Objective To construct a lentiviral vector of RNA interference (RNAi) of murine triggering receptor expressed on myeloid cells-1 (TREM-1) gene and to explore the effect of TREM-1 on the inflammatory response caused by Bacteroides fragilis. Methods Four target sequences were selected according to murine TREM-1 mRNA sequence, and then 4 pairs of double-strand DNA oligo according to these target sequences and one pair of negative control double-strand DNA oligo were designed and synthesized. These fragments were subcloned into pGCSIL-GFP/Lenti plasmid. After being identified by PCR and sequencing, these plasmids were cotransfected into 293T cells to package lentiviral particles. The lentiviral vector particles were transfected into Raw 264. 7 cells and TREM-1 expression in the transfected cells was assayed by real-time PCR and ELISA. Different concentrations of Bacteroides fragilis lipopolysaccaride (LPS) were administered in the Raw264. 7 cells, and the cells were stimulated with LPS for 12 h. TREM-1 expression was determined by real-time PCR and ELISA at the time points. Results PCR and sequencing confirmed that lentiviral vectors had the correct structure and could express high titer of virus. After being transfected into Raw264. 7 cells, TREM-1 expression was knocked down significantly by all of these lentiviral vectors at both protein and mRNA levels, and the pGCSIL-GFP/Lenti-1 had the most efficient interference. TREM-1 was upregulated in the presence of Bacteroides fragilis LPS, and this increase was partly abrogated in the TREM-1 siRNA-treated cell models of endotoxemia, depending on the sequence. Conclusion The lentivirus RNAi vector of TREM-1 was constructed successfully. The lentivirus RNAi vector of TREM-1 can inhibit the expression of TREM-1 in the murine endotoxemia model caused by Bacteroides fragilis LPS.

OBJECTIVETo observe the three-dimensional distribution of vessels, and to establish a new method for measurement of blood flow velocity in mice cerebral cortex using two-photon laser scanning microscopy and fluorescence probe labeling technique.

METHODSThe mouse was made cranial window surgery and injected Texas-Red through tail vein after anesthetized. The three-dimensional imaging of vessel was obtained through z-stack scanning, and blood flow velocity was quantified through line scanning.

RESULTSWe could detect vascular distribution for more than 500 µm depth using two-photon microscopy. The velocity of blood flow was (0.59 ± 0.12) mm/s in capillary.

CONCLUSIONThe method for observing the brain blood flow by two-photon microscopy was established, which could achieve quantification of single vascular blood flow velocity and provide experimental evidence for basic research and medical applications.

Animals ; Blood Flow Velocity ; Brain ; blood supply ; Capillaries ; Cerebrovascular Circulation ; Fluorescent Dyes ; Hemodynamics ; Mice ; Microscopy, Fluorescence

The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E. coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired. Beclin1 mRNA and Beclin1 protein were detected by RT-PCR and Western blot analysis respectively. The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1. Two SH-SY5Y transfected cell lines were successfully selected. Compared with the control group, RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28% ± 1.45% (P < 0.05)and 75.50% ± 2.63% (P < 0.05), respectively. The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SYSY cell lines with inhibited Beclin1 expression were established. It provides a useful cell model for studying the biological function of Beclin1.
Apoptosis Regulatory Proteins ; genetics ; metabolism ; Beclin-1 ; Cell Line, Tumor ; Down-Regulation ; Escherichia coli ; Gene Silencing ; Humans ; Membrane Proteins ; genetics ; metabolism ; Neuroblastoma ; Plasmids ; RNA, Messenger ; RNA, Small Interfering ; Transfection

