Life Science Journal Acta Zhengzhou University Overseas Edition Life Science Journal L I F E S C I E N C Marsland Press E J O U

Volume 10, Number 1 (Cumulative No.32) Par t 2 March 25, 2013 ISSN:1097-8135 Volume 10, Number 1, Par t 2 March 25, 2013 ISSN:1097-8135

Life Science Journal Acta Zhengzhou University Overseas Edition Life Science Journal L i f e S c i e n c Marsland Press e J o u

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ISSN 1097-8135 Website: http://www.lifesciencesite.com

9 771097 813002 Volume 10, Number 1, Par t 2 March 25, 2013 ISSN:1097-8135

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Life Science Journal - Acta Zhengzhou University Oversea Version ISSN: 1097-8135 Life Science Journal, the Acta Zhengzhou University Oversea Version, is an international journal with the purpose to enhance our natural and scientific knowledge dissemination in the world under the free publication principle. The journal is calling for papers from all who are associated with Zhengzhou University-home and abroad. Any valuable papers or reports that are related to life science - in their broadest sense - are welcome. Other academic articles that are less relevant but are of high quality will also be considered and published. Papers submitted could be reviews, objective descriptions, research reports, opinions/debates, news, letters, and other types of writings. Let's work together to disseminate our research results and our opinions. Editor-in-Chief: Shen, Changyu, Ph.D., Professor, Chinese Academy of Sciences Associate Editors-in-Chief: Ma, Hongbao; Cherng, Shen; Xin, Shijun Editorial Boards: Aghdam, Hashemi; An, Xiuli; Cao, Zhaolong; Chandra, Avinash; Chen, George; Dong, Ziming; Duan, uangcai; Edmondson, Jingjing; Fu, Qiang; Fu, Xianzhen; Gao, Danying; Han, Quancheng; Han, Zhijiang; Hosseini, Seyyed Mousa; Huang, Shuan-Yu; Kan, Quancheng; Lang, Jinghe; Li, Hui; Li, Xinhua; Li, Yan; Li, Yuhua; Li, Yusheng; Li, Zhen; Lindley, Mark; Liu, Hongmin; Liu, Hua; Liu, Xinjing; Ma, Changsheng; Ma, Margret; Ma, Yongsheng; Maio, Zhongrong; Ming, Gongguang; Mousavi, Amin; Nie, Wei; Qi, Yuanming; Qiao, Nancy X; Qin, Jie; Qin, Qin; Qin, Yuhua; Ouyang, Da; Sabyasachi Chatterjee; Seryasat, Omid Rahmani; Shang, Fude; Shi, Lifeng; Song, Chunpeng; Song, Yongping; Spurr, Lydia; Sun, Yan; Sun, Yingpu; Sun, Yiwu; Tan, Song; Venkatesh, Jaganathan; Wang, Jianliu; Wang, Lidong; Wang, Longde; Wang, Xiaoying; Wang, Yilong; Wang, Yongjun; Wang, Zhonggao; Wen, Jianguo; Wu, Ziying; Xu, Cunshuan; Xu, Yuming; Xue, Changgui; Yang, Qian; Young, Jenny; Zaki, Mona; Zhang, Guangxian; Zhang, Hong; Zhang, Jianying; Zhang, Kehao; Zhang, Liusuo; Zhang, Mingzhi; Zhang, Rui; Zhang, Shengjun; Zhang, Shuijun; Zhang, Suming; Zhang, Wei; Zhang, Xueguo; Zhang, Yanzhou; Zhang, Zhan; Zhang, Zhao; Zhang, Zhenxiang; Zhu, Huaijie. Introductions to Authors 1. General Information: (1) Goals: As an international journal published both in print and on internet, Life Science Journal is dedicated to the dissemination of fundamental knowledge in all areas of nature and science. The main purpose of Life Science Journal is to enhance our knowledge spreading in the world under the free publication principle. It publishes full-length papers (original contributions), reviews, rapid communications, and any debates and opinions in all the fields of nature and science. (2) What to Do: The Life Science Journal provides a place for discussion of scientific news, research, theory, philosophy, profession and technology - that will drive scientific progress. Research reports and regular manuscripts that contain new and significant information of general interest are welcome. (3) Who: All people are welcome to submit manuscripts in life science fields. Papers of other fields are also considered. (4) Copyright and Responsibility of Authors to their Articles: When the manuscript(s) is submitted to the journal, the authors agree the following: All the authors have participated sufficiently in this work; The article is not published elsewhere; Authors are responsibility on the contents of the article; The journal and author(s) have same right for the copyright of the article and either of the journal or author(s) can use it by anyway without noting the other party. (5) Publication Costs: US$640 per article to defray costs of the publication will be paid by the authors when it is received. (6) Advertisements: The price will be US$400/page, and we normally accept one advertisement for one page. Other size of the advertisement please contact with our editorial office through email. 2. Manuscript Preparation - Each manuscript is suggested to include the following components but authors can do their own ways: (1) Title: including the complete article title; each author’s full name; institution(s) with which each author is affiliated, with city, state/province, zip code, and country; and the name, complete mailing address, telephone number, facsimile number (if available), and at least one email address for author(s). (2) Abstract: including Background, Materials and Methods, Results, and Discussions. (3) Key Words. (4) Introduction. (5) Materials and Methods. (6) Results. (7) Discussions. (8) Acknowledgments. (9) References. 3. Manuscripts Submission: (1) Submission Methods: Submission through email ([email protected] or [email protected]) is encouraged. (2) Software: The Microsoft Word file. (3) Font: Normal, Times New Roman, 10 pt, single space. (4) Page Setup: Paper-Letter size; Margins-2.54 cm each side; Header/Footer-1.27 cm. 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(11) Submission Address: [email protected] or [email protected], Marsland Press, PO Box 180432, Richmond Hill, New York 11418, USA, 347-321-7172.

