Objective To prepare the recombinant Echinococcus granulosus Polo-like kinase 2 (rEgPLK2) protein and evaluate its immunoprotective efficacy against cystic echinococcosis, so as to provide insights into research and development of novel vaccines against echinococcosis. Methods The Polo-like kinase (PLK) protein sequences were retrieved from 12 species in the NCBI protein database, including E. granulosus and E. multilocularis. Multiple sequence alignment was performed using the Clustal Omega program, and structural visualization and homology analysis were conducted using the ESPript 3.2 program. The recombinant plasmid pET-30a-EgPLK2 was transformed into BL21(DE3) competent cells. Protein expression was induced with isopropyl-β-D-thiogalactoside (IPTG), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to characterize the expression and molecular weight of the rEgPLK2 protein. The purified rEgPLK2 protein was thoroughly emulsified with Freund’s complete adjuvant at a 1 : 1 volume ratio. Two New Zealand white rabbits were immunized with multipoint subcutaneous injection on the back at a dose of 300 μg per rabbit for primary immunization. For booster immunizations, the protein was emulsified with Freund’s incomplete adjuvant at a 1 : 1 volume ratio and administered on days 14, 28, and 42 after the primary immunization at a dose of 150 μg per rabbit. Serum was sampled from the rabbit ear vein on day 7 after the final immunization to yield anti-rEgPLK2 polyclonal antibodies. Antibody titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and antibody specificity was verified by Western blotting. The tissue localization of the EgPLK2 protein was detected in E. granulosus protoscoleces and adult worms using immunofluorescence assay (IFA). Eighteen 6- to 8-week-old female SPF-grade BALB/c mice were randomly divided into three groups, including the blank control group, rEgPLK2-ISA immunization group, and PBS-ISA adjuvant control group, of 6 mice each group. Mice in the rEgPLK2-ISA immunization group and PBSISA group received three primary immunizations via intramuscular injection, and animals in the rEgPLK2-ISA immunization group was inoculated with immunogens prepared by emulsifying rEgPLK2 protein with ISA 201 adjuvant at a 1 : 1 volume ratio (6 μg per mouse), while mice in the PBS-ISA adjuvant control group received an equal volume of PBS emulsified with ISA adjuvant at a 1 : 1 volume ratio. A fourth booster immunization was administered via intraperitoneal injection. Mice in the rEgPLK2-ISA immunization group received a booster immunization with 8 μg of rEgPLK2 protein per mouse, and animals in the PBS-ISA group received an equal volume of PBS, with immunizations given at 2-week intervals. Mice in the blank control group were given no treatment, and housed under standard conditions. Tail vein blood was collected from all mice 7 days after the final immunization, and levels of specific anti-rEgPLK2 IgG antibody and its subclasses (IgG1, IgG2a, IgG2b, IgG3) were measured by indirect ELISA. E. granulosus infection was modelled in mice through injection with 1 000 E. granulosus protoscoleces via intrahepatic portal vein in the rEgPLK2-ISA immunization group and PBS-ISA adjuvant control group 2 weeks after the last immunization. All mice were sacrificed and dissected. The number of cysts was counted in mouse livers, and the cyst reduction rate was calculated. Liver tissues were processed for paraffin sectioning and stained with hematoxylin and eosin (HE), and histopathological changes were examined under a light microscope. Results Sequence analysis revealed that EgPLK2 shared a high amino acid sequence homology with E. multilocularis PLK2 (EmPLK2) and contained the typical domains of the Polo-like kinase family, including the serine/threonine protein kinase catalytic domain (STKc) and Polo-box. The IPTG-induced rEgPLK2 protein was mainly expressed in the form of inclusion bodies, and the purified rEgPLK2 protein showed a relative molecular mass of approximately 70 kDa. The prepared rabbit anti-rEgPLK2 polyclonal antibody had a titer of 1 : 256 000, and Western blotting assay showed that this anti-body specifically recognized