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Clarifying the temporal and spatial changes in the carbon budget in the Chongqing metropolitan area and exploring the spatial correlation of land use carbon budget are of great significance for realizing the regional "double carbon" goal. Using 21 districts and counties in Chongqing metropolitan area as the research scale, the IPCC inventory method, carbon emission coefficient method, Gini coefficient, gravity model, and social network analysis were used to estimate the net carbon emissions from land use in Chongqing metropolitan area from 2000 to 2020, and the spatial correlation of the carbon budget was obtained. The results revealed that： ① In the past 20 years, the carbon budget of the Chongqing metropolitan area showed an overall upward trend, with an average annual growth rate of 2.83%, and the spatial distribution was "higher in the north and south, higher in the east and west, and lower in the middle." ② During the past 20 years, the spatial difference of net carbon emissions in the Chongqing metropolitan area became highly average, and the overall Gini coefficient decreased by 11.42%, whereas the intra-group difference was the largest in key development zones. ③ In the past 20 years, the overall structure of the spatial correlation network of land use carbon budget in the Chongqing metropolitan area has become stable and complex, and the network density and network correlation number have increased by 0.43 and 180, respectively, the network correlation degree has increased to 1, and the network health degree has improved. ④ In the individual network structure of land use carbon budget in the Chongqing metropolitan area, the degree centrality of each district and county has increased, and the increase in the central urban area was the most significant, with an increase of 81, whereas the decline in intermediate centrality and proximity centrality has promoted the regional coordinated development and integration process in the metropolitan area. ⑤ In the past 20 years, the density of net carbon emissions in the Chongqing metropolitan area has increased as a whole, with the density of the core area increasing by 0.35 and the density of the core-edge area increasing by 0.34. By exploring the change and spatial correlation of land use carbon budget in the Chongqing metropolitan area, this study clarified the spatial distribution difference of carbon budget and provided support for regional green development.

By collecting and analyzing the explanation/conception, acupoint name, acupoint location, indications, acupuncture and moxibustion techniques and contraindications of he -sea point recorded in Neijing ( Inner Canon of Yellow Emperor ) and Nanjing ( Yellow Emperor ' s Classic of Eighty-one Difficult Issues ), it is found that conception of he -sea point of five- shu points has been specifically formed, the names of he -sea points of eleven meridians, the locations and the ways to locate them were recorded. But, the summaries relevant to the indications of the many he -sea points have not been included in teaching materials, thus, it needs to be further analyzed. The acupuncture-moxibustion techniques at he -sea point are flexible and the attention should be paid to distinguish "needling he -sea point in autumn" from "needling he -sea point in winter". Besides, the consideration should be given on coma/fainting induced by arterial bleeding when needling he -sea point, as well as the joint motor impairment caused by over deep insertion in acupuncture.

Objective: To investigate the pathogen spectrum distribution and drug resistance of febrile neutropenic patients with hematological diseases in Shanghai. Methods: A retrospective study was conducted on the clinical isolates from the febrile neutropenic patients hospitalized in the departments of hematology in 12 general hospitals in Shanghai from January 2012 to December 2014. The drug susceptibility test was carried out by Kirby-Bauer method. WHONET 5.6 software was used to analyze pathogenic bacteria and drug susceptibility data. Results: A total of 1 260 clinical isolates were collected from the febrile neutropenic patients. Gram-positive bacteria accounted for 33.3% and Gram-negative bacteria accounted for 66.7%. Klebsiella pneumoniae (12.5%) , Stenotrophomonas maltophilia (9.5%) , Escherichia coli (9.1%) , Pseudomonas aeruginosa (8.7%) , Acinetobacter baumannii (6.6%) , Staphylococcus aureus (5.6%) and Enterococcus faecium (5.0%) were ranked in the first 7 of all pathogens. In the respiratory tract secretions specimens, non-fermented strains accounted for 56.2%. Stenotrophomonas maltophilia accounted for 15.2%. Enterobacteriaceae and coagulase-negative Staphylococci accounted for 42.3% (104/246) and 32.6% (85/246) respectively in blood samples. Enterobacteriaceae and Enterococcus bacteria accounted for 39.4% (76/193) and 28.5% (55/193) respectively in pus specimens. The detection rates of methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase negative Staphylococci (MRCNS) were 54.3% and 82.5%, respectively. Staphylococcus bacterial strain was not found to be resistant to linezolid, vancomycin and teicoplanin. The detection rate of Enterococcus vancomycin-resistant strains was 8.9%. Enterococcus was not detected resistance to oxazolidinone strains. Enterobacteriaceae bacteria were highly sensitive to carbapenems. The resistance rate of Pseudomonas aeruginosa to imipenem and meropenem was 34.1% and 15.8%, respectively. Stenotrophomonas maltophilia was more sensitive to minocycline hydrochloride, levofloxacin and sulfamethoxazole. The resistance rate of Acinetobacter baumannii only to cefoperazone-sulbactam was less than 10.0%. The antibiotic resistance rate of Klebsiella pneumoniae , Stenotrophomonas maltophilia , Pseudomonas aeruginosa and Acinetobacter baumanii to most of common antibiotics was lower than that of the CHINET surveillance. Conclusions: The pathogenic strain distribution in common infection sites of febrile neutropenic patients was characterized. Bacterial resistance surveillance was better than the CHINET nationwide large sample surveillance in China.

Objective: To explore the clinical curative effect of anterolateral acromial approach in treating two-and three-part of proximal humeral fractures., Methods: Forty-two patients of proximal humeral fractures from January 2010 to June 2014 were analyzed retrospectively, including 23 males and 19 females with a mean age of 61.5 years old ranging from 40 to 76 years old. Among them, 22 cases were treated with anterolateral acromial approach and 20 cases were treated with deltopectoral approach. The operation time, intraoperative blood loss, postoperative hospitalization days, fracture healing time of two groups were compared. The shoulder pain after 1 week was assessed by the VAS score. The postoperative shoulder joint function was evaluated after 3 months and more than 6 months by Constant score., Results: The follow-up time was at final 14 months. There were significant differences in operation time( P =0.003), intraoperative blood loss( P =0.001), postoperative hospital day( P =0.013), postoperative shoulder pain after 1 week( P =0.026), postoperative Constant score after 3 months( P =0.014) between the anterolateral acromial approach group and the deltopectoral approach group. There were no significant differences in clinical union time of bone( P =0.462), postoperative constant score after more than 6 months( P =0.204) between the anterolateral acromial approach group and the deltopectoral approach group. There were no breakage of the internal fixation and humeral head osteonecrosis., Conclusions: It has some advantages with anterolateral acromial approach to treat Neer two-and three-part of proximal humeral fractures, such as short operation time, less intraoperative bleeding, lighter postoperative pain, quicker recovery of function., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Objective: To study the effect of smoking on resting energy expenditure (REE) and the relationships among REE, smoking, inflammation and oxidative stress in patients with diabetic kidney disease., Methods: A case control study of 31 smokers and 40 non-smokers with early stage of diabetic kidney disease (stage III) were performed to evaluate the chronic effect of smoking on REE. REE/fat free mass (FFM), biomarkers of oxidative stress malondialdehyde (MDA), superoxide dismutase (SOD) and inflammation high-sensitivity C-reactive protein (hs-CRP), adiponectin, TNFα were also measured in these subjects. Data were analyzed by Pearson correlation analysis., Results: Compared with non-smokers, REE/FFM in smokers group was significantly increased by 15.96% (P = 0.001). Pearson analysis showed that smoking was significantly correlated with REE/FFM (t = 0.395, P = 0.001). There were significantly different between smokers and non-smokers in MDA, SOD and hs-CRP (P < 0.05). But no difference between two groups in adiponectin and TNFα (P > 0.05). No significant relationships between REE/FFM and MDA, SOD, hs-CRP, adiponectin, TNFα was found (P > 0.05)., Conclusion: Chronic smoking can lead to increased REE, arouse oxidative stress and inflammatory in patients with early stage of diabetic kidney disease. However, there is no relationship between increased REE due to smoking and oxidative stress and inflammatory.

Objective: To study the effects of salidroside-pretreatment on changes of neuroethology in rats with global cerebral ischemia-reperfusion injury so as to investigate its probable mechanism., Methods: Sixty SD male rats were randomly divided into sham-operated group, untreated group and salidroside-pretreated group. The rats in salidroside-pretreated group were intraperitoneally administered with salidroside for seven days. The dose of salidroside was 12 mg/(kg.d). Thirty minutes after the last administration, the acute global cerebral ischemia-reperfusion in rats of the untreated group and the salidroside-pretreated group was induced by using the modified Pulsinelli's 4-vessel occlusion method. Five rats in each group were killed to obtain their brains 24 hours after reperfusion. The water content in the right brain was measured by calculating the ratio of dry weight to wet weight of the right brain. Activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) in hippocampus of the rats were measured. Then neurological severity scores (NSSs) of the other 15 rats in each group were observed respectively before and 6, 12, 24, 48 and 96 h after reperfusion. At the fifth day after reperfusion, the test of Morris water maze was carried out to examine the memories and learning abilities of the rats., Results: The content of MDA, the activity of SOD, the NSS, the mean incubation period and the ratio of time in the second quadrant in the untreated group were significant different from those in the sham-operated group (P<0.05). Compared with the untreated group, the brain water content, the content of MDA and the NSS degraded, and the mean incubation period shortened in salidroside-pretreated group. The activity of SOD and the ratio of residence time in the second quadrant increased in salidroside-pretreated group as compared with the untreated group (P<0.05)., Conclusion: Salidroside can reduce the degree of cerebral edema of rats with global cerebral ischemia-reperfusion injury, relieve the metabolism abnormity of free radical and improve the function of cognition.

Objective: To determine the frequency of mce gene in Leptospira interrogans, and to investigate the gene transcription levels of L. interrogans before and after infecting cells., Methods: The segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of mce gene was constructed; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum, the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR., Result: mce gene was carried in all tested L.interrogans strains, but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc I. The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences (GenBank accession No: NP712236) were 99.02%-100% and 97.91%-100%, respectively. The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce. After infecting J774A.1 cells, transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated., Conclusion: The constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.

Objective: To determine the involvement of FasL/Fas pathway in apoptosis of J774A.1 cells induced by Leptospira interrogans., Methods: The cell infection model was established with mouse monocyte-macrophage J774A.1 cells infected by L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601. The morphological characteristics of apoptotic J774A.1 cells were observed by DAPI staining method, and the apoptosis rate was quantitatively determined by flow cytometry. FasL neutralizing antibody was applied to block the apoptosis. Expression of FasL or Fas in the L.interrogans strain 56601-infected J774A.1 cells was detected by flow cytometry using PE-conjugated monoclonal antibody., Result: Chromatin condensation and marginalization were found in J774A.1 cells infected by L.interrogans strain 56601 for 4 h, which became more predominant for 24 h and karyorrhexis was present in some cells. When J774A.1 cells were infected for 4 h and 24 h, the apoptosis rates were 53.6% and 64.31%, respectively. However, the apoptosis rates were decreased to 10.27% and 15.9% after the cells were pre-treated with FasL neutralizing antibody. When J774A.1 cells were infected for 4 h and 24 h, FasL expression rates were increased to 21.69% and 65.70% from that of 4.19% before infection, and Fas expression rates were risen to 91.96% and 88.01% from that of 12.88% before infection., Conclusion: Inducement of cell apoptosis is an important mechanism of L.interrogans strain 56601 injuring J774A.1 cells. The strain of L.interrogans is able to up-regulate FasL/Fas expression levels of host cells and induce apoptosis of the cells via FasL/Fas pathway.

