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2019 Vol. 49, No. 4 Published: 2019-04-25   235 Cholangiocarcinoma derived exosomes break down vascular integrity and cause metastasis by triggering endoplasmic reticulum stress in vascular endothelial cells LI Zhengzheng, SUN Hongwei, ZHANG Nan, SU Huafang Objective: To explore the effects of cholangiocarcinoma exosomes on the endothelial tight junction and the possible underlying mechanism. Methods: Exosomes were isolated from the supernatant of cholangiocarcinoma cell line CCLP and human intrahepatic bile duct epithelial cells (HIBEC) by ultracentrifugation according to the well-established protocol and were administrated to human umbilical vein endothelial cells (HUVEC), PBS was chosen as control. Endothelial monolayer permeability was assessed in vitro and tight junction proteins (TJPs) were evaluated by Western blot and immunofluorescence. BALB/c null mice were divided as three groups (PBS, exoCCLP and exoHIBEC), which were administrated with PBS, CCLP exosomes or HIBEC exosomes via tail vein respectively for indicated durations. FITC-Dextran or CCLP were then injected intravenously and leakage of FITC-Dextran in lung and liver, or pulmonary metastases were assessed. The endoplasmic reticulum stress related genes and proteins were analyzed by real-time PCR and Western blot. Endothelial PERK, ATF6 or IRE1α were partially knocked down by siRNA before HUVECs were treated with CCLP exosomes, and TJPs were evaluated afterwards. Results: CCLP exosomes downregulated the expression of TJPs in HUVECs, and increased the permeability of endothelial monolayer in vitro. In vivo experiments indicated that compared to 2019 Vol. 49 (4): 235-242 [Abstract] ( 714 ) HTML (1 KB)  PDF (2347 KB)  ( 936 ) 243 Metabonomics analysis of colorectal carcinoma cell lines SW480 and SW620 with different metastatic potentials NIU Yan, ZHOU Yi, ZHENG Hong, ZHAO Liangcai, LI Chen, GAO Hongchang. Objective: To study the metabolic profiles of human colorectal carcinoma cell lines with different metastatic potentials and analyze the relationship between metabolites and the metastasis of colorectal carcinomas. Methods: The characteristic alterations in metabolites of two isogenic colorectal cancer cell lines (highly metastatic SW620 and nonmetastatic SW480) were explored by the PLS-DA model based on the 1H NMR spectra. Moreover, Seahorse XF analyzer was used to assess ECAR and OCR. Results: The metabolic profile of SW620 and SW480 cell lines displayed obvious differences along in the PC1 direction. Compared with SW480, lactate, succinic, isoleucine, acetate, glycine and glutamine were increased, and phosphorylcholine, ATP, inositol and taurine were reduced in SW620 cells (P<0.05). SW620 cells with high metastatic potential have higher levels of glycolysis, TCA cycle activity, ECAR and OCR levels. Conclusion: The metabolic profile of SW620 cells with high metastatic potential shows clear differences from SW480 cells, and a variety of metabolites can be involved in the metastasis of colorectal carcinoma. Compared with SW480 cells, SW620 has a higher metabolic base potential, suggesting that it needs energy for high metastatic ability. Meanwhile, sw620 can adapt to its microenvironment more quickly, suggesting that the occurrence of metastasis is closely related to metabolism. 2019 Vol. 49 (4): 243-248 [Abstract] ( 792 ) HTML (1 KB)  PDF (1554 KB)  ( 902 ) 249 Preparation of a novel iron-containing polymeric nanobubbles ultrsound contrast agents and its acoustic properties LI Jiaping, JIANG Yan, WANG Doudou, YU Fangfang, CHENG Yueyue, LIU Zhe Objective: To prepare iron-containing polybutyl cyanoacrylate (PBCA) hybrid nanobubbles (UPNBs) and to explore the properties and ultrasonic imaging intensity of hybrid nanobubbles under different conditions. Methods: The nanobubbles (NBs) were produced via emulsion polymerization of PBCA and were purified by a simple mechanical agitation, and the UPNBs were prepared via an oil-in-water (O/W) encapsulation of iron oxide nanoparticles in the bubble shell. The mean diameter and concentration of UPNBs were analyzed by using a Beckmann Coulter Counter. The iron concentration in different UPNBs batches was determined by inductively coupled plasma mass spectrometer (ICP-MS). Furthermore, the contrast of UPNBs in ultrasound (US) imaging was evaluated by an ultrasound scanner. Results: These polymeric NBs exhibited strong contrast in US imaging. Moreover, with the iron concentration increasing, the size of UPNBs changed slightly and they had excellent backscattering capability in US imaging. Conclusion: These nanobubbles are candidates as hybrid imaging agents for US molecular imaging, which may be potential vehicles for MRI-guided US-mediated drug and gene delivery. 2019 Vol. 49 (4): 249-252，257 [Abstract] ( 642 ) HTML (1 KB)  PDF (1526 KB)  ( 1028 ) 253 Conservative analysis in CTL epitopes of HBV S protein between B and C genotypes CHEN Qingxin, LI Wenshu, Chen Zhuoyan, HE Jiali, Wang Wenhuan, Lin Mao, YE Juxiu Objective: To analyze the conservative property in CTL epitopes of HBV large S proteins and figure out the stable epitopes between B and C genotypes. Methods: A pair of primers for S gene of HBV was designed, the S gene amplified by PCR and construed into pIRES vector. According to large S protein from B and C genotypes of HBV, specific CTL epitopes in large S protein of HBV had been analyzed and screened by BIMAS, NetMHC and SYFPEITHI. Conservative property analysis was made by comparing candidate CTL epitopes with large S protein database of HBV B and C genotypes conducted by BLAST. Results: HBV S gene was cloned into pIRES expression vector successfully. Seventeen CTL epitopes of large S protein were screened according to scores of BIMAS, NetMHC or SYFPEITHI. High conservative property in B and C genotypes of HBV (>80%) was showed in 6 CTL epitopes which were not identified now. 17 CTL epitopes, including 6 identified CTL epitopes, were not conservative in B and C genotypes of HBV. Conclusion: The specific CTL epitopes of large S protein of HBV show high differences between B and C genotypes. Six CTL epitopes of HBV large S protein are obtained for further immunological studies. 2019 Vol. 49 (4): 253-257 [Abstract] ( 691 ) HTML (1 KB)  PDF (1283 KB)  ( 703 ) 258 Roles of microRNA-17-5p in the resistance of MMQ cells to Cabergoline GUAN Jiaqing, XU Jiadong, WANG Chunyong, SU Zhipeng, CAI Lin, CHEN Xianbin, ZHENG Weiming Objective: To study the roles of microRNA-17-5p may play in the resistance of MMQ cells to dopamine agonists. Methods: MMQ cells were either treated with miR-17-5p mimics or miR-17-5p inhibitor to regulate the expression of miR-17-5p in vitro qPCR was used to verify the transfection efficiency. After treatment, cell viability and its response to CAB was determined with CCK-8 assays. The target gene of miR-17-5p was predicted and confirmed via bioinformatics analysis and luciferase reporter assays. The expression of PTEN was analyzed by qPCR and Western Blotting. In addition we deployed CCK-8 assay to evaluate the response to CAB of MMQ cells which were initially treated with lentiviral vector containing PTEN. Results: Overexpression of miR-17-5p could promote the proliferation of MMQ cells and suppress CAB cytotoxicity. On the contrary, down-regulated of the expression of miR-17-5p could effectively inhibit proliferation and boost susceptibility to CAB in MMQ cells. The bioinformatics database identified the potential target of miR-17-5p and the luciferase reporter assay confirmed that PTEN was the direct target of miR-17-5p. While the expression of PTEN was down-regulated in miR-17-5p over-expressed MMQ cells, the expression of PTEN was up-regulated in miR-17-5p knockdown MMQ cells. Furthermore, PTEN over-expression was able to reverse the drug resistance induced by miR-17-5p on CAB treatment. Conclusion: miR-17-5p promotes MMQ proliferation and mediates CAB resistance by targeting PTEN. 2019 Vol. 49 (4): 258-262,280 [Abstract] ( 665 ) HTML (1 KB)  PDF (1531 KB)  ( 605 ) 263 Study of anti-liver fibrosis effect of astralosides via circular RNA34116 ZHOU Yuping, YANG Ping, TANG Chunlan, QU Hui, LYU Xueyou, HU Guimei Objective: To study the mechanism underlying Astragalosides in the inhibition of hepatic fibrosis via circularRNA. Methods: Mice were randomly divided as normal group, CCl4 model group, Astragalosides group and sorafenib group, with 8 mice in each group. A CCl4 liver fibrosis model was established. After drug intervention with Astragalosides, the serum ALT and TBIL, pathological changes (HE staining and Masson staining) of the liver and relative mmu_circ_34116 expressionwere detected and observed by RT-PCR. Results: Histopathological results showed that, compared with the normal group, liver tissue inflammation and collagen deposition were significantly increased Astragalosides and sorafenib significantly reduced inflammation and collagen deposition. The results of serum liver function showed that Astragalosides and sorafenib significantly reduced the level of ALT and TBIL after intervention in the model mice. RT-PCR results showed that, compared with the normal group, the expression of mmu_circ_34116 in liver tissue was significantly reduced in CCl4 model group, and its expression was significantly increased after intervention with Astragalosides in the model, but sorafenib had no significant effect on its expression. Conclusion: Astragalosides significantly inhibited the development of liver fibrosis. The mechanism may be associated with over expression of  mmu_circ_34116. 2019 Vol. 49 (4): 263-266 [Abstract] ( 757 ) HTML (1 KB)  PDF (1448 KB)  ( 651 ) 267 Preparation of agarose/ε-PLL/CHA barrier membranes and evaluation of its antibacterial and biological properties HE Zhiqi, QIAN Qiuping, DENG Hui, HU Rongdang Objective: To prepare a novel periodontal guided tissue regeneration (GTR) barrier membranes of agarose/ε-PLL/CHA and evaluation of it with excellent biocompatibility and antibacterial properties. Methods: CHA was prepared by the hydrothermal method. The characterization of the CHA were analyzed by Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The different concentrations of CHA and ε-PLL were added to the agarose to prepare the barrier membranes. The mechanical strength of barrier membranes was assessed through rheometer. Live/Dead staining was used to detect the biocompatibility of barrier membranes. The effect of antibacterial of barrier membranes was confirmed by inhibition zone. Results: SEM and FTIR indicated successful synthesis of hollow columnar CHA. Barrier membranes have good biocompatibility. CHA could enhance the mechanical properties of the barrier membranes. Live/Dead staining showed a good biocompatibility. There were obvious inhibition zones found around the barrier membranes. Conclusion: The agarose/ε-PLL/CHA barrier membrane was successfully prepared, and the membrane was proved to have good biocompatibility and antibacterial properties. 2019 Vol. 49 (4): 267-271 [Abstract] ( 693 ) HTML (1 KB)  PDF (1506 KB)  ( 814 ) 272 The effect and mechanism of salvianolic acid A on retinal pigment epithelial cells injured by ultra-violet radiation via upregulating SIRT3 expression LI Xia, WANG Yanfang, CHEN Zhanqiao, YU Songping. Objective: To investigate the effect and mechanism of salvianolic acid A (Sal A) on retinal pigment epithelial cells (RPE) injured by ultra-violet (UV) radiation via upregulating SIRT3 expression. Methods: ARPE-19 cells were divided into five groups: control group, UV group, Sal A+UV group, Sal A+negative control si-RNA+UV group, Sal A+si-SIRT3+UV group. The dose of UV irradiation was 30 mJ/cm2 and the concentration of Sal A was 50 μmol/L. The cell viability was tested by MTT. The ROS content was detected by DCFH-DA straining. The activity of SOD2 was analyzed by CuZn/Mn-SOD activity detection kit (WST-8 method). The expression of SIRT3, SOD2, cleavage-Caspase 3 and cytochrome c (Cyt c) was analyzed by Western blot. The expression of SIRT3 and SOD2 mRNA was detected by RT-qPCR. Results: The expression of SIRT3 protein and mRNA in ARPE-19 cells was decreased by UV irradiation. Pretreatment with 5, 25 and 50 μmol/L reversed the expression of SIRT3 protein and mRNA by UV. Compared with the control group, the apoptosis rate, ROS content, the expression of Bax, cleaved-caspase 3 and Cyt c in UV group were significantly increased, while the cell survival rate, the expression of SOD2 and Bcl-2, the activity of SOD2 were significantly decreased in UV group. Compared with UV group, the apoptosis rate, ROS content, the expression of Bax, cleaved-Caspase 3 and Cyt c were significantly decreased, while the cell survival rate, the expression of SOD2 and Bcl-2, the activity of SOD2 were significantly increased in Sal A+UV group. Compared with Sal A+UV group, the expression of Bax, cleaved-Caspase 3 and Cyt c in UV group were significantly increased, while the cell survival rate, the expression of SOD2 and Bcl-2, the activity of SOD2 were significantly decreased in Sal A+siSIRT3+UV group. Conclusion: Sal A inhibited cell damage induced by UV via up-regulating SIRT3 expression and reversing SOD2 activity. 2019 Vol. 49 (4): 272-276 [Abstract] ( 632 ) HTML (1 KB)  PDF (1543 KB)  ( 720 ) 277 Phenotypic and genetic analysis of a pedigree with inherited protein C deficiency LIU Meina, SU Kankan, ZHANG Haiyue, JIN Yanhui, YANG Lihong, LI Xiaolong, WANG Mingshan Objective: To ascertain the mutant gene of a family with hereditary protein C (PC) deficiency and to explore the molecular pathogenesis. Methods: The plasma protein C activity (PC:A), protein C antigen (PC:Ag) and other coagulation index of the family members were measured. For the proband, potential mutations in exons, flanking introns and 5’, 3’untranslated regions of PROC gene were screened by direct DNA sequencing. The Mutational site was further detected in the other family members. The ClustalX-2.1-win software and the online bioinformatics tool PolyPhen-2 were used to checke the conservatism and the possible impact of the mutation respectively. Structural analysis of the mutational site was processed with software Swiss-PdbViewer and PIC program. Results: The proband, her son and sister had reduced level of PC:A and PC:Ag among 39%~58%. Gene sequencing revealed that three people carried a heterozygous mutation c.997G>A in exon 9 of PROC gene resulting in a substitution of Alanine 291 by Threonine (p.Ala291Thr). According to the results of bioinformatics software analysis, p.Ala291Thr was “probably damaging” and Ala291 was not highly conserved. Model analyzing illuminated that replacement of Ala291 with Thr291 led to additional hydrogen bonding between Thr291-Pro327, affecting the normal spatial conformation and stability of protein. Conclusion: The proband carried a heterozygous mutation c.997G>A in exon 9 resulting in p.Ala291Thr which is a novel mutation. The p.Ala291Thr mutation could potentially account for the reduced activity of PC in this pedigree. 2019 Vol. 49 (4): 277-280 [Abstract] ( 747 ) HTML (1 KB)  PDF (1372 KB)  ( 843 ) 281 The effect of sclerostin-antibody or sclerostin-antibody with Astragalus membranaceus on ovariectomized osteoporosis: a comparative study SUN Shuaibo, CHEN Shaomin, LIN Jinti, ZHOU Chengwei, SHUI Xiaolong Objective: To compare the therapeutic effect of Sclerostin-antibody and Sclerostin-antibody with Astragalus membranaceus on osteoporosis in OVX rats. Methods: Bilateral ovariectomy (OVX, n=25) and sham (Sham, n=5) operation were performed in 30 healthy female SD rats at random. After 12 weeks, 5 rats in each group were executed and their femurs were taken for micro-CT to detect whether the osteoporosis animal model was established. When the model of osteoporosis is established successfully, all animals from OVX (n=20) group randomly divided into two groups:group Scl-Ab+Astragalus membranaceus and group Scl-Ab. Then group Scl-Ab+Astragalus membranaceus was given Scl-Ab (12.5 mg/kg, two times a week) by hypodermic injection and Astragalus membranaceus extract [200 mg/(kg·d), once a day] by gavage, Scl-Ab group was given Scl-Ab (25 mg/kg, two times a week) by hypodermic injection and saline [200 mg/(kg·d), once a day] by gavage. The femurs of rats were harvested for evaluation. The results of treatment for osteoporosis were evaluated by micro-computerized tomography and undecalcified sliced. Results: Compared with group Scl-Ab, the distal femurs from group Scl-Ab+astragalus membranaceus have a higher BMD, BV/TV, Tb.Th, Tb.N, Conn. D bone mineral apposition rates and a lower Tb.Sp. Conclusion: The results of this study indicates that Sclerostin-antibody with Astragalus membranaceus is superior to Sclerostin-antibody only in the treatment of ovariectomized osteoporosis in rats. 2019 Vol. 49 (4): 281-284,288 [Abstract] ( 777 ) HTML (1 KB)  PDF (1413 KB)  ( 729 ) 285 Enhanced osseointegration of nano textured Ti6Al4V implants by etching WU Weili, LUO Jun, YING Yukang, ZHU Haiqian, TAO Dan, XU Panshu, YU Weiqiang Objective: To evaluate the removal torque and bone gene expression of nano textured