Fluorescence resonance energy transfer (FRET) is the energy transfer from an activated donor fluorophore to a receiving fluorophore. The efficiency of the energy transfer is the function of the distance between the two fluorophores, and is very sensitive to the distance. Its effective response distance is between 1~10 nm, thus it can be used to measure the distance between atoms or molecules. The feature of FRET is very useful in researches on conformational changes, interaction between macromolecules and signal transductions within live cells, and FRET has become a powerful tool in biomedical investigations. However, biological processes often involve interactions between more than two macromolecules, and FRET using two color fluorophores cannot meet the research demand. Recently, two research groups made breakthrough, establishing a novel FRET technique using three color fluorophores based on confocol microscopy and flow cytometry, respectively. The invention will significantly advance researches in biological and related fields.

Objective To explore the mechanism of cell apoptosis of immortalized human keratinocytes (HaCaT cells) and protein expression related to this process after long term exposure to sodium arsenite (NaAsO2,1.0 μmol/L).Methods Malignant transformation model was set up through long-term exposure of HaCaT cells to 1.0 μmol/L NaAsO2.Cell passage for 0,1,7,14,21,28 and 35 generations in the process of malignant transformation were collected for measurement of cell apoptosis rate by flow cytometry,and apoptosis related proteins by Western blotting,including activation of cysteine protease 3,8 (cleaved-caspase-3,8),C/EBP homologous protein (CHOP),B-cell leukemia/lymphoma 2 (Bcl-2),and Bcl-2 associated X protein (Bax).Results Along with the arsenite treatment,the apoptosis levels were significantly decreased (F =26.770,all P ＜ 0.05),the apoptosis levels (0.307 ± 0.049,0.213 ± 0.055,0.163 ± 0.057,0.147 ± 0.035,0.053 ± 0.012) of the 7th,14th,21st,28th and 35thgenerations of cells after arsenite treatment were lower than that of control group of the 0th generation (0.393 ±0.021,all P ＜ 0.05).Compared between generations,there were statistical differences of the protein expression levels of cleaved-caspase-3,Chop,Bax and Bcl-2 in arsenite group (cleaved-caspase-3:1.000 ± 0.000,1.030 ± 0.027,1.104 ± 0.069,1.016 ± 0.087,0.838 ± 0.075,0.753 ± 0.082,0.677 ± 0.073;Chop:1.000 ± 0.000,1.059 ± 0.018,0.934 ± 0.095,0.976 ± 0.216,0.793 ± 0.136,0.651 ± 0.042,0.564 ± 0.056;Bax:1.000 ± 0.000,1.069 ± 0.037,1.028 ± 0.042,0.954 ± 0.118,0.641 ± 0.135,0.531 ± 0.132,0.429 ± 0.085;Bcl-2:1.000 ± 0.000,1.072 ± 0.023,1.249 ± 0.134,1.334 ± 0.143,1.633 ± 0.221,1.507 ± 0.152,1.461 ± 0.145,F =7.730,7.355,27.802,12.438,all P ＜ 0.05),compared with control group of the 0th generation (1.000 ± 0.000) and the same generation control group (1.000 ± 0.000),after the 21st generation,the differences were statistically significant (all P ＜ 0.05),while there was no difference of the protein expression levels of cleaved-caspase-8 (F =0.832,P ＞ 0.05).Conclusion In the process of malignant transformation,the apoptosis levels of HaCaT cells are inhibited after long term sodium arsenite exposure through mitochondria and endoplasmic reticulum (ER) stress signaling pathways.

