OBJECTIVES: To investigate the effects of formulated granules of Tianma Gouteng Yin (TGY) on motor deficits in a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced subacute Parkinson's disease (PD) and explore the possible molecular mechanisms. METHODS: Ninety C57BL/6 mice were randomized equally into 6 groups, including a control group, a PD model group, a NEC-1 (6.5 mg/kg) treatment group, two TGY treatment groups at 5 and 2.5 g/kg, and a Madopar (76 mg/kg) treatment (positive control) group. Mouse models of PD were established by intraperitoneal injection of MPTP (30 mg/kg) for 5 consecutive days with the corresponding treatments for 15 days. The mice were randomly selected for motor function tests. Western blotting was used to detect the changes in expressions of TH, α-syn, RIPK1, RIPK3 and MLKL in the striatum of the mice. Network pharmacology analysis and molecular docking studies were performed to explore TGY-mediated regulation of the necroptosis pathway for PD treatment. RESULTS: Compared with those in the control group, the PD model mice exhibited obvious motor deficits with significantly increased α-syn protein expression and lowered TH protein expression in the striatum. Treatment with NEC-1 obviously improved motor deficits, inhibited the necroptosis pathway, and alleviated the changes in TH and α‑syn proteins in PD mice. Network pharmacology and molecular docking analyses suggested that the therapeutic effect of TGY in PD was associated with the modulation of RIPK1, a key protein in the necroptosis pathway. In PD mouse models, TGY treatment at the two doses significantly improved motor deficits of the mice, increased TH expression, and decreased the expressions of α-syn and necroptosis-related proteins in the striatum. CONCLUSIONS TGY can effectively inhibit the necroptosis pathway, increase TH expression and decrease α-syn expression in the striatum to improve motor deficits in PD mice.
Animals ; Mice, Inbred C57BL ; Mice ; Necroptosis/drug effects* ; Drugs, Chinese Herbal/therapeutic use* ; Parkinson Disease/drug therapy* ; Disease Models, Animal ; Male

Objective To investigate the association between microvariants at locus DYS570 and Y-SNPs haplogroup.Methods 89 Y-SNPs and 34 Y-STRs in AIYSNP42,AIYSNP47 and YfilerTM Platinum kits were used to detect the genotype of 116 microvariants at locus DYS570 in Kunming,and the Set-B kit was used to detect the core repeat sequences of the DYS570 locus.The data were statistically analyzed by direct counting method.Then,a network map was drawn by Network 10.2,in order to visualize the genetic information of the sample.Results The results demonstrated that 111 DYS570/18.3-21.3 samples had a core repeat sequence of TTT[TITC]18-21,belonging to subgroup O2a2b1a1a1a4-F14494.A DYS570/20.3 sample had a core repeat sequence of[TTTC]15TTC[TTTC]5,belonging to O2a1b1a1a1a1e-F1365 subgroup.A DYS570/17.1 sample had a core repeat sequence of[TTTC]17 T,belonging to the O2a1b1a1a1a-F11 subgroup.Three DYS570(19.2)samples had[TTTC]3 TT[TTTC]16,belonging to the D1a1a-M15 haplogroup.Conclusion The results indicated that the microvariant with the same core repeat structure at locus DYS570 was associated with haplogroups,and the ancestry origin of samples can be inferenced from microvariant characteristics during the practice of forensic medicine.

