BACKGROUND: Previous reseamh has proved that RNA interference can inhibit vascular endothelial growth factor (VEGF) gene expression of colon carcinoma, carcinoma of prostate, and retinoblastoma. However, RNA interference inhibiting VEGF of carcinoma of gallbladder was not reported. OBJECTIVE: To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting hVEGF165 mRNA. DESIGN, TIME AND SETTING: A gene engineering study was performed at National Hepatobiliary & Enteric Surgery Research Center, Xiangya Hospital, Central South University from 2008 to 2009.MATERIALS: Human GBC-SD was provided by Tumor Research Institute of Tongji University. METHODS: Four pairs of shRNAs that targeted at VEGF gene were designed. The eukaryotic expression plasmids (named shRNA1-4) were constructed and identified using restriction enzyme analysis. The plasmids were then transfected into GBC-SD cells via liposome2000. The transfection rate of recombinant plasmids was measured at 48 hours after transfection. MAIN OUTCOME MEASURES: Enzyme analysis of recombinant plasmid; transfection rate; VEGF mRNA expression determined using fluorescent polymerase chain reaction. RESULTS: shRNA plasmid vector targeting at VEGF gene was successfully constructed, in particular, pDC316-EGFP-U6-shRNA2 was the most effective. The expression plasmids were confirmed by restriction enzyme analysis. The transfection rate of recombinant plasmids in GBC-SD cells was approximately 58.6%. shRNA could inhibit VEGF mRNA expression, in particular, the inhibitory rate of RNA2 was the highest by 86%.CONCLUSION: The shRNA eukaryotic expression plasmid targeting at VEGF gene is constructed and selected successfully, and it can remarkably inhibit VEGF expression of GBC-SD cells. Additionally, the inhibitory effect of RNA2 is the greatest.

Objective By investigating the actualities of career development potential of eight-year program medical students, to explore a direction of training mode innovation aiming to improve career development potential. Methods Eight-year program medical graduates, their tutors, and di-rectors of their clinical department in a university hospital were the subjects of the current study. Questionnaire method was applied to evaluate professional competence including clinical skills, re-search capabilities and integrative ability . Anonymous questionnaires were dispensed to eight-year program medical graduates for self-evaluation. Distinct anonymous questionnaires were dispensed to tutors directors for evaluation. Traditional doctoral graduates (professional degree or academic degree) were set as standard for comparison of specific ability of eight-year program medical students. Results 74 out of 85 (87%) questionnaires for Eight-year program medical graduates were available. 53 out of 63 (84%) questionnaires for tutors were available. 21 out of 26 (81%) questionnaires for directors of clinical departments were available. In clinical capabilities, according to 43 percent (32 persons) of the eight-year MD graduates' self-assessment and the evaluation of 94%(50 persons) mentors and 81%(17 people) department directors, eight-year program medical graduates were regarded as lower-skilled clinicians compared with doctoral graduates with professional degree . Eight-year program medical graduates were also considered with worse research capabilities than doctoral graduates with profes-sional degree by themselves (76%, n=56), their tutors (91%, n=48), and their department directors (86%, n=18). Conclusions The survey shows that to some extent, the current eight-year clinical doctoral graduates lack of career development potential, reflecting the current training mode cannot lead to the given training goals. We suggest that both clinical skills and research capabilities should be enhanced to make sure that eight-year program medical graduates deserve the title of academic physicianorphysician scientists.

