post-operative observation and intervention are essential when patients with metabolic syndrome undergo therapeutic ERCP.

Objective To investigate the clinical features of initially treated pulmonary tuberculosis(TB)patients with rifampin dependent mycobacterium tuberculosis Rv0341 antibody positive.Methods Forty two initially treated pulmonary TB patients with rifampin dependent mycobacterium tuberculosis Rv0341 antibody positive were included as the study group and 42 initially treated pulmonary TB patients with rifampin dependent mycobacterium tuberculosis Rv0341 antibody as the control group The aspects of clinical symptoms,X-ray imaging manifestations,positive rate in sputum culture of Mycobacterium tuberculosis and rifampin dependent mycobacterium tuberculosis,treatment effects,etc.were compared between the two groups.Results The incidence rates of expectoration,hemoptysis and shortness of breath in the study group were significantly higher than those in the control group(P＜0.05);the X-ray image manifestations in the study group showed that the lesions were much more involved in two lungs and multiple lung fields(P＜0.05),while thlesions in the control group more confined to single lung and one lung field(P＜ 0.05),moreover the rate appearing pulmonary cavity in the study group was higher than that in the control group(P＜0.05);the sputum culture positive rate of Mycobacterium tuberculosis had no statistical difference between the two groups(P＞0.05),but the sputum culture positive rate of rifampin dependent mycobacterium tuberculosis in the study group was significantly higher than that in the control group(P＜0.05);as for the treatment effects,the cure rate in the control group was higher than that in the study group(P＜0.05),the ineffective ratio in the control group was less than that in the study group.Conclusion Initially treated pulmonary TB patients with rifampin dependent mycobacterium tuberculosis Rv0341 antibody positive have the characteristics of severe clinical symptoms,wide range lesions,easily appearing cavity,high culture positive rate of rifampin dependent mycobacterium tuberculosis and poor treatment effect.

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering

The expression cDNA library of A. tabescens was constructed by SMART technique, which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0 × 106pfu/mland 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1 × 108pfu/mland 4.2 × 1010. The library was used to provide expressed sequence tags (ESTs). 147 Expressed SequenceTaqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. Thesequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them werefound that they have significant similarity with data in GenBank. EST AJ620046 was has significantsimilarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA ofAJ620046 was successfully obtained. In order to initially characterize the biochemical properties ofAJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast.Recombinant pHIL-S1-AF constructed by inserting AF into pHIL-S1 was transformed into Pichia PastorisGS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinantyeast.