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Objective@#To analyze the drug resistance of clinical isolates of Candida tropicalis in patients with infectious diseases, and preliminarily study their molecular characteristics.@*Methods@#95 strains of Candida tropicalis were isolated from the fungal culture specimens of 87 patients with infectious diseases in Shanghai Public Health Clinical Center from 2012 to 2015. Meanwhile, basic clinical data of patients were collected. The drug resistance of the strains to fungal drugs was analyzed by ATB FUNGUS 3 drug sensitivity test strips. All strains were classified by Multilocus sequence typing(MLST). Then, homology analysis was conducted by MEGA 5.2 software, and the evolutionary tree was mapped by using UPGMA method.@*Results@#Patients distribution of strains was rendered as following: 31 strains from TB patients, 21 strains from HIV/AIDS patients, 19 strains from patients with liver disease, and 24 strains from rare cause infection or fever patients. The drug resistance rate to five antifungal drugs commonly used in clinical (amphotericin B, 5-fluorine cytosine, fluconazole, itraconazole, voriconazole) were 2.11% (2 strains), 0, 26.32% (25 strains), 26.32% (25 strains), and 26.32% (25 strains) respectively. Among the 25 azole-resistant strains: 14 strains were from rare cause infection or fever patients, 8 strains were from HIV/AIDS patients, and 3 strains were from tuberculosis patients. In MLST, 72 sequence types (ST types) were produced, 70 of which were new types. Evolutionary tree analysis showed that 95 strains of clinical strains distribute as three large clusters. 24 azole resistant strains (96.0%) were located in CLUSER Ⅰ.@*Conclusion@#The isolated Candida tropicalis were mainly resistant to azole drugs. MLST typing indicates that they was closely related to their genetic background.
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Objective To investigate the clinical value of coplanar template-assisted CT guided radioactive seeds implantation in the treatment of pancreatic carcinoma. Methods A total of 22 advanced pancreatic carcinoma patients underwent CT guided radioactive seeds implantation were retrospectively analyzed.Ten patients were treated with coplanar template-assisted with an average age of(65±10)years(48 to 77 years).Tweleve patients were treated without coplanar template assist with an average age of(68±13) years (47 to 84 years). The preoperative planning designs and postoperative dosimetry verifications were performed for all patients.The dose related parameters including D90,MPD,V100,V150and V200were compared between pre and post operation by t test. The operating time were also evaluated between the two groups. Results Overall the 22 patients were treated successfully without serious surgery-related complications. An average of 26 seeds were implanted in the coplanar template assisted implantation group,and 23 seeds were implanted in the non template-assisted implantation group. Preoperative V100in coplanar template group and non template group were(94.45 ± 1.32)% and(93.27 ± 1.37)% separately. Postoperative V100in both groups were(89.31 ± 2.58)% and(85.25 ± 4.35)% separately. Postoperative D90in both groups were (147.32±7.12)Gy and(149.25±4.86)Gy separately.Postoperative V150in both groups were(57.83±7.74)% and(63.97±7.75)% separately.Preoperative D90in both groups were(152.41±6.78)Gy and(153.30±7.79) Gy separately. Preoperative V150in both groups were(58.61 ± 14.11)% and(62.45 ± 6.49)% separately. Postoperative MPD in both groups were(87.64±10.60)Gy and(87.12±7.66)Gy separately.Postoperative V200in both groups were(34.12±7.67)%,(39.42±7.18)% separately.Preoperative MPD in both groups were (82.12±7.81)Gy and(83.43±4.86)Gy separately.Preoperative V200in both groups were(29.04±10.64)%, (36.11 ± 7.22)% separately. Compared with preoperative plans, the mean value of D90and V100decreased while the mean value of MPD and V200increased in postoperative verifications in both coplanar template assist CT guided radioactive seeds implantation group and non template-assisted group.However,there was no significant difference between pre and post operation except for V100(P<0.05). The operating time of coplanar template assist group and non template-assisted group were(44.3±12.4)min and(60.0±12.8)min respectively. The difference of operating time between two groups were statistically significant (P<0.05). Conclusion Compared with the treatment without template assist, coplanar template-assisted brachytherapy could be more accurate in preoperative plans optimization,and shorten the operation time and improve the patients'tolerance.
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Objective To evaluated the safety and clinical efficacy of CT-guided percutaneous radioactive 125I seeds implantation in treatment of spinal metastatic tumors.Methods Totally 20 cases (23 lesions) of spinal metastatic tumors with spinal compression and severe back pain were treated by CT-guided percutaneous 125I seeds implantation.Intractable pain and nerve function before and after therapy were evaluated.And the postoperative cumulative local tumors control rates and cumulative survival rates were calculated.Results The median follow-up period was 14 months (range 7-32months).There was no patient lost follow-up.And no severe complication occurred.Intractable pain significantly relieved one month after therapy.The neural retention rate of 12 patients with nerve function impairment was 85.00％ (17/20) and the neural function recovery rate was 30.00％ (6/20) three months after 125 I seeds'implantation.The local tumors control rates in 3-,6-,and 12-month were 100％,100％ and 90％,respectively.The survival rates in 6-and 12-month was 100％and 78.81％,respectively.Conclusion CT-guided percutaneous radioactive 125I seeds implantation for spinal metastatic tumors is safe and feasible.It can relief pain and improve neural function effectively.
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Objective To evaluate the efficacy and safety of biliary stent loaded with 125I seeds in treatment of hilar cholangiocarcinoma with malignant obstructive jaundice.Methods Totally 43 patients with malignant obstructive jaundice caused by cholangiocarcinoma were included.All the patients underwent percutaneous transhepatic puncture of the left and right side branch of the bile duct.In the hilar stenosis,the biliary stent with 125I seeds were implanted,and the biliary drainage tube had been kept in 3 to 5 days after procedures.The drainage tube was removed and the puncture road was closed after the patency of stents were confirmed by cholangiography.The changes of liver function before and after procedures were recorded,and the survival time was observed.Results Five biliary stents loaded with 125I seeds were implanted in type I (n=5),36 in type Ⅱ (n=18),8 in type Ⅲ (n=4) and 25 in type Ⅳ (n=16).The serum total bilirubin and direct bilirubin of patients before procedures were (145.54 ± 65.35) μmol/L and (124.73 ± 35.04) μmol/L,respectively,and (65.91±29.43)μmol/L and (35.50±15.12)μmol/L respectively after procedures.Compared with preoperative,the total bilirubin,direct bilirubin,alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,C-reactive protein and gamma glutamic transaminase decreased significantly (all P＜0.05).The lactate dehydrogenase had no significant difference before and after operation (P=1.050).The median survival time was 13 months (3.0 to 22.5 months).The serious complications such as biliary puncture,pancreatitis,severe biliary tract infection or biliary bleeding were not occurred.Conclusion Biliary stent loaded with 125 I seeds is an effective therapy to alleviate symptoms of jaundice and prolong the survival time of patients with malignant obstructive jaundice caused by hilar cholangiocarcinoma.
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Objective To investigate whether lipids and reagents would interfere the results when serum total bile acid(TBA) was measured by enzymatic cycling assay.Methods The serum TBA was measured by enzymatic cycling assay.The carry-over contaminations of high density lipoprotein(HDL-C),low density lipoprotein(LDL-C),cholesterol(TC),and triglyceride(TG)rea-gents were evaluated.In order to reduce the interference and carry-over contaminations,different washing procedures and detection sequence were set.Results By measuring the levels of TBA in pooled serums with low and high levels of lipids,the results showed that there was statistically significant difference between the groups with and without the addition of cleaning process before and af-ter TBA measurement(P <0.01).Cleaning with water might be more effective on reducing interference than those with acid solu-tion.Moreover,the mean of TBA levels in HDL-C,TC,TG and LDL-C reagents were (476.06 ± 1.88 ),(127.78 ± 1.18 ), (121.05±1.08),and (2.23±0.51)μmol/L,respectively.The stability of TBA level was greatly affected by HDL-C regents,fol-lowing by TC and TG reagents,and was little affected by LDL-C reagent.Setting up proper detection sequence and flushing proce-dures could obviously reduce the interference(P <0.01),but not completely rule out.Conclusion Analysis sequence and flushing procedures of biochemical analyzer as well as exogenous substance from reagents may seriously affect the accuracy of determination results.To ensure the accuracy and reliability of the results,it is necessary not only to set up reasonable irrigation and reaction se-quence,but also to master the instrument operation,to know the principle of test reaction and the components of reagents as well as equipment maintenance.
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Objective This study aims to synthesize a novel gadolinium loaded nanoprobe targeted to vascular endothelial growth factor (VEGF) and assess its clinical value for imaging micro hepatic carcinoma.Methods A carrier was made by the biocompatible polymer material polylactic acid-polyethylene glycol-poly-L-lysinenanoparticles (PLA-PEG-PLLs).The targeted nanoprobe was obtained with anti-VEGF antibody and gadolinium (Gd) bonding to the surface of the carrier.MRI in vitro determined the T1 relaxivity of the nanoprobe.A live cancer model enhanced MR scan was performed by injecting targeted nanoprobes into the tail vein of grafted H22 tumor mice.The enhanced characteristics of the subcutaneous tumors and micro-heatic carcinoma were then reviewed.Results The particle size of the VEGF-targeted PLA-PEG-PLL gadolinium loaded nanoprobe was 85.8±7.2 nm with a zeta potential of 21.63±2.4 mV.The R1 relaxivity of the targeted nanoprobe was 18.394 mmol/s at 3.0 T when its gadolinium concentration was 8.0 μmol/ml.The enhanced MR scan using targeted probes showed that the big and micro-subcutaneous cancer exhibited a specifically delayed enhancement with an enhanced peak value at 2 or 3 hours,rather than the enhancement of the tumor using the nontargeted nanoparticles.Conclusion In conclusion,the VEGF targeted PLA-PEG-PLL gadolinium loaded nanoprobe was synthesized successfully,showed a high relaxivity,achieved targeted imaging of the micro-hepatic carcinoma,and exhibits a promising potential in the detection of this liver cancer.
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Objective:To screen and clone a carrier molecule for the expression of small bioactive peptides at high levels. Methods: A carrier molecule, PaP3.30, was screened out from the genome of Pseudomonas aeruginosa phage PaP3 and its gene was cloned by PCR method and inserted into pQE 32 expression plasmid, this recombinant plasmid was named pQE PaP30. The peptide antibiotics hPAB ? gene was then inserted into pQE PaP30 and induced to express the fusion protein in Escherichia coli . The ability of PaP3.30 to express other bioactive peptides was evaluated by fusing 6 different origins, varies in sizes and isoelectric points selected peptides to it. Results: After fused to PaP3.30, the peptide antibiotics hPAB ? could express as fusion protein above 30% of total bacterial proteins. Six selected peptides were also expressed by the level of 35%～44% total bacterial proteins when fused to carrier molecule, PaP3.30. Conclusion: The new carrier molecular, PaP3.30, is versatile in the expression of small bioactive peptides.
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Objectives: To design the mutants of peptide antibiotics hPAB ? based on its molecular structure. Methods: The three dimension structure of hPAB ? was constructed by protein homology modeling method. The mutant molecules were designed and generated by PCR and inserted into pQE CP4 expression plasmid. The recombinant plasmids were identified by PCR and DNA sequencing and then transformed into Escherichia coli JM109 to express target fusion proteins. Results:Peptide hPAB ? shows one ? helical and three ? sheet in its structure. Its ? helical regions seem play a key role in the formation of active oligomer. Aside from positioning Thr 7 and Lys 10 into contact positions, the orientation of the ? helix is conserved about the oligome core, forming a ridge around it. Additionally, the dipoles of the helices would overlap to create a positively charged region near the core. These dipoles may be offset, however, by the presence of Asp 4 at the base of the helix. Two mutant molecules, hPAB ? 38 and hPAB ? 34, were designed by deleting N or/and C terminal 2～5 amino acid residues based on hPAB ? structure. The recombinant plasmids containing the mutants gene can express interest fusion proteins in E. coli JM109 successfully. Conclusions: Design, cloning and expression of the mutants of peptide antibiotics hPAB ? lay down the foundation for screening of the mutant of shorter peptide chain and having high or same antimicrobial activity.
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Objective: Identification of the attachment site of phage PaP3 within the genome of Pseudo-monas aeruginosa PAS. Methods:The full genome of lysogenic bacteria was cleaved by Pst Ⅰ and produce a large fragment of more than 45 000 bp, which was subsequently digested by EcoR Ⅰ. Then the fragment containing DNA sequence of phage and bacteria was cloned into pFastBacTMHT A vector, and the result of sequencing indicated the right hybrid site attR. AttL was isolated by PCR on the base of integration mechanism. And then attP and attB were indentified according to the nucleotide sequences of attR and attB. Results:A sequence of 21 bp(5'-GGTCGTAGGTTCGAATCCTAC-3') was defined to be the core site of integration, which was located at t-RNAPro gene in the genome of phage PaP3 and t-RNALys gene in the genome of Pseudomonas aeruginosa PA3. The attP and attB flanked with a set of inverted repeat and direct repeat. Conclusion:The integrated site of PaP3 within the genome of PA3 was identified and characteriged, which could be of value in investigating the mechanism of integration and gene flow between different species in the natural world.
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Objective To investigate the binding ability of the terminase large subunit of Pseudomonas aeruginosa bacteriophage PaP3 to the cos site. Methods The gene tls was amplified from the genome of bacteriophage PaP3 by PCR and subcloned into pMD18-T vector. Then the gene tls cut down from the vector was inserted into the plasmid pQE31 which could give a 6-His tag at the N-terminal of the expressed protein. The recombinant vector pQE-tls was transformed to E.coli. JM109, after induction with IPTG, the expressed bacteria were resuspended and sonicated, then after centrifugation, the inclusion body was obtained. The inclusion body was dissolved with lysis buffer, then the tagged protein was purified by Ni-NTA affinity chromatography and renatured by dialysis. Finally the DNA-binding ability of the fusion protein rTLS was determined by EMSA. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. After purification and renaturation, the fusion protein rTLS can partially bind the cos fragment. Conclusion The fusion protein rTLS was successfully expressed, purified and renatured. The rTLS has the specific DNA-binding activity. The present work lays the foundation for the further research of the gene tls.
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Objective In order to explore the effects of trauma on MAC and its mechanism, the changes in MAC of sevoflurane and plasma ? endorphin were investigated in state of trauma. Methods Twenty New Zealand rabbits were randomly divided into control group and trauma group. MAP,CVP, P ET CO 2 and ECG were monitored continuously. The MAC of sevoflurane was measured in the two groups. In trauma group, the left femur of rabbits was fractured combined with parenchyma damage.Plasma ? endorphin concentration was assaied at various times in two groups. Results MAC of sevoflurane was 2 2%?0 2% in control group and 1 7%?0 2% in trauma group. Trauma induced increase of plasma ? endorphin concentration significantly 5min and 30min after trauma,? endorphin concentration increased by 44% and 52% in trauma group, respectively. Conclusions MAC of sevoflurane in rabbits is 2 2%?0 2%.Trauma can reduce MAC of inhalational anesthetic significantly. The endogenous ? endorphin releasing enormously induced with trauma may be one of the mechanisms to decrease MAC of inhalational anesthetics.
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Objective To evaluate the influence of propofol on the permeability of the blood brain barrier(BBB) in adult and aged rats.Methods Aged or adult rats were given two doses propofol in 1 hour,respectively.BBB permeability was examined by optical microscopy,electromicroscopy and Evans blue(EB) staining.Results (1)Brain EB staining was not seen in aged or adult groups that at either dose of propofol.(2)In all groups of aged and adult rats,the structure of the blood vessels was normal and lanthanum was not seen outside the blood vessels.(3)There were no significant changes in the central nervous system under microscope or electromicroscope in any groups.Conclusions Propofol at the two doses has no significant effect on BBB permeability or on the central nervous system morphology in aged and adult rats.
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The aim was to determine the antimicrobial activities of peptide antibiotics hPAB ? and construct the recombinant baculovirus of its mutants. Four mutants of hPAB ? were designed based on molecular autosyndetic modeling and its genes were cloned by PCR. The transfer plasmid and recombinant baculovirus were constructed. The results showed that the hPAB ? cloned previously, is a good peptide antibiotics. Four mutant genes of hPAB ? were inserted into pFAST HTa plasmid and the recombinant pFAST hPAB ? were screened by restriction enzymes analysis and DNA sequencing. The recombinant baculovirus was obtained after transforming pFAST hPAB ? into Escherichia coli DH10Bac. Our work lays a good foundation for further research.
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To observe the protective effect of lidocaine on acute lung injury(ALI) following intestinal ischemia/reperfusion(I/R). Adult SD rats were randomly divided into 4 groups ( n =8): control group, with super mesenteric artery isolation only; I/R group, intestinal I/R (60/180min); two lidocaine treated groups, in which lidocaine in a dose of 2mg/kg was administered intravenously immediately or 60min after reperfusion, respectively. Intestinal I/R resulted in deterioration of MAP, increased the lung permeability index, serum TNF ?level and lung TNF ?mRNA expression ,and produced histopathological changes in the lung. Lidocaine given immediately after reperfusion could attenuate these changes, while lidocaine 60min group showed no effects on the changes in MAP, serum TNF ? and pathological changes in the lung. These data suggested that lidocaine could attenuate lung injury following intestinal I/R,in part by inhibiting the sequestration of neutrophils and the production of TNF ?. Lidocaine given early after reperfusion seemed to be more effective .