• Title/Summary/Keyword: heparin

Search Result 446, Processing Time 0.024 seconds

Studies on the Effects of Estrous Cow Serum, Follicular Fluids and Matured Cumulus Cells on In Vitro Maturation and Fertilization of Bovine Follicular Oocytes (발정우 혈청, 난포액 및 난구세포의 첨가가 우난포란의 체외성숙 및 수정에 미치는 영향에 관한 연구)

  • 김상근;이만휘;김무강;박항균;한방근
    • Korean Journal of Animal Reproduction
    • /
    • v.14 no.3
    • /
    • pp.183-190
    • /
    • 1990
  • These studies were carried out ot investigate the effects of estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3-5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48 hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18$^{\circ}C$20 hrs. with motile capacitated sperm in the TCF(Tyrode calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes harvested, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 33.3%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5%~20% ECS and FCS were 74.0%~80.6, 26.2%~30.0% and 71.7%~76.9%, 51.9%~58.0%, and those values were higher the supplement of ECS than FCS. 3. The maturation rate(68.0%~64.6%) and fertilization rate(59.6%~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 4. The maturation rate(76.5%) and fertilization rate(61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$106/ml cumulus cells were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$104~5/ml and 1$\times$108/ml cumulus cells.lus cells.

  • PDF

Hemato-Biochemical Indices of Hanwoo Cattle Raised at Different Altitudes (서로 다른 고도에서 사육된 비육한우의 혈액-혈청 지수)

  • Ji, Joong-Ryong;Alam, Md. Rafiqul;Na, Chong-Sam;Kim, Nam-Soo
    • Journal of Veterinary Clinics
    • /
    • v.28 no.2
    • /
    • pp.219-224
    • /
    • 2011
  • The health status of three groups of Hanwoo steers (n=157) aged 2-3 years, raised at three different altitudes (600 m, n=50; 200-400 m, n=58; plane land, n=49) and environment with more or less similar management have been evaluated through hematological, biochemical and globulin examinations in order to determine the optimum environment suitable for raising cattle while at the same time minimizing the risk of disease. Five mL of blood samples from each animal were collected by jugular veinipuncture and 2 mL was transferred to a tube containing EDTA for complete blood count (CBC) and 3 mL in lithium heparin for chemistry screening (CS) and immunoassay. Among the CBC parameters a significantly higher white blood cell count (tWBC), total red blood cell count (tRBC), hemoglobin (Hb) and packed cell volume (PCV) were noticed in the high altitude groups, whereas those were lower in the plane land group. In the CS parameters higher levels of aspartate aminotransferase (AST), creatinine, lactate dehydrogenase (LDH) and total bilirubin (TBL) were found in the plane land group, whereas those were lower in the high altitude groups. The total protein (significantly) and globulins were higher in the 600 m group. The results of this study revealed that the overall health status of the Hanwoo cattle based on the hemato-biochemical indices was superior in the highest altitude and inferior in the plane land group but all the parameters were within the reference range in all the groups. Therefore, for recommendation of a suitable environment at an appropriate altitude for raising cattle there need to be further studied along with the hemato-biochemical parameters; considering, breeding, feeding, management, marketing, waste disposal and other factors.

Effects of established cell lines on bovine embryo development during in vitro culture (계대세포를 이용한 소 수정란의 체외배양 체계에 관한 연구)

  • Joo, Suk-chun;Lee, Byeong-chun;Lee, Won-yu;Choi, Yun-seok;Hwang, Woo-suk
    • Korean Journal of Veterinary Research
    • /
    • v.37 no.3
    • /
    • pp.647-659
    • /
    • 1997
  • To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

  • PDF

Anticoagulant Activity of Sulfated Polysaccharides Isolated from Codium fragile (청각 산추출물에서 정제한 함황다당류의 항응고활성)