Objective: To investigate the apoptosis of J774A.1 cells induced by Leptospira interrogans and the effect of caspase-3, -6 activation on the apoptosis., Methods: Mouse monocyte-macrophage like cell line J774A.1 was infected by L.interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae Lai strain 56601. The apoptosis or necrosis of infected cells was examined by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The activity of caspase-3, -6, and their cleaved substrates PARP and Lamin A/C were measured by fluorometry and Western Blotting, respectively., Result: L. interrogans strain Lai was able to induce apoptosis of J774A.1 cells and the maximal apoptotic rate was(48.81+/-5.95)% when microbe: cell ratio was 100: 1. The maximal activities of caspase-3 and -6 in the infected J774A.1 cells were (1453.41+/-36.07) and (618.65+/-39.82) FU, respectively, which were 16.38- and 9.98-fold of those uninfected cells. PARP and Lamin A/C in the infected cells were detected. Caspase-3 and -6 inhibitors remarkably blocked the L. interrogans-induced apoptosis in J774A.1 cells., Conclusion: L. interrogans is able to induce the apoptosis of J774A.1 cells and intracellular caspase-3 and -6 are closely associated with the apoptosis.

Objective: To clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis., Methods: The whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni-NTA affinity chromatography was applied to extract rTpN17 and rTpN47, while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen, respectively, ELISAs (rTpN17-ELISA and rTpN47-ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA., Result: The sequence similarity of the cloned tpn17 and tpn47 genes was 100 % compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2 % and 26.8 % of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum.The positive detection rate of TPHA (99.1%) was the highest (P<0.001). The positive detection rates of rTpN17-ELISA (85.3 %) and rTpN47-ELISA (84.3 %) were similar (P>0.05). The positive detection rates of TRUST (72.5 %) was lower than that of rTpN17-ELISA (P=0.001) but similar to that of rTpN47-ELISA (P=0.014). The detection results of all the serum samples from healthy individuals, RA patients and SLE patients were negative, whereas 7.1 % (3/42) of the samples from RA or SLE patients were positive., Conclusion: rTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.

Whether the hepatic pipestem fibrosis induced by schistosomiasis japonica can result in cirrhosis, confusion exists among parasitologists in China. Evidence from national and international pathologists and clinicians confirmed that the pipestem fibrosis could develop into cirrhosis undoubtedly. Owing to different pathogenic causes, the characters of cirrhosis are different. To re-understand cirrhosis induced by Schistosomiasis japonica is of significance for the diagnosis and treatment of the advanced patient.

Objective: To study the immunogenicity and immunoprotection of the recombinant expressing product (rSpaO) of S. paratyphi A spaO gene, and to demonstrate the frequencies of spaO gene carrying and expressing in S. paratyphi A isolates., Methods: The spaO gene of a clinical S. paratyphi A strain JH01 was amplified and then cloned. After sequencing of the cloned spaO gene, a prokaryotic expression system of the gene was constructed. SDS-PAGE were applied to examine the rSpaO expression. Ni-NTA affinity chromatography was performed to collect rSpaO. Immunogenicity of rSpaO was determined by Western blot assay. A PCR assay and an ELISA were established to respectively detect the carrying and expressing frequencies of the spaO genes in 98 S. paratyphi A isolates. The immunoprotective effects of rSpaO in S. paratyphi A strain 50001 infected mice were observed., Results: In comparison with the reported corresponding sequences, the nucleotide and putative amino acid sequence homologies of the cloned spaO gene were 99.45%-99.89% and 99.01%-100%, respectively. The expression output of rSpaO was approximately 75% of the total bacterial proteins. S. paratyphi A antiserum could recognize as well as combine with rSpaO. rSpaO could efficiently induce rabbits to produce specific antibody. 94.9% (93/98) of the S. paratyphi A isolates had spaO gene and 91.4% (85/93) of the spaO+ strains could express SpaO. 58.3% and 50.0% of the mice that oral-taken or subcutaneous injected with 500 microg of rSpaO for immunization were survival after challenged by lethal dose of S. paratyphi A strain 50001. When co-immunized with 5 microg rLTB, the survival rates of the mice increased to 88.3% and 75.0%, respectively., Conclusion: The S. paratyphi isolates had relatively high carrying and expressing frequencies of spaO gene. rSpaO showed a fine immunogenicity and a certain immunoprotective effect, which could be used as an antigen candidate for developing genetic engineering vaccine of S. paratyphi.

