A gene appA encoding a novel phytase was firstly cloned from Hafnia alvei by PCR and sequenced. The gene was consisted of 1335 bp, encoding 444 amino acids. The calculated molecular weight of the mature APPA was about 45.2 kD. The gene appA was expressed in E. coli BL21 (DE3). Recombinant APPA was purified and its enzymatic properties were determined. The optimum pH for the enzyme was 4.5 and the optimum temperature was 60 degrees C. The pH stability of r-APPA is good, the relative phytase activity was above 80% after treated in buffers of pH 2.0-10.0. The specific activity of r-APPA is 356.7 U/mg, and the Km value was 0.49 mmol/L and Vmax of 238 U/mg. The enzyme showed resistance to pepsin and trypsin treatment.
6-Phytase ; biosynthesis ; genetics ; isolation & purification ; Amino Acid Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Hafnia ; enzymology ; genetics ; Hydrogen-Ion Concentration ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Temperature

In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZalpha-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZalpha is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZalpha to construct the recombinant vector pAOXZalpha-appA-m. The recombinant Pichia pastoris 74#, which already contains one copy of appA-m and its fermentation potency exceeded 7.5 x 10(6) IU/mL, was used as the host strain for the transformation of pAOXZalpha-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supernatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2 x 10(7) IU/mL, which was about 1.6-fold compared with that of the host strain 74#. Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.
6-Phytase ; genetics ; Fermentation ; Gene Dosage ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombination, Genetic

Isotemporal substitution model is a powerful tool to explore the real association between physical behavior and health outcomes, which has the potential of the application in large-scale cohort study. This paper systematically introduces the principle of isotemporal substitution model and its implementation method in specific analysis to provide analytical ideas for the epidemiological research related to physical behavior in China. The baseline data of Regional Ethic Cohort Study in Northwest China conducted in Shaanxi province were used to analyze the relationship between physical behavior and cardiovascular disease with single-factor model, partition model and isotemporal substitution model. The advantages and disadvantages of different models were compared, and the advantages of isotemporal substitution model in quantifying physical activity health risk were introduced. Isotemporal substitution model could qualify physical behavior and health outcomes, which has wide application value in epidemiological research.
Humans ; Cohort Studies ; Epidemiologic Studies ; Cardiovascular Diseases ; China/epidemiology*

OBJECTIVETo analyze the measles immunity level of persistent population in Beijing.

METHODSA total of 2125 objects from 10 age groups, who had been living in Beijing for over 6 months, were selected from urban and rural areas in Beijing in 2012. Demographic characteristics, history of measles and vaccine immunization were investigated by questionnaire. 5 ml blood sample of each subject was collected, and the Measles IgG antibody was measured by ELISA assay.

RESULTSPositive rate of measles antibody was 84.71% (1800/2125) and standardized positive rate was 88.07% . Median of antibody was 960.46 IU/L. Positive rate and median of measles antibody were significantly different between population from different age groups (χ(2) = 341.60, P < 0.01; H = 216.27, P < 0.01). Antibody positive rate and median were lowest in the <1 year age group, which were separately 43.06% (90/209) and 185.80 IU/L; and highest in the 1-4 (97.31% (181/186) and 2448.81 IU/L) and 5-9 years age group (96.46% (218/226) and 1910.72 IU/L). The range of antibody positive rate and median in adults of ≥ 15 years were 81.98%-90.14% and 744.38-1474.84 IU/L. Antibody positive rate and median in persistent population, which were separately 82.45% (883/1071) and 899.82 IU/L, were lower than those in migrant population, which were 87.00% (917/1054) and 166.19 IU/L, respectively (χ(2) = 8.51, P < 0.01;U = 538 704.00, P < 0.01). Antibody positive rate and median in population with vaccination history, which were separately 91.95% (891/969) and 1443.11 IU/L, were higher than those population without vaccination history and people whose history unknown (32.95% (57/173) , 127.33 IU/L; 86.67% (852/983) , 923.73 IU/L). The difference showed statistical significance (χ(2) = 399.92, P < 0.01; H = 202.11, P < 0.01).

CONCLUSIONAmong the persistent population in China, measles antibody level among the children aging 1-9 years old was high enough to prevent outbreak and epidemic of measles. However, we should try our best to strengthen the measles antibody level among the babies younger than 1 year old and the migrant population aging between 15 and 40 years old.

Adolescent ; Adult ; Antibodies, Viral ; blood ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Male ; Measles ; epidemiology ; immunology ; prevention & control ; Measles virus ; Young Adult

OBJECTIVEDiscussing the natural history and the influencing factors of HIV infection among former commercial blood and plasma donors engaged in unsafe blood donation practices in China.

