• Title/Summary/Keyword: Infection times

Search Result 1,019, Processing Time 0.041 seconds

Establishment of a Dental Unit Biofilm Model Using Well-Plate (Well-Plate를 사용한 치과용 유니트 수관 바이오필름 모델 확립)

  • Yoon, Hye Young;Lee, Si Young
    • Journal of dental hygiene science
    • /
    • v.17 no.4
    • /
    • pp.283-289
    • /
    • 2017
  • The water discharged from dental unit waterlines (DUWLs) is heavily contaminated with bacteria. The development of efficient disinfectants is required to maintain good quality DUWL water. The purpose of this study was to establish a DUWL biofilm model using well-plates to confirm the effectiveness of disinfectants in the laboratory. Bacteria were obtained from the water discharged from DUWLs and incubated in R2A liquid medium for 10 days. The bacterial solution cultured for 10 days was made into stock and these stocks were incubated in R2A broth and batch mode for 5 days. Batch-cultured bacterial culture solution and polyurethane tubing sections were incubated in 12-well plates for 4 days. Biofilm accumulation was confirmed through plating on R2A solid medium. In addition, the thickness of the biofilm and the shape and distribution of the constituent bacteria were confirmed using confocal laser microscopy and scanning electron microscopy. The average accumulation of the cultured biofilm over 4 days amounted to $1.15{\times}10^7CFU/cm^2$. The biofilm was widely distributed on the inner surface of the polyurethane tubing and consisted of cocci, short-length rods and medium-length rods. The biofilm thickness ranged from $2{\mu}m$ to $7{\mu}m$. The DUWL biofilm model produced in this study can be used to develop disinfectants and study DUWL biofilm-forming bacteria.

Identification of multiple key genes involved in pathogen defense and multi-stress tolerance using microarray and network analysis (Microarray와 Network 분석을 통한 병원균 및 스트레스 저항성 관련 주요 유전자의 대량 발굴)

  • Kim, Hyeongmin;Moon, Suyun;Lee, Jinsu;Bae, Wonsil;Won, Kyungho;Kim, Yoon-Kyeong;Kang, Kwon Kyoo;Ryu, Hojin
    • Journal of Plant Biotechnology
    • /
    • v.43 no.3
    • /
    • pp.347-358
    • /
    • 2016
  • Brassinosteroid (BR), a plant steroid hormone, plays key roles in numerous growth and developmental processes as well as tolerance to both abiotic and biotic stress. To understand the biological networks involved in BR-mediated signaling pathways and stress tolerance, we performed comparative genome-wide transcriptome analysis of a constitutively activated BR bes1-D mutant with an Agilent Arabidopsis $4{\times}44K$ oligo chip. As a result, we newly identified 1,091 (562 up-regulated and 529 down-regulated) significant differentially expressed genes (DEGs). The combination of GO enrichment and protein network analysis revealed that stress-related processes, such as metabolism, development, abiotic/biotic stress, immunity, and defense, were critically linked to BR signaling pathways. Among the identified gene sets, we confirmed more than a 6-fold up-regulation of NB-ARC and FLS2 in bes1-D plants. However, some genes, including TIR1, TSA1 and OCP3, were down-regulated. Consistently, BR-activated plants showed higher tolerance to drought stress and pathogen infection compared to wild-type controls. In this study, we newly developed a useful, comprehensive method for large-scale identification of critical network and gene sets with global transcriptome analysis using a microarray. This study also showed that gain of function in the bes1-D gene can regulate the adaptive response of plants to various stressful conditions.

Microbial Risk Assessment in Treated Wastewater Irrigation on Paddy Rice Plot (하수처리수를 관개한 후 벼재배 시험구에 대한 미생물 위해성 평가)

  • Jung, Kwang-Wook;An, Youn-Joo;Jang, Jae-Ho;Jeon, Ji-Hong;Yoon, Chun-Gyeong
    • Korean Journal of Ecology and Environment
    • /
    • v.38 no.2 s.112
    • /
    • pp.225-236
    • /
    • 2005
  • The protection of public health In wastewater reclamation and reuse is one of the most important issues. Monitoring data of Escherichia coli were collected from paddy rice plots in 2003 and 2004 experiments. Five treatments were used and each one was triplicated to evaluate the changes of E. coli: surface water, biofilter effluent (secondary level), UV-disinfected water and pond treatment. Microbial risk was quantified to assess human health risk by exposure to E. coli in paddy rice plots, which were irrigated with reclaimed wastewater. Beta-Poisson model was used to estimate the microbial risk of pathogen ingestion that may occur to farmer and neighbor children. Monte-Carlo analysis (10,000 trials) was used to estimate the risk characterization of uncertainty. In the following analysis, two scenarios were related to the reduction of risk against direct ingestion and exposure times. Scenarios A and B were assumed that the risk was 1,000 and 10,000 times lower than direct ingestion.'Golfers were assumed to be 0.001 L of reclaimed water by contact with balls and their cloths. Opportunity of contact in paddy rice field with pathogens was more frequent than handing golf balls, because of agricultural activity was practiced in ponded water in paddy rice culture. As a result of microbial risk assessment using total data of experimental period, risk value of E. coli in 2003 and 2004 experiment ranged from $10^{-5}$ to $10^{-8}$ and $10^{-4}$ to $10^{-8}$, respectively. The risk values in biofilter effluent irrigation was the highest, which is $10^{-4}$ in 2003 and $10^{-5}$ in 2004 experiments with scenario A. Ranges of $10^{-6}$ to $10^{-8}$ were considered at reasonable levels of risk for communicable disease transmission from environmental exposure and the risk value above $10^{-4}$ was considered to be attributable to the risk of infection. Irrigation with UV-disinfected water in the paddy field during the agricultural Period showed significantly lower microbial risk than others, and their levels of risk value were within the range of actual paddy rice field with surface water.

The pathogenicity of Streptococcus parauberis isolated from cultured olive flounder Paralichthys olivaceus (양식 넙치, Paralichthys olivaceus에서 분리한 연쇄구균증 원인균, Streptococcus parauberis의 병원성)

  • Choi, Hee-Jung;Cho, Mi-Young;Lee, Jae-Il;Kwon, Mun-Gyeong;Choi, Dong-Lim;Kim, Jin-Woo;Han, Myoung-Chul;Lee, Deok-Chan
    • Journal of fish pathology
    • /
    • v.22 no.3
    • /
    • pp.263-273
    • /
    • 2009
  • There is an increasing number of reports describing Streptococcus parauberis as an important pathogen of cultured olive flounder Paralichthys olivaceus and starry flounder Platichthys stellatus in Korea. We tried to determine the effects of water temperature (14${^{\circ}C}$ and 21${^{\circ}C}$) on the pathogenicity in Streptococcal disease caused by S. parauberis. We have challenged 180 olive flounder by i.p injection to $2.0{\times}10^{7}$ live cells/fish. Mortality was monitored for 21 days post challenge. And histopathological characterizations as infection degree, tissue degeneration and/or bacterial distribution were investigated with H&E stain and in situ hybridization technique. Fifty percent and 16.7% of mortality occurred within 21 days at 21${^{\circ}C}$ and 14${^{\circ}C}$ water temperature, respectively. In most cases, the typical symptoms of olive flounder infected with S. parauberis were darkness of the skin, lethargy, mild abdominal distension cause by ascites, splenomegaly, congested liver and internal organs paleness. The pericardial sac contained large amounts of cloudy fluid. Numerous whitish nodules, which were variable in size and often confluent, were randomly scattered throughout the myocardium. Especially, pericarditis and/or myocarditis was observed in all tested fishes after death. Positive in reaction with S. parauberis were found in all tissues in situ hybridization analysis. The relative numbers of S. parauberis in heart were much more than in liver, spleen, kidney and stomach. We evaluated that S. parauberis strain causes serious damage in the pericardium, shortness of breath and the blood disorder. Therefore, pericarditis and myocarditis caused by S. parauberis were closely related to mortality of olive flounder.

Development of assay method for the activities of new compounds, and the effect of several fungicides against spore germination, adhesion, and myceial growth of Colletotrichum sp. causing red pepper anthracnose (고추 탄저병균의 포자 발아와 부착, 균사 생장에 미치는 화합물의 활성 검정법 확립 및 살균제의 효과)

  • Kim, Jae-Jeung;Kim, Joon-Tae;Park, Sung-Woo;Park, Eun-Suk;Kim, Heung-Tae
    • The Korean Journal of Pesticide Science
    • /
    • v.7 no.3
    • /
    • pp.159-168
    • /
    • 2003
  • With microtiter plate, the assay method was developed for detecting the fungicidal activity of new compounds against spore germination, spore adhesion and mycelial growth of Colletotrichum sp. JC24 cal1Sing red pepper anthracnose. Also, the effects of some commercialized fungicides on fungal development like above mentioned were investigated by measuring the optical density of mycelia grown into wells of microtiter plate. For the standardization of assay method, some factors, such as the treatment of MTT and/or propanol, inodulum density and incubation period, affecting on mycelial optical density were investigated. For obtaining precise and consistent mycelial optical density, it was necessary the treatment of MTT for 12 hrs and propanol for 1 hr. inoculum density adjusted to $1\times10^5$ spores/mL and incubation period for 36 hrs at $25^{\circ}C$. For fungicidal activities, 6 protective fungicides, 6 ones inhibiting sterol biosynthesis, and one inhibiting respiration were used in this study. While mancozeb, chlorothalonil and dithianon among 6 protective fungicides inhibited strongly spore germination, adhesion, and mycelial growth at $6.25{\mu}g/mL$, propineb, iminoctadine and fluazinam inhibited intermediately spore germination and mycelial growth at $100{\mu}g/mL$. Washing above 3 fungicides with new PD broth, their activity against spore adhesion decreased. With hexaconazole, tebuconazole and myclobutanil, the tendency of the activity against fungal differentiation of the early infection stage was similar to the latter group of protective fungicides, showing the decrease of the inhibitory activity against spore adhesion by washing 2 hrs after incubation. However, kresoxim-methyl inhibited spore adhesion distinctly, depending on the applied concentrations. Based on these results, it might be able to assess the fungicidal activity of many compounds against spore germination, adhesion and mycelial growth by the use of microtiter plate in vitro. Using the assay developed in this report, it was possible to investigate the inhibitory activity of some commercialized fungicides, too.

Preparation of $^{99m}Tc-HYNIC-PEG-liposomes$ for Imaging of the Focal Sites of Infection (농양 진단을 위한 $^{99m}Tc-HYNIC-PEG-liposomes$의 제조)

  • Hong, Jun-Pyo;Awh, Ok-Doo;Kim, Hyun-Suk;Lee, Eun-Sook;Lee, Tae-Sup;Choi, Tae-Hyun;Choi, Chang-Woon;Lim, Sang-Moo
    • The Korean Journal of Nuclear Medicine
    • /
    • v.36 no.6
    • /
    • pp.333-343
    • /
    • 2002
  • Purpose: A new linker, hydrazino nicotinamide (HYNIC), was recently introduced for labelling of liposome with $^{99m}Tc$. In this study we synthesized HYNIC derivatized PEG (polyethylene glycol)-liposomes radiolabeled with $^{99m}Tc$. Materials and Methods: In order to synthesize HYNIC-DSPE (distearoyl phosphatidyl ethanolamine) which is a crucial component for $^{99m}Tc$ chelation, first of all succinimidyl 6-BOC-hydrazinopyridine-3-carboxylic acid was synthesized from 6-chloronicotinic acid by three sequential reactions. A DSPE derivative of succinimidyl 6-BOC-hydrazinopyridine-3-carboxylic acid was transformed into HYNIC-DSPE by HCI/dioxane. HYNIC-PEG-liposomes were prepared by hydration of the dried lipid mixture of EPC (egg phosphatidyl choline): PEG-DSPE : HYNIC-DSPE:cholesterol (1.85:0.15:0.07:1, molar ratio). The HYNIC-PEG-liposomes were labeled with $^{99m}Tc$ in the presence of $SnCl_2{\cdot}2H_2O$ (a reducing agent) and tricine (a coligand). To investigate the level of in vivo transchelation of $^{99m}Tc$ in the liposomes, the $^{99m}Tc$-HYNiC-PES-liposomes were incubated with a molar excess of DTPA, cysteine or glutathione solutions at $37^{\circ}C$ for 1 hour. The radiolabeled liposomes were also incubated in the presence of human serum at $37^{\circ}C$ for 24 hours. Results: 6-BOC-hydrazinopyridine-3-carboxylic acid was synthesized with 77.3% overall yield. The HYNIC concentration in the PEG-coated liposome dispersion was 1.08 mM. In condition of considering the measured liposomal size of 106 nm, the phospholipid concentration of $77.5\;{\mu}mol/m{\ell}$ and the liposomal particle number of $5.2{\times}10^{14}$ liposomes/ml, it is corresponded to approximate 1,250 nicotinyl hydrazine group per liposome in HYNIC-PEG-liposome. The removal of free $^{99m}Tc$ was not necessary because the labeling efficiency were above 99%. The radiolabeled liposomes maintained 98%, 96% and 99%, respectively, of radioactivity after incubation with transchelators. The radiolabeled liposomes possessed above 90% of the radioactivity in serum. Conclusion: These results suggest that the HYNIC can be synthesized easily and applied in labelling of PEG-liposomes with $^{99m}Tc$.

Evaluation of Cryptosporidiurn Disinfection by Ozone and Ultraviolet Irradiation Using Viability and Infectivity Assays (크립토스포리디움의 활성/감염성 판별법을 이용한 오존 및 자외선 소독능 평가)

  • Park Sang-Jung;Cho Min;Yoon Je-Yong;Jun Yong-Sung;Rim Yeon-Taek;Jin Ing-Nyol;Chung Hyen-Mi
    • Journal of Life Science
    • /
    • v.16 no.3 s.76
    • /
    • pp.534-539
    • /
    • 2006
  • In the ozone disinfection unit process of a piston type batch reactor with continuous ozone analysis using a flow injection analysis (FIA) system, the CT values for 1 log inactivation of Cryptosporidium parvum by viability assays of DAPI/PI and excystation were $1.8{\sim}2.2\;mg/L{\cdot}min$ at $25^{\circ}C$ and $9.1mg/L{\cdot}min$ at $5^{\circ}C$, respectively. At the low temperature, ozone requirement rises $4{\sim}5$ times higher in order to achieve the same level of disinfection at room temperature. In a 40 L scale pilot plant with continuous flow and constant 5 minutes retention time, disinfection effects were evaluated using excystation, DAPI/PI, and cell infection method at the same time. About 0.2 log inactivation of Cryptosporidium by DAPI/PI and excystation assay, and 1.2 log inactivation by cell infectivity assay were estimated, respectively, at the CT value of about $8mg/L{\cdot}min$. The difference between DAPI/PI and excystation assay was not significant in evaluating CT values of Cryptosporidium by ozone in both experiment of the piston and the pilot reactors. However, there was significant difference between viability assay based on the intact cell wall structure and function and infectivity assay based on the developing oocysts to sporozoites and merozoites in the pilot study. The stage of development should be more sensitive to ozone oxidation than cell wall intactness of oocysts. The difference of CT values estimated by viability assay between two studies may partly come from underestimation of the residual ozone concentration due to the manual monitoring in the pilot study, or the difference of the reactor scale (50 mL vs 40 L) and types (batch vs continuous). Adequate If value to disinfect 1 and 2 log scale of Cryptosporidium in UV irradiation process was 25 $mWs/cm^2$ and 50 $mWs/cm^2$, respectively, at $25^{\circ}C$ by DAPI/PI. At $5^{\circ}C$, 40 $mWs/cm^2$ was required for disinfecting 1 log Cryptosporidium, and 80 $mWs/cm^2$ for disinfecting 2 log Cryptosporidium. It was thought that about 60% increase of If value requirement to compensate for the $20^{\circ}C$ decrease in temperature was due to the low voltage low output lamp letting weaker UV rays occur at lower temperatures.

Antitumor and Antimetastatic Effects of Toxoplasma Gondii in Mice with Lewis Lung Carcinoma (Lewis 폐암 마우스 모델에서 톡소포자충에 의한 항암 및 항전이 효과)