  • Park, Mee-Kyung;Kweon, Mee-Hyang;Cho, Hong-Yon;Yang, Han-Chul
    • Applied Biological Chemistry
    • /
    • v.42 no.2
    • /
    • pp.140-146
    • /
    • 1999
  • We have isolated two anticoagulant polysaccharides from an acidic extract of Codium fragile. The purification was conducted using three consecutive chromatographies of DEAE-Toyopearl 650C, Sephadex G-100 (G-75), and Sepharose CL-6B by measuring activated partial thromboplastin time (APTT). The two purified anticoagulant polysaccharides, CF-1-VIa-1 and CF-1-VIIa-1, were found to be nearly homogenous on HPLC using a gel permeation column and appeared to have molecular weights of about 80,000 Da and 40,000 Da, respectively. The polysaccharides consisted mainly of arabinose and galactose in a molar ratio of about 2 : 1, and also comprised 12-13% of sulfates at their constituent sugars. CF-1-VIa-1 and CF-1-VIIa-1 inhibited blood coagulation via both the intrinsic and the extrinsic pathways. The polysaccharides unlike heparin showed an inhibitory activity on thrombin when a pure fibrinogen without antithrombin III was used as a substrate. Structural modifications using sulfation and desulfation affected the anticoagulant activities directly, suggesting that the content of sulfate plays an important role in the blood coagulation cascade. The polysaccharides may inhibit some proteases involved in the blood coagulation cascade, judging from the independence of calcium concentrations in their anticoagulant activity.

  • PDF

The National Survey of Acute Pulmonary Thromboembolism in Korea (급성 폐혈전색전증 전국 실태 조사 보고)

  • Scientific Committee for National Survey of Acute Pulmonary Thromboembolism, Korean Academy of Tuberculosis and Respiratory Diseases
    • Tuberculosis and Respiratory Diseases
    • /
    • v.54 no.1
    • /
    • pp.5-14
    • /
    • 2003
  • Background : According to the study in ICOPER (International Cooperative Pulmonary Embolism Registry), the overall mortality rate of acute pulmonary thromboembolism (APTE) at 3 months is 17.4%. According to the study for current status of APTE in Japan, the hospital mortality rate is 14%. Although the incidence and mortality rate of APTE has been increasing, patient characteristics, management strategies, and outcome of APTE in the Korean population have not yet been assessed in large series. We therefore performed the national survey for the current status of APTE in the Korean population. Methods : 808 registry patients with APTE were analyzed with respect to clinical characteristics, risk factors, diagnostic procedures, treatment, and clinical outcome. Results : Main risk factors were immobilization, recent major surgery, and cancer. Common symptoms were dyspnea and chest pain. Common signs were tachypnea and tachycardia. The majority of registry patients underwent lung perfusion scanning. Spiral CT was used in 309 patients(42.9%), and angiography in 48 patients(7.9%). Heparin was the most widely used treatment. On multivariate logistic regression analysis, onset in hospital (odds ratio 1.88, p=0.0385), lung cancer (odds ratio 9.20, p=0.0050), tachypnea (odds ratio 3.50, p=0.0001), shock (odds ratio 6.74, p=0.0001), and cyanosis (odds ratio 3.45, p=0.0153) were identified as significant prognostic factors. The overall mortality rate was 16.9% and mortality associated with APTE was 9.0%. Conclusions : The present registry demonstrated the clinical characteristics, diagnostic strategies, management and outcome of patient with APTE in Korea. The mortality rate was 9.0%, and the predictors of mortality were onset in hospital, lung cancer, tachypnea, shock, and cyanosis. These results may be important for risk stratification as well as for the identification of potential candidates for more aggressive treatment.

A Study on the Types and Causes of Medication Errors and Related Drugs - by Analyzing AJNs Medication Error 73 Cases - (임상에서의 투약오류원인과 관련 의약품 분석 - AJN에 기고된 Medication Error 기사의 73사례를 중심으로 -)

  • Cho Won Sun
    • Journal of Korean Public Health Nursing
    • /
    • v.16 no.1
    • /
    • pp.176-189
    • /
    • 2002
  • The purpose of this study were to illustrate the various medication error types and causes and identified to related drugs to provide basic data for preventing nurses' medication error by analysing 73 cases of AJN 'medication Error' column(1993, Oct -2000, Nov). Nurses' types of medication error were classified into 7 types. The most frequent error types are wrong medication$(21.9\%)$ and the wrong dose$(21.9\%)$ together. The others are wrong $time(4.1\%)$, $omission(2.7\%)$, mechanical $error(2.7\%)$, incorrect IV $rate(1.4\%)$. wrong route $administration(1.4\%)$ in order. Nurses' causes of medication error were 9 kinds. The most frequent type is confusing between similar drug shape, color, size, name, injection devices and patient's $name(43.9\%)$ and the others are lack of knowledge about $drugs(26.8\%),\; slips(7.3\%),\; miscalculating\;dose(4.9\%)$, incorrect adjusts $devices(4.9\%)$, difficulty to read or illegible decimal $point(4.9\%),$ $abbreviation(2.4\%)$, fatigue with $overwork(2.4\%)$ and no communication with $patient(2.4\%)$ in order. Related drugs with medication error are as follows. - dose unit(IU. minims. mcg/min. mEq) : Heparin. insulin. synthetic calcitonin, some enzymes and hormones, vitamins, some antibiotics, tuberculin injection. MgSO4 injection. nitroglycerin - similar size, color and shape drug : $0.9\%$ N/S and acetic acid $0.25\%$ for irrigation. premixed 2mg lidocaine sol. and $0.9\%$ N/S, gentamycin 20mg/2mL for children and 80mg/2mL for adult, dextroamphetamine 5mg and 10mg capsule. sedatives chloral hydrate 250mg/5mL and 500mg/5mL - similar name :Aredia(pamidronate disodium) and Adriamycin(doxorubicin), Lamictal (lamotrigine) and Lamisil 250mg. Elderpryl and enalapril, cefotaxime and cefoxitin, carboplatin and cisplatin, sumatriptan and zolmitriptan, Celebrex and Celexa, Humulin and Humalog, Percodan and Percocet, Diabeta and Diabinese, Epivir and Retrovir, Xanax(alprazolam) and Zantac(ranitidine) - decimal point : low molecular weight warfarin, methotrexate - unfamiliar drug uses of familiar drug ; methotrexate. droperidol, imipramine, propranolol - number of drug name(misleading chemical name) : 6-thioguanine, 6-mercaptopurine, 5-fluorouracil - type of administration route : Oxycodone(OxyContin). - administration time : acarbose(Precose). - injection way (Z-track method): hydroxyzine - epidural cathether : LMWHs(enoxaparin, dalteparin), - ADD Vantage self contained delivery system : ceftriaxone(Rocephin)

  • PDF

AN EXPERIMENTAL STUDY ON THE BONE INDUCTION CAPACITY OF THE PORCINE BONE MATRIX-DERIVED BONE MORPHOGENETIC PROTEIN (돼지의 골기질유도 골형성단백질의 골유도능에 관한 실험적 연구)

