Gene targeted mutagenesis and replacement can be used to modify gene sequence in genomic background without position effect or insertion inactivation in transgenic plants. Targeted mutagenesis organism has little biosafety concerns free of transgenes or marker genes. Gene targeted mutagenesis and replacement in high plants now appears to be a potential tool for gene functional analysis in situ, crops genetic improvement and molecular design. Zinc finger nuclease(ZFN)is most important and would be widely used in gene targeted mutagenesis and replacement through introducing double-strand breaks in genome. Strategies for gene targeted mutagenesis and replacement in plants is discussed. ZFN is described in detail from its structure, operation model and application in plants. Developmental prospect of ZFN in plant gene targeted mutagenesis and replacement is also discussed.

OBJECTIVETo explore the underwater decompression schedule for 100 m Trimix conventional diving operations and evaluate its safety through a simulated rabbits Trimix conventional diving.

METHODSAccording to the Haldane theory, the assumed time units, the classification of tissue compartments, the nitrogen super-saturation safety coefficient and the selection of methods used for the calculation of the simulated 100 m Trimix conventional diving schedule were properly selected, and the calculating method for the dive decompression schedule was thus firmly established. In our experiments, five tissue compartments were selected during the calculation of decompression schedule: 5 min, 10 min, 20 min, 40 min and 75 min, and the nitrogen super-saturation safety coefficient was calculated by 1.6. Eight New Zealand rabbits were performed a simulated 100 m Trimix dive program which was established according to the Haldane theory, and eight rabbits for intact group. The tissues wet/dry ratio and ethology were detected and observed before and after the simulated diving to evaluate the safety of decompression schedule.

RESULTSBy using the developed underwater decompression schedule, abnormal ethology changes in rabbits could not be observed after compression and decompression to the surface; and the tissues wet/dry ratio of simulated diving rabbits had no significant changes compared with the intact group (P > 0.05).

CONCLUSIONThe decompression schedule calculated by Haldane theory seemed to be safe and reliable, the diving breathing gas concentration did not cause oxygen toxicity and nitrogen narcosis among the dive rabbits, and dive efficiency was greatly improved by using enriched oxygen gas in UPTD safety range during decompression.

Animal Experimentation ; Animals ; Decompression ; Diving ; Helium ; Nitrogen ; Oxygen ; Rabbits

GJ-4 is crocin enrichments extracted from Gardenia jasminoides J. Ellis, and our previous studies have shown that GJ-4 significantly improved learning and memory impairment induced by Aβ in mice. Herein, a memory deficit model was developed by injecting okadaic acid (OA) into the lateral ventricle of mice, and the neuroprotection and underlying mechanism of GJ-4 on neuronal injury caused by Tau hyperphosphorylation were investigated. The Animal Care & Welfare Committee, Institute of Materia Medica, CAMS & PUMC has approved all procedures (No.00000318). GJ-4 at different doses was intragastric administration to mice for 16 days. Step-down test and Morris water maze test showed that GJ-4 could significantly improve OA-induced memory impairment in mice, and reduced the loss of Nissl bodies in the hippocampus of mice. GJ-4 could also decrease the phosphorylation level of Tau protein at Ser396, Thr231 and Ser404 via increasing protein phosphatase 2A (PP2A) activity and inhibiting glycogen synthase kinase-3β (GSK-3β) activity. Besides, further researches indicated that GJ-4 could inhibit the level of oxidative stress in the brain of OA mice, reduce neuronal apoptosis and inhibit the neuroinflammation mediated by activation of astrocytes in the hippocampus of mice, and eventually achieve its effects in improving learning and memory impairment in mice. According to these findings, we anticipated that GJ-4 might be a potential therapeutic drug for Alzheimer's disease.

In order to investigate the sleep quality and influencing factors in patients with Parkinson's disease (PD),201 PD patients were enrolled and underwent extensive clinical evaluations.Subjective sleep evaluation was assessed using the Pittsburgh Sleep Quality Index (PSQI),and the Epworth Sleepiness Scale (ESS).It was found that poor sleep quality (77.11％) and excessive daytime sleepiness (32.34％) were commonly seen in PD patients and positively correlated with disease severity.Then 70 out of the 201 PD patients and 70 age-and sex-matched controls underwent a polysomnographic recording.The parameters were compared between PD group and control group and the influencing factors of sleep in PD patients were analyzed.The results showed that sleep efficiency (SE) was significantly decreased (P＜0.01),and sleep latency (SL) and the arousal index (AI) were increased (P＜0.05) in the PD group as compared with those in the control group.SE and total sleep time (TST) were positively correlated with the Hoehn and Yahr (H&Y) stage.There was significant difference in the extent of hypopnea and hypoxemia between the PD group and the control group (P＜0.05).Our results indicate that PD patients have an overall poor sleep quality and a high prevalence of sleep disorder,which may be correlated with the disease severity.Respiratory function and oxygen supply are also affected to a certain degree in PD patients.