Life Science Journal - Acta Zhengzhou University Overseas Edition (Life Sci J), ISSN: 1097-8135 PO Box 180432, Richmond Hill, New York 11418, USA http://www.lifesciencesite.com; http://www.sciencepub.net; [email protected]; [email protected]

22 The Protective Effect of Pomegranate(Punica granatum) Juice against Carbon 151-158 Tetrachloride-Induced Oxidative Stress in Brain Tissue of Adult Male Albino Rats Sahar M. Mahmoud andAhmed E. Abdel Moneim

23 Study of Coumarin Content ofPelargonium fragrans -Willd. Root Grown in Egypt 159-164 K.M. Meselhy

24 Use of oligomer modifier to enhance the fracture characteristics of glass reinforced 165-169 composites for civil and medicine Fakhrodin Alimoradi, Mohammad R. Aabaadiaan, Ali Heidari

25 Effect of reactive vinyl-based modifier on thermal and failure resistance of reinforced 170-174 epoxy used in pipelines Ahmad Fakhar, Mohammad Aabaadiaan Mohamad Farhat

26 Comparison betweenmethods used for manufacturing ofaluminum foam 175-185 Beigi kheradmand, S. Otroj, Z. Soleimanpour, M. Beigyfar

27 Simulating the buckling deflection of carbon nanotube-made detectors used in medical 186-191 detections by applying a continuum mechanics model Alireza Vahdati, Mehdi Vahdati, R. A. Mahdavinejad

28 Protective effects of (-)-epigallocatechin gallate on blue light-induced damage in 192-198 retinoblastoma Y79 cells by activating estrogen receptor pathway Mei-Ling Peng,Ching-Ju Lee, Chung-Liang Chien,Chun-Lan Liu, Cheng-Yu Tsai, Yang-Cheng Wen, Kuang-Wen Tseng

29 Biomass-density relationships and self-thinning of shell and tissue in marine intertidal 199-203 barnacles Claver Sibomana and Gen-Xuan Wang

30 Assessment of Zinc and Copper Contents in the Hair and Serum and Also Superoxide 204-209 Dismutase, Glutathion Peroxidase and Malondi Aldehyde in Serum in Androgenetic Alopecia and Alopecia Areata Mehdi Amirnia, Sima Sinafar, Hassan Sinafar, Mohammad Nuri, Abdoreza Taban Sadeghi

31 Correlation between body mass index (BMI) and immunologic Response to Hepatitis B 210-213 Vaccine Mojtaba Varshochi and Sepehr Taghizadeh

32 Isolation ofCryptococcusneoformansfrompigeonexcretainQazvin 214-219 Fatemeh Samiee Rad

33 Hypodense area within epidural hematoma in brain CT scan; Prediction of active bleeding 220-222 in epidural hematoma Mahboub Pouraghaei, Payman Moharamzadeh, Sima Dibazar, Ali Taghizadieh, Alireza Ala, Parham Maroufi, Reza Rikhtegar Ghiasi

34 Time Frequency of Guillain-Barre Syndrome in Northwest of Iran 223-225 Mehdi Farhoudi, Hormoz Ayromlou, Amir Mohammad Bazzazi, Farrokh Bakht Shadi, Samad EJ Golzari, Kamyar Ghabili, Marjan Dehdilani

35 Analysis of Various Anti-Windup Schemes used to Control PMDC Motors employed in 226-230 Orthopedic Surgical Simulators G. Murugananth, S. Vijayan , S. Muthukrishnan

36 Knowledge and Practice of Female Employee About Premenstrual Syndrome and its 231-243 Effect on Daily Life Activities in EL-Minia University Mona A.Abd EL-Hamid,Diaa Ahmed El Moghazy, Manal F..Moustafa and.Ekbal A. Emam.

37 An Outlier Based Bi-Level Neural Network Classification System for Improved 244-251 Classification of Cardiotocogram Data Sundar Chinnasamy, Chitradevi Muthusamy and Geetharamani Gopal

38 The impact of descriptive evaluation on knowledge and achievement of Meta cognitive of 252-255 the elementary students of Boushehr city Fatemeh Mahzounzadeh

39 Consistency of linguistic domain of the Quran of Quds with Dehei dialect in central 256-261 plateau of Iran Ali Akbar Nargesian

40 Heat Shock Protein-70 and -27 Expressions as Parameters of Early Diagnosis and Disease 262-268 Progression in Hepatocellular Carcinoma Amal Fawzy, Hatem Attia,Fatma A Khalaf, Eman Abd El Sameea,Mahmoud A El Tahawy,Mohamed Farag andFatma Younis

41 Use of Fourier Transform Infrared Spectroscopy to Study Cadmium-Induced Changes in 269-276 Strongylocentrotus nudus gonad ZhenlinWei

42 An Epidemiological Study of Nosocomial Infections in Tabriz Children’s Hospital Based 277-279 on National Nosocomial Infection Surveillance System (NNIS) Shahram Abdoli Oskouie, Mohammad Ahangarzadeh Rezaee, Kamyar Ghabili, Farahnaz Firoozi

43 Promoting the recovery of neurologic function and antagonizing cell apoptosis in ICH 280-284 model rats byInduced Pluripotent Stem CellTransplantation Jing Qi,Jie Qin, Guangming Gong,Bo Song, Huili Zhang, Yanlin Wang, Haiyun Qi, Bo Yang,Yi Zhang,Yuming Xu, Shilei Sun

44 Contact lens care and disease characteristics in keratoconus 285-287 John Ching-Jen Hsiao,Shyan-Tarng Chen

45 The Influence of Fermentation by Different Lactobacillus on the Free Radical Scavenging 288-292 Activity of Burdock and Variations of Its Active Components Chi-Ting Horng,Shih-Chiang Lee, Rei-Chu Chang,Wan-Ping Lee, Feng-Lang Lin, Chin-Wen Hsu,Fu-An Chen

46 Study the sources of mercury vapor in atmosphere as a threatening factor for human 293-296 health and bio-filtering methods for removal of toxic pollution Maryam K. Hafshejani, Farzaneh Khandani, Ramin Heidarpour, Armin Arad, Sadegh Choopani

47 The Age Effects of Traffic Signs on Visual Performance 297-302 Chun-Wen Chao, Cheng Hung Huang,Tienwei Tsai

48 Withdrawn 303-305

II Life Science Journal 2013;10(1) http://www.lifesciencesite.com

The Protective Effect of Pomegranate (Punica granatum) Juice against Carbon Tetrachloride-Induced Oxidative Stress in Brain Tissue of Adult Male Albino Rats

Sahar M. Mahmoud1 and Ahmed E. Abdel Moneim2

1 Department of Zoology, Faculty of Science, Cairo University, Egypt. 2 Department of Zoology and Entomology, Faculty of Science, Helwan University, Egypt. [email protected]

Abstract: This study was undertaken to evaluate the effect of pomegranate (Punica granatum, P.g.) juice on the oxidation state of the brain tissue of adult male albino rats and whether P.g. juice administration improves the activity of the antioxidant enzymes after carbon tetrachloride (CCl4) treatment as an oxidative stress agent. Rats were divided into four groups, Group I (control) was received intraperitoneal (i.p.) injection of 300µl saline solution (0.9% NaCl) for 12 weeks; Group II (CCl4 group) was injected i.p. once per week for 10 weeks with CCl4 (2ml/kg body weight); Group III (P.g. group) was administered P.g. juice orally for 12 weeks and Group IV (P.g. + CCl4) was received P.g. 2 weeks before CCl4 injection and continued for 10 weeks; CCl4 was administered as shown in group II, decapitation of all groups were carried out one week after the last dose. Determination of glutathione (GSH) content and the activity of the antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR), also malonaldhyde (MDA) and nitric oxide (NO) levels were carried out. The present results demonstrate that treatment with CCl4 induced biochemical changes in brain tissue as induction of (MDA) and nitrite/nitrate level accompanied with significant decrease in GSH content, SOD, CAT, GPx, GR and GST activities. Administration of P.g. juice alone enhanced the activities of enzymes under investigation and decreased the level of NO. P.g. Juice administration before and during CCl4 treatment attenuated the decrease in the enzymes activities and GSH content of the brain, if compared to CCl4 group. The present study suggest that P.g. juice has a potential protective effect as it can elevate antioxidant defense system, clean up free radicals, lessens oxidative damages and protect the brain tissue against CCl4-induced toxicity. [Sahar M. Mahmoud and Ahmed E. Abdel Moneim. The Protective Effect of Pomegranate (Punica granatum) Juice against Carbon Tetrachloride-Induced Oxidative Stress in Brain Tissue of Adult Male Albino Rats. Life Sci J 2013;10(1):151-158] (ISSN:1097-8135). http://www.lifesciencesite.com. 22

Keywords: pomegranate (Punica granatum); carbon tetrachloride; oxidative stress; brain; rats.

1. Introduction and Far East countries. This botanic isolation is Oxidative stress results from the imbalance of coincident with a unique biochemistry, the seeds reactive oxygen species (ROS) and defense contain an oil of which about 80% is a rare trans 18 mechanisms, which results in cell damage (Hartly et carbon fatty acid (punicic acid) (Schubert et al., al., 1999). Animal tissues are constantly coping with 1999). There have been numerous reports on the in highly (ROS), such as superoxide anion, hydroxyl vivo properties of P.g., namely on anti-atherosclerotic radicals, hydrogen peroxide, and other radicals capacity (Aviram and Dornfeld, 2001); anti- generated during numerous metabolic reactions proliferative and pro-apoptotic activities of (Melin et al., 2000). Oxidative stress plays a critical pomegranate extract (Seeram et al., 2005); anti- role in cancer, inﬂammatory, cardiovascular and inflammatory activity (Adams et al., 2006) as well as neurodegenerative diseases as well as in aging (Storz, chemopreventive and chemotherapeutical potential 2005). towards prostate cancer (Malik et al., 2005). The enhanced production of free radicals and Therefore, this study was performed in order to oxidative stress can be induced by a variety of factors investigate the effect of P. g. juice on general such as ionizing radiation or exposure to drugs and oxidation status in brain tissue of adult male albino xenobiotics (e.g., CCl4). It is well documented rat, and whether it protects the brain from cellular (Halim et al., 1997) that the treatment with damage resulting from oxidative stress after CCl4 antioxidants such as vitamins C and E or S- intoxication. adenosylmethionine can modulate the toxic effect of 2. Materials and Methods CCl4 on liver and kidneys. Animals: Pomegranate (Punica granatum) is one of the Adult male Wistar albino rats weighing 120- oldest edible fruit and belongs to the Punicacea 150g (7-9 weeks) were obtained from the Holding family (Fadavi et al., 2006). P.g. is extensively Company for Biological Products and Vaccines, cultivated in the Mediterranean area and most Near- (VACSERA), Giza, Egypt. After acclimatization for a