  • Park, Young-Wook;Lee, Jong-Ho;Kim, Soo-Kyeong
    • Maxillofacial Plastic and Reconstructive Surgery
    • /
    • v.19 no.3
    • /
    • pp.265-286
    • /
    • 1997
  • Bone morphogenetic proteins(BMPs) are a group of transforming growth factor beta(TGF-${\beta}$)-related factors and multifunctional proteins, especially the only known biologic factors capable of inducing endochondral bone formation at an extraskeletal site. This study was performed to investigate the effect of the partially purified porcine BMP(pBMP) at an ectopic site. PBMP was partially purified from porcine bone matrix and its activity was monitored by an in vivo bioassay. The purification method utilized extraction of the bone-inducing activity with 4M guanidine, followed by chromatography on heparin-Sepharose. Active fractions were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. And the fractions were reconstituted with inactive insoluble collagenous bone matrix from rats, acid soluble type I collagen from rat tail and chondroitin-6-sulfate sodium salt and implanted into the pectroralis muscle pouches of Sprague-Dawley rats. And the carrier complex was implanted on the opposite side as control. The rats were sacrificed at the day of 1st, 3rd, 5th, 7th, 11th, 14th and 21st after implantation and examined histologically, radiologically and biochemically. And alkaline phosphatase activity and calcium content were used as indices of bone formation. The results were as follows ; 1. Active fractions were localized in a zone between 31 and 40 KDa on SDS-PAGE. 2. The implanted 3.0mg of the partially purified pBMP induced cartilage and bone in the muscle tissue of rats through an endochondral ossification process. 3. Inactive insoluble bone matrix, type I collagen and chondroitin-6-sulfate have functioned as carriers for pBMP, but revealed some foreign body reactions. 4. Soft X-ray didn't reveal significant change between the experimental and the control group. 5. The alkaline phosphatase activities in the experimental group of 5th, 7th, 11th, 14th and 21st were increased significantly compared with control (p<0.01) with the peak in the group of 11th day. 6. With time, the calcium content of the experimental group increased. And the calcium contents in the experimental group of 11th, 14th and 21st were increased significantly compared with control (p<0.01).

  • PDF

EFFECTS OF LOCAL IRRIGATION AND/OR INTRAVENOUS ADMINISTRATION OF ARGATROBAN ON THROMBOSIS IN MICROVASCULAR ANASTOMOSES OF FEMORAL VEIN OF RABBIT (가토대퇴정맥에서 미세혈관문합시 Argatroban의 국소세척 및 전신투여가 혈전형성에 미치는 영향)

  • Kook, Min-Suk;Park, Hong-Ju;Oh, Hee-Kyun
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
    • /
    • v.31 no.4
    • /
    • pp.300-305
    • /
    • 2005
  • Purpose: For the reconstruction of maxillofacial defect created by trauma, infection, or tumor etc, the role of microvascular anastomosis or vessel graft has been increased. Many methods has been tried to increase the success rate of microvascular anastomosis. Various anticoagulants and thrombolytic agents have been used to reduce the failure rate of microvascular anastomosis and avoid re-operation. Many drugs, however, have been used in the limited cases because most of these drugs may cause complications, such as allergy, fever or systemic bleeding. This study was performed to evaluate the influence of the Argatroban on patency and thrombosis in microvascular anastomosis when it is used for local irrigation or general administration. Materials & methods: Eight mature rabbits, weighing 2kg, were used. After exposing both femoral veins, the artificial thrombotic model was made by crushing injury using a smooth needle holder, and the transverse incision were made on femoral vein. The animals were divided into 4 groups according to Argatroban administration methods; control group (n=4), topical irrigation of lumen with saline solution; experimental group 1 (n=4), topical irrigation of lumen with Argatroban saline solution; experimental group 2 (n=4), topical irrigation of lumen with heparin followed by intravenous injection of Argatroban; experimental group 3 (n=4), topical irrigation of lumen with Argatroban followed by IV of Argatroban. Microvascular anastomosis was done with 10-0 Ethilon. The patency was evaluated by empty-and-refill test 30 minutes and 3 days after microanastomosis. The thrombus formation was examined 3 days after microanastomosis by surgical microscope. The histologic findings were also examined. Results: 1. Thirty minutes after microvascular anastomosis, the patency of all experimental groups was better than that of control group, but there was no significant difference among groups. 2. Three days after microvascular anastomosis, the patency of all experimental groups was more improved than that of control group (p<0.05). There was no significant difference among experimental groups. 3. Three days after microvascular anastomosis, the amount of thrombus in all experimental groups was less than that of control group (p<0.05). There was no significant difference among experimental groups. 4. Histologically, a lot of luminal thrombus was observed around sutured area in control group. Few luminal thrombus was observed in all experimental groups. The necrotic changes were observed on the sutured vein wall in all specimens. Conclusion: These results indicate that topical irrigation and/or intravenous administration of Argatroban is effective in improving patency and preventing thrombus formation after microvascular anastomosis.

