Objective To develop a BP26 recombinant BCG (rBCG-BP26) vaccine,and to observe the effects of rBCG-BP26 on CD4+,CD8+ T cells in immunized mice.Methods The recombinant shuttle vector pMV261-Ag85B-BP26 was constructed by using traditional molecular biological technology.The recombinant strains were obtained by kanamycin resistance screening and PCR identification after electroporation.Western blotting was used to detect the expression of recombinant BP26 vaccine in immunized mice.Safety experiment was carried out in three different groups:the target experiment(rBCG-BP26) group,the positive control(BCG) group and the negative control(PBS) group,15 BALB/c mice in each group.Intradermal inoculations of 100 μl rBCG-BP26 [containing 106 colony forming units(CFU)],BCG,and PBS were carried out,respectively.Signs of mice in each group were observed.After immunization for 10,20,30,and 40 days,body weight was weighed,and tail blood was collected to observe the change of peripheral blood CD4+ and CD8+ T cells by flow cytometry.Results The rBCG-BP26 was successfully constructed.The expression of BP26 protein was detected in the liquid medium and the bacteria cells.The results of safety test analysis showed that there were no significant differences in signs and body weights(F=2.468,0.331,1.520,0.739,all P＞ 0.05),between PBS group[ (19.24 ± 0.54),(21.37 ± 0.66),(22.83 ± 0.62),(25.06 ± 0.37)g],BCG group[ (19.90 ± 0.02),(21.53 ± 1.57),(21.95 ± 0.55),(24.70 ± 0.39)g]and rBCG-BP26 group[ (19.16 ± 0.55 ),(20.89 ± 0.20),(22.15 ± 0.76),(24.60 ± 0.64)g].The results of flow cytometry showed that the percentages of CD4+ T cell level were lower in BCG group(26.70％,33.07％) and rBCG-BP26 group( 13.40％,26.70％) than that of the PBS group(33.85％,29.33％) and the values of CD4+/CD8+ T cells increased in rBCG-BP26 group (0.69％,1.27％,1.57％,1.70％ ) 10,20 and 30 days after immunization.Conclusions Recombinant BCG-BP26 vaccine strain can express brucella BP26 protein efficiently.Furthermore,its virulence is mild,and it can activate CD4+,CD8+ T cells in the body.It can be used as one of candidate vaccine strain against brucellosis.

Objective To construct the bp26 deletion mutant of Brucella vaccine strain M5-90 (M5-90Δbp26),to compare its immunogenicity with parental strain(M5-90),and to develope a new candidate vaccine strain of Brucella melitensis with reduced virulence that can be used to distinguish vaccinated livestock from infected animals.Methods Mutant vaccine strain of Brucella melitensis was constructed by conventional molecular biology techniques then the genetic stability of mutant M5-90Δbp26 was tested and its conventional bacteriological nature was identified; 1.0 × 109 CFU/2 ml doses of M5-90Δbp26 strain and the parental strain were used to vaccinate 3 sheep; sera were analyzed for reactivity against BP26 by Western blotting and for agglutination activity; to analyze the virulence of mutant and parental strain,mice were injected with 1.0 × 106,6.0 × 106 and 2.0 × 107 CFU/0.2 ml doses of M5-90 and M5-90Δbp26,respectively,and clinical symptoms were monitored and the death of mice was recorded.Results The M5-90Δbp26 was successfully generated and reversion was not observed in 15 generations.The size of PCR products was 629 bp while the parental strain was 1279 bp.The sequence analysis showed a 650 bp missing in M5-90Δbp26.The conventional bacteria identification tests confirmed that the mutant was depth variant strain,including mono-specific antiserum M type transformed into R,and the BK2 phage based splitting assay converted from the positive to the negative.Western blotting showed the purified BP26 protein was recognized by the serum against the parental strain while not by the serum against M5-90Δbp26 strain.Agglutination test showed the level of the serum antibody induced by M5-90Δbp26 strain(1:50) was significantly lower than that of serum induced by parental strain(＞ 1:800).Virulence test showed that M5-90Δbp26 strain was less virulent than parental strain.Conclusions M5-90Δbp26 has been successfully constructed.M5-90Δbp26 of Brucella melitensis has the characteristic of reduced virulence and has a potential as brucellosis candidate vaccine strain permitting serological discrimination between diseased and vaccinated livestock.

