OBJECTIVE To study the immunotherapeutic effect of chimeric virus-like particles(VLPs) based on hepatitis E virus(HEV) against human papillomavirus type 16(HPV 16) tumor. METHODS HPV16 E7 was inserted into the p239 protein of HEV to form the recombinant chimeric protein p239-HPV16 E7. The constructed recombinant protein was expressed by Escherichia coli, purified, and then refolded, and the protein was detected by electron microscopy and dynamic light scattering to confirm size and shape. Then, the C57B/L mice were immunized with the protein grain, and the lymphocyte differentiation of mouse spleen was detected by flow cytometry and enzyme-linked immune spot immunoassay; in addition, TC-1 tumor cells were used to construct tumor models in C57B/L mice to evaluate the anti-tumor immune effect of protein particles in mice. RESULTS After refold in vitro, the structure of chimeric protein was observed under electron microscopy, and the size of particle was 22.80 nm. The obtained protein particles induced favorable specific cellular immune response in C57B/L mice. Compared with the control group, the proportions of CD3+/CD4+ and CD3+/CD8+ in spleen lymphocytes of experimental groups were significantly different(P＜0.05), and effector T cells secreting IFN-γ interferon were also increased remarkably. At the same time, the obtained protein particles could effectively inhibit the growth of tumor cells in TC-1 tumor-bearing mice, and the mice did not die during the experimental period, while the tumors in the control mice grew rapidly and all died after 6 weeks. CONCLUSION Chimeric protein p239-HPV16E7 which was expressed in prokaryotes can form virus-like particles and effectively induce anti-tumor immunity against HPV16.

Objective:To evaluate the effect of eye-covering pretreatment on acute delirium in ophthalmology preschool-age children who underwent binocular and monocular surgery by general anesthesia.Methods:The 300 preschool-age children who underwent general anesthesia of elective ophthalmic surgery in the Second Affiliated Hospital, Zhejiang University School of Medicine, from August 2019 to February 2021 were selected as the research object. They were divided into control group and blindfold group with 150 cases each by random number-table. Children in the control group received regular education on cartoon animation videos before surgery; children in the blindfold group received eye-covering pretreatment on the basis of cartoon animation videos(monocular surgery with monocular cover, binocular surgery with binocular cover). The Modified Yale Preoperative Anxiety Scale (m-YPAS) , the Nursing Delirium Screening Scale(NU-DESC), the incidence rate of delirium and the score of postoperative nursing difficulty were compared between two groups.Results:The 271 cases were completed in this study, including 129 cases(monocular surgery 66 cases, binocular surgery 63 cases) in the blindfold group and 142 cases (monocular surgery 73 cases, binocular surgery 69 cases) in the control group. The preoperative m-YPAS score, the postoperative NU-DESC score, the incidence rate of acute delirium and postoperative nursing care difficulty score of monocular surgery in the blindfold group , monocular surgery was (40.28 ± 15.02) points, 1.00 (0.00, 2.00) points, 27.3%(18/66), 1.00 (1.00, 2.00) points,and binocular surgery was (41.69 ± 16.35) points, 1.00 (0.00, 2.00), 39.7%(25/63), 1.00(1.00, 2.00); in the control group, monocular surgery was (46.28 ± 15.76) points, 2.00 (1.00, 3.00) points, 67.1% (49/73), 2.00 (1.00, 3.00) points, and binocular surgery was (47.77 ± 14.82) points, 3.00 (2.00, 4.00) points, 82.6% (57/69) and 2.00 (1.50, 3.00) points respectively. The difference between the two groups was statistically significant ( t= -2.29, -2.24, Z values were -5.74 - -2.95, χ2= 32.94, 25.78, all P<0.05). The preoperative m-YPAS score, the postoperative NU-DESC score, the incidence rate of acute delirium and postoperative nursing care difficulty score of monocular surgery patients in the blindfold group had no significantly statistical difference with that of binocular surgery patient (all P>0.05) . Conclusions:Monocular/ binocular eye-covering pretreatment can effectively decrease the preoperative m-YPAS score, the postoperative NU-DESC score, incidence rate of acute delirium and the postoperative nursing care difficulty in preschool-age children who underwent general anesthesia both monocular or binocular surgery. There was no difference in the application effect of monocular or binocular surgery.

Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)‍-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.
Animals ; Apoptosis ; Chlorocebus aethiops ; Gene Expression Profiling ; Humans ; Mammals/genetics* ; Toxoplasma/genetics* ; Toxoplasmosis/pathology* ; Vero Cells ; ras GTPase-Activating Proteins/genetics*

Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.

We discussed the influence of liquid nitrogen cryopreservation to survive capability of Babesia microti standard strain.The whole blood of mice infected with Babesia microti was put in liquid nitrogen to cryopreservation for 1 month,3 months,6 months,9 months,the whole blood was get out respectively and recovery at room temperature,and infected 3 mice respectively,100 μL/ mouse (the first generation after redissolution,the experiment group).In the same time,3 mice were also infected with Babesia microti as the animal conservation control group.When the infection rate was at a high level,the whole blood of the experiment group mice were injected into 3 normal BALB/c mice (the second generation after redissolution),to observe the changes of the Babesia microti form and proliferation situation,and also to observe the infection rate of the first and the second generation after redissolution in different conserving time.Compared with Babesia microti of animal subcultivation,the form of Babesia microti of liquid nitrogen cryopreservation changed a little.Small trophozoites,annular trophozoites,schizont and immature and mature merozoite and other form can also be seen.Compared with Babesia microti of animal subcultivation,the first time to see the worms and the time attaining to the high infection level were 1 to 2 days later,but for the second generation after redissolution,it is the same.There was no significant difference in different conserving time of 1,3,6,9 months.The influence of liquid nitrogen cryopreservation to survive capability and worm form of Babesia microti is a little,so liquid nitrogen cryopreservation can be a better way to conserving Babesia microti.

Objective To analyze the fractional proteins and immunoreactivity of the soluble antigens from Babesia microti (B.microti),and find the candidate antigens for diagnosis with high sensitivity and specificity.Methods BALB/c mice were inoculated with B.microti-infected red blood cells by intraperitoneal injection.The B.microti were collected from the infected red blood cells when the infection rate reached its peak (infection rate ＞70％),then the soluble antigens were extracted by repeated freezing-thawing and ultrasonic method.The mice sera before and after the infection with B.microti for 7,14,21,28,35,42,49 and 56 days were also collected.The polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein components of the soluble antigens of B.microti and the Western blot was used to analyze the immunoreactivity of the soluble antigens with the pooled mice sera before and after the infection.The specific positive protein bands were identified by Liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS),and the amino acid sequences of the proteins were analyzed by bioinformatics tools.Results The results from SDS-PAGE analysis indicated that the soluble antigens of B.microti showed distinct protein bands with the range between 12 and 185 × 103 (kDa,relative molecular mass,Mr),among which 9 main bands and 12 minor bands were obtained.In the Western blot analysis,the protein bands with Mr at 40 and 45 kDa could be recognized by pooled mice sera 7 days after infection;the protein bands with Mr at 40,45,54 and 95 kDa could be recognized by pooled mice sera 14 days after infection;the protein bands with Mr at 27,40,45,54,95 and 110 kDa could be recognized by pooled mice sera 21 days after infection.While,the protein bands with Mr at 27,40,45,54,95,1 10 kDa and other weak-reactive bands were recognized by pooled mice sera 28-56 days after infection,and the reaction became stronger with the infection continued.There were 336 proteins,including surface antigen,heat shock protein 70,seroreactive antigen,Eta subunit of chaperonin containing t-complex polypeptide 1 and unnamed protein products,were identified as the components of soluble antigens after mass spectrometry and sequence analysis.Conclusion The 40,45 and 54 kDa protein components from the soluble antigens of B.microti may be ideal candidate antigens for diagnosis,andtheir potential applications in diagnosing of human babesiosis deserve further study.