Objective This study aims to examine the development of the posterior lateral line of the zebrafish and establish ant model to study the process of hair cell differentiation and regeneration .Methods We observed the posterior lateral line system formation by DAPI immunohistochemistry and whole mount in situ hybridization .We further evaluated hair cells differentiation within neuromast by using Transgenic Tg (Brn3c:mGFP) zebrafish and stained the functional hair cells by the mechanotransduction marker FM 1 -43FX .We labelled proliferating cells in primordium and neuromast by addition of BrdU to the system water .Results The posterior lateral line primordium originated from a sensory placode and started its journey at around 20 hours post fertilization to migrate along the horizontal myoseptum to the tail -tip with a constant speed (1 .7somite/hour) .The primordium depositd five or six neuromasts spaced along the body ,and two or three terminal neuromasts at the tail -tip at 48 hours post fertiliza-tion .At 3 ,5 and 7 days post fertilization ,zebrafish contained 5 .68 ± 1 .46 ,10 .1 ± 0 .99 ,and 12 .45 ± 1 .32 hair cells per neuromast ,respectively .Furthermore ,the average number of FM1-43FX stained hair cells within each neuro-mast were 3 .68 ± 1 .11 ,8 .18 ± 1 .86 ,and 10 .22 ± 1 .24 ,respectively .Conclusion We establish the development model of hair cells in zebrafish lateral line neuromast and suggest that 3 to 7 days post fertilization is an important period for lateral line neuromast differentiation .This study would be useful for underlying the mechanisms of hair cell differentiation and regeneration .

BACKGROUND: Previous studies demonstrated that proliferation of cancer cells can be inhibited via RNA interference on the expression of vascular endothelial growth factor (VEGF). However, few studies report RNA interference on the expression of VEGF in gallbladder carcinoma, OBJECTIVE: To design and screen shRNA targeting VEGF, and to observe the effect of small interfering RNA targeting on proliferation of gallbladder cancer cells. METHODS: The VEGF-shRNA fragment was synthetized and connected with pCYU6/GFP/Neo-shRNA plasmid vector, shRNA was transfected into gallbladder cancer cells. The gallbladder carcinoma models of nude mice were prepared and randomly divided into blank control, negative control and experimental groups, With 6 animals in each group. ShRNA was injected into tumor. Cell growth was detected by fluorescence microscope MTT. The RNA interference efficiency was examined by fluorescent quantitative RT-PCR. Changes of tumor volume were also observed. RESULTS AND CONCLUSION: Gallbladder cancer cells ware shrunk with round shapes and a part of cells were dead after RNA interference on VEGF. shRNA-VEGF1 and shRNA-VEGF2 could signiticently inhibit mRNA gene expression of VEGF, the inhibition ratio was 86% and 82%, respectively. The tumor volume of the experimental group was smaller than the other groups, with slowly growth (P < 0.05). No obvious changes were found in the blank control and negative control groups. The constructed hVEGF-shRNA vector markedly decreases VEGF gene expression and inhibits cellular proliferation, eventually, to treat gallbladder cancer.

Objective To investigate changes of the interhemispheric coordination integrity in patients with hemi-parkinsonism using resting-state functional magnetic resonance imaging(rs-fMRI) homotopy technique called voxel-mirrored homotopic connectivity(VMHC).Methods Sixteen Parkinson disease(PD) patients with right body side motor symptom onset(RPD),15 patients with left side onset(LPD),and 19 age-,gender-,and education-matched healthy controls(HC) were included in this study.rs-fMRI scanning and pre-processed the raw data were performed.Then by using analysis of variance(ANOVA) and two sample t tset,we performed VMHC analyses on rs-fMRI data of these participants(P＜0.05,corrected with AlphaSim,clusters≥16 voxels).Exploratory linear correlations analyses were performed between the VMHC of regions showing significant group differences and the clinical features of LPD or RPD patients.Results Compared with HCs,patients with LPD had significantly reduced VMHC in visual regions,sensorimotor regions,and the cerebellar cortex(voxels size of 17-77,t=-5.06--3.42,P＜0.05).Patients with RPD exhibited decreased VMHC in the prefrontal cortex and sensorimotor regions.Both LPD and RPD groups had increased VMHC in subcortical regions.When compared with the RPD group,the LPD group displayed decreased VMHC in the visual regions,sensorimotor regions(voxels=16 and 18; t=-3.68and-3.87,respectively,both P＜0.05) and increased VMHC in the supramarginal gyrus(voxels=4,t=4.72,P＜0.05);ROI-based correlation analyses indicated that the VMHC in the inferior occipital gyrus and the postcentral gyrus was negatively correlated with the BDI-Ⅱ scores in the LPD group(r=-0.58 and-0.59,respectively; both P＜0.05),and positively correlated with the MMSE scores(r=0.56 and 0.52,respectively;both P＜0.05).In the RPD group,a positive correlation was found for the VMHC in the supramarginal gyrus and the illness duration(r=0.56,P＜0.05) and for the VMHC in the postcentral gyrus and the(mini-mental state exam) MMSE(r=0.53,P＜0.05).Conclusions The differential pattern of deficits in the interhemispheric coordination integrity in hemi-parkinsonism reflected by VMHC may provide insights into the neurological pathophysiology underlying the asymmetry of symptom appearance in PD.

Objective To investigate the immediate effects of lower limb with open chain weight-adding on joint position sense and gait symmetry in stroke patients. Methods From January, 2016 to January, 2017, 39 stroke patients were included. Their joint position sense and gait symmetry were compared before and after weight-adding. The joint position senses of active reproduction of active positioning (ARAP) and passive reproduction of passive positioning (PRPP) were assessed during lower limb straight leg raise. The gait symmetry was also as-sessed and three indexes were recorded including the symmetry of foot rotation angle, step length and percentage of single leg support phase. Results After weight-adding, the position sense of PRPP did not improve (t=0.832, P=0.832), nor of the symmetry of foot rotation an-gle (t=-0.704, P=0.483) and percentage of single leg support phase (t=0.381, P=0.702);the position sense of ARAP improved (t=3.158, P=0.011), as well as the symmetry of step length (t=2.022, P=0.041). Conclusion The lower limb with open chain weight-adding could im-prove the active joint position sense and symmetry of step length.

Quite a few orthopedics experts have fabricated some novel bone scaffolds with nanotechnology and have carried out some researches on nano-biological effects. The study of the biological effects about nano-biomaterials can facilitate the understanding of the interaction between the biomaterials and the organism, and provide research ideas and direction to construct new biomaterials with physiological function. To better understand the interaction of nano biomaterials with protein, cells and bio-security, this review presents recent advances of biological effects about nano scaffold for bone tissue engineering.
Biocompatible Materials ; metabolism ; Bone Substitutes ; Bone and Bones ; Humans ; Nanostructures ; Nanotechnology ; Surface Properties ; Tissue Engineering ; methods ; Tissue Scaffolds

Histone deacetylases are involved in many biological processes and have roles in regulating cell behaviors such as cell cycle entry, cell proliferation and apoptosis. However, the effect of histone deacetylases on the development of hair cells (HCs) has not been fully elucidated. In this study, we examined the influence of histone deacetylases on the early development of neuromasts in the lateral line of zebrafish. Hair cell development was evaluated by fluorescent immunostaining in the absence or presence of histone deacetylase inhibitors. Our results suggested that pharmacological inhibition of histone deacetylases with inhibitors, including trichostatin A, valproic acid and MS-275, reduced the numbers of both HCs and supporting cells in neuromasts. We also found that the treatment of zebrafish larvae with inhibitors caused accumulation of histone acetylation and suppressed proliferation of neuromast cells. Real-time PCR results showed that the expression of both p21 and p27 mRNA was increased following trichostatin A treatment and the increase in p53 mRNA was modest under the same conditions. However, the expression of p53 mRNA was significantly increased by treatment with a high concentration of trichostatin A. A high concentration of trichostatin A also led to increased cell death in neuromasts as detected in a TUNEL assay. Moreover, the nuclei of most of these pyknotic cells were immunohistochemically positive for cleaved caspase-3. These results suggest that histone deacetylase activity is involved in lateral line development in the zebrafish and might have a role in neuromast formation by altering cell proliferation through the expression of cell cycle regulatory proteins.
Animals ; Apoptosis ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor Proteins/genetics/metabolism ; Histone Deacetylase Inhibitors/*pharmacology ; Histone Deacetylases/*metabolism ; Histones/metabolism ; Larva/growth & development/metabolism ; Lateral Line System/cytology/*growth & development/metabolism ; Mechanoreceptors/drug effects/*metabolism/physiology ; RNA, Messenger/genetics/metabolism ; Zebrafish ; Zebrafish Proteins/*metabolism