151 Life Science Journal 2013;10(1) http://www.lifesciencesite.com period of one week, the animals were housed in cages Determination of Nitrite/Nitrate Concentration in a room under standard conditions of illumination and MDA Level: with a 12-hours light-dark cycle, at room temperature Nitrite/nitrate (breakdown products of NO) 25±1ºC. They were provided with water and a concentration and MDA level were assayed balanced standard pellet obtained from VACSERA calorimetrically in brain homogenate according to the used as a diet and water ad libitum. The animals were methods of Berkels et al. (2004) and Ohkawa et al. kept in clean and low stress environment and an (1979), respectively. enclosed door provided a substantial amount of sound Estimation of Glutathione and Anti-Oxidant proofing. All animals received care in compliance Enzymes: with the Egyptian rules for animal protection. The brain glutathione (GSH) was determined by Pomegranate Juice Preparation: the method of Ellman (1959). This method is based Ten kg of P.g. were washed and manually on the reduction of Ellman's reagent (5, 5` dithiobis peeled, without separating the seeds. Juice was (2-nitrobenzoic acid, "DTNB") with GSH to produce obtained using a commercial blender (Braun, a yellow compound measured at 405nm. In addition, Germany), filtrated with a Buchner funnel and the level of brain antioxidant enzymes as catalase immediately diluted with distilled water to a volume (CAT) was assayed by the method of Aebi (1984). of 1:3 and stored at 4 ºC for no longer than 2 months Brain superoxide dismutase (SOD) activity was (Faria et al., 2007). assayed by the method of Nishikimi et al. (1972). Experimental Protocol: Glutathione-S-transferase (GST) activity in the brain To study the protective effect of P.g. juice on was assayed by the method of Habig et al. (1974). brain tissue of CCl4-induced toxicity in rat, twenty Brain glutathione peroxidase (GPx) activity was four adult male Wistar albino rats were randomly measured by the method of Paglia and Valentine allocated to four groups of six rats each. Group I (1967). Glutathione reductase (GR) activity in the (Control) served as control and received i.p. injection brain was assayed by the method of Factor et al. of 300µl saline for 12 weeks. Group II (CCl4) (1998). received weekly i.p. injection of 2ml CCl4/kg body Flow Cytometry: weight for 10 weeks as described by Sohn et al. Tissue samples were prepared by manual (1991). Group III (P.g.) received P.g. juice supplied disaggregation procedure. Briefly, a few drops of on dark water bottles and renewed every 2-3 days RPMI media were added to tissue and then minced (Faria et al., 2007) for 12 weeks. Their average daily until complete tissue disaggregation was achieved. intake of fluid was 5.2 to 6.5ml. The animals of Suspended cells were filtered using a 50μm pore size Group IV (P.g. + CCl4) were received P.g. juice as mesh and then centrifuged at 1000 rpm for 10 min. described in group III for 2 weeks before CCl4 i.p. Cells were re-suspended in protein bovine serum, injection (2ml/kg body weight) for 10 weeks. P.g. counted and washed by calcium buffer then juice supplementation to rats of this group was also centrifuged at 1500 rpm for 5 min. The pellet was re- continued during the whole 10 weeks. After one week suspended and then cells were counted. Annexin V of the last i.p. injection of CCl4, the animals of all and propidium iodide apoptotic assay was carried out groups had cervical dislocation. Half of each rat brain using IQP-120F Kit (IQ Products, Groningen, was weighed and homogenized immediately to give Netherlands). FAC scan Becton-Dickinson (BD) flow 50% (w/v) homogenate in ice-cold medium cytometer was used and data were analyzed using cell containing 50mM Tris-HCl and 300mM sucrose Quest software. (Tsakiris et al., 2004). The homogenate was Statistical Analysis: centrifuged at 500g for 10 min. at 40C.The The obtained data were presented as means ± supernatant (10%) was used for the various standard error. One-way analysis of variance biochemical determinations. The second half of the (ANOVA) was carried out. The statistical rat brain was freshly prepared to be used in flow comparisons among the groups were performed with cytometer investigation. Duncan's test (1955) using a statistical package Chemicals: program (SPSS version 17.0). All p-values are two- Carbon tetrachloride (CCl4), tris HCl buffer and tailed and p<0.05 was considered as significant for all 50, 50 dithiobis-2-nitrobenzoic acid (DTNB), were statistical analysis in this study. purchased from Sigma (St. Louis, MO, USA). 3. Results Perchloric acid, thiobarbituric acid (TBA) and In a trial to evaluate the ability of P.g.juice in trichloroacetic acid (TCA) were purchased from modulating the oxidative-antioxidative state in the Merck. All other chemicals and reagents used in this brain tissue, the data obtained in the present study study were of analytical grade. Double-distilled water (Table 1) concerning treatment of adult male Wistar was used as the solvent. rats with P.g. juice and/or CCl4 (2ml/kg body weight)

152 Life Science Journal 2013;10(1) http://www.lifesciencesite.com

revealed that CCl4 injection for 10 weeks caused a Injection of CCl4 induced a significant significant decrease at p<0.05 in GSH, GST, GPx and inhibition (p<0.05) in SOD and CAT activities in the GR levels as compared to control group. The level of brain tissue as compared to control group (Figure 1). oxidative markers assessed in P.g. juice-treated rats The supplementation of P.g. juice to rats showed a group showed a significant increase at p<0.05 in significant enhancement at p<0.05 in CAT activity as GSH, GST levels coupled with a non significant compared to control group. In P.g. juice & CCl4- change in GPx and GR levels. In P.g. juice and CCl4- treated rats, although CCl4 effect was evident in treated rats group, the data showed the toxic inhibiting both SOD and CAT activities versus the manifestations of CCl4 and the recovery pattern after control group, P.g. juice supplementation before and P.g. juice administration. The co-administered group during treatment of CCl4 indicated a significant exhibited an increase in GSH, GST, GPx and GR elevation (p<0.05) in both enzymes activities when levels, if compared to CCl4-treated group, the increase compared to CCl4 group. was significant at p<0.05 in GSH, GPx and GR.

Table (1): The effect of P.g. juice and/or CCl4 (2ml/kg body weight) on the levels of GSH, GST, GPx and GR in brain tissue of adult male albino rats. Groups GSH (mmol/g tissue) GST (μmol/h/g tissue) GPx (U/g tissue) GR (μmol/ g tissue) Control 32.38±0.35 0.36±0.05 1945.3±61.2 8.24±0.53 a a a a CCl4 22.09±0.48 0.21±0.03 1074.1±73.1 4.96±0.75 P.g. 39.23±0.48a 0.49±0.04a 1891.3±55.9 10.72±1.86 ab ab b P.g. + CCl4 29.16±0.42 0.29±0.04 1482.2±32.6 6.36±0.1.21 Values are means ± S.E. (n=6). a: Significant change at p<0.05 with respect to Control group. b: Significant change at p< 0.05 with respect to CCl4-treated group.

Figure (1): The effect of P.g. juice and/or CCl4 Figure (2): The effect of P.g. juice and/or CCl4 (2ml/kg body weight) on SOD and CAT activities in (2ml/kg body weight) on MDA and NO levels in brain tissue of adult male albino rats. brain tissue of adult male albino rats.

Flow cytometric analysis was performed to The data represented in Figure (2) indicated that investigate the anti-apoptotic effects of P.g. juice on CCl treatment induced a significant increase 4 brain cells against CCl -induced apoptosis and (p<0.05) in MDA and NO contents in the brain as 4 necrosis. Cells were dual-stained with Annexin V and compared to control group. The supplementation of propidium iodide (Figure 3 & Table 2). In vitro, P.g. juice to rats showed a significant decrease treatment of brain cells with CCl caused apoptosis (p<0.05) in NO level in the brain tissue versus control 4 with a percent of (62.3%). Apoptotic cells in CCl4- group. On the other hand, P.g. juice supplementation treated group showed a significant (p<0.05) increase before and during CCl4 injection reduced the versus control group. The protective effect of P.g. enhanced MDA and NO levels in the brain tissue with juice on CCl4-induced apoptosis in neurons were a significant change at p<0.05, if compared to CCl4- abolished by decreasing the number of apoptotic cells treated rats group. (44.7%), whereas, increased the number of viable

cells (49.5%) when compared with CCl4-treated group.

153 Life Science Journal 2013;10(1) http://www.lifesciencesite.com

Figure (3): Assessment of apoptosis by annexin V and propidium iodide staining on brain tissue of rats treated with P.g. juice and/or CCl4 (2ml/kg body weight).

Table (2): The effect of P.g. juice and/or CCl4 (2ml/kg body weight) in assessing of apoptosis by annexin V and propidium iodide staining on brain tissue of adult male albino rats. Groups Viable Neurons Apoptotic Neurons Dead Neurons Necrotic Neurons Control 44.6±1.9 49.5±1.0 4.5±0.2 1.2±0.05 a a a CCl4 34.2±1.5 62.3±1.8 2.6±0.1 0.9±0.02 P.g. 64.2±2.1a 30.2±1.0a 5.2±0.3 0.4±0.01a b b b P.g. + CCl4 49.5±1.7 44.7±0.8 4.5±0.3 1.3±0.02 Values are means ± S.E. (n=6). a: Significant change at p<0.05 with respect to Control group. b: Significant change at p< 0.05 with respect to CCl4-treated group.

4. Discussion distributed and deposited to organs such as the liver, The present results demonstrated that treatment brain, kidney, and heart. The time courses of uptake with CCl4, induced biochemical signs of brain tissue and elimination of CCl4 appeared to be governed injury, evidenced by decreased GSH content, largely by the rate of blood perfusion and lipid increased MDA and NO levels and inhibited the content of the tissue. CCl4 is rapidly taken up by liver enzymes activity of SOD, CAT, GPx, GST and GR and brain (Sanzgir et al., 1997). which were considered as an index of lipid The reactive metabolite trichloromethyl radical peroxidation. (CCl3) has been formed from the metabolic Carbon tetrachloride, a typical hepatotoxic, conversion of CCl4 by cytochrome P-450. These free exerts its toxic effects by the generation of free radicals initiate the peroxidation of membrane poly- radicals (Melin et al., 2000). The lipid solubility of unsaturated fatty acids which results in the generation CCl4 allows it to cross cell membranes; it is and excess production of reactive oxygen Species

154 Life Science Journal 2013;10(1) http://www.lifesciencesite.com

(ROS) (Hartly et al., 1999 and Melin et al., 2000), all time points studied. The high concentration of finally cell necrosis. However, when oxidative stress polyunsaturated fatty acids and aerobic metabolic reaches a certain limit, defense mechanisms against activity of the brain increase the susceptibility of this ROS become insufficient and led to a decrease in the organ to peroxidative damage induced by reactive intracellular concentration of GSH and antioxidant oxygen species after CCl4ingestion. It has been enzymes (Manna et al., 2011). reported (Del Maestro and Mc Donald, 1987 and Oxidative stress can produce major interrelated Somani et al., 1995) that the brain content of derangements of cellular metabolism, including cytochrome P-450 and arachidonic acid is lower than alteration of protein and nucleic acid structure, that in the liver, demonstrating that the antioxidant increase in intracellular free calcium, damage to defense system has limited capacity in the brain. membrane ion transport and permeability and Szymonik-Lesiuk et al. (2003) suggest that GPx is destruction of the cells by lipid peroxidation more important than catalase for detoxification of (Reckmage et al., 1989). Lipid peroxidation has hydrogen peroxide in the brain. The brain contains attracted much attention because of its association small amounts of catalase, and GPx can also interact with a number of abnormal physiological processes directly with lipid peroxides (Adams et al., 1991). (Reckmage et al., 1989 and Hartly et al., 1999). Szymonik-Lesiuk et al. (2003) also suggested that In the present study, a significant elevation of brain have different coping mechanisms to deal with MDA (lipid peroxidation product) has been observed oxidative stress than liver. in the brain tissue of CCl4-treated group. Evidence of Moreover, Ichi et al. (2009) reported that lipid peroxidation by increased MDA level is one of after CCl4 (4ml/kg body weight) treatment, the the primary means of associated oxidative processes enhanced activity of neutral sphingomyelinase with an overall decrease in cellular function. The (SMase), which was significantly increased in the increase in MDA which indicates lipid peroxidation is liver, kidney, and brain, was accompanied by a an index to identify free radicals-induced injuries decrease of GSH. Thus, the activation of neutral (Rosenblat et al., 2006). Moreover, Jayakumar et SMase via oxidative stress induced the increase of al. (2008) indicated that male Wistar rats exposed ceramide, a biologically active lipid causing apoptosis to CCl4 revealed elevated levels of MDA and lowered in a variety of cells during CCl4 intoxication, in not levels of GSH, accompanied by lower activities of the only the liver but also other tissues. The authors antioxidant enzymes, CAT, SOD, GPx and GST in found that, in the brain, the total ceramide, the kidneys, heart and brain. dramatically, increased at 36h, after CCl4 Previous studies have demonstrated the different injection. Ichi et al., (2009) suggested that the responses of the antioxidant enzymatic system in increase in ceramide, in plasma was transferred to the different tissues during oxidative stress (Jones et al., kidney and the brain. This may explain the increase in 1986). The difference in the enzyme responses may apoptotic cells noticed in the brain of CCl4-treated be in part due to the differences in the amount, nature, group when compared to control group. and activities of these enzymes in particular tissues. Administration of CCl4 enhanced the induction The particular enzyme associated with the different of MDA and reduced GSH levels as well GST sub-cellular fractions can exert different effects. activity in brain as reported by Soliman and Fahmy Results obtained by Zhang et al. (1989) demonstrated (2011). The decrease in GST transcription was a tissue- and age-specific expression of GPx, the accompanied by a decrease in GST activity, as highest activities of this enzyme being in liver and confirmed by Faria et al. (2007). In addition, kidney but the lowest in brain and muscle. Several Moreno and Muriel (2006) indicated that Cirrhosis studies have shown that natural antioxidant defense produced by i.p. administration of CCl4 did not systems have limited capacity in the brain as produce a significant effect in nitrite/nitrate level but, compared to peripheral tissues (Del Maestro and Mc reduced GSH and increased lipid peroxidation. Donald, 1987 and Somani et al., 1995). The brain The findings of the present study indicated that contains small amounts of catalase and different brain CCl4 enhances NO generation in the brain tissue of regions contain different activities of antioxidant rat. This elevation in NO production may inhibit key enzymes (Somani et al., 1995). enzymes of energy metabolism, damage DNA, Vohra and Hui (2001) found deplete intracellular glutathione and react with that, CCl4 intoxicated cultured neurons increased lipid superoxide to form the much more powerful oxidant peroxidation product and decreased GPx activity, in a peroxynitrite (Bashkatova et al., 2004). Peroxynitrite concentration dependent manner. Szymonik-Lesiuk is a key element in resolving the contrasting roles of et al. (2003) found that, SOD and CAT activities NO in physiology and pathology. The generation of decreased, in the brain, after 24h of CCl4 ingestion, peroxynitrite over long periods of time will result in but GPx activity significantly increased statistically at substantial oxidation and potential destruction of host