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology

Objective: To explore the relationship between fathers′ nursing time and maternal parenting stress of children with autism spectrum disorder(ASD). Method: Mothers of 98 ASD children who were first diagnosed in the department of Child Health Care, Children′s Hospital of Fudan University during June 2015 to January 2016 were included in the ASD group, with mothers of 92 typical children from a Community Maternal and Child Health Hospital and a kindergarten in the control group. The evaluation of parenting stress, parents′ nursing time and other related factors were cross-sectionally analyzed. Interview was conducted with the following tools: Parental Stress Index-Short Form(PSI-SF)for maternal parenting stress, and self-made General Parenting Information Questionnaire for nursing time of both parents and other related factors. The relationships were analyzed by Multiple Linear Regression analysis and Wilcoxon Rank-Sum test. Result: Maternal parenting stress of ASD children had a significant negative correlation with father′s nursing time in total score of parenting stress, PCDI domain and PD domain (t=-2.76, -2.98, -2.79; P=0.007, 0.004, 0.006), within which PD domain also included family annual income and mothers′ nursing time (R²=0.22, 0.24, 0.25); while no such correlation was found in control group in terms of father′s nursing time(P=0.22, 0.42, 0.06). Wilcoxon Rank-Sum test showed that in 62 (63.3%) double-income ASD families and 72(78.3%) double-income typical families, there were significant differences between ASD fathers′ and ASD mothers′and typical fathers′nursing time(2.0(0.5, 2.1)vs. 3.5(2.4, 6.0)vs. 3.0(2.0, 4.7)h, t=-86.32、-49.65, all P<0.01). Conclusion Lack of fathers′ involvements was common in ASD children′s families. Increasing these fathers′ nursing time, as well as their enthusiasm and initiative in the family intervention could relieve maternal parenting stress and improve the intervention pattern of ASD children.

Neural development is a complex process.Its development is affected by a variety of molecular signals such as signals from intestinal microorganisms.Recent animal research showed that microorganisms play an important role in neurogenesis,myelination,microglial cell maturation and formation of blood-brain barrier,and the regulation of animal behavior from many aspects.This article reviewed the impact of prenatal and postnatal gut microbes on the development and function of the nervous system,and the relationship between gut microbes and autism spectrum disorder as well.

Objective:To investigate the correlation between liver stiffness and histopathological changes in a rat model of acute hepatitis using virtual touch tissue imaging quantification (VTIQ) technology.Methods:A total of 100 SPF-grade SD rats were randomly divided into 3 groups: control ( n=30), low-dose ( n=35), and high-dose ( n=35) groups. Acute hepatitis models were induced in the low-dose and high-dose groups using 400 mg/kg and 600 mg/kg of Thioacetamide (TAA), respectively. Liver stiffness parameters of the right median lobe and right lobe were measured using VTIQ technology, Mean-H and Mean-L represent the liver lobes with higher and lower liver stiffness measurments, respectively, while Mean represent the average of the measurements from both liver lobes. Comparative analyses of liver stiffness parameters were performed across three groups and between the two lobes of the liver. The correlations between the Mean values of liver stiffness and semi-quantitative histopathological data were investigated. Ten rats were randomly selected from each of the 3 groups to test the repeatability of VTIQ values before and after euthanasia with intraperitoneal anesthesia. Subsequently, 10 rats after euthanasia from each 3 group were randomly chosen to assess the repeatability of VTIQ measurements for inter-observer and intra-observer variabilities. Results:VTIQ results showed statistically significant differences in Mean, Mean-H, and Mean-L among the 3 groups (all P<0.01). The high-dose group had higher measurements compared to the low-dose and control groups, with significant intergroup differences (all P<0.01). Significant differences in Mean-H and Mean-L were observed between the two liver lobes in both low and high-dose groups (all P<0.01). The Mean value showed significant positive correlations with semi-quantitative histopathological data of hepatocellular edema, periportal inflammatory cell infiltration, macrophage proliferation, and bile duct proliferation ( r=0.391, 0.648, 0.577, 0.542; all P<0.01). Multivariate linear regression analysis indicated that hepatocellular edema, eosinophilic change, and bile duct proliferation significantly and positively predicted the Mean value (β=-0.278, -0.196, -0.333; all P<0.05). There were no significant differences of VTIQ measurements befor and after euthanasia (all P>0.05), with repeatability coefficients of 0.166, 0.182, 0.185 for Mean, Mean-H, and Mean-L, respectively. Post-euthanasia, inter- and intra-observer VTIQ differences remained non-significant (all P>0.05), with Mean, Mean-H, Mean-L coefficients of 0.114, 0.194, 0.165 and 0.206, 0.322, 0.268, respectively. Conclusions:VTIQ technology demonstrates potential clinical value in assessing a rat model of acute hepatitis, offering a new perspective for non-invasive evaluation of acute hepatitis. However, its clinical application requires further validation.