OBJECTIVE： To establish a method for simultaneous determination of contents of β-pinene， linalool， L-camphor， L-borneol， β-caryophyllene and xanthoxylin in the oil of Blumea balsamifera. METHODS： GC method was adopted. The determination was performed on RTX-1701 capillary column （programmed temperature）. The FID detector was controlled at 240 ℃. The inlet temperature was set at 240 ℃. The carrier gas was high-purity nitrogen 3 mL/min. The the sample size was 0.5 μL， and split ratio was 50 ∶ 1. RESULTS： The linear range of β-pinene， linalool， L-camphor， L-borneol， β-caryophyllene and xanthoxylin were 0.029 7-0.267 1 mg/mL （r＝0.999 9）， 0.024 3-0.218 9 mg/mL （r＝0.999 9）， 0.126 0-1.134 0 mg/mL （r＝0.999 9）， 0.217 2-1.954 8 mg/mL （r＝0.999 9）， 0.136 3-1.226 9 mg/mL （r＝0.999 9）， 0.044 5-0.400 3 mg/mL（r＝0.999 5）， respectively. The limits of quantitation were 0.028 5， 0.008 7， 0.018 6， 0.016 8， 0.014 5， 0.042 1 mg/mL； the limits of detection were 0.009 4， 0.002 9， 0.006 1， 0.005 5， 0.004 8， 0.013 9 mg/mL， respectively. RSDs of precision， stability， reproducibility and durability tests were all lower than 3％. The average recoveries were 98.13％-101.30％（RSD＝1.20％，n＝9），98.44％-101.81％（RSD＝1.28％，n＝9），98.26％-101.05％（RSD＝1.19％，n＝9），99.08％-101.58％（RSD＝0.89％，n＝9），98.66％-101.66％（RSD＝1.17％，n＝9），98.84％-103.60％（RSD＝0.96％，n＝9）， respectively. The contents of 6 components in the sample were 14.552-46.766， 16.951-22.096， 80.597-113.115， 205.224-242.537， 47.761-135.697， 26.493-45.771 mg/g， respectively. CONCLUSIONS： The established method is simple， accurate， precise and reproducible， which can be used for simultaneous determination of contents of 6 components in the oil of B. balsamifera. It can provide reference for comprehensive evaluation and extraction technology study of the oil of B. balsamifera.

Objective To evaluate the therapeutic effect of antibiotics combined with recombinant tuberculosis vaccine AEC/BC02 on Mycobacterium tuberculosis infection in guinea pigs. Methods Two weeks after guinea pigs were challenged subcutaneously with a high dose of Mycobacterium tuberculosis,the guinea pigs with the positive skin test responses to the recombinant ESAT6-CFP10 allergen were randomly di-vided into four groups:normal saline (NS),AEC/BC02,antibiotics and antibiotics+AEC/BC02. In antibiotics+AEC/BC02 group,guinea pigs firstly received isoniazid ( INH) and rifapentine ( RFT) treatment once a week for a total of three times,and then were immunized with a single dose of AEC/BC02 vaccine six times at 10-day intervals. Guinea pigs in AEC/BC02 and antibiotics groups were respectively vaccinated with AEC/BC02 vaccine and given INH and RFT treatment at the same dose and frequency as given to antibiotics+ AEC/BC02 group. Thirteen weeks after challenge,all guinea pigs were sacrificed for necropsy. Results The gross pathological scores of NS,AEC/BC02,antibiotics and antibiotics+AEC/BC02 groups were 83±8,77± 22,45±28 and 19±14,respectively. Antibiotics+AEC/BC02 group had a significantly lower gross pathological score than antibiotics,AEC/BC02 and NS groups (P<0. 05,P<0. 01,P<0. 01,respectively). Moreover,the gross pathological scores of antibiotics and AEC/BC02 groups were significantly decreased as compared with that of NS group (both P<0. 01). However,there was no significant difference between AEC/BC02 and NS groups. The spleen bacterial load of antibiotics+AEC/BC02 group was (2. 50±1. 26) lg CFU,which was sig-nificantly lower than those of NS [(4. 92+0. 52) lg CFU],AEC/BC02 [(4. 78+0. 84) lg CFU] and antibi-otics [(4. 39+0. 50) lg CFU] groups (P<0. 01). Compared with NS group,antibiotic and AEC/BC02 groups showed no significant difference in spleen bacterial load. Histopathological changes indicated different levels of granulomatous lesions appeared in lung tissues of all groups and the most severe change was ob-served in AEC/BC02 group,followed by that in NS,antibiotics and antibiotics+AEC/BC02 groups. Conclu-sion INH and RFT treatment in combination with AEC/BC02 vaccine in the treatment of guinea pigs with Mycobacterium tuberculosis infection was superior to either treatment alone as it significantly alleviated organ lesions and lowered the bacterial loads in spleen and lung.

Objective To identify the cross-reactive antigens shared by Mycobacteria smegmatis(MS) and Mycobacteria tuberculosis(MTB) and to analyze their antigenicity.Methods Bacterial antigens were extracted from strains of MS and MTB by ultrasonication.Western blot assay was performed to analyze common antigens that reacted with both of the antiserum samples against MS and MTB.The extracted bacterial antigens were mixed with incomplete Freund′s adjuvant and then were injected into muscles of mice.Cytokines secreted by murine spleen lymphocytes following stimulation with various antigens of MS and MTB were determined by ELISPOT and flow cytometry on the 7th day.IgG levels in serum samples were detected by ELISA 7 days after injection.Results There were cross-reactive antigens shared by MS and MTB.Potent humoral immune responses and cellular immunity against both MS and MTB could be induced by those cross-reactive antigens after sensitization the mice by either MS or MTB antigens.Cytokines of IL-2 and IFN-γ in CD4+ and CD8+T cells of mice stimulated with MS or MTB antigens were significantly increased as compared with those of non-sensitization group and those of Brucella antigens stimulation group.ConclusionCross-reactive antigens shared by MS and MTS can effectively promote specific immune reactions to the infection of MTB, which provides a scientific basis for the development of tuberculosis vaccines.