OBJECTIVETo explore the differential gene expressions of chronic hepatitis B (CHB) of Gan depression Pi deficiency syndrome (GDPDS) and Pi-Wei damp-heat syndrome (PWDHS).

METHODSThe ulnar venous blood was withdrawn from healthy subjects (26 cases), patients of GDPDS (35 cases) and PWDHS (34 cases) on an empty stomach. The total RNA was extracted using Trizol method. The differential genes were detected using Aglient expression profile chip and screened using randomized variance model. The results were analyzed using GO, Pathway, GeneBank, NCBI, and Geneontology.

RESULTSThere were 125 differential genes between CHB patients of GDPDS and those of PWDHS (including 66 up-regulated genes and 59 down-regulated genes), mainly involving in functions of transmembrane transport, response to selenium ion, and regulation of calcium ion-dependent exocytosis. The signal pathway participated in mainly includes cell adhesion molecules, calcium ion signaling pathway, leukocyte trans-endothelial migration. We present gene co-expression networks to find 9 interactions among genes (LOC340508, HIST2H2BE, MPL, FLJ22536, TUBA8, NT5M, EGFL7, PTPRF, TSPAN33), which were mainly involved in immune response, cell growth, DNA damage, signal transduction, inflammatory reaction, and so on.

CONCLUSIONSThe differential expression genes existed between CHB patients of GDPDS and those of PWDHS, indicating that Chinese medicine syndrome classification has its own basis for gene expression profile. The genomics research method is expected to provide an objective basis for Chinese medicine syndrome typing.

Adult ; Case-Control Studies ; Female ; Gene Expression ; Hepatitis B, Chronic ; diagnosis ; genetics ; Humans ; Male ; Medicine, Chinese Traditional ; methods ; Oligonucleotide Array Sequence Analysis ; Transcriptome

Objective To observe the correlation between conventional coagulation tests and Dabigatran concentration in order to find proper test method to predict the bleeding risk of patients receiving Dabigatran.Methods The clinical data of forty-nine non-valvular atrial fibrillation (NVAF) patients who took Dabigatran in Peking University Third Hospital from 2015 to 2017 were analyzed and the bleeding rates were calculated.The plasma samples from twenty healthy volunteers were collected and mixed up into normal pooled plasma (NPP).Different doses of active Dabigatran were mixed with NPP,making Dabigatran concentrations from 0 to 1 000 ng/mL.Prothrombin time (PT),activated partial thromboplastin time (APTT),thrombin time (TT),fibrinogen (Fib) and diluted Russell viper venom time (dRVVT) were determined.Fresh whole blood samples from three normal volunteers were collected and mixed with Dabigatran in the same way to perform thromboelastogram (TEG).Results The total bleeding rate of NVAF patients receiving Dabigatran was 28.6％,among which gastrointestinal bleeding accounts for the most.APTT,dRVVT,R time (R) and clot index (CI) showed a good linear correlation with Dabigatran concentration.The sensitivity of dRVVT was the best,followed by APTT.R and CI showed low sensitivity compared with APTT and dRVVT.Conclusion APTT and dRVVT should be good parameters for monitoring the bleeding risk of Dabigatran and helpful for physicians to choose proper point-in-time for withdrawal of Dabigatran and reduce bleeding events.

AIMTo investigate the effects of Angelica sinensis on the expression of Flt-1, Flk-1 mRNA after the ischemic brain injury in rats.

METHODSWistar rats randomly divided into two groups: group A rats underwent middle cerebral artery occlusion (MCAO) for 2 hours by suture, group B rats underwent MCAO for 2 hours meanwhile received treatment with Angelica sinensis (5g/kg). At 1 st d, 3 rd d and 7 th d after reperfusion, 36 rats( n = 18 in each group) were assessed by neurological scale and brain tissue was taken to assess the lesion ration with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The other rats (n = 3 at different time points in each group) were decapitated at 3 h, 6 h, 12 h , 1 st d, 3 rd d, 7 th d after reperfusion. Quantitative reverse transcription and polymerase chain reaction (RT-PCR) technique was used to examine the gene expression of Flt-1, Flk-1.

RESULTSThe neurologic deficit score of rats in group B decreased significantly compared with group A at the same time point (P < 0.05). The infarct volume of group A was significant greater than group B at the same time point after reperfusion (P < 0.01). The results of RT-PCR revealed that the gene expression of Flt-1, Flk-1 in the two groups increased from 3 h after reperfusion and reached its peak at the time of 3 rd d after reperfusion, then declined gradually. The gene expression of Flt-1, Flk-1 in the group B was significantly increased than group A at the same time point (P < 0.01). The gene expression of Flk-1 was positive correlated with Flt-1 in two groups (r = 0.957).

CONCLUSIONThe increasing amount of Flt-1, Flk-1 expression was enhanced by Angelica sinensis following transient interruption of cerebral blood flow in rats.

