OBJECTIVE: The population density and diversity of Sinomenium acutum (Menispermaceae) have been greatly reduced recently by overharvesting for medicinal purposes in China. Therefore, it is urgent that the remaining populations are investigated, and that strategies for the utilization and conservation of this species are developed. This study aimed to find the possible glacial refugia and define the genetic diversity of S. acutum for its proper utilization and conservation. METHODS: A total of 77 S. acutum samples were collected from four locations, Qinling Mountains, Daba Mountains, Dalou Mountains, and Xuefeng Mountains, in subtropical China. Genetic diversity among and between these populations were phylogenetically analyzed using four chloroplast DNA molecular markers (atpI-atpH, trnQ-5'rps16, trnH-psbA and trnL-trnF). RESULTS: A total of 14 haplotypes (C1 to C14) were found in collected samples. Haplotypes C1 and C3 were shared among all populations, with C3 as the ancestral haplotype. Haplotypes C11 and C12 diverged the most from C3 and other haplotypes. No obvious phylogeographic structure was found in four locations using the GST/NST test. There is no evidence of rapid demographic expansion in S. acutum based on the mismatch distribution, and the results of Tajima's D test, and Fu's FS test. Our analyses of molecular variance revealed a high level of genetic variation within populations. In contrast, the genetic differentiation among S. acutum populations was low, indicating frequent gene flow. CONCLUSION Xuefeng, Dalou, and Daba Mountains were possible glacial refugia for the populations of S. acutum. C1, C3, C11 and C12 haplotypes of S. acutum should be carefully preserved and managed for their genetic value.

Objective To evaluate the instant effects and five-year clinical outcomes of coronary artery disease patients complicated with diabetes mellitus after StentBoost-optimized percutaneous coronary intervention (PCI). Methods From March 2009 to July 2010, 184 patients undergoing PCI at our hospital were found stent underexpansion or malapposition by StentBoost after stents implantation and were divided into the diabetic (=73, 39.67%) and the non-diabetic group (=111, 60.33%). All patients received StentBoost-guided post-dilatation after stent implantation. The instant procedural results were measured and clinical outcome after five-year follow-up was analyzed in each group. Between-group comparisons were performed using Chi-square test or Student's test. Multivariate logistic regression analysis was carried out to reveal the independent predictors for long-term clinical outcomes of StentBoost-optimized PCI . Results After StentBoost-guided post-dilatation, the minimum diameter (MinLD), maximum diameter (MaxLD) and average diameter in both groups increased significantly than before (<0.001), the (MaxLD-MinLD)/MaxLD ratio and the in-stent residual stenosis decreased accordingly (<0.001). The five-year follow-up showed similar mortality rate (4.92% . 2.86%, =0.67) and major adverse cardiac event rate (11.48% . 11.43%, = 1.0) between the diabetic and the non-diabetic group, whereas the recurrence of angina pectoris was higher in the diabetic group compared to the non-diabetic group (47.54% . 29.52%; =0.02). A multivariate logistic regression analysis revealed that age and left ventricular ejection fraction rather than diabetes mellitus were independent predictors for long-term clinical outcomes. Conclusions StentBoost could effectively improve instant PCI results; the long-term clinical outcomes of StentBoost-optimized PCI were similar between diabetic and non-diabetic patients. Age and left ventricular ejection fraction were the independent predictors for long-term clinical outcomes.

Aim To study whether there was arterial heterogeneity and association with L-type calcium channel (LCC) in different parts of arteries in re-sponse to certain vasoconstrictor. Methods The aor-ta, renal arteries and coronary arteries were dissected from rats. Arterial ring contractions induced by pheny-lephrine (Phe), 5-hydroxyl tryptamine (5-HT) or U46619 in concentration-dependent manner were meas-ured using the Multi Myograph system and the response to nifedipne was observed. Results (1) Phe had no obvious effect on the tension of coronary artery,but in-duced concentration-dependent vasoconstriction in aor-ta and renal artery,and pEC50of aorta was significantly higher than that of renal artery (P<0.05). The inhi-bition rate of nifedipine on the aortic contractile re-sponses was significantly higher than that of renal arter-y (P<0.05). (2) The contraction induced by 5-HT on aorta was not obvious, but was significant on renal artery and coronary artery. The inhibitory rate of nife-dipine on coronary artery vasoconstriction was signifi-cantly higher than that of renal artery (P <0.05). (3) U46619 could induce aorta,renal artery and coro-nary artery concentration- dependent contraction, but the Emaxof them were both higher than that of renal ar-tery (P<0.05). And the pEC50of aorta was the lar-gest (P<0.05). Nifedipine significantly inhibited the contraction of aorta, renal artery and coronary artery induced by U46619 with the greatest inhibitory rate on the coronary artery vasoconstriction and minimal inhibi-tion on aortic vasoconstriction. Conclusions The re-sponse to certain vasoconstrictor is different among aor-ta, renal artery and coronary artery in rats, and the contraction mediated by L-type calcium channel is also different.