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Animals ; Antibody Affinity ; Cells, Cultured ; Cytidine Deaminase ; genetics ; metabolism ; HEK293 Cells ; Humans ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Mutation ; Single-Chain Antibodies ; chemistry ; genetics ; immunology ; Somatic Hypermutation, Immunoglobulin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Objective: To understand the infection status, characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli (STEC) in animal feces in Shandong Province. Methods: From 2015 to 2016, convient sampling method was used to collect 1 022 fresh feces of animals in Weishan county and Laizhou city, and 24 non-O157 STEC were isolated. The serotypes of non-O157 STEC strains were confirmed through serum agglutination test. The susceptibility was explored through the antimicrobial sensitivity experiments. ESBLs activity was confirmed by double-disc diffusion. PCR method was used to detect the resistance genes. PFGE typing was operated to assess the relatedness and variability of the strains. The multi-locus sequence typing (MLST) was adopted to get the allelic profile and ST sequence of strains. Analysis was made on the evolutionary relationship between different ST groups was made through CLC Sequence Viewer and Counting Express. Results: A total of 24 non-O157 STEC were isolated from animal feces. 23 strains were from pig feces, and 1 strain was from cow feces, and the serotypes were more dispersed. All of the 24 strains carried stx2 genes. The highest resistance rate was sulfamethoxazole(22 strains), the mount of cotrimoxazole and nalidixic acid was 18 strains, chloramphenicol was 13 strains, tetracycline was 19, and there was a phenomenon of multiple drug resistance. The drug resistance spectrum was sulfamethoxazole tetracycline-compound novammin-naphthidine-chloramphenicol. All strains were sensitive to cefepime and imipenem. The ESBLs confirmatory test showed that 4 strains of non O157 STEC produced beta lactamase. PCR detected 7 resistance genes, and 4 tetracycline resistance genes (Tet A, Tet B, tetC and tetD) were detected. The beta lactamase resistance genes (blaSHV-1, bla CTX-M, bla TEM) were all negative. 24 strains were divided into 15 PFGE types, and their clustering results were more dispersed and no dominant PFGE type. There were 11 kinds of MLST types, most of them are ST540 and ST5133 types, each of which was 4 strains, and clustered into 1 MLST genomes. Conclusion The serotypes of non-O157 STEC in animal feces O157 STEC were dispersed, and the resistant rate to common antibiotic was high. MLST typing results presents obvious polymorphism. Surveillance and manage ment of these strains should be strengthened.

Objective To investigate the effect of berberine chloride on autophagy and β-secretase (BACE) level in mice with traumatic brain injury (TBI).Methods Eighteen female healthy C57BL/6 mice were randomly divided into three groups:control group,model group and berberine group (n=6).TBI models in the later two groups were established by a weight-drop hitting device and mice in berberine group were administered intragastricly with berberine chloride (50 mg/kg) once daily for 21 d.Immunofluorescent staining were used to assess LC3 and BACE expressions in ipsilateral cortex or thalamus,and then,their mean fluorescence intensities were calculated and compared among these three groups.Results LC3 expression in the ipsilateral cortex and thalamus and BACE expression in the ipsilateral cortex (0.02±0.01,0.06±0.02 and 0.04±0.01 in the control group,model group and berberine group) showed significant difference among the three groups (P＜0.05):LC3 expression in ipsilateral cortex and thalamus and BACE expression in the ipsilateral cortex of the model group were significantly increased as compared with those of the control group (P＜0.05);the LC3 expression in the ipsilateral cortex and thalamus and BACE expression in the ipsilateral cortex of the model group were significantly decreased as compared with those of the berberine group (P＜0.05).Conclusion Autophagy is over-activated in the ipsolateral cortex and thalamus and BACE is over-activated in the ipsolateral cortex after TBI,and these changes are significantly suppressed by berberine chloride.

Objective:To study the safety factors of Fuyanyu mixture. Methods: The contents of antiseptic and residual ethanol were determined by HPLC and GC, respectively, and those of heavy metals and harmful elements were detected by ICP-MS. Results:The contents of benzoic acid in 6 batches of samples were less than 0. 3%. In two thirds of samples, ethanol residue was more than 0. 5%. The total mercury exceeded 15μg/day in one of the samples, and the contents of Pb, Ge, Gr, As, Ni and Cu met the limits described in USP<232> in all of the last samples. Conclusion: The methods are accurate and reliable. It is urgent to improve the preparation process so as to reduce the residual amount of ethanol according to the detection results. It is recommended to increase the testing items that affect the safety of the preparation so as to control the preparation quality strictly.