Objective To master the epidemic prevalence and the risk factors of brucellosis in animals and human,and to provide a basis for making prevention strategies and measures to brucellosis in Chongqing Municipality.Methods Qijiang and Wansheng district of Chongqing Munipality were selected as investigation points.Feeding status of goats in feedlots was investigated,and blood samples of goats were collected for laboratory testing.Epidemiological survey in employees on feedlots,family members closely exposed with goats,and other focus groups such as stockbreeding and veterinary was conducted,and blood samples was collected for laboratory testing according to the principle of informed consent.Clinical features of infested persons were investigated.Blood samples were screened by plate agglutination test (RBPT) and tube agglutination test (SAT).Results A total of 582 households in the two districts were investigated.The number of positive households was 40 households.The positive rate of the households was 6.87％.A total of 20 105 goat blood samples were tested.Of them,989 blood samples tested positive.The positive rate was 4.92％.A total of 337 blood samples of the risk population from 22 towns of the two districts were tested.Of them,45 samples were positive,and the positive rate was 13.35％.Eleven people were active patients of brucellosis.The epidemic sites were distributed in 16 towns,which accounted for 73.73％.The difference of the positive rates between the two districts was not significant (x2 =0.37,P ＞ 0.05).The positive rate of brucellosis among male and female was 11.57％ (28/242) and 17.89％ (17/95),respectively.The age of brucellosis infection distributed from 2 years old to 83 years old.One preschool child and 3 students were identified positive.The positive rate of brucellosis among feeder was the highest,which accounted for 31.75％ (40/125).The difference of positive rate of brucellosis among different professions was significant (x2 =63.40,P ＜ 0.05).Conclusions There is not a brucellosis case among animals or people reported in Chongqing non epidemic areas of brucellosis,but there are lots of infection.Surveillance and prevention of brucellosis should be strengthened in Chongqing.

The bioconversion of lignocellulosic materials for organic acid had great meaning to both economic and social benefit,owning to its renewable character,abundance and low price.In order to find out the key to the efficient utilization of lignocellulose,kinds of fermentation strategies to product organic acid(such as loctic acid,funaric acid)from lignocellulcsic were discussed,also the xylose metabolic pathway of Rhizopus sp.was extensively reviewed,and it find out that the low conversion of xylose was the bottle-block.

OBJECTIVETo discuss a method for reconstruction of deeply burned of extremities' functional regions with local skin flaps.

METHODSNinety-eight cases (with 126 place) of deeply burned wound of extremities were reconstructed with fasciocutaneous flaps.

RESULTSThe survival rate of 162 flaps was 93.2%. The movement of 112 joints (88.9%) was restored to normal, and the movement of 14 joints was mild limitation, none of the cases was severe obstacle.

CONCLUSIONSThe method is simple and with satisfactory results, which can decrease the rate of disability suffered from burn.

Adolescent ; Adult ; Burns ; surgery ; Child ; Extremities ; surgery ; Fascia ; Female ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Surgical Flaps ; blood supply

OBJECTIVETo clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents.

METHODThe cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied.

RESULTThe cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences.

CONCLUSIONThe intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

Base Sequence ; Cloning, Molecular ; Computational Biology ; Evolution, Molecular ; Gene Expression Regulation, Plant ; Lonicera ; enzymology ; genetics ; Methyltransferases ; genetics ; Nucleotide Motifs ; Phylogeny ; Sequence Alignment ; Sequence Analysis

OBJECTIVETo study a feasible method of measuring right ventricular pressure by catheterization in mice.

METHODSMeasuring the right ventricular pressure and the pulmonary artery pressure by homemade PE pipe through venous cannula in external jugular vein, using catheterization in mice with powerlab multimodal biometric signal recording system.

RESULTSForty-six out of 51 mice were experimented with this method smoothly and got a total success rate of 90.2%. Thirty of 33 normal mice and 16 of 18 mice with pulmonary arterial hypertension (PAH) were catheterized successfully. The right ventricular pressure were as follow: systolic blood pressure: (23.4 +/- 5.7) mmHg in normal group vs (32.2 +/- 2.8) mmHg in mice with PAH, diastolic blood pressure: (3.7 +/- 2.6) mmHg vs (3.8 +/- 2.0) mmHg, mean pressure: (12.0 +/- 3.7) mmHg vs (14.9 +/- 2.3) mmHg. After autopsy for those 5 failed cases, we found that 2 cases were into the inferior vena cava, another 2 cases pierced the right auricle and the last one punctured the axillary vein into the chest wall.