155 Life Science Journal 2013;10(1) http://www.lifesciencesite.com cellular constituents, leading to the dysfunction of From the present results the P.j. juice was critical cellular processes, disruption of cell signaling active and possessing very potent and novel pathways, and the induction of cell death through therapeutic agents for scavenging of NO. P.g juice both apoptosis and necrosis (Pacher et al., 2007). may also exert its effects on the regulation of There is an intimate relationship between pathological conditions caused by excessive nutrition and the antioxidant defense system, as some generation of NO and its oxidation product- exogenous low molecular weight antioxidants may be peroxynitrite. The protective effect of P.g. juice supplied by the diet. These two main systems of the treatment agreed with Aviram et al. (2000) who antioxidant defense act in coordination, their levels reported that P.g. reduced the oxidative stress. being regulated by each other, to avoid oxidative However, polyphenols are known to modulate stress events (Masella et al., 2005). In the past few the transcription and expression of proteins related to years, a considerably large group of molecules the endogenous antioxidant defense by interacting widespread in plants has come into focus. with antioxidant response elements in gene promoter Herbal medicine has been used for more than regions of genes encoding proteins related to 5000 years. The interest in polyphenols has grown oxidative injury management (Myhrstad et al., 2002 considerably because of their high capacity to trap and Moskaug et al., 2005). In addition, in the present free radicals associated with different diseases. study, P.g.juice supplementation considerably Phenols and flavonoids are very important plant increased the formation of antioxidant products which constituents because of their antioxidant activity. The may be regarded to the phenolic constituents and its plant phenolics are commonly present in fruits, antioxidant activity. Flavonoids present in P.g. juice vegetables, leaves, nuts, seeds, barks, roots and in have been shown to alleviate the oxidative stress by other plant parts (Lansky and Newman, 2007). The increasing the endogenous antioxidant status, antioxidant activity of phenolic compounds is mainly protecting cells against free-radical damage by due to their redox properties which play an important increasing resistance to oxidative stress (Li et al., role as free radical scavengers, reducing agents, 2006 and Bachoual et al., 2011). quenchers of singlet oxygen and complexes of pro- It could be concluded that, P.g. juice reversed oxidant metals (Mustafa et al., 2010). the deteriorated antioxidant enzymes activity in the The present study showed that P.g. juice brain tissue of adult male albino rats intoxicated with supplementation before and during treatment of CCl4 CCl4 through scavenging free radicals, decreasing exhibited an increase in GSH, GST, GPx, GR levels, lipid peroxidation, attenuating the brain susceptibility SOD and CAT activities, while reduced the enhanced to oxidative stress thus, improving cellular membrane MDA and NO levels, if compared to CCl4-treated rats and organ functioning more profoundly and brought group. variables towards control values. As depicted from the present results, treatment of P.g. juice with CCl4 resulted in a reduction in Corresponding author: oxidative stress clearly indicating the antioxidant Mahmoud, S.M. ability of P.g. which was attributed to diverse Department of Zoology, Faculty of Science, Cairo phenolic compounds (Kaur et al., 2006) present in University, Cairo, Egypt. [email protected] P.g. juice, including punicalagin isomers, ellagic acid derivatives and anthocyanins (Seeram et al., 2005). 5. References These compounds are known for their properties in Aebi, H. (1984): Catalase in vitro. Methods Enzymol.; scavenging free radicals and inhibiting lipid 105:121-126. oxidation. Adams, L.S.; Seeram, N.P.; Aggarwal, B.B.; Takada, Another reliable oxidative stress indicator is the Y.S. and Heber, D. (2006): Pomegranate juice, total measure of GSH level. Glutathione is an antioxidant pomegranate ellagitannins, and punicalagin suppress marker which plays a protective role in the inﬂammatory cell signaling in colon cancer cells. J. metabolism of a large number of toxic agents. It acts Agric. Food Chem.; 54: 980-985. as a free radical trapping agent and preserves Adams, J.D.; Klaindman, L.K.; Odunze, L.N.; Shen, cytochrome P-450 by blocking lipid peroxidation H.C. and Miller, C.A. (1991): Alzheimer’s and Parkinson’s dissease. Brain levels of glutathione, (Yuan et al., 2008). In the present study, P.g. juice glutathione disulﬁde and vitamin E. Mol. Chem. markedly increased the brain GST activity and Neuropathol.; 14: 213-226. maintained GSH level, when compared to CCl4- Aviram, M. and Dornfeld, L. (2001): Pomegranate treated rats group. The mechanism of protection by juice consumption inhibits serum angiotensin P.g. juice against CCl4 toxicity might be due to converting enzyme activity and reduces systolic restoration of the GSH level. blood pressure. Atheroscler.; 158:195-198.

