Objective To determine the effect of knocking down zebrafish faf1 gene by CRISPR/Cas9 editing technique.Methods gRNA was designed and prepared for the faf1 gene of zebrafish,and gRNA was mixed with Cas9 mRNA by microinjection into zebrafish single cell embryos.The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing.The mutant F0 was genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish,and the genotype of zebrafish was detected by microscopic observation.Results The faf1 gRNA and Cas9 mRNA were successfully prepared.The gRNA (gRNA6) located in the exon 6 of faf1 could shift the faf1 gene into frameshift mutations.The mutation type MU1 was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish.At 4 d post fertilization (dpf),there were sarcomeric dysplasia and head shrinkage,increased hyoid angle and other craniofacial cartilage deformities.And the zebrafish died at 8 ～9 dpf.Conclusion CRISPR/Cas9 knocking out thefaf1 gene produces a new phenotype for zebrafish,with delayed pigment deposition and nodule-like change in tail muscle section.

Air Pollutants, Occupational ; analysis ; Solvents ; analysis

OJECTIVETo explore whether different acupuncture signals were afferent to the cerebellar fastigial nucleus (FN) neuron and to find out their corresponding effect features through observing the effect of spontaneous discharge of cerebellar FN neuron by needling at different acupoints.

METHODSTotally 120 male SD rats were anesthetized by 20% urethane and their right cerebellar FN were positioned (AP 11. 6 mm, RL 1. 0 mm, H 5. 6 mm). Extracelluar discharge was recorded by glass microelectrode (AP: -11. 6 mm, R: 1. 0 mm, H: 5.7 -7. 0 mm), using extracellular microelectrode recording method, recording the spontaneous discharge of cerebellar FN neurons as a baseline. Random order of needling at zusanli (ST36), quchi (Lil1), weishu (BL21), and zhongwan (CV12) were compared with the baseline before each acupuncture. Their effects on the discharge of cerebellar FN neurons were observed and compared with baselines.

RESULTSThe frequency of FN neuronal discharge could be elevated by needling at zusanli (ST36), quchi (LiI), weishu (BL21), and zhongwan (CV12) (P <0. 01, P <0. 05). The response rate of needling at Zhongwan (CV12, 56. 00%) was higher than that of needling at Zusanli (ST36), Quchi (Ll1), and Weishu (BL21) (35. 00%, 34. 62%, 36. 63%, respectively) with statistical difference (P <0. 05). The response rate of needling at zhongwan (CV12) was obviously higher than that of needing at other points (F = 2. 101, P < 0. 05).

CONCLUSIONSNeedling at zusanli (ST36 ), quchi (Lil), weishu (BL21), and zhongwan (CV12) could elevate the spontaneous discharge frequency of cerebellar FN neurons. Needling at Zhongwan (CV12) had advantageous roles in regulating cerebellar FN.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Cerebellar Nuclei ; physiology ; Male ; Microelectrodes ; Neurons ; Rats ; Rats, Sprague-Dawley

OBJECTIVEEndothelial apoptosis plays an important role in the initiation of atherosclerosis. It would be useful to clarify whether activation of non-neuronal muscarinic receptor (NNMR) could prevent endothelial apoptosis and atherosclerosis. We investigated the effects of NNMR activation on regulating rat aortic endothelial cells (RAECs) apoptosis induced by homocysteine, an independent risk factor of atherosclerosis, and further studied its molecular mechanism.

METHODSRAECs were incubated using homocysteine at the concentration of 2.7 mmol/L for 36 h. RAECs were also pre-treated with carbachol or arecoline to examine their effects. RT-PCR was used to assess changes in the gene expression related to cell apoptosis.