Ti6Al4V implants in vivo. Methods: The implants were divided as group of nano textured Ti6Al4V implants by etching and as the control of the machined implants. All the implants were placed in the tibia of rabbits. After four weeks, removal torque was analyzed by a digital torque gauge. New bone was found with fluorescence microscope. The gene expression of Runx2, Col-1 and OCN was examined using real-time PCR. Results: Nano textured Ti6Al4V implants by etching showed significantly bigger removal torque (P<0.05) and higher Runx2, Col-1 and OCN gene expression (P<0.05) than the machined implants. In addition, more new bone was found around nano textured Ti6Al4V implant around. Conclusion: The nano textured Ti6Al4V implants by etching could improve the early osseointegration processin vivo, which may be applied clinically in the future. 2019 Vol. 49 (4): 285-288 [Abstract] ( 702 ) HTML (1 KB)  PDF (1347 KB)  ( 795 ) 289 The effect of low tube voltage in combination with iterative reconstruction algorithm on image quality and radiation dose in upper limb intravascular fistula CT angiography CHEN Yonghua, FU Gangzhe, PAN Kehua, CHEN Cong, CAO Guoquan Objective: To study the effect of low-tube voltage (80, 100 kV) combined with the iterative algorithm (IR) on image quality and radiation dose in upper limb intravascular fistula CT angiography. Methods: 72 cases of suspected  upper limb arteriovenous fistula (AVF) dysfunction were randomized into 120 kV, 100 kV and 80 kV voltage groups (24 cases per group). The CTA was undertaken with a 320-row scanner (Toshiba Aquilion ONE), and the low tube voltage groups combined with standard adaptive iterative dose reduction technique (AIDR 3D). The states of AVF were observed by using axial images combined with CPR, VR and other reconstruction techniques. The general conditions, vessel density (VD), SNR, CNR, subjective image quality score and radiation dose of the three groups were compared respectively. Results: CTDIvol and DLP in the 100 kV and 80 kV groups were significantly lower than those in the conventional 120 kV group (P<0.05), but the difference between the 100 kV and 80 kV groups was not significant (P>0.05). Combined with the standard iterative reconstruction, VD increased significantly with tube voltage reduction (P<0.05); There was no significant difference in image noise between the 100 kV group and the 120 kV group (P>0.05); When the tube voltage rduced to 80 kV, the image noise increased comparing with the 100 kV and 120 kV groups (P<0.05). SNR and CNR in the 80 kV group were the highest, 67.5% and 78.6% higher than those in the 120 kV group, but the difference between the 80 kV and 100 kV groups was not significant (P>0.05). There was no significant difference in subjective score between the low-tube voltage group and the conventional 120 kV group (P>0.05), and all the three groups had good image quality. Conclusion: Low tube vohage (100 kV) combined with standard IR algorithm can significantly improve the image quality and reduce radiation dose in upper limb intravascular fistula CT angiography. It has certain clinical application value. 2019 Vol. 49 (4): 289-293,297 [Abstract] ( 687 ) HTML (1 KB)  PDF (1383 KB)  ( 672 ) 294 The value of small dose pituitrin in conservative laparoscopic surgery for ectopic pregnancy XU Lixia, SU Aifang, ZHOU Xiaohong. Objective: To investigate the value of small dose of pituitrin administered in conservative laparoscopic surgery for ectopic pregnancy. Methods: A total of 124 patients who were confirmed with ectopic pregnancy and underwent laparoscopic tubal incision were reviewed retrospectively in Jinhua Maternal and Childcare Hospital. Sixty-three patients included in the study group received injection of 2 U pituitrin into mesosalpinx at the implantation site intraoperatively; Sixty-one patients in the control group underwent routine laparoscopic tubal incision without pituitrin injection during the operation. The operation time, intraoperative blood loss, postoperative persistent ectopic pregnancy rate, postoperative uterine fallopian tube angiography, postoperative pregnancy rate and postoperative ectopic pregnancy rate were observed. Results: The operation time of the study group was (47.0±7.5) min, significantly shorter than that in the