Objective:To investigate the changes of brain energy metabolism and cognitive function in mice with specifically knocking out AMP-activated protein kinase α1 subunit ( AMPKα1) gene in the excitatory neurons by Cre-loxP recombination system. Methods:Sixteen 6-month-old mice with genotype AMPKα1 flox/flox/Camk2a-Cre/ERT2 obtained by hybrid breeding were randomly divided into AMPKα1 knockout group ( n=8) and AMPKα1 wild-type group ( n=8). Mice in the AMPKα1 knockout group were intraperitoneally injected 0.1 mL tamoxifen (20 mg/mL, dissolved in corn oil) daily for a consecutive 5 d to control AMPKα1 gene knockout in the excitatory neurons; and mice in the AMPKα1 wild-type group were intraperitoneally injected 0.1 mL corn oil daily for a consecutive 5 d. Seven d after that, Morris water maze and T maze experiments were employed to detect the spatial learning and memory abilities and spatial working memory of these mice; chemical exchange saturation transfer imaging (CEST) was used to observe the glucose metabolism in the hippocampus and cortex surrounding the hippocampus; Western blotting was used to detect the AMPKα1 and glutamate receptor 1 (GluR1) protein expressions in the hippocampus and cortex surrounding hippocampus of two groups. Results:(1) Morris water maze showed that, as compared with those in the AMPKα1 wild-type group, mice in the AMPKα1 knockout group had significantly prolonged escape latency ([13.90±3.72] s vs. [22.40±6.28] s; [11.95±3.86] s vs. [22.39±9.77] s]) on the 3 rd and 4 th d of experiment, statistically decreased times crossing the platform ([5.25±1.83] times vs. [1.75±1.28] times, P<0.05). (2) T-maze experiment showed that as compared with that of the AMPKα1 wild-type group, the free alternation rate in mice of the AMPKα1 knockout group was significantly decreased ([73.21±9.16]% vs. [48.21±11.29]%, P<0.05). (3) CEST showed that the glucose metabolism levels in the hippocampus and cortex surrounding the hippocampus of AMPKα1 knockout group were significantly lower than those in AMPKα1 wild-type group (1.51±0.81 vs. 2.77±0.67; 1.31±0.83 vs. 2.42±0.95, P<0.05). (4) Western blotting showed that the AMPKα1 and GluR1 protein expressions in the hippocampus and cortex surrounding the hippocampus of the AMPKα1 wild-type group were significantly higher than those of the AMPKα1 knockout group (AMPKα1: 0.70±0.05 vs. 0.49±0.03, 0.98±0.04 vs. 0.64±0.06; GluR1: 1.22±0.18 vs. 0.60±0.11, 0.96±0.08 vs. 0.79±0.04, P<0.05). Conclusion:Specifically knocking out AMPKα1 in excitatory neurons can result in abnormal glucose metabolism in the brain of mice, and thus cause cognitive dysfunction, whose mechanism may be related to excitatory synaptic disorder caused by energy metabolism disorder.

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median (P₂₅-P₇₅) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group (P<0.001). DNA methylation levels of P16, RASSF1A and MGMT were 2.04±0.41, 2.19 (1.94-2.51), 2.22 (1.94-2.46)%5mC in exposure group, and 2.19±0.40, 2.41 (2.11-2.67), 2.44 (2.15-2.91)%5mC in control group. DNA methylation levels were lower in exposure group (P values were 0.005, 0.002 and 0.001, respectively). Spearman correlation analysis showed that DNA methylation levels of P16, RASSF1A, and MGMT were negative associated with urinary εdA level (r values were -0.155, -0.137, and -0.198, respectively, P<0.05). No significant correlation was observed between the εdC level and any measured DNA methylation levels (P>0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95%CI) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95%CI) was -0.094 (-0.179--0.008), P=0.032). Conclusion DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A and MGMT.