AIM: To observe the effect of LPS on CIAS1 gene expression and to analyze the function-structure relationship of its mutation region, the NACHT domain of cryopyrin. METHODS: RT-PCR was used to detect the expression of CIAS1 gene in human leucocytes in the presence of LPS. The methods of molecular modeling and bioinformatics were used to observe the relationship of the structure-function of NACHT domain in human CIAS1 gene. RESULTS: LPS increased CIAS1 mRNA expression in a dose-dependent manner. However, the stimulation with LPS at a concentration of 100 mg/L at different time points showed the pattern of early down-regulation and later up-regulation of CIAS1 mRNA expression. The sequence analysis suggested that the motifs Walker A and Walker B, to which the ATP and Mg~ 2+ can bind, were found in the NACHT domaim of CIAS1 gene encoded product cryopyrin. The main points of the disease-associated mutation showed the close relation in sequence and structure to these motifs. The binding sites of Ca~ 2+ and polysaccharide were observed in the leucine rich repeats region of cryopyrin. CONCLUSION: Cryopyrin may act as an important regulator in inflammation. LPS induces human leukocytes to express the CIAS1 gene product.

OBJECTIVE:To offer the literature basis for clinical safe drug use by literature research about ADR induced by Yishen juanbi pill. METHODS:Using“Yishen juanbi pill”as searching word,related literatures about ADR induced by Yishen juanbi pill were collected from CNKI,and then the occurrence of ADR was summarized and analyzed. RESULTS:A total of 15 literatures were included,involving 58 patients. Primary disease were mainly rheumatoid arthritis (28 cases,48.28%);organs/systems involved in ADR were digestive system (77 cases, 76.24%). Main clinical manifestations were epigastric discomfort, pernicious vomiting,diarrhea,etc. No obvious ADR was found. ADR-inducing dose was mainly 8 g,tid(47 cases,92.16%);ADR-inducing drug combination were two-drug combination (33 cases,56.90%). Fifty-eighe cases of ADR were recovered after treatment,and main treatment was drug withdrawal or symptomatic treatment. CONCLUSIONS:Although Yishen juanbi pill may induce ADR,those ADR can disappear spontaneously after drug withdrawal or the symptoms are recovered after symptomatic treatment. Yishen juanbi pill is a relatively safe Chinese patent medicine of anti-inflammatory,but ADR monitoring should be strengthened during application.

Objective To evaluate the effect of pediatric renal transplantation using donation after cardiac death (DCD).Methods The male DCD meeting Chinese standard Ⅲ (C-Ⅲ) was 49 years old,and the recipient with chronic renal failure was 14 years old.The right kidney of the donor was transplanted to the recipient.The renal artery and renal vein of the donor were end-to-side anastomoscd to the common iliac artery and common iliac vein of the recipient,respectively.The graft was transplanted into the fight iliac fosse.Warm ischemia time was 12 min,and cold ischemia time was 2 h.Immunity induction therapy was performed with basiliximab.Tacrolimus + mycophnolate mofetil + Pred were used as immunosuppressive regimen.Results The transplantation was done successfully.One day after operation,ALT was increased dramatically.The recipient was diagnosed as acute drug-induced liver injury.There was no occurrence of acute rejection and delayed graft function.The recipient was discharged one month after the operation,and followed up for 6 months with normal graft function.Conclusion Pediatric renal transplantation using DCD is effective and safe,even though the long-term effect still needs to be further observed.

Objective To analyze and evaluate the characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase(ESBL) with Pro167 residue substitution. Methods By molecular cloning and PCR techniques, CTX-M-14 gene was directionally cloned into plasmid pET28a( + ) from a clinical E. coli isolate and formed an expression vector to transform competent E. coli BL21 (DE3 ). Prol67 residue substitutions of P167G, P167Q, P167S and P167T were introduced to CTX-M-14 by site-directed muta-genesis based on overlap extension PCR with the former recombinant plasmid as PCR template, respectively.The wild-type CTX-M-14, recombinant CTX-M-14 protein and its variants were expressed and purified, then their steady-state kinetic parameters (Kcat, Km and Kcat/Km ) against β-lactam antibiotics were determined.Results The kinetic parameters of wild-type and recombinant CTX-M-14 had no statistically significant differences (P>0.1). The 1/Km, Kcat and Kcat/Km values of P167S variant against ceftazidime were 16-fold, 2.87-fold and 43.6-fold higher than those of recombinant CTX-M-14, respectively, and the Kcat/Km value of P167S variant against penicillin, ampicillin, cefazolin, cefuroxime, ceftriaxone, cefotaxime decreased( < 0.05). Compared with the kinetic parameters of recombinant CTX-M-14, the kinetic parameters of P167T variant against ceftazidime had no significant change, but the Kcat values of P167Q and P167G variants decreased dramatically(P<0.01). Conclusion There was no difference between the enzyme activities of wild-type and recombinant CTX-M-14. P167S variant could not only promote the enzyme affinity of CTX-M-14 to ceftazidime but also improve the conversion rate of enzyme-substrate complex in the ceftazidime hydrolysis. The comparison of the kinetic parameters of CTX-M-14 and its variants with Pro167 residue substitution showed that the increased activity of CTX-M ESBL variants against ceftazidime could not be simply explained with the enlarged cavity in active site that may be caused by the replacement of Pro167 residue by smaller amino acids.

Jingui Yaolue is a part ofTreatise on Cold-Attack and Miscellaneous Diseases. Because of the archaic words, students lost the interest on it. So it became the questions that how to make students interested in learning theJingui Yaolue and to make traditional Chinese medicine(TCM) classical guide clinical treatment. Thus, a try has been made to teach the TCM classic in the clinical practice apprentice setting rather than the classrooms. Here, some experiences about apprentice teaching in clinical practice of Jingui Yaolue were shared.

Objective:To investigate the effect of CIRc_0000267 on proliferation, migration, invasion and apoptosis of gastric cancer cells in vitro.Methods:Gastric cancer cell lines with circ_0000267 knockdown and miR-661 overexpression were constructed in vitro, and the expressions of circ_0000267, miR-661 and NGAL mRNA in gastric cancer tissues and cells were detected by qRT-PCR. Cell proliferation, migration, invasion and apoptosis were detected by clonogenesis, Transwell chamber and flow cytometry, and related protein expression was detected by Western blot. Online prediction combined with double luciferase assay and RNA pull down assay was used to verify the targeting relationship between circ_0000267, miR-661 and NGAL. Tumogenesis assay in nude mice was used to observe the effect of circ_0000267 knockout on the growth of gastric cancer cells in vivo. Results:Circ_0000267 was highly expressed in gastric cancer tissues and cells. Knockdown circ_0000267 inhibited the proliferation, migration and invasion of gastric cancer cells and promote apoptosis. Circ_0000267 targeted miR-661, and inhibition of miR-661 partially reversed the effect of circ_0000267 on gastric cancer cells. NGAL was the target gene of miR-661, and overexpression of miR-661 regulated the proliferation, migration, invasion and apoptosis of gastric cancer cells by inhibiting NGAL. Knockdown circ_0000267 targeting miR-661 inhibited NGAL expression in gastric cancer cells; Knockdown circ_0000267 inhibited the growth of gastric cancer cells in vivo.Conclusions:Circ_0000267 may regulate the proliferation, migration, invasion and apoptosis of gastric cancer cells and inhibit the growth of tumors in vivo by regulating the expression of miR-661/NGAL.