METHODSUsing ambispective cohort study, with data obtained from ten counties (districts) from six provinces in the National AIDS Control Demonstration Area. HIV/AIDS cases were found and confirmed prior to July 24, 2006 being former commercial blood. Plasma donors were selected and data regarding infection, incidence, death, and influencing factors was collected. Analysis was performed using SPSS 12.0 statistical analysis software.

RESULTS(1) In 7551 cases of HIV infection, there were 6533 typical progressors (86.52%, 4757 cases of AIDS), 108 rapid progressors (1.43%), 910 long-term non-progressors (12.05%) with 4865 cases progressed to AIDS (64.43%). The median incubation period for HIV progression to AIDS was nine years (95% CI:8.96-9.04). (2) According to data, from a total of 1157 AIDS cases without ARV therapy (23.78% of total AIDS cases), there were 283 confirmed AIDS-related deaths, of which the median survival time was 6 months (95% CI:4-7) and the two and three year fatality rates were 95% and 99%, respectively. (3) The duration of HIV incubation period was irrespective to gender and age at the time of HIV infection (P > 0.05). Length of survival for untreated AIDS showed correlation to gender (P < 0.05) but no correlation with culture, marital status or age at the time of diagnosis of AIDS (P > 0.05).

CONCLUSIONCompared with the UNAIDS theory regarding slow disease progressors among adults, our study showed a longer AIDS incubation period and shorter outlook for untreated survival, but a similar incubation period for other routes of HIV infection.

Acquired Immunodeficiency Syndrome ; epidemiology ; mortality ; Adolescent ; Adult ; Aged ; Blood Donors ; statistics & numerical data ; China ; Female ; HIV Infections ; epidemiology ; mortality ; Humans ; Infectious Disease Incubation Period ; Male ; Middle Aged ; Young Adult

Objective To investigate the degrees of injury severity of sepsis models made by different kinds of Escherichia coli. Methods The 152 mice were randomly divided into control group, DH5α group, 44102 group, and 25922 group, with 38 rats in each group. DH5α group, 44102 group and 25922 group were intraperitoneally injected with 300 μL of Escherichia coli DH5α, 44102 and 25922 at the concentration of 1.0 × 109CFU/kg to prepare sepsis models of different kinds of Escherichia coli. Control group was injected intraperitoneally with the same amount of normal saline. (1) After 8 h, four mice were taken from each group for peripheral blood bacterial culture . (2) After 12 h, ten mice in each group were used for measuring serum levels of TNF-α and IL-6 by enzyme-linked immunosorbent assay (ELISA). (3) Western blot assay was used to determine the serum levels of high-mobility group protein (HMGB1) in four mice of each group. (4) Ten mice in each group were used to measure serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (CR) and blood urea nitrogen (BUN) by automatic biochemical analyzer. (5) After liver, lung and kidney tissues were fixed with formaldehyde, hematoxylin-eosin (HE) staining was performed (n=10 for each group). Results In DH5α group, 44102 group and 25922 group, bacteria, inflammatory cytokines TNF-α, IL-6 and HMGB1 protein, liver and kidney indicators ALT, AST, CR and BUN showed a sequential increasing trend (P＜0.01). The severe degrees of alveolar structure damage, hepatic cell infiltration and renal glomerular atrophy were DH5α group, 44102 group and 25922 group in turn. There were no obvious damages of lung, liver or kidney tissues in control group. Conclusion Escherichia coli 25922 induces severe sepsis injury and can be used to study the animal models of the initial inflammatory phase of sepsis. Escherichia coli 44102 induces moderate damage of sepsis and can be used in animal models that do not require definitive sepsis staging experiments. Escherichia coli DH5α induces less damage of sepsis and can be used to explore immunosuppressive therapy of the animal model of sepsis.

Objective:To explore whether CD5 + CD19 + B cells has the function of secreteing interleukin-10 (IL-10) in vitro, and to further investigate its possible effects and mechanisms on CD8 + cells in the process of hepatitis B virus (HBV) infection. Methods:From July 2017 to June 2018, at Wuxi Second People′s Hospital Affiliated to Nanjing Medical University, 23 patients with chronic hepatitis B (chronic hepatitis B group), 18 patients with liver cirrhosis (liver cirrhosis group) and 19 healthy individuals in the same period as healthy controls (healthy control group) were enrolled. Peripheral blood mononuclear cell (PBMC) were isolated and cultured. CD5 + CD19 + B cells were isolated. The cells were analyzed by flow cytometry. The ratio of high CD5 + CD19 + B cells content (>6 % of lymphocytes), the secretion of IL-10 by CD5 + CD19 + B and the ratio of high IL-10 + cells content (>4 % of lymphocytes) of three groups were compared. The effects and possible mechanisms of CD5 + CD19 + B cells on the secreting of interferon-γ (IFN-γ) by CD8 + cells were analyzed. Liver biopsy and immunohistochemistry examination were conducted in 18 patients (13 patients with chronic hepatitis B and 5 patients with liver cirrhosis) and the expression of CD5 + CD19 + B cells in human liver tissues was analyzed. Chi square test and Fisher exact probability test were used for statistical analysis. Results:The ratio of high CD5 + CD19 + B cells content of liver cirrhosis group was higher than that of healthy control group (8/18 vs. 2/19) and the difference was statistically significant (Fisher exact probability test, P=0.029). The precentage of CD5 + CD19 + B cells in healthy control group ( n=10), chronic hepatitis B group ( n=23) and liver cirrhosis group ( n=18) accounted for 81.6%, 82.3% and 70.1%of IL-10 + cells, respectively, and the number of patients with high IL-10 + cells precentage was 2, 7 and 2, respectively. There were no statistically significant differences among three groups (all P>0.05). After stimulated with lipopolysaccharide and cultured for 48 hours, the precentage of CD8 + IFN-γ + cells in lymphocytes of healthy control group ( n=10), chronic hepatitis B group ( n=10) and liver cirrhosis group ( n=10) were compared, and the differences were not statistically significant (all P>0.05). After CD5 + CD19 + B cells were eliminated, the precentage of CD8 + IFN-γ + cells in lymphocytes increased in 5, 4 and 4 patients of healthy control group ( n=10), chronic hepatitis B group ( n=10) and liver cirrhosis group ( n=10). After adding IL-10 receptor blocker, the precentage of CD8 + IFN-γ + cells in lymphocytes in PBMC increased compared with that before the addition of IL-10 receptor blocker (7.23% vs. 6.87%). The results of immunohistochemistry examination of liver biopsy indicated that CD4 + and CD8 + cells were strong expressed in portal area of liver tissue of patients, while CD5 + and CD19 + were less expressed. Conclusions:CD5 + CD19 + B cells do not show obvious quantitative and functional differences in the process of chronic HBV infection, however the ability of CD8 + cells to secrete IFN-γ, which may be achieved by secreting IL-10 rather than by direct contact between cells.

This paper reviews the researches on acupuncture preconditioning before surgery in recent years and explores its application value from three aspects, i.e. relieving preoperative anxiety, preventing from postoperative cognitive dysfunction, and preventing from postoperative gastrointestinal dysfunction. As a relatively safe non-drug treatment, acupuncture has the underlying advantages in participating into multidisciplinary coordination in the enhanced recovery after surgery (ERAS). By building up higher-quality medical evidences and revealing the effect mechanism of acupuncture from multi-dimenisonal aspects, it is expected that acupuncture technology can be coordinated with ERAS to optimize the clinical path in the perioperative period, and boost the development of the perioperative medicine ultimately.
Humans ; Acupuncture Therapy ; Anxiety ; Anxiety Disorders ; Enhanced Recovery After Surgery ; Perioperative Period

Adult ; Female ; Humans ; Hyperthyroidism ; drug therapy ; metabolism ; Iodine Radioisotopes ; pharmacokinetics ; therapeutic use ; urine ; Male ; Middle Aged ; Models, Biological ; Radiation Dosage ; Radiation Monitoring ; Radiopharmaceuticals ; pharmacokinetics ; therapeutic use ; urine ; Radiotherapy Dosage ; Young Adult

This paper explored the chemical constituents of Boswellia carterii by column chromatography on silica gel, Sephadex LH-20, ODS column chromatography, and semi-preparative HPLC. The structures of the compounds were identified by physicochemical properties and spectroscopic data such as infrared radiation(IR), ultra violet(UV), mass spectrometry(MS), and nuclear magnetic resonance(NMR). Seven diterpenoids were isolated and purified from n-hexane of B. carterii. The isolates were identified as(1S,3E,7E,11R,12R)-11-hydroxy-1-isopropyl-4,8,12-trimethyl-15-oxabicyclo[10.2.1]pentadeca-3,7-dien-5-one(1),(1R,3S,4R,7E,11E)-4,8,12,15,15-pentamethyl-14-oxabicyclo[11.2.1]hexadeca-7,11-dien-4-ol(2), incensole(3),(-)-(R)-nephthenol(4), euphraticanoid F(5), dilospirane B(6), and dictyotin C(7). Among them, compounds 1 and 2 were new and their absolute configurations were determined by comparison of the calculated and experimental electronic circular dichroisms(ECDs). Compounds 6 and 7 were obtained from B. carterii for the first time.
Molecular Structure ; Boswellia/chemistry* ; Diterpenes/chemistry* ; Mass Spectrometry