Nanobodies are derived from the variable domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of Camelidae. They are the smallest antibody fragments capable to bind antigens. With the characteristics of their increased solubility, increased domain stabilities, nanomolar affinities, easy crossing the blood-brain barrier, easy generation, engineering, optimization and tailoring, easy humanization, nanobodies have extensive application prospects in diagnosis and detection. Although nanobody has demonstrated tremendous success, a number of practical challenges limit its broader applications in disease diagnosis and detection, including construction of a phage library and selection of nanobody fragments with high affinity and immunogold labeling technique. Here, we review several recent findings on the use of nanobodies in molecular diagnostics and suggest some practical strategies in resolving the current challenges in this attractive research area, particularly to optimize the affinity, solubility, humanization of nanobodies.
Humans ; Immunoglobulin Heavy Chains ; chemistry ; Single-Domain Antibodies ; chemistry ; drug effects

Objective To establish A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. Methods The characteristics of A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentra-tion of A1E3-HRP. Under the optimal conditions,the serum samples of 20 acute schistosomiasis cases,46 chronic schistosomiasis cases,and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy-two antibody positive serum sam-ples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit,to evaluate the detection effects of this method. Results The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 contained a clear heavy chain with molecular weight of 88 000 and 52 000 respectively and had the same light chain with molecular weight of 20 000;while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schis-tosomiasis cases. The SEA-based ELISA demonstrated the titers of B1C4 and A1E3 were 1∶105 and 1∶30 000,respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA for detecting the circulating antigen were 45.8%and 43.1%respectively,and there was no significant difference between the results of the two methods. Conclusion A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.

Objective To investigate characteristics of glucose metabolism of non-obese and obese women with polycystie ovary syndrome(PCOS).Methods From March 2008 to July 2011,118 PCOS patients were enrolled in this study and were divided into 53 cases (body mass index(BMI) ≥25 kg/m2) in obese group and 65 cases (BMI ＜ 25 kg/m2) in non-obese group.Participants' clinical data,glucose tolerance and insulin release test results were retrospectively reviewed.The prevalence of type 2 diabetes mellitus (T2DM),results of oral glucose tolerance test,impaired fasting glucose (IFG),impaired glucose tolerance (IGT) were compared between the two groups.Results (1) Blood glucose levels:at the time points of 30,60,120 and 180 minutes,the levels of fasting glucose were (5.2 ± 1.1),(8.5 ± 2.8),(8.1 ± 2.4),(6.3 ± 2.0) and (4.8 ± 1.5)mmol/L in non-obese group and(5.4 ±0.9),(9.1 ± 1.8),(9.3 ±0.6),(7.6 ± 1.0) and (5.4 ±0.8) mmol/L in obese group.Statistical difference was observed between obese and non-obese groups at each time point (t =-6.125,-4.005,-6.074,-6.751 and-4.512 respectively,P ＜0.01).(2) The level of insulin:at the time points of 30,60 and 120 min,the level of fasting insulin were (8 ± 4),(55 ± 21),(65 ± 14) and (45 ±18)mU/L in non-obese group and (13 ± 8),(85± 30),(105± 54) and (76 ± 46)mU/L in obese group.There were significant statistical difference between the two groups at each time point (t =-17.024,-12.540,-15.791 and-16.149 respectively,P ＜ 0.01).However,at the time point of 180 minutes,the level of insulin did not exhibit significant difference between obese and non-obese groups (P ＞ 0.05).(3) The prevalence of abnormal glucose metabolism:The rates of IGT were 13.85 ％ (9/65) in non-obese group and 24.53 ％ (13/53)in obese group,which also showed remarkable difference (x2 =18.446,P ＜ 0.01).The rates of T2DM were 1.54％ (1/65) in non-obese group and 7.55％ (4/53) in obese group,which reached significant difference (x2=16.005,P ＜ 0.01).Conclusion Abnormal glucose metabolism was observed more frequently in overweight or obese PCOS women.