Effect of Equilibration Time and Cell Stage on the Survival of IVF Bovine Embryos Cryopreserved by Vitrification (한우 체외수정란의 동결보존시 평형시간과 배 발달단계가 생존성에 미치는 영향)

  • 공일근;주영국;이은봉;김용권;박충생
    • Journal of Embryo Transfer
    • /
    • v.9 no.1
    • /
    • pp.7-14
    • /
    • 1994
  • The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

  • PDF

Studies on the Hemodilution Perfusion with Rygg-Kyvsgaard Oxygenator (혈액희석 체외순환법에 관한 임상적 관찰 -상온하 Rygg-Kyvsgaard 산화기 및 Sigmamotor pump 사용예를 중심으로-)

  • 손광현
    • Journal of Chest Surgery
    • /
    • v.3 no.2
    • /
    • pp.73-90
    • /
    • 1970
  • Clinical perfusion data on 16 cases of cardiopulmonary bypass using Sigmamotor pump and RyggKyvsgaard Oxygenator which performed at Seoul National University Hospital during the period of Aug. 1968 to Aug. 1970 was analized. AIl cases were hemodiluted and the perfusion was carried out under the normothermic condition. The age of the patients ranged between 6 and 43 years. The b:dy weight varied between 18.3 and 54.0 kg and the body surface area between 0.78 and 1. 59$M^2$. The priming solution was consiste:I with fresh ACD blood. Hartmann solution and Mannitol. The average amount of priming was approximately 2242 ml. The average hemodilution rate was 17%. The flow rate ranged from 1.7L to 3.5L/Min/$M^2$ and averaged 2.4L/Min/$M^2$ or 78mI/Min/kg. The duration of perfusion varied from 22 to 110 min with average of 56.9 minutes. Some hemodynamic responses were observed. The arterial pressure dropped immediately after the initiation of partial perfusion and was more marked after the total perfusion foIlowed by gradual increase to the safety level. The central venous pressure reflected the reduced blood volume especially in the cases of prolonged perfusion which lasted over 60 min. In most of the cases, red blood cell count decreased and white blood ceIl count increased after the perfusion. Hemoglobin level was decreased, averaging of 12.5mg%, Hct 3.3% and platelets count of 18% postoperatively. Plasma hemoglobin increased mildly, from pre-perfusion average value of 4. 06mg% to postperfusion value of 22.5mg%. Serum potassium was 4.4mEq/L pre-operatively and was decreased to 3.7mEq/L postoperatively. Five cases showed definite hypopotassemia immediately after the operation. Sodium and chloride decreased mildly. These electrolyte changes are thought to be related with hemodilution. diuretics and reduced blood volume during and after the perfusion. Arterial blood pH value revealed minimal to moderate elevation from preperfusion average value of 7.376 to 7.461 during perfusion and then 7.365 after perfusion. The pC02 and hicarbonate showed minimal to moderately lowered values. The total CO2 was decreased. Buffer base decreased during perfusion (Av. 42.6mEq/L) and further decreased after the perfusion (Av. 40.8mEq/L). These arterial blood acid base changes suggested that the metabolic acidosis was accompanied by respiratory alkalosis during and immediately after the perfusion. Authors belived that the acidosis could more effectively be corrected with the more additional dose of bicarbonate than we used by this study. The chest tune drainage during the first 24 hours following operation was 1158 ml in average. One case (Case No. 15) showd definite bleeding tendency and it was believed that the cause might be due to the defect of heparin and protamine titration. The average urinary out put during 24 hours post-perfusion was 1291ml. One case (Case No. ]) showed definite post perfusion oliguria. As conclusion hemodilution using fresh ACD blood. Hartmann and Mannitol solution added with Bivon and high flow rate unler normothermia. was thought to amelioratc the severity of mctabolic acidosis during and after perfusion with relatively satisfactory effect on the diuresis and bleeding tendency.

  • PDF