the rEgPLK2 protein with a relative molecular mass of approximately 70 kDa. Immunofluorescence assay showed that the EgPLK2 protein was localized in the excretory bladder and rostellum of E. granulosus protoscoleces, as well as the tegument, suckers, and inter-proglottid junctions of adult worms. Immunoprotective assay showed that the serum levels of specific anti-rEgPLK2 IgG, IgG1, IgG2a, and IgG2b antibodies were 2.92 ± 0.49, 0.33 ± 0.10, 0.31 (0.36), and 3.12 (1.73) in mice in the rEgPLK2-ISA immunization group, which were all significantly higher than those in the PBS-ISA adjuvant control group (0.14 ± 0.04, 0.07 ± 0.01, 0.12 ± 0.04, and 0.11 ± 0.04, respectively) (t = 19.28 and 8.46, Z = 3.75 and 4.15; all P values < 0.001); however, there was no significant difference in the serum anti-IgG3 antibody level between the rEgPLK2-ISA immunization group and the PBS-ISA adjuvant control group [0.07 (0.01) vs. 0.073 (0.07); Z = 0.69, P > 0.05)]. In the mouse model of E. granulosus infections, the area of hepatic lesions was reduced and the inflammatory infiltration was alleviated in the rEgPLK2-ISA immunization group than in the PBS-ISA adjuvant control group, and the number of hepatic cysts was higher in the PBS-ISA adjuvant control group than in the rEgPLK2-ISA immunization group [8.00 (2.00) vs. 1.00 (0.75); Z = −2.93, P < 0.01], with a cyst reduction rate of 80.40%. Indirect ELISA assay measured higher serum levels of specific anti-rEgPLK2 IgG (3.28 ± 0.48 vs. 0.11 ± 0.04; t = 15.86, P < 0.01), IgG1 (0.29 ± 0.02 vs. 0.09 ± 0.01; t = 15.67, P < 0.01), IgG2a [3.71 (1.09) vs. 0.08 (0.03); Z = 2.88, P < 0.01], and IgG2b antibodies [3.34 (1.01) vs. 0.08 (0.03); Z = 2.88, P < 0.01] in the rEgPLK2-ISA immunization group than in the PBS-ISA adjuvant control group, and there was no significant difference in the serum level of the specific anti-rEgPLK2 IgG3 antibody between the rEgPLK2-ISA immunization group and the PBS-ISA adjuvant control group (0.07 ± 0.01 vs. 0.07 ± 0.01; t = 1.29, P > 0.05). Conclusions The prokaryotic expression system has been successfully constructed for the EgPLK2 gene and the anti-rEgPLK2 polyclonal antibody has been obtained. The rEgPLK2 protein exhibits a high immunogenicity, and is effective to protect against E. granulosus infection, and inhibits cyst development, which is a promising candidate vaccine target against cystic echinococcosis.

Objective To detect the positive rates of antibodies against avian influenza virus (AIV) subtypes H5, H6, H7 and H9 among people in poultry occupations in Guangdong province and to analyze the transmission of various subtypes of AIV from poultry to human contacts for the prevention and control of novel AIV infection in human beings.Methods Serum specimens were collected from 1066 peo-ple in poultry occupations ( occupational group) and 205 people not in poultry occupations ( non-occupational group) in 10 cities of Guangdong province.The inactivated AIV strains, isolated from poultry or environment of Guangdong province, were used as antigens to detect antibodies against AIV subtypes H5, H6, H7 and H9 by using the hemagglutination inhibition ( HI) assay.Results The positive rates of antibodies against AIV subtypes H5, H6, H7 and H9 carried by people from the occupational group were respectively 0.44%, 0%, 0.30%and 0.30%in 2013 and 1.08%, 0.0%, 0.0%and 0.27%in 2014.Only the anti-H9 anti-bodies were detected in serum samples collected form people in the non-occupational group in 2013 with a positive rate of 0.95%.No significant differences with the positive rates of anti-AIV antibodies were found between the occupational group and the non-occupational group.However, the geometric mean titer ( GMT) of anti-AVI antibodies in people from the occupational group was higher than that of the non-occupational group.Conclusion Although a grand spread of AIV from avian to human is not likely to happen yet, con-tacting with poultry is the risk factor for AIV infection in Guangdong population.A long-term surveillance of anti-AIV antibodies in serum should be strengthened among people in poultry occupations for the timely pre-vention and control of novel AIV outbreak.