Objective To evaluate the left ventricular systolic function of patients with type 2 diabetes combined with microangiopathy using two dimensional speckle tracking imaging. Methods A total of 29 simple diabetes patients from January 2016 to December in Zhejiang Chinese Medicine University Affiliated Jiaxing Hospital of Traditional Chinese Medicine (DM group), 35 diabetes patients with microvascular disease (diabetic microangiopathy group), and 35 healthy volunteers (healthy control group) were enrolled in present study. The conventional echocardiography was used to evaluated the left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVEDS), end diastolic ventricular septal thickness (IVSDD), left ventricular posterior wall end diastolic thickness (LVPWTD), left ventricular ejection fraction (LVEF), and left atrial volume index (LAVI). Using pulsed Doppler technique, the mitral early diastolic and late diastolic blood flow velocity ratio (E/A), E peak deceleration time (DCT), the organization of the mitral E wave velocity and the Doppler organization Doppler e' ratio (E/e) were measured. Automatic functional imaging was used to calculate the left ventricular apical axis, the four cavities of the apex, the two cavities of the apex and the overall mean longitudinal strain of the left ventricle. The clinical data, routine echocardiographic parameters and strain parameters among 3 groups were compared by one-way ANOVA. The Tukey HSD method was used to compare the differences between two groups. Pearson correlation analysis was used to analyze the correlation between the total mean longitudinal strain of left ventricle and the concentration of glycosylated hemoglobin, triglyceride concentration and systolic pressure. Results Among the 3 groups, the subject age, heart rate, body mass index had no significant difference. In diabetic microangiopathy group, the glycosylated hemoglobin concentration, triglyceride, systolic blood pressure were higher than that in healthy control and diabetic groups, and the differences were statistically significant (t=16.135, 8.947; t=8.777, 10.947; t=13.806, 8.278, all P＜0.05). The 3 groups had no significant differences in LVEDD, LVEDS, IVSDD, LVPWTD, DCT and LVEF. Compared with healthy controls, patients with diabetes mellitus showed decreased E/A and increased E/e; while patients with diabetic microangiopathy had decreased E/A and increased LAVI and E/e (t=13.786, 13.565; t=9.571, 11.267, 8.351, all P＜0.05). In the left ventricular apical long axis view, four chamber view, two chamber view and left ventricular overall average longitudinal strain values, increasing trend was found from healthy control group, to DM group, and to diabetic microangiopathy group, and the differences between any 2 groups were statistically significant (t=5.491, 10.907, 6.076; t=4.276, 7.011, 3.250; t=10.445, 11.633, 3.683; t=10.746, 18.731, 9.532; all P＜0.05). Pearson correlation analysis showed that the mean longitudinal strain value of left ventricle was negatively correlated with glycosylated hemoglobin concentration (r=-0.746, P=0.000), and there was no correlation with the triglyceride concentration and systolic blood pressure (r=0.079, P=0.438; r=0.067, P=0.416). Conclusion The two-dimensional speckle tracking imaging technique can effectively evaluate the left ventricular systolic function in patients with type 2 diabetic microangiopathy.

Objective:To investigate the effects of serum exosomes of patients with gastric cancer on cisplatin resistance, clonal formation, migration, invasion and apoptosis of the AGS gastric cancer cells, and the corresponding molecular mechanisms.Methods:The experimental study was conducted. The exosomes of patients with gastric cancer was separated from their serum, and the expression of lncRNA HOTTIP was analyzed using the quantitative real time polymerase chain reaction (qRT-PCR). Normal gastric epithelial cell line GES1, gastric cancer cell line AGS and human embryonic kidney cell 293T were cultured in vitro. AGS cells were incubated with exosomes (Exo),with phos-phate buffered saline (PBS) treatment as control, and transfected with si-NC or si-HOTTIP-3, named as Exo group, PBS group, si-NC+Exo group, and si-HOTTIP-3+Exo group. The AGS cells were trans-fected with si-NC, si-HOTTIP-1, si-HOTTIP-2, si-HOTTIP-3, oe-HOTTIP, vector, oe-HOTTIP+miR-138-5p mimic, oe-HOTTIP+mimic NC, miR-138-5p inhibitor, inhibitor NC, miR-138-5p inhibitor+si-TJP1 and miR-138-5p inhibitor+si-NC. They were recorded as si-NC group, si-HOTTIP-1 group, si-HOTTIP-2 group, si-HOTTIP-3 group, oe-HOTTIP group, vector group, oe-HOTTIP+miR-138-5p mimic group, oe-HOTTIP+mimic NC group, miR-138-5p inhibitor group, inhibitor NC group, miR-138-5p inhibitor+si-TJP1 group and miR-138-5p inhibitor+si-NC group. The 293T cells transfected with mimic NC+HOTTIP wt, miR-138-5p mimic+HOTTIP wt, mimic NC+HOTTIP mut, miR-138-5p mimic+HOTTIP mut, mimic NC+TJP1 3'UTR wt, miR-138-5p mimic+TJP1 3'UTR wt, mimic NC+TJP1 3'UTR mut, miR-138-5p mimic+TJP1 3'UTR mut were recorded as the mimic NC+HOTTIP wt group, miR-138-5p mimic+HOTTIP wt group, mimic NC+HOTTIP mut group, miR-138-5p mimic+HOTTIP mut group, mimic NC+TJP1 3'UTR wt group, miR-138-5p mimic+TJP1 3'UTR wt group, mimic NC+TJP1 3'UTR mut group, miR-138-5p mimic+TJP1 3'UTR mut group. The cell counting kit-8 (CCK8) was used to analyze the cisplatin sensitivity of gastric cancer cells. The colony formation experiment was used to analyze the colony formation of gastric cancer cells. The Transwell experiment was used to analyzed cell migration and invasion of gastric cancer cells. The flow cytometry experiment was used to analyze cell apoptosis of gastric cancer cells. The Western bolt assay was used to analyze the expression of exosome marker proteins, including the CD63 and CD81, and the protein of TJP1, the drug-resistance related proteins, including the P-gp and MCL-1. The dual-luciferase assay was used to analyze the targeted relationships among lncRNA HOTTIP, miR-138-5p and TJP1. Observation indicators: (1) expression of lncRNA HOTTIP; (2) resistance of gastric cancer cells to cisplatin regulated by exosome-mediated lncRNA HOTTIP; (3) regulation of cisplatin resistance in gastric cancer cells mediated by miR-138-5p through lncRNA HOTTIP overexpression; (4) targeting of TJP1 gene 3′-untranslated region (UTR) by miR-138-5p; (5) regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR-138-5p inhibition. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. The one-way ANOVA was used for comparison for multiple groups and the Tukey′s test was used for further pairwise compari-son. Count data were described as absolute numbers, and the chi-square test was used for comparison. Correlation analysis was conducted using the Pearson′s test. Results:(1) Expression of lncRNA HOTTIP. The expression of lncRNA HOTTIP in the serum exosome of patients with gastric cancer was higher than that in healthy volunteers, showing a significant difference ( P<0.05). Results of transmi-ssion electron microscopy examination showed that the serum exosomes were circular or oval in shape. Results of Western bolt assay showed the expression of marker proteins of CD63 and CD81 in serum exosomes. (2) Resistance of gastric cancer cells to cisplatin regulated by exosome-mediated lncRNA HOTTIP. Compared with the PBS group, the biochemical half maximal inhibitory concentra-tion (IC50), the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the Exo group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the si-NC+Exo group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the si-HOTTIP-3+Exo group decreased, while the cell apoptosis rate increased, showing significant differences between them ( P<0.05). (3) Regulation of cisplatin resistance in gastric cancer cells mediated by miR-138-5p through lncRNA HOTTIP overexpression. Compared with the vector group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the oe-HOTTIP group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the oe-HOTTIP+mimic NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the oe-HOTTIP+miR-138-5p mimic group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). (4) Targeting of TJP1 gene 3′-UTR by miR-138-5p. Results of dual-luciferase assay showed that the luciferase activity in 293T cells treatment with mimics of control+vectors of wild type of TJP1 gene 3′-UTR and 293T cells treatment with mimics of miR-138-5p+vectors of wild type of TJP1 gene 3′-UTR was 1.00±0.09 and 0.21±0.03, respectively, showing a significant difference between them ( t=15.02, P<0.05). (5) Regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR-138-5p inhibition. Compared with the inhibitor group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the miR-138-5p inhibitor group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the miR-138-5p inhibitor+si-NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the miR-138-5p inhibitor+si-TJP1 group decreased, while the cell apoptosis rate increased, showing significant differences between them ( P<0.05). Conclusion:Serum exosomes-mediated lncRNA HOTTIP can promote cisplatin resistance, clonal formation, migration, invasion and apoptosis of gastric cancer cells and inhibit cell apoptosis of gastric cancer cells through regulating the expression of miR-138-5p/TJP1.