AIM:To investigate the changes of cardiac structure and function in rats with type 2 diabetic melli-tus(T2DM),and to explore the mechanisms underlying diabetic cardiomyopathy.METHODS:The cardiac structure and function were measured by echocardiography in Zucker diabetic fatty(ZDF)rats and their control Zucker lean(ZL)rats. The size of the cardiomyocytes was determined by wheat germ agglutinin staining.The protein expression of atrial natriuretic peptide(ANP),β-myosin heavy chain(β-MHC), receptor for advanced glycation end products(RAGE), L-type cal-cium channel α1C subunit(CaV1.2)and Orai1 was assessed by Western blot.RESULTS:Compared with the ZL control rats,the thickness of left ventricular wall,ejection fraction(EF),fractional shortening(FS)and the sizes of cardiomyo-cytes were significantly increased,and diastolic function was decreased in the ZDF rats(P<0.05).The protein expression of β-MHC, ANP, RAGE and Orai1 was increased, while the expression of Ca V1.2 was decreased in ZDF rats(P <0.05).CONCLUSION:T2DM rats show the prominent features including cardiomyocyte hypertrophy,ventricular hyper-trophy and compensatory enhancement of cardiac function, and the Ca2+handling and increase in RAGE expression may play important roles in the processes.

AIM:To investigate the role of microRNA (miR)-199a-5p in myocardial fibrosis and the potential target of miR-199a-5p.METHODS:C57BL/6 mouse cardiac fibroblasts were isolated and cultured for cellular experimen-tal study.Dual-luciferase reporter assay was performed to confirm the interaction between miR-199a-5p and the 3'-untrans-lated region (3'-UTR) of silent information regulator 1 (SIRT1).The expression of SIRT1 and fibrosis markers collagen (Col) 1a1, Col3a1 and α-smooth muscle actin (α-SMA) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively.RESULTS:The expression levels of miR-199a-5p, Col1a1, Col3a1 andα-SMA were marked-ly increased in cardiac fibroblasts after treatment with angiotensin Ⅱ(AngⅡ).The over-expression of miR-199a-5p signif-icantly increased the expression of Col1a1, Col3a1 andα-SMA in cardiac fibroblasts.Moreover, the results of dual-lucifer-ase reporter assay revealed that miR-199a-5p interacted with the 3'-UTR of SIRT1.miR-199a-5p inhibited SIRT1 expres-sion at post-transcriptional level.Meanwhile, miR-199a-5p mimic, in parallel to SIRT1 siRNA, inhibited SIRT1 expres-sion, increased the expression of Col1a1, Col3a1 and α-SMA in cardiac fibroblasts.Inactivation of NF-κB signaling con-tributed to the decrease in miR-199a-5p in Ang II-treated cardiac fibroblasts .CONCLUSION:SIRT1 is a target gene of miR-199a-5p, which mediates the pro-fibrotic effect of miR-199a-5p on cardiac fibroblasts .