Objective To evaluate the efficacy and safty of antiviral treatment for chronic hepatitis B ( CHB ) patients in second trimester of pregnancy.Methods Seventy-nine CHB patients in second trimester of pregnancy were collected from Hangzhou First People’ s Hospital and Xixi Hospital of Hangzhou during January 2010 to December 2013.Patients were divided into antiviral treatment group ( n=47) and the control group (n=32) according to their own wishes.Patients in antiviral treatment group were given lamivudine or telbivudine treatment plus hepatoprotective medication, while those in control group were only given hepatoprotective medication.All pregnant women were observed for 12 weeks after childbirth and the neonates were followed-up for 6 months after birth.The liver function, HBV DNA loads, HBV serological markers were measured;adverse effects during pregnancy, blocking rates of mother-to-child transmission and the growth of neonates were documented.t test or Chi-square test was used for statistical analysis.Results Alanine aminotransferase ( ALT) normalization rate and HBV DNA negative rate in antiviral treatment group before childbirth were 88.6%(39/44) and 84.1%(37/44) , while those in the control group were 60.0%(18/30) and 0 (χ2 =8.27 and 50.46, P<0.05).After 12 weeks of childbirth, ALT normalization rate and HBV DNA negative rate in antiviral treatment group were both 100.0% (44/44), which were higher than those in control group (90.0%and 0) (χ2 =4.59 and 74.00, P<0.05).HBeAg seroconversion was observed in 1 (2.8%) and 4 (11.1%) patients in antiviral treatment group before and 12 weeks after childbirth, but it was not observed in the control group.The difference in HBeAg seroconversion rate bwteen two groups was not of statistical significance (P>0.05).No patient in antiviral treatment group terminated pregnancy due to abnormal liver function or adverse effect of drugs, while 2 out of 30 patients (6.7%) in the control group terminated the pregnancy, but the difference between two groups was not of statistical significance (χ2 =1.01, P >0.05).Mother-to-child transmission of HBV was successfully blocked in antiviral treatment group, while 3 cases (11.5%) in control group were failed (χ2 =5.19, P<0.05).No significant differences in gestational age, body mass, body length, Apgar score between two groups were observed (t=0.65, 0.84, 0.25 and 0.77, P>0.05).Conclusion Antiviral treatment can improve liver function, inhibit HBV replication and reduce the risk of mother-to-child transmission, and is safe for CHB patients in second trimester of pregnancy.

Alzheimer′s disease ( AD) is a type of neurodegener-ative disease. Recent studies indicate that neuronal degeneration and loss triggered by tau, APP and Aβare the probable risks for AD. Neurofibrillary tangles are formed after tau truncated by ac-tivated caspases and subsequently induced tau aggregates, which causes neuronal degeneration and loss. In addition, caspases are crucial components in the biological functioning in the apoptosis pathways. Apoptosis pathway involves activation of upstream ini-tiator caspase-8 and downstream executor caspase-3/-6/-7. After the actions of β- and γ-secretase, APP transforms into sAPPβand Aβ40/42 . Aggregated Aβ42 can activate apoptosis pathway through DR4/5 interaction. C-APP is truncated into C31 frag-ments by caspases and cell apoptosis is facilitated. N-APP, a product of sAPPβhydrolysis, can promote the abnormal develop-ment of neurons mediated by DR6. Caspase activates γ-secre-tase-activating protein to regulate activity ofγ-secretase, and the production of C31 and Aβ40/42 , which, then, causes the occur-rence of AD. This brief review summarizes the specific roles of caspases and the concerning apoptosis pathways on the mecha-nisms of neuronal degeneration and loss, and how they impact the occurrence of AD in the hope of uncovering additional poten-tial therapeutic targets that can be employed in drug development and clinical therapy for AD.

Animals ; Asian Continental Ancestry Group ; Chickens ; China ; epidemiology ; Cytokines ; metabolism ; Genetic Variation ; Genotype ; Humans ; Influenza A Virus, H7N9 Subtype ; classification ; genetics ; pathogenicity ; Influenza A Virus, H9N2 Subtype ; genetics ; Influenza in Birds ; transmission ; virology ; Influenza, Human ; ethnology ; transmission ; virology ; Mice, Inbred BALB C ; Molecular Sequence Data ; Orthomyxoviridae Infections ; metabolism ; mortality ; virology ; Phylogeny ; Seasons ; Survival Rate ; Virulence ; genetics