Angelica sinensis ; Animals ; Brain Ischemia ; metabolism ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

BACKGROUNDGalectin-3 is the most recently identified advanced glycosylation end products (AGEs) binding protein. This study aimed to investigate the effects of AGEs and rosiglitazone on the expression and secretion of galectin-3 in cultured human renal mesangial cells (HRMCs).

METHODSHRMCs were incubated with different concentrations of AGE-bovine serum albumin (BSA) (0, 50, 100, 200, and 400 mg/L) for different time (0, 24, 36, 48, and 72 hours), and exposed to AGE-BSA in the presence of different concentrations of rosiglitazone (1, 10, and 100 micromol/L). The mRNA and protein expression of galectin-3 in HRMCs were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The culture medium of HRMCs was collected and concentrated, and the content of galectin-3 in the medium was detected by Western blotting.

RESULTSBoth RT-PCR and Western blotting revealed that AGE-BSA up-regulated the expression of galectin-3 in HRMCs in a concentration- (P < 0.05) and time-dependent (P < 0.05) manner compared with the control. Compared with the control, AGE-BSA elevated the content of galectin-3 in the culture medium of HRMCs time- and concentration-dependently (P < 0.05, respectively). Both protein and mRNA expression of galectin-3, and its content in the medium of HRMCs exposed to different concentrations of rosiglitazone in the presence of AGE-BSA were increased compared with those of cells exposed to AGE-BSA alone (P < 0.05). Rosiglitazone increased the expression and secretion of galectin-3 in a dose-dependent manner (P < 0.05).

CONCLUSIONSAGEs up-regulates the expression and secretion of galectin-3 in HRMCs. Rosiglitazone further enhances the upregulation of galectin-3 in HRMCs induced by AGEs, which suggests that rosiglitazone may play a role of reno-protection via up-regulation of galectin-3.

Blotting, Western ; Cell Line ; Galectin 3 ; genetics ; secretion ; Glycation End Products, Advanced ; pharmacology ; Humans ; Hypoglycemic Agents ; pharmacology ; Mesangial Cells ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Serum Albumin, Bovine ; pharmacology ; Thiazolidinediones ; pharmacology

BACKGROUNDPrenatal hyperglycaemia may increase metabolic syndrome susceptibility of the offspring. An underlying component of the development of these morbidities is hepatic gluconeogenic molecular dysfunction. We hypothesized that maternal hyperglycaemia will influence her offsprings hepatic peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) expression, a key regulator of glucose production in hepatocytes.

METHODWe established maternal hyperglycaemia by streptozotocin injection to induce the maternal hyperglycaemic Wistar rat model. Offspring from the severe hyperglycemia group (SDO) and control group (CO) were monitored until 180 days after birth. Blood pressure, lipid metabolism indicators and insulin resistance (IR) were measured. Hepatic PGC-1α expression was analyzed by reverse transcription polymerase chain reaction and Western blotting. mRNA expression of two key enzymes in gluconeogenesis, glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK), were analyzed and compared.

RESULTSIn the SDO group, PGC-1α expression at protein and mRNA levels were increased, so were expression of G-6-Pase and PEPCK (P < 0.05). The above effects were seen prior to the onset of IR.

CONCLUSIONThe hepatic gluconeogenic molecular dysfunction may contribute to the metabolic morbidities experienced by this population.

Animals ; Female ; Hyperglycemia ; chemically induced ; physiopathology ; Insulin Resistance ; physiology ; Liver ; metabolism ; Male ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Peroxisome Proliferator-Activated Receptors ; metabolism ; Pregnancy ; Prenatal Exposure Delayed Effects ; RNA-Binding Proteins ; Rats ; Rats, Wistar ; Streptozocin ; toxicity ; Transcription Factors

Objective To understand the awareness of HIV/AIDS-related knowledge and characteristics of sexual behaviors among men who have sex with men (MSM) in Changsha City. Methods By a snowball sampling method, volunteers were recruited in two social public welfare organizations in Changsha (Qingcai and Zhongda Sunshine) and interviewed by anonymous electronic questionnaires. Data were analyzed using software SPSS 19.0. Results Among 150 MSM, the overall awareness rate of HIV/ AIDS-related knowledge was 86.0% (129/150). For different demographic characteristics, higher age group, higher education level and higher income groups had significantly higher rates of awareness about HIV/AIDS related knowledge, compared with the reference groups, respectively. For the sexual behaviors, 32.7% of the investigated MSM population had their first MSM sexual intercourse at age of <18 years old, the rate of ≥18 years old group was significantly higher than the <18 years old group (2=4.315, P=0.038), 46.7% of the MSM population had more than 1 sexual partner during the past six months, the ratio of MSM used condoms in the sexual intercourse occasionally or never was 29.3% and 6.7%, respectively. Conclusions Young age, relatively low educational level and low income MSM in Changsha had a relative low awareness of HIV/AIDS-related knowledge. MSM in Changsha City had first MSM sexual intercourse at a very younger age. There is an urgent need to take well-targeted measures to improve the HIV/AIDS-related knowledge with special MSM population, and develop effective intervention measures for the high-risk sexual behaviors among MSM.