CONCLUSIONMeasuring the right ventricular pressure through venous cannula in external jugular vein with homemade PE pipe in mice gets not only a high success rate but also help to save time. Moreover, this method can be popularized easily. It is a good and feasible method for measuring right ventricular pressure in mice.

Animals ; Cardiac Catheterization ; methods ; Jugular Veins ; Male ; Mice ; Mice, Inbred BALB C ; Ventricular Pressure

BACKGROUND: Skeletal muscle satellite cells have been found to possess the potential of stem cells that have the ability to self-renew with the roles of repair and regeneration. Therefore, it can be used to treat myofibrosis. OBJECTIVE: To explore the function, molecular regulation and related mechanisms of skeletal muscle satellite cells, and to promote the repair and regeneration of skeletal muscle fibers, thereby providing new ideas and intervention strategies for the rehabilitation and treatment of skeletal muscle injury. METHODS: A computer-based online search was conducted in PubMed, Mendeley, Google Scholar, and CNKI databases from 1995 to November 2016 for the relevant literature using the keywords of "skeletal muscle,satellite cells, stem cell, muscle repair". Data screening, processing, and summarization were performed. RESULTS AND CONCLUSION: According to the inclusion criteria, 51 eligible articles were enrolled. Analysis results show that skeletal muscle satellite cells are closely related to muscle fiber lesions. Based on the mechanism of skeletal muscle lesions, the regeneration and repair mechanisms of skeletal muscle satellite cells are explored. Skeletal muscle tissue is a functional and sound system that can regenerate and repair through skeletal muscle satellite cells. The function of skeletal muscle satellite cells is controlled by intracellular and extracellular information networks, which can enhance endogenous satellite cell viability. When the skeletal muscle is injured or malnourished, the viability of skeletal muscle satellite cells is inhibited, further inducing skeletal muscle fiber degeneration. Therefore, skeletal muscle fiber lesions can be treated through skeletal muscle satellite cell transplantation and stimulating the viability of endogenous skeletal muscle satellite cells, which provides intervention strategies for skeletal muscle repair and regeneration.

OBJECTIVE: To evaluate the spontaneous brain activity alterations in liver transplantation (LT) recipients using resting-state functional MRI. MATERIALS AND METHODS: Twenty cirrhotic patients as transplant candidates and 25 healthy controls (HCs) were included in this study. All patients repeated the MRI study one month after LT. Amplitude of low-frequency fluctuation (ALFF) values were compared between cirrhotic patients (both pre- and post-LT) and HCs as well as between the pre- and post-LT groups. The relationship between ALFF changes and venous blood ammonia levels and neuropsychological tests were investigated using Pearson's correlation analysis. RESULTS: In the cirrhotic patients, decreased ALFF in the vision-related regions (left lingual gyrus and calcarine), sensorimotor-related regions (left postcentral gyrus and middle cingulate cortex), and the default-mode network (bilateral precuneus and left inferior parietal lobule) were restored, and the increased ALFF in the temporal and frontal lobe improved in the early period after LT. The ALFF decreases persisted in the right supplementary motor area, inferior parietal lobule, and calcarine. The ALFF changes in the right precuneus were negatively correlated with changes in number connection test-A scores (r = 0.507, p < 0.05). CONCLUSION: LT improved spontaneous brain activity and the results for associated cognition tests. However, decreased ALFF in some areas persisted, and new-onset abnormal ALFF were possible, indicating that complete cognitive function recovery may need more time.
Ammonia ; Brain* ; Cognition ; Fibrosis ; Frontal Lobe ; Hepatic Encephalopathy ; Humans ; Liver Transplantation* ; Liver* ; Magnetic Resonance Imaging ; Motor Cortex ; Neuropsychological Tests ; Occipital Lobe ; Parietal Lobe ; Rabeprazole ; Somatosensory Cortex