Objective To test and analyze the genetic background of highly immunodeficient mice from different sources.Methods Four highly immunodeficient mouse strains from different sources of NOD background were collected. 30 microsatellite DNA sites were detected, and the genotype can be displayed by gel electrophoresis and STR scanning. Results 17 microsatellite sites exhibit polymorphism in 20 mice of the four groups.There were 30 homozygous loci in the mice of groups A and B, and heterozygous in the other two groups.The genetic distance is minimum between groups A and B, showing a higher genetic similarity.Conclusions The genetic backgrounds are different in highly immunodeficient mice from different sources.

Objective To obtain the basic growth parameters of a self-established F1 hybrid CB6F1 C57-ras transgenic mouse model, and to provide basic information for commercialization of this mouse model. Methods F1 hybrid mice (CB6F1) were produced by crossing C57-ras heterozygous transgenic (c-Ha-ras+/-) male mice and wild-type BALB/cJ female mice.The average litter size, weaning rate, sex ratio, growth performance and C57-ras transgenic positive rate were recorded and analyzed.Results The average litter size was eight, weaning rate was 90%, and sex ratio was approximately in accordance with prediction.The average body weight of newborn mice was 1.73 ±0.05 g.The average body weight of 10-week old c-Ha-ras transgenic female and male mice in CB6F1 background was 24.38 ±1.74 g and 29.42 ±1.72g, respectively, which had a significant difference (P<0.01).The c-Ha-ras transgenic positive rate was 46.9%. which was in accordance with genetic rules.Conclusions The F1 hybrid mice (CB6F1) produced by crossing C57-ras heterozygous transgenic ( c-Ha-ras +/-) male mice and wild-type BALB/cJ female mice show normal growth performance and development characteristics, and it can be used for large-scale commercial supply.

Due to the minimum free energy model, it is very important to predict the RNA secondary structure accurately and efficiently from the suboptimal foldings. Using clustering techniques in analyzing the suboptimal structures could effectively improve the prediction accuracy. An improved k-medoids cluster method is proposed to make this a better accuracy with the RBP score and the incremental candidate set of medoids matrix in this paper. The algorithm optimizes initial medoids through an expanding medoids candidate sets gradually. The predicted results indicated this algorithm could get a higher value of CH and significantly shorten the time for calculating clustering RNA folding structures.
Algorithms ; Cluster Analysis ; Nucleic Acid Conformation ; RNA ; chemistry

Objective To discuss the effect of in vitro fertilization ( IVF) and mouse sperm cryopreservation , to establish a simple and economic frozen system for the genetically engineering mice preservation .Methods Sperm from genetically engineering mice were cryopreserved , IVF was performed using post-thawed sperm, then embryo transfer, to compare the effects of cryopreservation medium、age of male mice and sperm preincubation medium .Results Using CPA as sperm cryopreservation medium , when PM was used thawed-sperm preincubation in IVF , the fertility rates were from 82.49%to 91.43%, when HTF was used thawed-sperm preincubation in IVF , the fertility rates were from14.46%to 27.38%, there was a signification difference between PM and HTF sperm preincubation medium;10 to 35 weeks male genetically engineering mice sperm were succeed cryopreservation , and positive mice were procreated after 2-cell embryos were transferred;R18S3、CPM and CPA was used to freeze sperm , the fertility rates were 75.85%、88.89%to 94.27%, positive mice were procreated after 2-cell embryos were transferred;2-cell embryos after IVF were freezed , then thawed and positive mice were procreated after 2-cell embryos were transferred .Conclusion Using CPA as sperm cryopreservation medium , when PM was used thawed-sperm preincubation in IVF , genetically engineering mice sperm were succeed cryopreservation .

Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem ( ES) cells.Meth-ods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6( Cg)-Tyrc-2J and BALB/c mouse blastocysts, respectively.The efficiency was statistically evaluated according to three aspects:blastocyst collection, chimera production and germline transmission.Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyrc-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts.Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P =0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice ( P<0.01).Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse.Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.