Objective: To determine the immunoprotective effects of rUreB and rHpaA bivalence genetic engineering vaccine with inner adjuvant of rLTB on BALB/c mice against H.pylori strain SS1 infection., Methods: rUreB, rHpaA, rLTB-rUreB-rHpaA, rLTKA63, rLTB and rLTB were collected by NTA-Ni affinity chromatography. Western blot was applied to demonstrate the immunoreactivity of the recombinant protein antigens. Adjuvant activities of rLTB, rCTB and rLTB-rUreB-rHpaA were determined by GM1-ELISA. H.pylori strain SS1-infected BALB/c mouse modal was established to measure immunoprotective effects of different compositions of antigens and adjuvants. H.pylori in gastric biopsy specimens was examined by routine isolation method and silver staining method. Two ELISAs were established to detect specific S-IgA in gastric juices and specific IgA in sera of the immunized mice., Results: rUreB, rHpaA and rLTB-rUreB-rHpaA were recognized by commercial antibody against whole cell of H.pylori and were able to combine to the bovine GM1. The protective rate in the mice immunized with single rUreB or rHpaA was lower than 70%. When using rUreB or rHpaA plus rLTB or rCTB, the positive rates increased to 75.0%-83.3%. With different combination of antigens and adjuvants, the immunoprotective rate of rLTB-jrUreB-rHpaA was as high as 100%, and then was 91.7% for rUreB+rHpaA+rCTB+rLTKA63 and was 90.9% for rUreB+rHpaA+rLTB. Both the rLTB and rCTB showed remarkable effects to induce specific IgA in sera and specific S-IgA in gastric juices of the immunized mice, and the former showed stronger S-IgA-inducing ability than the latter. The positive rates of specific IgA were basically identical to the corresponding mouse immunoprotective rates. S-IgA positive rates specific to rUreB and rHpaA in rLTB-rUreB-rHpaA immunized mice were 100% and 91.7%, respectively., Conclusion: The rUreB and rHpaA possess qualified immunoreactivity and antigenicity. The rLTB and rCTB can show adjuvant activity of mucosal immunization. The high immunoprotective rate of Helicobacter pylori UreB and HpaA bivalence recombinant vaccine with inner adjuvant in mice is associated with high dosage of rLTB, larger molecular weight of the antigen and the high levels of local specific S-IgA.