RESULTSIncubation of RAECs with homocysteine at the concentration of 2.7 mmol/L resulted in morphologic changes, such as cellular shrinkage, membrane blebbing, chromatin condensation and margination. These could be attenuated by pretreatment with carbachol and arecoline at the concentration of 10 micromol/L for 12 h. Homocysteine induced apoptosis in RAECs and the molecular mechanisms were associated with the regulation of fas, fas-L and caspase-8 in the death receptor pathway, bcl-2, bcl-xL and bax in the mitochondrial pathway, caspase-12 in the endoplasmic reticulum pathway and caspase-3, caspase-6 and p53 as downstream effectors. Carbachol and arecoline attenuated the effects of homocysteine on genes in the death receptor pathway, in the mitochondrial pathway and in the downstream pathway. Atropine could reverse all of the effects of arecoline.

CONCLUSIONActivation of NNMR by carbacol and arecoline inhibits homocysteine-induced endothelial cell apoptosis mainly through regulation of death receptor pathway, mitochondrial pathway and downstream effectors.

Animals ; Aorta ; cytology ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Arecoline ; Carbachol ; Cell Cycle ; Endoplasmic Reticulum ; metabolism ; Endothelial Cells ; cytology ; drug effects ; Homocysteine ; adverse effects ; Mitochondria ; metabolism ; Rats ; Receptors, Muscarinic ; metabolism

Female ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Infant, Newborn ; Lung ; metabolism ; pathology ; Macrophage Migration-Inhibitory Factors ; analysis ; genetics ; Male ; Respiratory Distress Syndrome, Adult ; blood ; genetics ; pathology

Although previous reports showed drug-eluting stent (DES) could effectively inhibit neointima formation, in-stent restenosis (ISR) remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, proliferation of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1-S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.

BACKGROUND:Laminoplasty and laminectomy are the most commonly used treatment methods for multilevel cervical spondylotic myelopathy, which is more common in elderly patients. C5 nerve root palsy is the common postoprative complication after posterior cervical repair. OBJECTIVE:To compare the incidence of C5 nerve root palsy after laminoplasty with mini-titanium plate fixation and laminectomy with internal fixation in repair of multilevel cervical spondylotic myelopathy. METHODS:Total y 134 patients with multilevel cervical spondylotic myelopathy from August 2010 to December 2014 were enrol ed, and then divided into laminoplasty group (n=45) and laminectomy group (n=89) owing to different ways of repair. Patients in the laminoplasty group were treated with laminoplasty with mini-titanium plate fixation, and patients in the laminectomy group were treated with laminectomy with internal fixation. C5 nerve root palsy condition after repair was recorded and evaluated. The cervical lordosis angle (Cobb angle) and cervical curvature index were compared. The Japanese Orthopaedic Association score was used for neurological assessment. RESULTS AND CONCLUSION:Al patients in both groups were fol owed up for more than 6 months. There were no significant differences in cervical lordotic angle and cervical curvature index at the first week before and after the treatment between these two groups (P>0.05). The Japanese Orthopaedic Association scores of patients after 6 months of treatment were significantly improved compared with that before treatment in these two groups (P<0.05). There were 2 cases of C5 nerve root palsy after the treatment of laminoplasty, the occurrence rate was 4%(2/45);there were 10 cases of C5 nerve root palsy after the treatment of laminectomy, the occurrence rate was 11%(10/89);there was significant difference between these two groups (P<0.05). These results suggest that compared with laminectomy fixation, the incidence of C5 nerve root palsy was lower after the laminoplasty with mini-trianium plate fixation, which can be widely used in decompression repair treatment of multilevel cervical spondylotic myelopathy.

OBJECTIVETo explore the feasibility of inducing human umbilical cord mesenchymal stem cells (HuMSCs) to differentiate into Leydig cells through conditioned medium derived from Leydig cells.

METHODSHuMSCs and Leydig cells were obtained by tissue blocks culture attachment and enzymatic digestion respectively. HuMSCs were induced by conditioned medium of Leydig cells as an experiment group while those before induction were cultured as a control group. The expressions of LHR, 3β-HSD and StAR in the induced HuMSCs were determined by RT-PCR after 3, 7 and 10 days of culture; those of CYP11A1, CYP17A1 and 3β-HSD measured by immunofluorescence staining after 2 weeks; and that of 3β-HSD detected by Western blot after 4 weeks.

