AIM:To observed the protective effect of diminazene aceturate ( DIZE) , an angiotensin-converting enzyme 2 (ACE2) activator, on diabetic nephropathy (DN) rats.METHODS:Male Wistar rats (n=30) were randomly divided into normal control (NC) group, DN group and DIZE group (each group consisted of 10 rats).The rats in DN group and DIZE group were induced by intraperitoneal injection of streptozotocin at dose of 65 mg/kg.After 12 weeks, the rats in DIZE group and DN group received subcutaneous injection of DIZE (15 mg· kg -1 · d-1 ) or vehicle for 4 weeks. The samples of blood and urine were collected at week 16, and ratio of kidney weight to body weight (KW/BW), plasma glucose (GLU), 24 h urinary protein (24UP) and serum creatinine (SCr) were measured.The renal pathological changes in each group were observed by periodic acid-Schiff ( PAS) staining and immunohistochemistry .The levels of AngⅡ and Ang-(1-7) in the plasma, and TGF-β1 and VCAM-1 in the renal tissues were measured by ELISA .The mRNA and protein levels of collagen I and FN were determined by quantification real-time PCR and immunohistochemistry .The effects of DIZE on the expression of ACE2 in DN rats were determined by Western blot .RESULTS:DIZE remarkably increased the expression of ACE2 and Ang-(1-7) in DN rats.Compared with NC group , the GLU, KW/BW, 24UP, SCr, and the ex-pression of collagen I , FN, TGF-β1 and VCAM-1 in DN group and DIZE group were increased .However , after treatment of the DN rats with DIZE, these indicators were decreased except the KW/BW.The GLU showed no significant change . CONCLUSION:DIZE raised the activity of ACE2 and increased the expression of Ang-(1-7), thus alleviating fibrosis and inflammation in the kidney and having therapeutic potential for diabetic nephropathy .

OBJECTIVE:To investigate the effect of Wuzhi soft capsule on the pharmacokinetics of sirolimus in rats. METH-ODS:Wistar rats were randomly divided into 5 groups,with 6 rats in each group. They were given very low-dose,low-dose,me-dium-dose and high-dose of Wuzhi soft capsule(67,134,268 and 536 mg/kg)ig or blank solvent,respectively;and then given si-rolimus 0.4 mg/kg. The blood 100 μl was sampled by capillary eyes before giving sirolimus(0 h)and 0.5,0.75,1,1.5,2,2.5, 3,4,6,8,12,24 and 36 h after giving sirolimus and put into edetic acid anticoagulation tube. Blood concentration of sirolimus was assayed by LC-MS/MS. The pharmacokinetic parameter was calculated by DAS 2.0 software using non-compartment model. RESULTS:The pharmacokinetic parameters of sirolimus in very low-dose,low-dose,medium-dose and high-dose groups and blank solvent group were cmax of(6.79±1.15),(17.40±3.13),(21.24±3.14),(22.06±4.82),(2.80±0.72)ng/ml;t1/2 of(11.01± 0.82),(12.20±2.02),(12.28±2.38),(12.36±0.73),(10.59±0.69)h;AUC0-∞ of(85.79±15.26),(162.18±41.75),(273.12± 73.69),(268.79±28.46),(36.72±3.01)ng·h/ml,respectively. Compared with blank solvent group,cmax and AUC0-∞ of sirolimus were all increased in very low-dose,low-dose,medium-dose and high-dose groups,but t1/2 changed slightly. CONCLUSIONS:Wu-zhi soft capsule can substantially enhance the absorption of sirolimus in rats.

BACKGROUND: That the nerve growth factor(NGF) is capable of treating peripheral nerve injury has been broadly acknowledged. But it is not sure whether it is able to pass through blood brain barrier(BBB) to act on central nerve system. In this study, the NGF encapsulated in liposome was compared with NGF alone in their abilities of passing through BBB.OBJECTIVE: To compare the dedicated distribution of NGF in different forms using single photon emission computerized tomography(SPECT).DESIGN: It is a randomized controlled study with New Zealand rabbits as subject.SETTING: An affiliated hospital of Nanjing Medical College.MATERIALS:The trial was conducted in Nanjing Senke Medical Company and the Nuclear Medicine Department of the First Hospital of Nanjing Medical College from June 2003 to May 2004. The subjects were 19 New Zealand rabbits of either sex, weighting (2.0 ±0. 2) kg, from Anlimo Technology Company. [99Tcm] -NGF(labeling rate 98.9%, purity 99.7% )was made by Senke Medical Company. Liposome was provided by Shengyang Pharmaceutical College. Urethine solution(200 g/L) was from Pharmacy Department of NanJing Medical Collage.METHODS: [99Tcm]-NGF was encapsulated in liposome and was treated as the following: The liposomesA containing 1.48 × 108Bq[99Tcm] -NGF were injected into rabbits and its distribution percentage was analyzed with SPECT. The same amounts of[99Tcm] -NGF and[99Tcm] -NGF-ordinary liposomesB were treated in the same way.MAIN OUTCOME MEASURES: The ratio of concentration and radiation percentage of NGF in brain to those in the whole body.RESULTS: The[99Tcm]-NGF encapsulated in self-made liposomeA presented high radiation in the brain. But[99Tcm] -NGF alone was almost completely excreted through urinary system and[99Tcm] -NGF encapsuled in ordinary liposomeB was mostly phagocytized by liver reticuloendothelial system.CONCLUSION: The self-made NGF-liposomeA is brain-dedicated, which set a basis for drugs to pass through BBB.

To identify SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of the recombinant protein as a new vaccine candidate for schistosomiasis, polymerase chain reaction (PCR) technique was used to amplify the cDNA of the gene and real-time RT-PCR was used to analyze the transcription profiles of SJCHGC01743 at different development stages. Recombinant plasmid was successfully constructed and transformed into competent Escherichia coli BL21 (DE3). Then the recombinant protein was expressed, purified and emulsified with ISA206 adjuvant to immunize BALB/c mice for three times. The immunogenicity was confirmed by Western blotting and tissue localization was detected by indirect immunofluorescent assay. The specific antibody level was detected by ELISA. The immunoprotection of rSjOST48 was evaluated by the reduction in worm and egg counts in mice. A cDNA with 1 248 nucleotides was isolated from 28-day-old schistosomes cDNAs by PCR. Sequence analysis revealed that SJCHGC01743 was a 48-kDa subunit of the oligosaccharyltransferase complex (OST48) and named as SjOST48. Real-time PCR analysis indicated that this gene was expressed in all investigated stages and had the highest expression level in 28 d worms, the level of gene transcription in female worms was significantly higher than that of male worms. Then recombinant plasmid pET28a(+)-SjOST48 was successfully constructed and expressed in E. coli BL21 (DE3). Western blotting analysis showed that rSjOST48 had good immunogenicity. Indirect immunofluorescent analysis revealed that SjOST48 was mainly distributed on the tegument of the worms. The result of ELISA indicated that the rSjOST48 vaccinated group could induce a significant increase in the level of specific IgG, IgG1 and IgG2a. An immunoprotection experiment showed that the vaccination of rSjOST48 in mice induced 32.62% (P < 0.05) reduction in the numbers of worms and 57.61% (P < 0.01) in eggs in liver, compared with that of the control group. This study provides the foundation for proceeding further research on the biological function of SjOST48 and screening new vaccine candidates for schistosomiasis.
Animals ; Antibodies, Helminth ; blood ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Female ; Genes, Helminth ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; Schistosoma japonicum ; genetics ; Schistosomiasis japonica ; prevention & control ; Vaccination

To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Humans ; Leptin ; chemistry ; genetics ; metabolism ; Molecular Weight ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism

High speed data transmission rotating connector system for signal high-speed transmission used in the fixed end and rotating end, it is one of the core component in the CT system. This paper involves structure design and analysis of the retaining ring in the CT high speed data transmission rotating connector system based on the principle of off-axis free space optical transmission. According to the problem of the actual engineering application of space limitations, optical fiber fixed and collimator installation location, we designed the structure of the retaining ring. Using the static analysis function of ANSYS Workbench, it verifies rationality and safety of the strength of retaining ring structure. And based on modal analysis function of ANSYS Workbench, it evaluates the effect of the retaining ring on the stability of the system date transmission, and provides theoretical basis for the feasibility of the structure in practical application.

ObjectiveTo obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. MethodsThe COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsSequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of ＞1×108. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P＜0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P＜0.05), suggesting that COX-2-sgRNA was active. ConclusionA CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2-/- cells with stable COX-2 gene knockout are obtained.

Antibiotic-associated diarrhea(AAD) is a frequent adverse effect of antibiotic in children.AAD is associated with longer hospitalization, higher healthcare cost and even lead to death.Pediatricians usually do not pay enough attention to AAD.Domestic experts from pulmonary medicine, infection and gastroenterology are organized to develop the consensus, to improve the diagnosis, treatment and prevention of AAD, and contribute the children health in future.

Objective: To analyze the epidemiological characteristics of coronavirus disease 2019 ( COVID-19 ) clusters in Lishui, so as to provide basis for the prevention and control of COVID-19 clusters. Methods: The data of COVID-19 clusters in Lishui from January 23 to March 29, 2020 were collected through China Disease Control and Prevention Information System-Public Health Emergency Information System, and analyzed time, space, scale, source of infection, exposure and transmission route by descriptive epidemiological method. Results: There were 31 cases in 8 clusters ( about 4 cases per cluster ), with no death. The report time was bimodal, peaked first from January 20 to February 10 with 4 clusters imported from domestic and peaked second from March 1 to 29 with 4 clusters imported from overseas. Qingtian County reported 4 clusters, Liandu District, Yunhe County, Qingyuan County and Jingning County each reported 1 cluster. Thirteen cases were restaurant employees, accounting for 41.94%. The cases were mainly occurred in the condition that exposed in the same family ( 6 clusters ), in the same dinner and car ( 1 clusters ), and in the same party ( 1 clusters ). The exposure modes that caused more cases infected were through the same family (9 cases) and through the same dinner and car ( 6 cases ). There were 3 clusters with first-generation cases, 3 clusters with second-generation cases and 2 clusters with third-generation cases. The recurrence rate of the 8 clusters ranged from 1.49% to 7.69%, with a median of 3.47%. Conclusions The COVID-19 clusters in Lishui imported from domestic in the early stage and later from overseas. Most cases were reported from Qingtian County, were engaged in catering business, and exposed by living with families.

Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a(+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control
Animals ; Antibodies, Helminth ; blood ; Blotting, Western ; Cloning, Molecular ; DNA Repair Enzymes ; genetics ; metabolism ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Genetic Vectors ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Schistosoma japonicum ; genetics ; metabolism ; Schistosomiasis japonica ; prevention & control ; Vaccines ; immunology