Objective: To construct prokaryotic expression systems of ltB/ctB-lipL41/1 fusion genes, identify immunogenic and adjuvant activities of the products as well as to understand the frequencies of lipL41 gene that carrying and expressing in L. interrogans wild strains and specific antibody levels in sera from patients with leptospirosis., Methods: Polymerase chain reaction (PCR) with linking primer was applied to construct the fusion genes ltB-lipL41/1 and ctB-lipL41/1. By routine molecular biological techniques, prokaryotic expression systems of the two fusion genes were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine expression of the target recombinant proteins rLTB-rLipL41/1 and rCTB-rLipL41/1. Both western blot and Ganglioside-enzyme linked immunosorbent assay (GM-ELISA) were used while immunogenic and adjuvant activities of rLTB-rLipL41/1 and rCTB-rLipL41/1 were measured. PCR and MAT were performed to detect lipL41 gene and expression of the gene in 97 wild strains of L. interrogans, respectively. Antibodies against product of lipL41 gene in serum samples from 228 leptospirosis patients were detected by ELISA., Results: In comparison with reported corresponding sequences, the similarities of ltB-lipL41/1 and ctB-lipL41/1 fusion genes to nucleotide and putative amino acid sequence were 99.6%-99.9% and 99.8%-100%, respectively. Expression outputs of both rLTB-rLipL41/1 and rCTB-rLipL41/1, mainly presenting with inclusion body, consisting approximate 10% of the total bacterial proteins. Both rLTB-rLipL41/1 and rCTB-rLipL41/1 could combine rabbit anti-rLipL41/1 serum as well as bovine GM1, respectively. 87.6% of the L. interrogans wild strains(85/97) having lipL41 gene while 84.5% (82/97) of the wild strains with rLipL41/1 or rLipL41/2 antiserum were positive for MAT with titers of 1:4-1:128. 84.6% (193/ 228), 78.5% (179/228) from the patients' serum samples were positive for rLipL41/1 and rLipL41/2 antibodies, respectively., Conclusion: ltB-lipL41/1 and ctB-lipL41/1 fusion genes and their prokaryotic expression systems were successfully constructed in this study. The two expressed fusion proteins showed qualified immunogenic and adjuvant activities. lipL41 gene was extensively distributed and frequently expressed in different serogroups of L. interrogans. rLTB-rLipL41/1 or rCTB-rLipL41/1 seemed to have had good potential to serve as an antigen in L. interrogans genus-specific vaccine.

Objective: To construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products., Methods: The fusion genes ltB-ompL1/1 and ctB-ompL1/1 were constructed using linking primer PCR method. SDS-PAGE was used to examine expression of the target recombinant proteins rLTB-rOmpL1/1 and rCTB-rOmpL1/1. Western blot and GM1-ELISA were used to measure the immunogenic and GM(1)-binding activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1, respectively. PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains. Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method., Results: The homogeneity of nucleotide and putative amino acid sequence of ltB-jompL1/1 and ctB-ompL1/1 fusion genes were 99.7 % - 99.9 % and 99.5 % - 100 %, in comparison with the reported corresponding sequences. Expression outputs of both rLTB-rOmpL1/1 and rCTB-rOmpL1/1, mainly present in inclusion body, accounted for 10% of the total bacterial protein. Both rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbit anti-rOmpL1/1 serum and bovine GM(1). 89.7 % of L.interrogans wild strains had ompL1 gene. 87.6% of the wild L.interrogans strains presented positive results for MAT (titers :1:4-1:256) with the rabbit anti-rOmpL1/1 or anti-rOmpL1/2 sera. 86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies, respectively., Conclusion: The fusion proteins, rLTB-rOmpL1/1 and rCTB-rOmpL1/1, showed high immunogenic and GM(1)-binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross-antigenicity.

Objective: To determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence., Methods: A special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively., Results: The adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis., Conclusion: The established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.

Objective: To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients., Methods: lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method., Results: The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable., Conclusion: lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

Objective: To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products., Methods: PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively., Results: Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1., Conclusion: An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.