Objective To determine the difference of macrophage RAW264.7 apoptosis induced by Brucella melitensis virulent strain 16M and attenuated strain M5-90 and elucidate the regulatory role of caspases 3,8 and 9.Methods The best multiplicity of infection (MOI) was determined through kinetic analysis of Brucella melitensis strain 16M and M5-90 induced mouse macrophages apoptosis(bacterium ∶ cell =100 ∶ 1,50 ∶ 1,10 ∶1).The infection model was established using the best MOI =50 ∶ 1.The numbers of in vivo bacteria by colony formation units were calculated after macrophages were infected for different times,including 2,4,8,12,24 and 48 h,and the infected cells were collected.The ratios of apoptosis were detected and the regulation of caspases 3,8 and 9 in apoptosis pathway was elucidated by flow cytometry.Results The numbers of 16M in vivo bacteria were 105.4,104.8,105.8,106.5,108.0 and 109.0,respectively and of M5-90 were 106.1,106.2,106.4,106.3,106.1 and 105.0,respectively.The number of in vivo bacteria of 16M was significantly increased than that of M5-90 after infected for 24 h to 48 h.The ratios of apoptosis induced by 16M after infected for 2,4,8,12,24 and 48 h was (2.67 ± 0.09)％,(13.13 ± 0.30)％,(6.56 ± 0.42)％,(6.49 ± 0.28)％,(16.07 ± 0.86)％ and (24.23 ± 1.67)％,respectively,and by M5-90 was (3.62 ± 0.02)％,(32.01 ± 2.59)％,(17.58 ± 0.44)％,(16.09 ± 0.10)％,(62.53 ± 2.70)％ and (85.53 ± 0.15)％,respectively,and by control group was [(1.90 ± 0.20)％,(1.92 ±0.16)％,(1.99 ± 0.03)％,(2.48 ± 0.11)％,(3.56 ± 0.07)％,(5.26 ± 0.33)％].The differences were statistically between groups in same time.The Brucella melitensis vaccine strain M5-90 was more powerful than virulent strain 16M in respect of inducing macrophage apoptosis after infected for 24 to 48 h.Twenty-four hours after infection,the expression of caspases 3,8 and 9 was (1.47 ± 0.05)％,(1.52 ± 0.02)％ and (2.47 ± 0.12)％,respectively,in control group and the expression was (9.70 ± 0.46)％,(6.08 ± 0.56)％ and (35.08 ± 1.64)％,respectively,after infected for 24 h induced by M5-90.The expression of caspases 3,8 and 9 was significantly higher than that control group (P ＜ 0.01).Twenty-four hours after given caspases 3,8 and 9 inhibitor,apoptosis rate in control group was (66.72 ± 1.28)％,in M5-90 group was (22.58 ± 0.55)％,(53.15 ± 1.85)％ and (29.18 ± 0.23)％,respectively,and compared with control group,apoptosis rate of caspases 3,8 and 9 was significantly lower(P ＜ 0.01).Conclusions Apoptosis of macrophage can be induced by Brucella melitensis virulent vaccine strain 16M and attenuated strain M5-90.M5-90 is stronger than that of strain 16M.Caspases 3,8 and 9 can regulate macrophage apoptosis after M5-90 infection.

OBJECTIVETo compare the efficacy of different points combination in the treatment of menopausal insomnia.

METHODSNinety-six cases of menopausal insomnia were randomized into 3 groups, Xinshu (BL 15), Shenshu (BL 23), Sishencong (EX-HN 1), Shenmen (HT 7), Sanyinjiao (SP 6) were chosen in the restore interaction between the heart and the kidney group (group A, 32 cases); Zhaohai (KI 6), Jiaoxin (KI 8), Shenmai (BL 62), Pucan (BL 61) were chosen in the acupuncturing qiao mai group (group B, 32 cases); auricular Shenmen (TF4) and sensitive spot at the distribution area of auricular vagus nervus were chosen in the ear acupuncture group (group C, 32 cases). Six days made one session and the treatments were finished after 4 courses. The polysomnography (PSG) and Pittsburgh sleep quality index (PSQI) were employed before and after treatment to evaluate the alleviation of insomnia.

RESULTSThe parameters of the sleep latency (SL), rapid wave sleep latency (RL) and sleep efficiency (SE) were significantly improved in the three groups, and the differences were statistically significant (P < 0.05, P < 0.01). The SL and awaking time (AT) in group C [SL (401.08 +/- 16.54) min and AT (4.87 +/- 2.64) times] were significantly superior to those in the other two groups [SL (50.36 +/- 18.47) min, (54.87 +/- 20.92) min, AT (5.98 +/- 2.11) times, (6.13 +/- 3.04) times, all P < 0.05]. The S(3+4) (%) in group C was also significantly higher than those in the other two groups (both P < 0.05). It was indicated by PSQI that the sleep quality of group C (0.78 +/- 0.12) was significantly superior to that in group B (1.32 +/- 0.29), the total score and cured and markedly effective rate in group C [(4.34 +/- 1.43), 68.8% (22/32)] were superior to those in group A [(7.48 +/- 3.09), 53.1% (17/32), both P < 0.05].

CONCLUSIONEar acupuncture has a better curative effect than the restore interaction between the heart and the kidney group and acupuncturing qiao mai group, it is worth of being promoted.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Female ; Humans ; Menopause ; psychology ; Middle Aged ; Sleep ; Sleep Initiation and Maintenance Disorders ; therapy ; Treatment Outcome

Objective: To examine the effect of functional sports training on the development of spatial awareness in children aged 6-8 years old,to provide a reference to improve children s ability of spatial sense. Methods: A class of 125 children aged 6-8 years from first, second, and third grades of an elementary school in Zhengzhou City were conveniently selected by stratified random sampling, who were divided into the experimental group ( n =62) and the control group ( n =63) by random number tables. The experimental group received functional sports intervention for 8 weeks,3 times a week,20 min each time, and the control group received traditional sports game program. Results: After the intervention,the error values of depth perception, orientation perception, and space perception in the experimertal group of 6 and 7 year old children reduced by 1.98 cm, 2.88°, and 22.00 cm (6 year old children) and 1.61 cm, 2.34°, and 17.99 cm (7 year old children) compared with the control group, respectively. Compared with those in the control group of 8 year old after the intervention, and the differences were of statistical signifiance( t =-3.07, -2.94, -3.07 ; -3.25, -3.29, -3.15, P <0.01). There was no significant difference in the error values of depth perception, orientation perception and space perception between the experimental group and the control group after intervention ( P >0.05). In the experimental group, the error values of depth perception, orientation perception and space perception reduced by 2.30 cm, 3.88°, 28.05 cm (6 year old children), 2.16 cm, 2.15°, 17.45 cm (7 year old children) and 1.16 cm, 1.81°, 9.10 cm (8 year old children) in children aged 6-8 years after intervention, significant improvement were observed compared with before intervention ( t = 8.50 , 9.04, 7.35; 7.39, 10.30, 11.05; 4.67, 4.46, 14.14, P <0.01). Compared with before the intervention, children aged 6-8 in the control group only had significant differences in space perception( t =4.13, 6.71, 8.93, P <0.01). Conclusion Functional sports games can improve depth perception, orientation perception and spatial perception for children aged 6-8 years. It can be integrated into children s daily activities to play a positive role in promoting the healthy growth of children.

OBJECTIVETo detect the expression of Per2 in non-small cell lung cancer (NSCLC), and analyze its clinical significance.

METHODSThe expression of Per2 was determined in 60 NSCLC and 20 normal lung tissues by immunohistochemical assay, and the relationship between Per2 expression and clinicopathological features was analyzed.

RESULTSThe positive expression rates of Per2 in NSCLC and normal lung tissues were 71.7% and 95.0%, respectively (P < 0.05). The expression of Per2 in NSCLC was correlated with pathological differentiation and TNM stage (P < 0.05).

CONCLUSIONThe expression of Per2 in NSCLC is decreased. The negative expression of Per2 may contribute to the development and invasion in NSCLC.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; surgery ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; pathology ; surgery ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Grading ; Neoplasm Staging ; Period Circadian Proteins ; metabolism ; Smoking

Objective: To measure and analyze the bone volume and thickness of mandibular angle area by computer-assisted CT. Methods: From January 2010 to January 2016, 94 female patients with mandibular angle hypertrophy and bilateral mandibular angle osteotomy were selected. Twenty female patients with non-mandibular surgery were selected as control group (patient examined after nasal augmentation surgery), aged 18 to 37 years, with an average of 27.5 years. Three dimensional CT data of maxillofacial bone of patients were collected. The three dimensional reconstruction and measurement software Mimics 16.0 was used. A three-dimensional model of mandibular angle region was established. The thickness and volume of mandibular angle were measured and analyzed. The total difference between hypertrophic group and normal group was compared by two independent t-test. Results: The thickness of mandibular angle area and mandibular angle area in hypertrophic group were (9.39±1.48) mm and (3 065.25±720.1) mm³ respectively, and the thickness of mandibular angle area and mandibular angle region in control group were (7.48±0.92) mm and (1 276.57±265) mm^3, respectively. There was a significant difference between the two groups (P < 0.05). Conclusions Based on CT scanning data, a method of measuring the bone volume and thickness of mandibular angle region is established, which is helpful to the three-dimensional measurement and analysis of mandibular shape in patients with mandibular angle hypertrophy.

Two new labdane diterpenoids were isolated from 95% ethanol extract of the leaves of Callicarpa formosana Rolfe by using silica gel column, MCI column, ODS column and HPLC. Their structures were elucidated by HR-ESI-MS, NMR and ECD spectral data. All of them are new compounds, named 13E-6β-hydroxylabda-8(17),13-dien-15-oic acid (1) and 13E-7α-hydroxylabda-8(17),13-dien-15-oic acid (2). Compounds 1 and 2 were tested for antioxidant activity, and none of them had obvious activity.

OBJECTIVE: To explore the clinical characteristics and genetic basis of a child with Kartagener syndrome (KTS). METHODS: Trio-whole exome sequencing was carried out for the child and his parents, and candidate variants were verified by Sanger sequencing. Changes in protein structure due to missense variants were simulated and analyzed, and the Human Splicing Finder 3.0 (HSF 3.0) online platform was used to predict the effect of the variant of the non-coding region. RESULTS: The child had featured bronchiectasis, sinusitis and visceral inversion. Genetic testing revealed that he has harbored compound heterozygous variants of the DNAH5 gene, namely c.5174T>C and c.7610-3T>G. Sanger sequencing confirmed the existence of the variants. The variants were not found in the dbSNP, 1000 Genomes, ExAC, ClinVar and HGMD databases. Protein structural analysis suggested that the c.5174T>C (p.Leu1725Pro) variant may affect the stability of local structure and its biological activity. The results of HSF 3.0 analysis suggested that the c.7610-3T>G variant has probably destroyed a splicing receptor to affect the transcription process. CONCLUSION The compound heterozygous variants of the DNAH5 gene probably underlay the pathogenesis in the child. Above finding may facilitate the understanding of the clinical characteristics and genetic basis of KTS, and further expand the spectrum of DNAH5 gene variants.
Male ; Humans ; Child ; Mutation ; Kartagener Syndrome/genetics* ; Genetic Testing ; Mutation, Missense ; Exome Sequencing ; Axonemal Dyneins/genetics*

Brucellosis is a zoonotic disease that causes animal and human diseases. Vaccination is a major measure for prevention of brucellosis, but it is currently not possible to distinguish vaccinated animals from those that have been naturally infected. Therefore, in this study, we constructed the Brucella (B.) abortus 2380 wbkA mutant (2308DeltawbkA) and evaluated its virulence. The survival of 2308DeltawbkA was attenuated in murine macrophage (RAW 264.7) and BALB/c mice, and it induced high protective immunity in mice. The wbkA mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon. Antibodies to 2308DeltawbkA could be detected in sera from mice, implying the potential for use of this protein as a diagnostic antigen. The WbkA antigen would allow serological differentiation between infected and vaccinated animals. These results suggest that 2308DeltawbkA is a potential attenuated vaccine against 16M. This vaccine will be further evaluated in sheep.
Animals ; Antibodies ; Brucella abortus* ; Brucella* ; Brucellosis ; Humans ; Immunization* ; Immunoglobulin G ; Interferons ; Macrophages ; Mice* ; Sheep ; Staphylococcal Protein A ; Vaccination ; Virulence ; Zoonoses