control group [(58.7±7.9)min] (P<0.01). The intraoperative blood loss in the study group was (17.3±7.3)mL, significantly less than the control group [(41.0±9.1)mL] (P<0.01); the incidence of persistent ectopic pregnancy (1.5%) in the study group was lower than that of the control group (3.3%) (P<0.05); the postoperative fallopian tube patency rate (55.6%) was higher than the control group (34.4%) (P<0.05) and  the pregnancy rate within 1 year after surgery was significantly higher in the study group (46.6%) than that in the control group (31.6%) (P<0.01), while the ectopic pregnancy rate in the study group (1.7%) was lower than that in the control group (7.0%) (P<0.05). Conclusion: Laparoscopic fallopian tube incision after small dose of pituitary vasopressin at the oviduct pregnancy site of the tubal pregnancy could significantly reduce intraoperative bleeding, reduce  the incidence of persistent ectopic pregnancy, protect postoperative fallopian tube function and improve postoperative pregnancy rate. 2019 Vol. 49 (4): 294-297 [Abstract] ( 706 ) HTML (1 KB)  PDF (1288 KB)  ( 666 ) 298 A comparison between 3D and 2D-DSA in the evaluation of nonthrombotic iliac venous lesions with chronic venous insufficiency LIU Xiaojun, XU Tingting, FU Xin, MEI Tong, CHEN Jinhui, LU Yongming Objective: To investigate the value of 3D-iliac vein DSA in nonthrombotic iliac venous lesions (NIVL) with chronic venous insufficiency (CVI). Methods: Clinical data of patients with NIVL with CVI experienced both 3D and 2D-distal subtraction angiography (DSA) examination in our department from January 2015 to December 2016 were analyzed. The differences of two methods in the diagnosis of NIVL with CVI were compared. Results: Altogether 175 cases were collected. Compared with DSA in the diagnosis of NIVL with CVI, sensitivity, specificity, Youden index, positive likelihood ratio and negative likelihood ratio was 91.1%, 91.2%, 0.823, 11.234 and 0.097 respectively. Kappa value was 0.825 and there was no significant difference (P>0.05, McNemar test) between the two groups. Both areas under curves (AUC) were>0.9, showed by receiver operating characteristic (ROC) curve; imaging contrast agents and imaging exposure time of 3D-DSA were less (P<0.05), with statistical difference. Conclusion: 3D-DSA is remarkable in sensitivity and specificity for NIVL with CVI, which is consistent with 2D-DSA. There is no significant difference between 3D and 2D-DSA in the diagnosis of NIVL with CVI. 3D and 2D-DSA is important in the diagnosis and preoperative assessment of NIVL with CVI, imaging contrast agents and imaging exposure time of 3D-DSA were less. 2019 Vol. 49 (4): 298-300 [Abstract] ( 626 ) HTML (1 KB)  PDF (1297 KB)  ( 771 ) 301 The effect analysis of shared decision-making nursing model on neurology patients WU Yan, LI Liang, LANG Chengyun Objective: To analyze the influence of shared decision-making model on the decision-making willingness and disease management of patients with cerebral infarction in the department of neurology. Methods: From June 2016 to May 2018, 104 patients were admitted to our hospital. Based on the number upon admission, the patients were randomly divided into the study group and the control group, with 52 cases in each group. The control group was implemented traditional nursing intervention, while a shared decision-making model was introduced in the study group. Then two groups of patients were compared in terms of decision-making willingness, information needs and nursing satisfaction. Results: The decision-making willingness of patients in the study group was stronger, and their demand for this information and satisfaction with the nursing service model were higher than the control group. The decision-making efficiency of both patients and nurses was significantly better than that of the control group (P<0.05). Conclusion: The shared decision-making model applied to patients with cerebral infarction can improve the decision-making willingness of these patients to take healthcare measures, help them to acquire better knowledge about the disease, facilitate disease management and improve nursing satisfaction. 2019 Vol. 49 (4): 301-305 [Abstract] ( 682 ) HTML (1 KB)  PDF (1270 KB)  ( 785 )

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