Objective: To determine the effects of leptospiral strains with different virulence on intracellular free Ca(2+)level and its relation with phospholipase C (PLC) activity of L.interrogans., Methods: L.interrogans-j infection cell modals were established with Vero and J774A.1 cell lines. Vero and J774A.1 cells were co-incubated with L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 (strong virulence) and serogroup Pomona serovar pomona strain 56608 (weak virulence) and L.biflexa serogroup Samaranga serovar patoc strain Patoc I (non-virulence). Intracellular free Ca(2+)levels were detected by laser scanning confocal microscopy with specific fluorescence labeling of fluoj3/AM. Using [(3)H] PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay., Results: The baseline intracellular free Ca(2+)levels in the normal Vero and J774A.1 cells were (102.3+/-8.2)% and (105.9+/-7.3)%,respectively. The fluorescence intensity in the two cell lines incubated with L.biflexa strain Patoc I were fluctuated in range of (102.3+/-8.2)%approximate, equals(102.2+/-8.3)% during the observation period. The intracellular free Ca(2+)levels in the two cell lines infected with L.interrogans strain 56601 showed elevation with double peak patterns, with first peaks of (430.5+/-35.7)%, (747.5+/-18.5)% and the second peak of (380.6+/-17.4)%, (804.6+/-22.4)%, respectively. When the cells were infected with L.interrogans strain 56608, the intracellular free Ca(2+)levels were rising slowly with a single slope-like pattern, with the maximal of (235.0+/-19.3)% in Vero cells and (402.4+/-17.4)% in J774A.1 cells, which were significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). The culture supernatants, and cytoplasma and cytomembrane proteins of all three strains displayed PLC activities (P<0.05)., Conclusion: The cells infected with L.interrogans of different virulence show distinct intracellular free Ca(2+)levels and peak patterns. The different host cell lines can affect the intracellular free Ca(2+)levels, which is not related to the PLC activity in the leptospiral strains.

Objective: To investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans., Methods: Ni-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA., Results: Under the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h., Conclusion: The expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.

Objective: To determine cagA/vacA dominant genotypes of Helicobacter pylori in patients suffering from chronic gastritis (CG) or peptic ulcer (PU), and to understand the correlation of different genotype H. pylori infection, coinfection and the gastroduodenal diseases., Methods: H. pylori strains were isolated from antrum and corpus samples on 42 patients with CG and 36 patients with PU. Polymerase chain reaction was used to detect cagA and the s and m regions of vacA in 156 H. pylori isolates from both antrum and corpus. The distribution of H. pylori genotypes and coinfection in CG and PU was analyzed., Results: Almost all of the isolated H. pylori strains were cagA positive. In region of vacA, only one genotype of signal region (s1a) and four genotypes of the middle region (m1, m2, m1b and m1b-m2) were found. The proportions of s1a/m1, s1a/m2, s1a/m1b, s1a/m1b-m2 and coinfection of multiple H. pylori strains in 78 isolates from antrum samples were 6.4%, 55.1%, 26.9%, 1.3% and 3.8%; and the related proportions of those from corpus samples were 6.4%, 53.8%, 25.6%, 3.8% and 5.1%, respectively. Sixteen (20.5%) patients had multiple H. pylori strains with different cagA and vacA genotypes, and multiple samples were better than single sample taken from one stomach to increase the positive proportion of coinfection., Conclusion: cagA(+) s1a/m2 was the dominant genotype of H. pylori in the CG or PU patients followed by cagA(+) s1a/m1b in the Zhejiang area of China. Some of the patients were coinfected with multiple H. pylori strains of different cagA and vacA genotypes. However, there was no significant correlation between the genotypes or mixed infection with multiple strains, CG or PU.

Objective: To clone Helicobacter pylori flagellin B gene (flaB) to construct prokaryotic expression system of the gene and to identify immunity of the fusion protein., Methods: The flaB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted flaB gene was constructed. FlaB fusion protein was expressed in E.coli strain BL21DE3 inducted by IPTG at different dosages. Western blot was applied to determine immunoreactivity and immunogenicity of the fusion protein and antibody against whole cell of H.pylori and rabbit antiserum immunized with the fusion protein, respectively., Results: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned flaB gene was from 96.31% approximate, equals 97.73%, while the homology of its putative amino acid sequence was as high as 99.41% approximate, equals 100%. The expression output of FlaB fusion protein in pET32a-flaB-BL21DE3 system was approximately 40% of the total bacterial proteins. FlaB fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce specific antibody with high titer after the animal was immunized with the protein., Conclusion: A prokaryotic expression system of H. pylori flaB gene with high efficiency has been established successfully. The expressed FlaB fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine and detect kit.

