Objective To construct the prokaryotic expression and purification protocols for Pseudomonas aeruginosa type E flagellin (FlgE) and to study its bioactivity.Methods With analysis of Pseudomonas aeruginosa flagellin FlgE sequences,the whole-length FlgE gene was amplified from Pseudomonas aeruginosa genomic DNA by using PCR and primers with proper restriction enzyme sites.The amplified FlgE fragment and prokaryotic expression plasmid pET24a were digested with Nde Ⅰ and Hind ⅢⅣ respectively.The target fragment and vector were recovered and ligated to obtain the recombinant plasmid pET24a-FlgE.DNA sequencing of positive clone confirmed that the target gene and the junctions with vectors were all correct.The plasmid pET24a-FlgE was transformed into BL21 bacteria.The culture conditions like temperature,rotation speed,inducer concentrations,time length were optimized to achieve maximal expression of the target recombinant FlgE with 6×His tag at C terminal.FlgE-His proteins were purified using His-Trap affinity chromatography columns and identified by SDS-PAGE.The purified proteins were further subjected to endotoxin elimination with proper kits.The purified recombinant FlgE was added to cultured corneal epithelial cells for 4 h and the expression of several inflammation-related molecules was examined by using real-time quantitative PCR.Results The recombinant plasmid pET24a-FlgE was successfully constructed and high level FlgE expression was achieved in BL21 with rotation at 16℃ and 1 mmol/L isopropyl-β-D-glucopyranosyl-galactosidase induction for 20 h.Purified recombinant FlgE-His was obtained and used for primary bioactivity assay.After treatment of corneal epithelial cells with 20 μg/ml FlgE for 4 h,the expression of inflammatory cytokines IL-6,IL-8 were significantly increased.Inactivation of the FlgE with ethanol abolished its stimulatory activity.Conclusion The prokaryotic expression and purification system for recombinant Pseudomonas aeruginosa flagellin FlgE was set up,and the recombinant FlgE stimulated the expression of inflammatory factors in corneal epithelial cells.

Objective To understand the characteristics of schistosome cercarial infested water contact of residents in Poyang Lake endemic areas after the operation of Three Gorges Project,so as to provide the evidence for formulating a well-directed con-trol strategy. Methods The residents in two villages in schistosomiasis endemic areas were selected and surveyed for their activi-ties of infested water contacts by questionnaire. Results The proportion and frequency of infested water contacts of residents in Poyang Lake region were high and the average rate of infested water contacts was 69.77%,and 98.96%of infested water contacts occurred in spring,summer and autumn. The main ways of infested water contacts were fishing/swimming/playing for adult men, washing clothes/fishing for adult women,and swimming/playing for school children,respectively. The rates of infested water con-tacts were 73.33%,71.63%and 61.87%in adult men,women and school children,respectively. The frequencies of infested water contacts>1 time/week,1 time/week-1 time/month,and<1 time/month were 62.61%,23.74%and 13.66%respectively. Conclu-sions The infested water contacts mainly occurs in spring,summer and autumn,and the main way of infested water contacts was fishing. Farming and mowing grass,which had been the primary way,now become the secondary way for the adult residents.

10.3969/j.issn.2095-4344.2013.24.020

Objective To explore the epidemical pattern of acute schistosomiasis in Poyang Lake area so as to provide the scientific evidence for control-strategy-making. Methods The data of acute schistosomiasis in 12 counties along Poyang Lake from 1960 to 2007, including the data of 2 special investigations from 1982 to 1984 and from 1987 to 1989 were collected and analyzed with the retrospective investigation methods. Results There were 29 864 cases of acute schistosomiasis in 12 counties of Poyang Lake area from 1960 to 2007, accounting for 85.5% of the total acute schistosomiasis cases of Jiangxi Province. The largest number of cases existed in Yugan County, accounting for 18. 7%. The peak of acute schistosomiasis prevalence was in 1987 with 2 088 cases. However, the case of acute schistosomiasis declined significantly, keeping at a low level, since 1990. During the 2 special investigations, the people whose age were lower than 30 years old, were the susceptible populations of acute schistosomiasis (86. 1% and 82. 9% , respectively) , most of them were peasants and students, and the infection rates were 57. 4% and 21.2%, 61.7% and 21. 8%, respectively. Fishing and swimming were the main infection ways, with the infection rates of 43.0% and 44.9% , respectively. The peak season of infection was from April to October. Conclusions The river bank and continent of Poyang Lake are the main infection areas, the spring and autumn are the main epidemic seasons and the susceptible populations are the peasants, fishmen and young students. It is very important to enhance the comprehensive prevention and treatment measures in the key areas and population for schistosomiasis control.

Objective To investigate the influence of murine cytomegalovirus on the expansion of CD+CD25+ Foxp3+ regulatory T cell (Treg) and the activation of CD4+ CD25+Foxp3 - effector T cell (TE) in vivo. Methods Forty-two BALB/c mice were intraperitoneally inoculated with appropriate amount ofMCMV Smith strain for establishing the model of infection, another 42 mice served as mocked-infected con-trois. Day 28 post MCMV infection was determined to be the demarcation point of the acute and chronic in- fection based on the viral load of major visceral organs. On day 1,3, 7, 14, 28, 45 and 60 post infection, splenocytes were prepared by means of routine method. The proportions of CD4+CD25+ Foxp3+Treg and CD4+CD25+Foxp3- activated TE in T lymphocyte were measured by flow cytometry. Results The propor- tion of CD+CD25+ Foxp3+ T cells in T lymphocytes was persistently suppressed since day 7 post infection, and fell to the lowest level on day 28 post infection (P <0.01), then zoomed and reached the peak value on day 60 post infection (P < 0.05). CD4+CD25+ Foxp3 - TE proportion was up to the highest on day 3 post infection(P < 0. 01), then suppressed and in significantly lower level since day 45 post infection (P < 0.05). Treg/CD+TE ratio was in lower level on day 3 to 14 post infection(P <0.05) ; but on day 45 and day 60 post infection Treg/CD+ TE ratio was markedly increased (P < 0.05). Conclusion MCMV infec- tion can increase the CD+CD25+Foxp3+ Treg proportion, and inhibit CD4+T cells activation in chronic in- fection phase, which is likely to suppress the function of antiviral immunity in the infected host to cause a persistent latent infection.

Aim To investigate the protective effect of flavonoids from Radix tetrastigmae (RTFs)on lipopo-lysaccharide (LPS)induced acute lung injury (ALI) of aged mice and the mechanism.Methods Aged C57BL/6J mice were bronchially instillated LPS to in-duce ALI.RTFs were orally administered to treat ALI. After 3 days,bronchoalveolar lavage fluid (BALF) was collected to enumerate leukocytes with Wright-Gi-emsa staining,and to detect inflammatory cytokines with ELISA.ELISA and Western blot methods were al-so used to detect the expression of MAPKs and NF-κB in lung tissues.The activity of NF-κB in nucleic pro-tein extract was detected with TransAMkit.Results
ALI models were successfully induced through LPS in-stillation.RTFs significantly reduced leukocyte,espe-cially neutrophil infiltration in BALF,inhibited IL-1 β, IL-6,IL-1 2p40,TNF-αand sTNF-R1 secretion,and improved pathohistological change of lung tissues.Be-sides,RTFs significantly attenuated the phosphoryla-tion of p38MAPK,NF-κB and the activity of NF-κB. Conclusion RTFs inhibits LPS-induced ALI through p38MAPK and NF-κB pathway and exhibits significant anti-inflammation effect on aged mice.

Chemotherapy is one of the traditional tumors treatment solutions.Chemotherapy has the feature of tissue non-specificity,which can cause side effects on normal cells while inhibiting tumor cell growth.Magnetic targeting drug delivery system (MTDDS) employs biocompatible and stable magnetic nanoparticles (MNP) as drug carries to transport and accumulate anticancer drugs to the specific tumor tissues under the guidance of external magnetic field.This technology not only improves the efficiency of drug delivery and antitumor activity,but also reduces the drug dosage and side effects.The properties of drug-loaded MNPs and the applied external magnetic field are the main factors that affecting the MNPs targeting to the tumor tissues.The effectiveness of the targeted delivery of the drug-loaded magnetic nanoparticles mainly depends on the form and strength of the magnetic field at the target site.That is,whether there is sufficient strength to attract and retain NMPs,and to promote antitumor drug release at the tumor region.In this paper,the research progress of static magnetic field targeting drug delivery system in tumor diagnosis and therapy was summarized,which can provide some basic information for the relative scientific researches.