  • Kim, Ju-Ock;Jung, Sung-Soo;Park, Hee-Sun;Kim, Myoung-Hoon;Kim, Sun-Young;Lee, Young-Ha
    • Tuberculosis and Respiratory Diseases
    • /
    • v.52 no.4
    • /
    • pp.317-329
    • /
    • 2002
  • Background : Immunotherapy is another treatment modality for various cancers. There is little information on the antitumor effects of immunotherapy on implanted lung cancer mouse models. Toxoplasma gondii is able to potently induce a nonspecific stimulation of the host immune system. Therefore, this study evaluated the antitumor and antimetastatic effect of nonspecific immune stimulation by T. gondii in a Lewis lung cancer mouse model. Methods : Female C57BL/6 mice were injected with either Lewis lung cancer cells ($1{\times}10^6$ per mouse) or 5 cysts from the T. gondii Me49 strain with various schedules. The number of survival days, the tumor size of the implanted muscle and the histopathological findings of each group were noted. In addition to these mice, the Toxoplasma antigen($50{\mu}g$ per mouse) or a lymphokine (0.5 ml per mouse) was added to boost the immunotherapy. Results : No mouse in the Toxoplasma-infected group had died, whereas the mice receiving only the cancer cells (cancer control) survived for $29.1{\pm}4.4$ days. Cancer cells were revealed from 1 week after cancer cell inceulation in the muscle and from 3 weeks in the lung of the cancer control, whereas cancer cells were found in both the preinfection control and coinfection control groups from 2 weeks and 4 weeks in the lung respectively. The in the number of survival days were $32.4{\pm}3.3$ in the mice receiving T. gondii 2 weeks prior to the cancer cells inoculation (preinfection control), $30.9{\pm}5.1$ in mice received both simultaneously (coinfection control), and $34.9{\pm}2.9$ in mice received T. gondii 2 weeks after cancer cells implantation (postinfection control). These 3 infection groups had significantly longer survival days and suppressed tumor growth than those of the cancer control. In addition to these mice, and injection with the Toxoplasma antigen alone or in combination with lymphokine resulted in a significant increase in the number of survival days. Conclusion : These findings suggest that an injection with T. gondii can induce the antitumor and antimetastatic effects in Lewis lung cancer mouse models. Moreover, these effects were increased with an injection of the Toxoplasma antigen alone or in combination with lymphokine. However, this therapy can not prevent the development of cancer.

Mumps- and Rubella-specific IgG Levels in Adolescents (청소년기의 연령증가에 따른 볼거리 및 풍진 항체가 변동)

  • Cheon, Hae Won;Shin, Young Kyoo;Lee, Kang Woo;Choung, Ji Tae;Tockgo, Young Chang
    • Pediatric Infection and Vaccine
    • /
    • v.5 no.1
    • /
    • pp.128-135
    • /
    • 1998
  • Purpose : This study was intended to measure seropositivities and the level of mumps- and rubella-specific IgG of MMR vaccinees from 12 to 17 years of age in Korea. Materials and Methods : From May 1996 to July 1996 we obtained sera from students of 1 middle and 2 high schools in Seoul, who were MMR vaccinees from 12 to 17 years of age and had no evidence of immunodeficiency. These 216 study population include 110 males and 106 females. Mumps- and rubella-specific IgG antibody levels were measured by ELISA. Cut-off values for seropositivity were 20 U(Gamma Unit) in mumps and over 0.17 in rubella. Results : 1) As age increased, seropositivities to mumps increased, being 68.4% in 12 year, 79.3% in 13 year, 72.2% in 14 year, 82.0% in 15 year, 87.5% in 16 year, 87.0% in 17 year, which however has no statistical significance. 2) As age increased, the level of mumps-specific IgG antibody(mean+standard deviation, GU) increased, being $52.0{\pm}49.2$ in 12 year, $65.9{\pm}51.4$ in 13 year, $71.1{\pm}66.0$ in 14 year, $67.8{\pm}53.6$ in 15 year, $82.8{\pm}67.8$ in 16 year, $92.0{\pm}68.9$ in 17 year, which however has no statistical significance. 3) As age increased, seropositivities of rubella-specific IgG increased significantly, being 26.3% in 12 year, 20.7% in 13 year, 50.0% in 14 year, 67.2% in 15 year, 66.7% in 16 year, 65.2% in 17 year(P<0.001). 4) As age increased, rubella-specific IgG increased significantly, being $0.13{\pm}0.145$ in 12 year, $0.087{\pm}0.101$ in 13 year, $0.194{\pm}0.168$ in 14 year, $0.260{\pm}0.187$ in 15 year, $0.305{\pm}0.213$ in 16 year, $0.325{\pm}0.221$ in 17 year(P<0.001). There was positive correlation between age and rubella-specific IgG titer(rubella-specific $IgG=0.0517{\times}age-0.5586$, r=0.3752, P<0.001). Conclusion : In adolescent, seropositivities and the level of mumps-specific IgG remained relatively high, but approximately 20% of study population showed seronegativity. Seropositivities and the level of rubella-specific IgG showed the lowest level at 13 years of age and were increased with age after 14 years of age. Further evaluation may be needed to elucidate the cause of these changes of rubella-specific IgG.

  • PDF

Development of validated Nursing Interventions for Home Health Care to Women who have had a Caesarian Delivery (조기퇴원 제왕절개 산욕부를 위한 가정간호 표준서 개발)

  • HwangBo, Su-Ja
    • Journal of Korean Academy of Nursing Administration
    • /
    • v.6 no.1
    • /
    • pp.135-146
    • /
    • 2000
  • The purpose of this study was to develope, based on the Nursing Intervention Classification (NIC) system. a set of standardized nursing interventions which had been validated. and their associated activities. for use with nursing diagnoses related to home health care for women who have had a caesarian delivery and for their newborn babies. This descriptive study for instrument development had three phases: first. selection of nursing diagnoses. second, validation of the preliminary home health care interventions. and third, application of the home care interventions. In the first phases, diagnoses from 30 nursing records of clients of the home health care agency at P. medical center who were seen between April 21 and July 30. 1998. and from 5 textbooks were examined. Ten nursing diagnoses were selected through a comparison with the NANDA (North American Nursing Diagnosis Association) classification In the second phase. using the selected diagnoses. the nursing interventions were defined from the diagnoses-intervention linkage lists along with associated activities for each intervention list in NIC. To develope the preliminary interventions five-rounds of expertise tests were done. During the first four rounds. 5 experts in clinical nursing participated. and for the final content validity test of the preliminary interventions. 13 experts participated using the Fehring's Delphi technique. The expert group evaluated and defined the set of preliminary nursing interventions. In the third phases, clinical tests were held at in a home health care setting with two home health care nurses using the preliminary intervention list as a questionnaire. Thirty clients referred to the home health care agency at P. medical center between October 1998 and March 1999 were the subjects for this phase. Each of the activities were tested using dichotomous question method. The results of the study are as follows: 1. For the ten nursing diagnoses. 63 appropriate interventions were selected from 369 diagnoses interventions links in NlC., and from 1.465 associated nursing activities. From the 63 interventions. the nurses expert group developed 18 interventions and 258 activities as the preliminary intervention list through a five-round validity test 2. For the fifth content validity test using Fehring's model for determining lCV (Intervention Content Validity), a five point Likert scale was used with values converted to weights as follows: 1=0.0. 2=0.25. 3=0.50. 4=0.75. 5=1.0. Activities of less than O.50 were to be deleted. The range of ICV scores for the nursing diagnoses was 0.95-0.66. for the nursing interventions. 0.98-0.77 and for the nursing activities, 0.95-0.85. By Fehring's method. all of these were included in the preliminary intervention list. 3. Using a questionnaire format for the preliminary intervention list. clinical application tests were done. To define nursing diagnoses. home health care nurses applied each nursing diagnoses to every client. and it was found that 13 were most frequently used of 400 times diagnoses were used. Therefore. 13 nursing diagnoses were defined as validated nursing diagnoses. Ten were the same as from the nursing records and textbooks and three were new from the clinical application. The final list included 'Anxiety', 'Aspiration. risk for'. 'Infant behavior, potential for enhanced, organized'. 'Infant feeding pattern. ineffective'. 'Infection'. 'Knowledge deficit'. 'Nutrition, less than body requirements. altered', 'Pain'. 'Parenting'. 'Skin integrity. risk for. impared' and 'Risk for activity intolerance'. 'Self-esteem disturbance', 'Sleep pattern disturbance' 4. In all. there were 19 interventions. 18 preliminary nursing interventions and one more intervention added from the clinical setting. 'Body image enhancement'. For 265 associated nursing activities. clinical application tests were also done. The intervention rate of 19 interventions was from 81.6% to 100%, so all 19 interventions were in c1uded in the validated intervention set. From the 265 nursing activities. 261(98.5%) were accepted and four activities were deleted. those with an implimentation rate of less than 50%. 5. In conclusion. 13 diagnoses. 19 interventions and 261 activities were validated for the final validated nursing intervention set.

  • PDF