Objective: To determine vacA dominant genotypes of Helicobacter pylori in patients with peptic ulcer (PU) or chronic gastritis (CG)., Methods: H.pylori strains were isolated from mucosa samples of gastric antrum and corpus of patients suffering from PU (n=29) or CG (n=34), 126 strains of H.pylori were selected for PCR to detect s and m regions in vacA gene of the isolates. Parts of the amplification products were sequenced after T A cloning. The correlation between infection or coinfection with different vacA genotypes of H.pylori and different gastroduodenal diseases was further analyzed., Results: The positive amplification products of vacA gene, s and m regions, were found in the DNA samples of all the isolates. In these products, s1a/m1, s1a/m2, s1a/m1b and s1a/m1b m2 genotypes of vacA gene were detected and s1b and s2m1a genotypes absent. Proportions of the s1a/m1, s1a/m2, s1a/m1b and s1a/m1b-m2 genotypes were 7.1% (9/126), 61.9% (78/126), 29.4% (37/126) and 1.6% (2/126), respectively. 17.5% (11/63) of the patients were confirmed to be coinfected with different genotype H.pylori strains. No statistical differences were found in the distribution of different genotype H.pylori strain infection in the gastric diseases (P>0.05). In comparison with the reported sequences of H.pylori strain 60190 with s1a genotype and strain 87-203 with m2 genotype, homologies of the nucleotide sequences of s1a PCR products from 6 strains of H.pylori isolates and m2 PCR products from 4 strains of H.pylori isolates were 93.15% approximate, equals 94.86%and 93.63% approximate, equals 97.61%, respectively., Conclusion: H.pylori with s1a/m2 or s1a/m1b are the dominant genotypes in the PU or GC patients in Zhejiang area. The nucleotide sequences of partial amplification products from the vacA dominant genotypes of H.pylori show high homology compared with the reported sequences. Part of the patients may be coinfected with different vacA genotypes of H.pylori.

Objective: To clone Helicobacter pylori ureB gene, to construct prokaryotic expression system of the gene and to identify immunogenicity of the fusion protein., Methods: The ureB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted ureB gene was constructed. ureB fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein., Results: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned ureB gene was from 96.88% approximate, equals 97.82%, while the homology of its putative amino acid sequence was as high as 99.65% approximate, equals 99.82%. The expression output of UreB protein in pET32a-ureB-BL21DE3 system was approximately 40%of the total bacterial proteins. UreB protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce high titer antibody after the animal was immunized with the protein., Conclusion: An expression system with high efficiency of H.pylori ureB gene has been established successfully. The expressed UreB protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

Objective: To clone Helicobacter pylori adhesin (hpaA) gene,to construct the expression vector of the gene and to identify immunogenicity of the fusion protein., Methods: The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted hpaA gene was constructed. hpaA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein., Results: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned hpaA gene was from 94.25% approximate, equals 97.32%, while the homology of its putative amino acid sequence was as high as 95.38% approximate, equals 98.46%. The expression output of HpaA fusion protein in pET32a-hpaA-BL21DE3 system was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to preduce high titer antibody after the animal was immunized with the protein., Conclusion: An expression system with high efficiency of H.pylori hpaA gene has been established successfully. The expressed HpaA fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

OBJECTIVE: To investigate the frequency of transfusion transmitted virus (TTV) infection in healthy blood donors in Hangzhou area and the mutation of TTV genomic fragment. METHODS DNA in serum samples of 203 healthy donors was extracted by phenol-chloroform method to detect TTV by semi-nested polymerase chain reaction and nucleotide sequences of partial amplification products were determined after T-A cloning. RESULTS TTV infection rate in 203 cases of blood donors in Hangzhou area was 15.3%. The homology of the amplified products of partial TTV positive samples compared with thereported nucleotide and putative amino acid sequences of TTV TA278 were 63.51% approximate, equals 67.12% and 59.46% approximate, equals 66.22% respectively. CONCLUSIONS TTV infection rate in the blood donors in Hangzhou is relatively high. The TTV infecting blood donors in the area may be a kind of novel genotype.