OBJECTIVE: To estimate the biological dose by the chromosome aberration analysis in a victim suffered in the radioactive source-loss accident in Nanjing City. METHODS: Peripheral blood sample of the victim was collected 6 days after radioactive exposure. Individual radioactive biological dose was estimated by identifying the aberration of the dicentrics plus centric rings in the metaphase chromosome of cells. The distribution of chromosome aberration was tested by the Poisson distribution. Impure Poisson distribution was used to estimate the radiation dose. RESULTS: Among the 353 cells observed at metaphase,75 aberration of dicentrics plus centric rings were found,43 cells had 1 aberration and 7 cells had 2 aberrations. Moreover,there were 3 multi-aberration cells with 3,4 and 11 aberrations respectively. The average individual radiation dose was estimated to be 1. 52 Gy,which was consistent with the clinical diagnosis as mild acute bone marrow radiation disease. The Poisson distribution u test revealed that the patient suffered from nonhomogeneous radiation.By the impure Poisson distribution test,the radiation exposure proportion of the whole body was estimated to be 55% and the average dose was 3. 02 Gy. CONCLUSION: The biological dose estimation was successfully performed,the results showed that this was a case of nonhomogeneous irradiation.

Objective To improve the method used for traditional compression test to reflect the compressive elastic modulus of trabecular bone in proximal femur more accurately and investigate its biomechanical properties to provide experimental evidences for clinical treatment. Methods The properties of trabecular bone from normal cadaveric proximal femur(45~60 years old) were measured by the micro material mechanics testing system. Results The elastic modulus in the direction of principle compression and principle tension on trabecular bone were (335.26±183.85) MPa and (59.27±23.88) MPa, respectively. The biomechanical properties of trabecular bone in the direction of principle compression were significantly higher than those in its vertical direction.The displacement and strain distribution profiles under the loading were recorded, which showed to be asymmetric. Conclusions It is feasible to test the biomechanical properties of trabecular bone more accurately by the micro material mechanics testing system. The compressive properties of trabecular bone in proximal femur are obviously anisotropic and heterogeneous.

OBJECTIVETo investigate whether Leptotrombidium scutellare could be naturally infected by both Hantaan virus (HV) and Orientia tsutsugamushi (OT) and transmission status by stinging.

METHODS3459 Leptotrombidium scutellares from mice bodies and 3265 which were free were collected in the epidemic area of hemorrhagic fever with renal syndrome (HFRS) and tsutsugamushi disease.15 days later, the suspensions of lung and spleen of mice with 6 in a group stung by 1, 5 or 10 infected mites were injected intra-cerebrally into other mice for the detection of HV and OT in the next 6 generations of the mice, with immunofluorescent antibody technique (IFAT) and Giemsa staining technique. The passages of Vero-E6 cells inoculated on the aseptic filtrations from different number of infected mites were used to detect HV and OT pathogens. HV-RNA and OT-DNA were detected by PCR.

RESULTSAfter passage, HV positive mouse body mite group out of both 5 and 10 mites in the sixth generation, OT positive mouse body mite group out of the 10 mites in the sixth generation, both HV and OT positive mouse body mite group out of 1 mite in the fifth and sixth generation, both HV and OT positive mouse body mite group out of 5 and 10 mites in the sixth generation, and free mites group out of 1, 5 and 10 mites in the sixth generation, were found one mouse infected by both HV and OT, respectively. Out of the fourth generation of Vero-E6 cells, one sample was found both HV and OT positive out of 5 and 10 HV and OT mouse body mite group, respectively. In the sixth generation, both HV and OT positive cells were detected in one mouse mite group and the 1, 5, 10 free mite groups, respectively. HV-RNA and OT-DNA were all detected by PCR.

CONCLUSIONBoth HV and OT could be coexisted in wild Leptotrombidium scutellare and transmitted by stinging.

Animals ; Hantaan virus ; Hemorrhagic Fever with Renal Syndrome ; transmission ; Insect Bites and Stings ; Mice ; Mice, Inbred Strains ; Mites ; parasitology ; virology ; Murinae ; Orientia tsutsugamushi ; Scrub Typhus ; transmission ; Trombiculidae

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.
Adolescent ; Adult ; Aged ; Anesthetics, Dissociative ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Heart Atria ; cytology ; Humans ; Ketamine ; administration & dosage ; pharmacology ; Male ; Middle Aged ; Myocytes, Cardiac ; cytology ; drug effects ; physiology ; Patch-Clamp Techniques ; Potassium Channels ; drug effects ; Young Adult

OBJECTIVETo establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.

METHODSBased on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.

RESULTSIn the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).

CONCLUSIONThe dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.

Animals ; Cell Line ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; Luciferases ; genetics ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection