Objective To cultivate and identify the transgenic affalfa containing Echinococcus granulosus Eg95 gene. Methods The alfalfa plants were transformed by co-cultivating alfalfa cotyledons via recombinant Agrobacterium tumefaciens LBA4404 harboring pBI-Eg95. The transgenic alfalfa explants were selected by kanamyein after calli formation, shoots and roots regeneration in the selective medium, the seedlings of transgenic plants were obtained which were finally transplanted into pots containing nutrient soil. After 2-3 months growth, the complete transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene were obtained. To identify the transgenic alfalfa plants, the total DNA, RNA and leaf protein were extracted from fresh leaf tissue of the transgenic alfalfa plants and confirmed by PCR, RT-PCR, SDS-PAGE and Western blot assay. Results A specific band around 471 bp was amplified by PCR with total DNA, and the same band was obtained by RT-PCR with total RNA, which confirmed that the Eg95 gene was stably integrated into the transformed alfalfa genome. SDS-PAGE analysis showed that the relative molecular mass(Mr) of the expressed protein was about 16.5×103, consistent with the Eg95 protein, and the level of Eg95 expression was up to 0.06% of total soluble leaf protein by Bio-Rad Quantity one assay. Western blot verified the expressed protein was reactive with the sera of mice infected with Echinococcus granulosus. Conclusion The transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene are successfully cultivated.

BACKGROUND:Taurine is an important non-enzymatic system antioxidant in the lens.The mechanism of its anti-oxidative effect is mainly to protect lens from oxidative injury by anti-lipid peroxidation.OBJECTIVE:This study was to observe the effect of exogenous taurine on lens epithefial cell apoptosis-induced by H2O2 in vitro.DESIGN:A randomized controlled animal experiment.SETTING:Department of Ophthalmology,Affiliated Hospital of Qingdao University Medical College.MATERIALS:Seventy-five adult New Zealand standard tabbits,of either gender,weighing 1.5-2.5 kg,were provided by Qingdao Laboratory Animal Center.Reagent kit for in situ detecting cell apoptosis(Sigma Company,USA),taurine and H2O2(Shanghai Guangda Chemical Reagent Factory,China)were included in this study.METHODS:This study was performed in the Department of Ophthalmology and Central Laboratory.Affiliated Hospital of Qingdao University Medical College from Mav 2005 to June 2007.Rabbit lenses were harvested and randomly divided into 3 groups:control group,in which,clear lenses were incubated in non-serum and non-phenolsulfonphthalein MEM medium which was renewed every 24 hours,H2O2 group,in which,clear lenses were incubated in non-serum and non-phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 with addition of 62 μL H202(30 g/L)every 6 hours,and H2O2+taurine group,in which,clear lenses were incubated in non-serum and non-phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 and 10 g/L taurine.which was renewed every 6 hours.The protocol was conducted in accordance with ethical guidelines for the use and care of animals.MAIN OUTCOME MEASURES:Observation of lens opacity 6.12,24.48 and 72 hours after culture;Lens epithelial cell apoptosis determined by DNA in situ end labeling and DNA fragment analysis.RESULTS:Lens opacity:The lens opacity in the H2O2 group was aggregrated gradually along with the time of oxidative injury.The lens opacity in the H2O2 group was severer than that in the H2O2+taurine group.Lens epithelial cell apoptosis:There were no apoptotic cells in the control group within 72 hours.The number of apoptotic cells in the H2O2 group was increased gradually with the prolonged time of oxidative injury.Till the 72nd hour,the cells were all tamed into apoptotic cells.A few aopototic celIs were found in the H2O2+taurine group since hour 24,and then were more and more,and the number of apoptotic cells accounted for about 30%at hour 72.The apoptotic rate in the H202+taurine group was significantly lower than that in the H2O group at each time point(q=8.6845,P＜0.01).There was no significant difference in apoptotic rate between the H202+taurine group and the control group (P＞0.05). Findings of DNA fragmentation assay:The DNA"ladder"was found in the H2O2 group at hours 24,36,48 and 72,while no DNA"ladder"but only normal electrophoresis straps were present in the other two groups 24 hours after culture.CONCLUSION:Taurine can inhibit the oxidative injury-induced apoptosis of rabbit lens epithelial cells,and alleviate lens opacity.

Objective To understand the differences of virulence genes and molecular typing in Campylobacterjejuni isolates from poultry products and diarrhea patients in Shenzhen.Methods According to specific primers,four virulence genes (cdtB,cadF,flaA,virB1 1 )of C.jejuni were detected by polymerase chain reaction (PCR).Molecular typing for C.jejuni strains was performed by Pulsed-field gel electrophoresis (PFGE).Results There were no differences of gene distribution (cdtB+,cadF+,virB1 1-)between isolates from poultry products and diarrhea patients.Two virulence genes of cdtB and cadF were found in all of ten C.jejuni strains lacking virB1 1 .The carriage rate of flaA in food-borne isolates (3/5 )was higher than those in patient isolates (2/5).In the PFGE map,the clustering analysis of C.jejuni strains showed that a total of 5 to 9 DNA bands were observed in ten strains through the digestion of Sam I.There was high homology (above 85%) between food-borne isolates and patients isolates,but the distribution of flaA in these highly homologous strains was differ-ent.Conclusion So far,C.jejuni strains with cdtB,cadF and flaA were present in Shenzhen,and showed high diversity and homology.This implies that the occurrence of diarrhea in patients with C.jejuni was associated with the contaminated poul-try products by this pathogen.Their findings can provide basic data and evidences about diarrheal disease caused by food-borne C.jejuni for the local region.

Aspergillosis, Allergic Bronchopulmonary ; diagnosis ; drug therapy ; Child ; Humans ; Male

Abstract: Objective　To analyze the clinical characteristics of Chlamydia psittaci pneumonia and improve the diagnosis and treatment skills of clinicians on this disease. Methods The clinical data of thirty-nine Chlamydia psittaci pneumonia cases detected by metagenomic next-generation sequencing (mNGS) from September 2020 to January 2022 at the Affiliated Hospital of Southwest Medical University were retrospectively analyzed. Results There was a history of poultry exposure in 89.7%(35 cases) of the patients. The most common clinical manifestations were high fever (92.3%, 36), cough (76.9%,30), muscle soreness (48.7%,19), headache (38.5%,15), etc. Laboratory examinations showed 76.9% of patients had a normal leukocyte count, and 76.9% had decreased lymphocyte count, often accompanied by elevated C-reactive protein (100%), procalcitonin (97.4%), interleukin-6 (95.8%), interleukin-10 (95.8%), alanine aminotransferase (74.4%), and aspartate aminotransferase (84.6%). Univariate analysis indicated that there were statistically significant differences in the levels of aspartate transaminase, blood urea nitrogen, C-reactive protein, and procalcitonin between severe pneumonia patients and non-severe pneumonia patients（P<0.05）. Multivariate logistic regression analysis showed that an elevated blood urea nitrogen (OR=4.899) had guiding significance for predicting the occurrence of severe pneumonia. Bronchoscopy examination showed no abnormalities in 53.6% of the patients. The imaging manifestations of pulmonary lesions were mainly lobar pneumonia (61.5%) and air bronchograms (94.9%). Therapeutically, it was sensitive to tetracyclines, macrocyclic lactones, and fluoroquinolones. A total of 84.6%(33 cases) of the patients were cured and discharged from the hospital at the end of the treatment. Conclusion Chlamydia psittaci pneumonia is a zoonotic disease that can be detected by mNGS. An elevated blood urea nitrogen level has guiding significance for predicting the occurrence of severe pneumonia. Empirically-selected regimens based on doxycycline are effective for the treatment of Chlamydia psittaci pneumonia.

Objective To investigate the causes of arterial reocclusion in diabetic feet patients after endovascular treatment and its remedial measures.Methods From January 2009 to October 2013,clinical data of 371 arterial reocclusion of diabetic feet patients after endovascular treatment in Tianjin First Central Hospital were reviewed retrospectively.We summarized the causes of reocclusion,treatment methods and the short term results.Results According to the Trans-Alantic Inter-Society Consensus (TASC) Ⅱ grading standards,the first time when the endovascular treatment started there were 37 cases of grade A,85 cases of grade B,143 cases of grade C,106 cases of grade D.Arterial re-occlusion developed from one day to 36 months,averaging at (21 ± 8) months.Causes of re-occlusion included intimal hyperplasia in 263 cases (70.9％),thrombosis in 65 cases (17.5％),dissection in 19 cases (5.1％),stent fracture in 17 cases (4.6％),vascular rupture in 7 cases (1.9％).Remedial therapy adopted for arterial reocclusion was repeated endovascular treatment in 327 cases (88.1％),arterial bypass surgery in 23 cases (6.2％),conservative treatment in 13 cases (3.5％),amputation (cut toe) in 4 cases (1.1％),4 cases (1.1％) died perioperatively.275 cases were followed up for 1 to 36 months,the average was (13 ± 8) months.patency rate was 82.9％,71.3％ and 63.0％ at 6 months,1 year and 2 years.Amputation rate was 1.1％,1.8％ and 2.5％ at 6 months,1 year and 2 years.Conclusions Intimal hyperplasia is to blame for arterial reocclusion after endovascular treatment of diabetic foot.In this case most patients still can benefit from second time endovascular treatment,with a satisfactory short term patency rate.

Objective To explore the feasibility of detecting atherosclerosis with 7.0T MR and Micro-PET. Methods Ten 46-week-old ApoE-/- mice with high lipid diet for 6 months were selected to establish atherosclerosis models. Among them, 5 mice underwent MRI before and 12 h, 24 h, 36 h after injection of SPIO, respectively, and the other 5 mice were injected with ~(18)F-fluorodeoxyglucose (~(18)F-FDG) through tail vein and observed with Micro-PET after 1 h, 2 h and 3 h. The specimens of abdominal aorta were taken for pathologic examination. Results Atherosclerotic plaques were observed in all animals with 7.0T MRI after 6 months high lipid diet. Thirty-six hours after the injection of SPIO, the high signal rings were thinner and the lumen of blood vessels were wider than those before injection on T2WI. Radioactive concentration was observed in abdominal aorta and both sides of iliac artery 3 h after the injection of ~(18)F-fluorodeoxyglucose (~(18)F-FDG). Pathological examination showed the formation of atherosclerotic plaques and the aggregation of the macrophages. Conclusion 7.0T MRI and Micro-PET can be used to observe the macrophage-rich plaque and to judge the vulnerability of plaque, thus provide theoretical basis for early detection, diagnosis and treatment of atherosclerosis.

Objectives To investigate the effects of lipoxin receptor agonist BML-111 on IFN-βand IE86 mRNA expression of macrophages infected by human cytomegalovirus (HCMV). Methods Macrophages were infected with HCMV (MOI=0.5), and the cultured cells were randomly divided into control group, HCMV group, HCMV+BML-111 group, and HCMV+MP group. The cells were collected at 0,1,2,4,8 and 12 h after infection, and the levels of IFN-βand IE86 mRNA were tested by real-time PCR. Results Compared with HCMV group, the levels of IFN-βmRNA in HCMV+BML-111 group increased significantly (P < 0.05), while the levels of IFN-βmRNA in HCMV+MP group decreased significantly (P < 0.05); Compared with HCMV group, there were no significant differences of the levels of IE86 mRNA in HCMV+BML-111 group (P>0.05), while the levels of IE86 mRNA in HCMV+MP group increased significantly (P < 0.05). Conclusion BML-111 exerts antiviral activity by promoting the expression of IFN-βmRNA at the early stage of HCMV infection.

BACKGROUND:The anti-inflammation and protective effects of lipoxin have been verified in several immunity-related disease models. Preliminary studies of our research group have shown that, lipoxin receptor agonist BML-111 has negative regulation effects on the human cytomegalovirus (HCMV)-induced immunological injury. However, the effect of BML-111 on the HCMV replication remains unclear.
OBJECTIVE:To observe the influence of lipoxin receptor agonist BML-111 on HCMV replication and proliferation in THP-1 macrophages and human embryonic lung fibroblasts.
METHODS:THP-1 macrophages were infected by HCMV AD169 strain, and were divided into three groups:mock infection, HCMV infection, HCMV+BML-111. The final concentration of BML-111 was 100 nmol/L. cells in each group were col ected at 0, 1, 2, 4, 12, 36, 48 hours, the mRNA levels of IE86 and pp65 in the THP-1 macrophages were tested by RT-PCR method. Human embryonic lung fibroblasts were infected with HCMV (MOI=0.1), and were divided into two groups:HCMV infection and HCMV+BML-111. The patho-morphous changes of human embryonic lung fibroblasts were observed under light microscope, and the cellnumber was measured. The infective virus titer changes in human embryonic lung fibroblasts were examined by plaque assay.
RESULTS AND CONCLUSION:After the macrophages were infected by HCMV, compared with the mock infection group, the mRNA levels of IE86 and pp65 in the HCMV group and HCMV+BML-111 group were increased significantly;compared with the HCMV infection group, the mRNA levels of IE86 and pp65 in the HCMV+BML-111 group were increased significantly in the early stage (within 4 hours) after infection, but the pp65 mRNA levels were decreased significantly in the medium and late stages (24-72 hours) after infection. After human embryonic lung fibroblasts were infected by HCMV, the degree of the patho-morphous in the HCMV+BML-111 group reached 100%2 days earlier than the of HCMV infection group. The infective virus titer reached the peak 2 days earlier than the HCMV infection group, but no significant difference was found between the two groups. BML-111 accelerates the replication of HCMV in the early stage of infection, but inhibits the expression of pp65 gene in the late stage. BML-111 has no impact on the proliferation of the infective HCMV titer in vitro.

ObjectiveTo investigate the prevalence and distribution characteristics of dental fluorosis of children aged 8 - 12 in Wushan and Fengjie counties of Chongqing and to provide a scientific basis for prevention and control of the disease.MethodsTwenty townships(towns) in Fengjie county and 18 in Wushan county were selected as survey points by random cluster sampling in 2010.Dental fluorosis of all the children aged 8 - 12 was examined with Dean index.The detection rate of children's dental fluorosis,defect rate and dental fluorosis index were compared between the two counties.Results Totally 38 209 children aged 8 - 12 were investigated.The total detection rate of dental fluorosis was 43.09％( 16 466/38 209) in the two counties.The detection rates of dental fluorosis in Wushan and Fengjie were 48.98％ (9397/19 186)and 37.16％(7069/19 023),respectively,and the difference was statistically significant(x2 =544.03,P ＜ 0.01 ).Total detection rates of dental fluorosis of the five age groups(8,9,10,11,and 12-year-old age groups) were 32.52％(2157/6632),40.07％(2672/6668),43.67％(3420/7831 ),46.01％ (3861/8391) and 50.14％ (4356/8687),respectively,and the difference was statistically significant (x2 =510.50,P ＜ 0.01),Dental fluorosis indexes in Wushan and Fengjie were 0.713 and 0.485,respectively.Defect rates of dental fluorosis in Wushan and Fengjie were 4.05％ (777/19 186) and 1.57％(298/19 023),respectively.Conclusions The total detection rate of dental fluorosis of the two counties is still high,which gradually increases with age.Wushan is still an endemic area of dental fluorosis,and Fengjie is at the edge of the