BACKGROUND: Conventional hemodialysis mainly for cleaning uremic micro molecule substance, such as urea nitrogen or creatinine; however, few hemodialyses can clean uremic middle molecule substances (MMS). With prolonged dialysis duration, MMS accumulates in vivo and induces a series of complications. OBJECTIVE: To compare the efficiency of adsorptive dialysis (hemoperfusion unites hemodialysis) and conventional hemodialysis in cleaning uremic MMS. METHODS: Totally 60 maintenance hemodialysis patients were averagely divided into the adsorptive dialysis group and conventional hemodialysis group. First of all, hemoperfusion apparatus and dialyser were connected in series to take the adsorptive dialysis in the adsorptive dialysis group (hemoperfusion apparatus were equipped before dialyser). 120 minutes later, the hemoperfusion apparatus was toke off and continues to hemodialysis for 120 minutes. Duration of conventional hemodialysis was 240 minutes. Changes in clinical symptoms and levels of liver function, kidney function, serum electrolytes, hemocytes and uremic MMS were observed prior to and after treatment. RESULTS AND CONCLUSION: Adsorptive dialysis could remove the MMS notably. Compared with the conventional hemodialysis group, a single 120 minutes treatment could decrease MMS significantly (P < 0.05). The platelet levels were obviously decreased in the adsorptive dialysis group after treatment (P < 0.05), which were significantly different from the conventional hemodialysis group (P < 0.05). There was no significant difference in liver function, kidney function or serum electrolytes concentration. But related symptoms, such as the skin itch, sleep disorders and myalgia, were relieved more or less.

In traditional Chinese medicine herbs (TCM), including Radix Salviae Miltiorrhizae (Danshen), Radix Puerariae Lobatae (Gegen), Radix Angelicae Sinensis (Danggui), and Rhizoma Chuanxiong (Chuanxiong) are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with Western drugs as a part of integrative medicine practice. Carboxylesterase 1 (CES1) plays a pivotal role in the metabolisms of pro-drugs. Since (S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydro-thiazole-4-carboxylate (NLMe) has recently been identified by us as a selective CES1 bioluminescent sensor, we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats. This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above. The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in the blood, liver, kidney and heart than that in the small intestine, spleen, lung, pancreas, brain and stomach. In addition, the four herbs showed tissue-specific effects on rat CES1 expression. Based on the CES1 biodistribution and its changes after treatment in rats, the possibility that Danshen, Gegen and Danggui might alter CES1 ac-tivities in human blood and kidney should be considered. In summary, a selective and sensitive biolu-minescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats.

Objective To summarize CT and MRI features of hyaline vascular type localized Castleman disease(LCD)and analyze the causes of misdiagnosis,to improve the preoperative diagnosis rate.Methods The clinical and imaging data of 7 patients with hyaline vascular type LCD confirmed by operation and pathology were analyzed retrospectively.Results (1)6 cases were misdiagnosed before operation,1 case was misdiagnosed as pancreatic neuroendocrine tumor,1 case as thymoma,1 case as neurogenic tumor,1 case as pheochromocytoma, 1 case as clear cell renal cell carcinoma and 1 case as small mesenteric stromal tumor.(2)1 case was located in the right neck,1 case in the anterior superior mediastinum,1 case in the neck of the pancreas,1 case in the upper part of the left kidney,2 cases in the retroperitoneum and 1 case in the lower abdomen.(3)3 cases were scaned by dynamic enhanced MRI,3 cases were scaned by dynamic enhanced CT, and 1 case was checked by plain CT and enhanced MRI.CT and MRI showed that 7 cases had a round or elliptical soft tissue mass, and 4 cases with well defined margin,3 cases were not clear in edge,2 cases with spot or strip calcification on CT images,4 cases had slightly longer T1 and longer T2 signal,4 cases were restricted of diffusion and had higher signal on DWI.All the lesions were enhanced in arterial phase,and went on in the delayed phase.There were 5 cases with distorted vascular shadow in the middle and/or around of the mass, 3 cases with strips,spoke-like low-density areas or low-signal areas,and some lesions were filled in delayed phase.Conclusion CT and MRI features of hyaline vascular type LCD have certain characteristics such as rich blood supply,enhancement in persistent,tortuosity of peripheral vascular,with some short strip calcification and high signal on DWI,which may be helpful for preoperative diagnosis.