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Aviram, M.; Dornfeld, L.; Rosenblat, M.; Volkova, Ichi, I.;Kamikawa, C.; Nakagawa, T.;Kobayashi, N.; Kaplan, M.; Coleman, R.; Hayek, T.; Presser, K.; Kataoka,R.;Nagata, E.; Kitamura, D. and Fuhrman, B. (2000): Pomegranate juice Y.; Nakazaki, C.;Matsura, T. and Kojo S. (2009): consumption reduces oxidative stress, atherogenic Neutral sphingomyelinase-induced ceramide modifications to ldl, and platelet aggregation: accumulation by oxidative stress during carbon Studies in humans and in atherosclerotic tetrachloride intoxication. Toxicol.; 261(1-2): 33- apolipoprotein e-deficient mice. Am. J. Clin. Nutr.; 40. 71(5): 1062-1076. Jayakumar, T.; Sakthivel, M.; Thomas, P.A. Bachoual, R.; Talmoudi; W.; Boussetta, T.; Braut, F. and Geraldine, P. (2008): Pleurotus ostreatus, an and El-Benna, J. (2011): An aqueous pomegranate oyster mushroom, decreases the oxidative peel extract inhibits neutrophil myeloperoxidase in stress induced by carbon tetrachloride in rat kidneys, vitro and attenuates lung inflammation in mice. Food heart and brain. Chem. Biol. Interact.; 176(2-3):108- Chem. Toxicol.; 49(6): 1224-1228. 20. Bashkatova, V.; Alam, M.; Vanin, A. and Schmidt, Jones, T.W.; Thor, H. and Orrenius, S. (1986): W.J. (2004): Chronic administration of rotenone Against toxic substances. Arch. Toxicol. Suppl.; 9: increases levels of nitric oxide and lipid peroxidation 259-271. products in rat brain. Exp. Neurol.; 186(2): 235-241. Kaur, G.; Jabbar, Z.; Athar, M. and Alam, M.S. Berkels, R.; Purol-Schnabel, S. and Roesen, R. (2006): Punica granatum (pomegranate) flower (2004): Measurement of nitric oxide by reconversion extract possesses potent antioxidant activity and of nitrate/nitrite to NO. Methods Mol. Biol.; 279: 1- abrogates Fe-NTA induced hepatotoxicity in mice. 8. Food Chem. Toxicol.; 44: 984-993. Del Maestro, R. and Mc Donald, W. (1987): Lansky, E.P. and Newman, R.A. (2007): Punica Distribution of superoxide dismutase, glutathione granatum (pomegranate) and its potential for peroxidase and catalase in developing rat brain. prevention and treatment of inflammation and Mech. Ageing. Dev.; 41: 29-38. cancer. J. Ethnopharmacol.; 109(2): 177-206. Duncan, D.B. (1955): Multiple F -test. Biometrics; Li, Y.; Guo, C.; Yang, J.; Wei, J.; Xu, J. and Cheng, 11:1-42. S. (2006): Evaluation of antioxidant properties of Ellman, G.L. (1959): Tissue sulfhydryl groups. Arch. pomegranate peel extract in comparison with Biochem. Biophys.; 82(1): 70-77. pomegranate pulp extract. Food Chem.; 96(2): 254- Factor, V.M.; Kiss, A.; Woitach, J.T.; Wirth, P.J. 260. and Thorgeirsson, S.S. (1998): Disruption of redox Malik, A.; Afaq, F.; Sarfaraz, S.; Adhami, V.M.; homeostasis in the transforming growth factor- Syed, D.N. and Mukhtar, H. (2005): Pomegranate alpha/c-myc transgenic mouse model of accelerated fruit juice for chemoprevention and chemotherapy of hepatocarcinogenesis. J. Biol. Chem.; 273(25): prostate cancer. Proc. Nat. Acad. Sci. USA; 102: 15846-15853. 14813-14818. Fadavi, A.; Barzegar, M. and Hossein, M. (2006): Manna, P.; Bhattacharyya, S.; Das, J.; Ghosh, J. and Determination of fatty acids and total lipid content in Sil, P. (2011): Phytomedicinal role of oilseed of 25 pomegranates varieties grown in Iran. Pithecellobium dolce against CCl4-mediated hepatic J. Food Comp. Analy.; 19: 676-680. oxidative impairments and necrotic cell death. Evid. Faria, A.; Monteiro, R.; Mateus, N.; Azevedo, I. and Based Compl. Alternat. Med.; 17 pp. Calhau, C. (2007): Effect of pomegranate (Punica Masella, R., Di Benedetto, R., Vari, R., Filesi, C. and granatum) juice intake on hepatic oxidative stress. Giovannini, C. (2005): Novel mechanisms of Eur. J. Nutr. 46(5): 271-278. natural antioxidant compounds in biological systems: Habig, W.H.; Pabst, M.J. and Jakoby, W.B. (1974): involvement of glutathione and glutathione-related Glutathione s-transferases. The first enzymatic step enzymes. J. Nutr. Biochem.; 16: 577-586. in mercapturic acid formation. J. Biol. Chem.; Melin, A.M.; Perromat, A. and Deleris, G. (2000): 249(22): 7130-7139. Pharmacologic aplication of Fourier transform IR Halim, A.B.; El-Ahmady, O., Hassab-Allah, S.; spectroscopy: in vivo toxicity of carbon tetrachloride Abdel-Galil, F.; Hafez Y. and Darwish, A. (1997): on rat liver. Biopolym.; 57(3):160-168. Biochemical effect of antioxidants on lipids and liver Moreno, M.G. and Muriel, P. (2006): Inducible nitric function in experimentally-induced liver damage. oxide synthase is not essential for the development Annu. Clin. Biochem.; 34(6): 656–663. of fibrosis and liver damage induced by CCl4 in Hartley, D.P.; Kolaja, K.L.; Reinchord, J. and mice. J. Appl. Toxicol.; 26(4): 326-32. Peterson, D.R. (1999): 4-Hydroxynonenal and Moskaug, J.O.; Carlsen, H.; Myhrstad, M.C. and malondialdehyde hepatic protein adducts in rats Blomhoff, R. (2005): Polyphenols and glutathione treated with carbon tetrachloride: immunochemical synthesis regulation. Am. J. Clin. Nutr.; 81: 277S- detection and lobular localization. Toxicol. Appl. 283S. Pharmacol.; 161 (1): 23-33. Mustafa, R.A.; Hamid, A.A.; Mohamed, S. and Bakar, F.A.(2010): Total Phenolic Compounds,