RESULTSThe experimental group showed positively expressed LHR, 3β-HSD and StAR at 3, 7 and 10 days, CYP11A1, CYP17A1 and 3β-HSD at 2 weeks, and 3β-HSD at 4 weeks, while the control group revealed negative expressions at all the time points.

CONCLUSIONInduced with conditioned culture medium derived from Leydig cells, HuMSCs are likely to differentiate into steroidogenic cells and eventually into Leydig cells.

Cell Differentiation ; Culture Media, Conditioned ; Humans ; Leydig Cells ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

OBJECTIVETo evaluate the reliability, validity, and responsiveness of traditional Chinese medicine (TCM) clinical outcomes rating scale for heart failure (HF) based on patients' report.

METHODSTCM clinical outcomes rating scale for HF (TCM-HF-PRO) were evaluated based on 340 HF patients' report from multiple centers. The completion of the investigation was recorded. Cronbach's α coefficient and split-half reliability were used for reliability analysis, and factor analysis was used to assess the construct validity of the rating scale. Pearson correlation analysis was then used for criterion validity analysis. Discriminant analysis was used to assess the responsiveness of the scale. All 340 HF patients having complete TCM-HF-PRO data were assigned to the treatment group and the control group by central randomization. The total TCM-HF-PRO scores of the two groups were compared using paired t-test to reflect the longitude responsiveness of the scale before treatment and at week 2 after treatment.

RESULTS(1) The recycling rate of the scale was 100.0%. One of them was not filled completely, which was rejected thereby. So the completion rate was 99.7%. The completion time for TCM-HF-PRO scale ranged 15 to 25 min. (2) The Cronbach's α coefficient of rating scale was 0.903, split-half reliability was 0.844 and 0.849. (3) Confirmatory factor analysis showed that 7 factors and items formed according to maximum load factor basically coincided with the construct of the rating scale, 7 factors accumulated contribution rate was 43.8%. TCM clinical outcomes rating scale for HF based on patients' report was relatively better correlated with the Minnesota living with HF questionnaire (r = 0.726, P < 0.01). (4) Discriminant analysis showed that the rating scale correctly classified more than 78.8% of case studies having confirmed initial differential diagnosis by experts. The total scale of the rating scale decreased more in the two group after treatment, with significant difference as compared with before treatment (P < 0.01.

CONCLUSIONTCM clinical outcomes rating scale for HF based on patients' report had good reliability, validity and responsiveness, hence it could be used to assess clinical efficacy for HF patients.

Diagnosis, Differential ; Discriminant Analysis ; Factor Analysis, Statistical ; Heart Failure ; diagnosis ; Humans ; Medicine, Chinese Traditional ; methods ; standards ; Reproducibility of Results ; Surveys and Questionnaires

The antitussive activity assay for the root extraction of Disporum cantoniense was carried out with coughing mice induced by ammonia liquor. The results showed that the ethanol and water extractions of D. cantoniense possess strong antitussive activity, and the high dose of the former was better than positive control, and then the constituents of the ethanol extraction were separated and purified by various modern chromatographic techniques. Their structures were identified by physico-chemical properties and spectroscopic data. As a result, eight compounds were isolated and identified as stigmast-4-en-3-one(1), (22E, 24R)-ergosta-5, 7, 22-trien-3beta-ol(2), obtucarbamate A(3), obtucarbamate B(4), neotigogenin(5), azo-2, 2'-bis[Z-(2,3-dihydroxy-4-methyl-5-methoxy) phenyl ethylene] (6),dimethyl {[carbonylbis (azanediyl)] bis( 2-methyl-5, 1-phenylene) j dicarbamate (7) , and quercetin-3-O-pB-D-glucopyranoside(8). All compounds were isolated from this plant for the first time, and the result of bioactivity-directed isolation showed that compounds 3, 4, and 6 had obvious effect on antitussive activity, and compound 6 had the same level as positive control.
Animals ; Antitussive Agents ; chemistry ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Ethanol ; chemistry ; Female ; Liliaceae ; chemistry ; Male ; Mice