Objective:To investigate the changes of podocyte circadian rhythm in the high-glucose environment and diabetic nephropathy (DN) mouse model and the protective effect of melatonin on podocyte injury in DN.Methods:Primary podocytes were cultured in vitro and divided into 3 groups: control group, high glucose (30 mmol/L) group and high glucose (30 mmol/L) treated with melatonin (0.1 mmol/L or 0.5 mmol/L) group. The podocytes were collected every 4 hours for 24 hours, synchronized with dexamethasone (100 nmol/L) for 2 hours, which was recorded as zeitgeber time 0 point. Male C57BL/6J mice aged 6-8 weeks and weighing about 20 g were randomly (randomized block) assigned to three groups: control group, DN (high-fat diet+streptozotocin 120 mg/kg intravenously injected) group, and DN (high-fat diet+streptozotocin 120 mg/kg intravenously) treated with melatonin (20 mg/kg intragastric treatment) group (melatonin group). Real-time quantitative PCR was used to detect the mRNA levels of rhythm genes in podocytes. Western blotting was used to detect the protein expression levels of circadian clock genes (Clock and Bmal1), podocyte marker proteins (Nephrin, Synaptopodin, WT1, and Desmin) and autophagy-related proteins (Beclin1, LC3Ⅱ/Ⅰ and P62). Immunofluorescence staining was used to detect the protein expression level of WT1, and immunohistochemistry was used to analyze the protein expression levels of P62 and cleaved-caspase-3 in renal tissues of mice. The pathological changes of renal glomerulus were observed under electron microscope. Results:(1) Dexamethasone reseted the expression and rhythmic oscillation of circadian clock genes in podocytes. The circadian rhythmic oscillations of Clock and Ck1e mRNA in the high glucose group were flattened compared to the control group, and the circadian rhythmic oscillations in Clock and Ck1e mRNA expression were partial recovery in high glucose treated with melatonin group (all P<0.05). (2) Compared with the control group, the protein expression levels of Nephrin, Synaptopodin and WT1 were lower while Desmin was higher in the high glucose group (all P<0.05). The protein expression levels of Nephrin, Synaptopodin and WT1 were higher and the protein expression level of Desmin was lower in the high glucose treated with melatonin (0.5 mmol/L) group compared with the high glucose group (all P<0.05). (3) The invivo experimental results showed that compared with the DN group, melatonin group had higher protein expression levels of glomerular Nephrin and WT1, and lower urinary albumin/creatinine ratio, width of foot process and thickness of glomerular basement membrane (all P<0.05). The protein expression levels of Beclin1 and LC3Ⅱ/Ⅰ in the DN group were lower than those in the control group, and the protein expression level of P62 was higher than that in the control group (all P<0.05). Compared with the DN group, the protein expression levels of Beclin1 and LC3Ⅱ/Ⅰ in the melatonin group were significantly higher, and the protein expression level of P62 was lower (all P<0.05). Conclusions:Melatonin can partially restore the circadian rhythm of clock genes in high-glucose environment, improve autophagy and alleviate injury in podocytes.

Objective To establish a nomogram for predicting the risk of post-hepatectomy complications (PHC) in hepatic echinococcosis by analyzing the risk factors for PHC in two types of hepatic echinococcosis, and to investigate its value in clinical practice. Methods A retrospective analysis was performed for the clinical data of 263 patients with two types of hepatic echinococcosis who underwent hepatectomy in Qinghai University Affiliated Hospital from January 2015 to August 2020, and among these patients, 93 were enrolled as PHC group and 170 were enrolled as control group. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups, and the independent samples t -test was used for comparison of normally distributed continuous data between two groups; the chi-square test and the Fisher's exact test were used for comparison of categorical data between two groups. Univariate and multivariate logistic regression analyses were used to screen out independent risk factors for PHC, and a nomogram risk prediction model was established based on the weight of each independent risk factor. The Bootstrap resampling method was used for internal verification of the model; the receiver operating characteristic (ROC) curve was plotted to evaluate the discriminatory ability of the model; calibration curve and the Hosmer-Lemeshow test were used to evaluate the consistency of the model; decision curve analysis (DCA) was performed to verify the clinical effectiveness of the model. Results Albumin-bilirubin (ALBI) score (odds ratio [ OR ]=3.694, 95% confidence interval [ CI ]: 1.860-7.336, P < 0.05), time of operation ( OR =2.848, 95%CI: 1.384-5.859, P < 0.05), intraoperative blood loss ( OR =4.832, 95%CI: 2.384-9.793, P < 0.05), and hydatid diameter ( OR =3.073, 95%CI: 1.528-6.177, P < 0.05) were independent risk factors for PHC in two types of hepatic echinococcosis. A nomogram risk prediction model was established based on the weight of the above four independent risk factors, and the model had an area under the ROC curve of 0.877 (95% CI : 0.831-0.923). The model had a consistency index of 0.871 after internal verification using the Bootstrap resampling method, suggesting that the model had good discriminatory ability. The fitting of the observed value and the actual value of the calibration curve and the Hosmer-Lemeshow test ( P =0.905) showed that the predicted value of the nomogram risk prediction model had good consistency with the actual observed value. When the threshold probability was 35.6%, DCA showed a net clinical benefit of 22%, and the model had good clinical applicability within the threshold probability ranging from 8% to 89%. Conclusion ALBI score, time of operation, intraoperative blood loss, and hydatid diameter are independent risk factors for PHC in patients with two types of hepatic echinococcosis, and the nomogram risk prediction model established based on these factors has good accuracy, consistency, and clinical practicability.