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Flavonoids, and Radical Scavenging Activity of 21 Sohn, D.H.; Yun, Y.P.; Park, K.S.; Veech, R.L. and Selected Tropical Plants. J. Food Sci.; 75: C28-C35. Song, B.J. (1991): Post-translational reduction of Myhrstad, M.C.; Carlsen, H.; Nordstrom, O.; cytochrome P-450 by CCl4, its substrate. Biochem. Blomhoff, R. and Moskaug, J.O. (2002): Biophys. Res. Commun.; 179(1): 449-454. Flavonoids increase the intracellular glutathione Soliman, A.M. and Fahmy, S.R. ( 2011): Protective level by transactivation of the gamma- and curative effects of the 15 KD isolated protein glutamylcysteine synthetase catalytical subunit from the Peganum harmala L. seeds against carbon promoter. Free Radic. Biol. Med.; 32: 386-393. tetrachloride induced oxidative stress in brain, tests Nishikimi, M.; Appaji, N. and Yagi, K. (1972): The and erythrocytes of rats. Eur. Rev. Med. Pharmacol. occurrence of superoxide anion in the reaction of Sci.;15(8):888-899. reduced phenazine methosulfate and molecular Somani, S.M.; Ravi, R. and Rybak, L.P. (1995): oxygen. Biochem. Biophys. Res. Commun.; 46(2): Effect of exercise training on antioxidant system in 849-854. brain region of rat. Pharmacol. Biochem. Behav.; Ohkawa, H.; Ohishi, N.; Yagi, K. (1979): Assay for 50(4): 635-639. lipid peroxides in animal tissues by thiobarbituric Storz, P. (2005): Reactive oxygen species in tumor acid reaction. Anal. Biochem.; 95(2): 351-358. progression. Front Biosci.; 10: 1881-1896. Pacher, P.; Beckman, J.S.; Liaudet, L. (2007): Nitric Szymonik-Lesiuk, A.; Czechowska, G.Y.; Stryjecka- oxide and peroxynitrite in health and disease. Zimmer, M.; Slomka, M.; Madro, M; Celinski, K. Physiol. Rev.; 87(1): 315-424. and Wielosz, M. (2003): Catalase, superoxide Paglia, D.E. and Valentine, W.N. (1967): Studies on dismutase, and glutathione peroxidase activities in the quantitative and qualitative characterization of various rat tissues after carbon tetrachloride erythrocyte glutathione peroxidase. J. Lab. Clin. intoxication. J. Hepatobil. Pancreat. Surg.; 10: 309– Med.; 70(1): 158-169. 315. Reckmage, R.O.; Glende, E.A.; Dolak, J.A. and Tsakiris, S.; Schulpis, K.; Marinou, K. and Behrakis, Waller, R.L. (1989): Mechanism of carbon P. (2004): Protective effect of L-cysteine and tetrachloride toxicity. Pharmacol. Ther.; 43: 139- glutathione on the modulated sucking rat brain Na+, 154. K+, ATPase and Mg2+-ATPase activities induced by Rosenblat, M.; Hayek, T. and Aviram, M. (2006): the in vitro galactosaemia. Pharmacol. Res.; 49: 475- Anti-oxidative effects of pomegranate juice (pj) 479. consumption by diabetic patients on serum and on Vohra, B.P. and Hui, X. (2001): Taurine protects macrophages. Atheroscler.; 187(2): 363-371. against carbon tetrachloride toxicity in the cultured Sanzgir, U.Y.; Srivatson, V.; Muralidhara, S.; Dallas, neurons and in vivo. Arch. Physiol. Biochem.; C.E.; Bruckner, J.V. (1997): Uptake, distribution 109(1): 90-94. and elimination of carbon tetrachloride in rats tissues Yuan, L.P.; Chen, F.H.; Ling, L.; Bo, H.; Chen, following inhalation and ingestion exposures. Z.W.; Li, F.; Zhong, M.M. and Xia, L.J. (2008): Toxicol. Appl. Pharmacol.; 134(1): 120-129. Protective effects of total flavonoids of Bidens Seeram, N.P.; Adams, L.S.; Henning, S.M.;Niu, Y.; bipinnata L. against carbon tetrachloride-induced Zhang, Y.; Nair, M.G. and Heber, D. (2005): In liver fibrosis in rats. J. Pharm. Pharmacol.; vitro antiproliferative, apoptotic and antioxidant 60(10):1393-1402. activities of punicalagin, ellagic acid and a Zhang, L.; Maiorino, M.; Roveri, A. and Ursini, F. totalpomegranate tannin extract are enhanced in (1989): Phospholipid hydroperoxide glutathione combination with other polyphenols as found in peroxidase: speciﬁc activity in tissues of rats of pomegranate juice. J. Nutr. Biochem.; 16: 360-367. different age and comparison with other glutathione Schubert, S.Y.; Lansky, E.P. and Neeman, I. (1999): peroxidases. Biochem. Biophys. Acta.; 1006:140- Antioxidant and eicosanoid enzyme inhibition 143. properties of pomegranate seed oil and fermented juice flavonoids. J. Ethnopharmacol.; 66: 11-17.

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Study of Coumarin Content of Pelargonium fragrans - Willd. Root Grown in Egypt

K.M. Meselhy

Department of Pharmacognosy and Medicinal Plants, Faculty of Pharmacy, Misr international and Cairo Universities, Cairo, Egypt. [email protected]

Abstract: Three coumarins viz; C1 (Umbelliferone), C2 (Umckalin) and novel coumarin glycoside C3, were isolated from Pelargonium fragrans Willd root. Isolated compounds were identified on the basis of their physicochemical, UV spectral data, 1H-NMR, 13C-NMR and EIMS. These compounds were isolated for the first time from the plant. Significant antiwormal effect on fresh earthworms (Allolobophora caliginosa), remarkable antioxidant activities and strong antifungal activity were recorded on the extract, fractions and isolated compounds. [K.M. Meselhy. Study of Coumarin Content of Pelargonium fragrans - Willd. Root Grown in Egypt. Life Sci J 2013;10(1):159-164] (ISSN:1097-8135). http://www.lifesciencesite.com. 23

Keywords: Coumarin, Pelargonium fragrans, antioxidant activities, antiwormal, antifungal.

1. Introduction was air-dried, reduced to fine powder and kept in The genus Pelargonium Family (Geraniaceae) is tightly closed amber colored glass containers. a rather big one and comprises over 200 species Voucher specimens are kept in the Department of (Bailey, 1953 and Leung and Foster, 1977). A paste Pharmacognosy, Faculty of Pharmacy, Cairo made from Pelargonium leaves is used to treat University. wounds and abscesses. A decoction from the roots is 2.2. Chemicals used to wash feverish patients and extracts of the 2.2.1 Reference samples: roots have found widespread usage against infections -Coumarins from Sigma (USA) and Aldrich of the sinus, throat and respiratory tract. In (Germany). aromatherapy, Pelargonium is used as sedative, in -Glucose, galactose, xylose, rhamnose and depression, confusion, panic and anxiety (Dweck glucouronic acid (E. Merck, Darmstadt, Germany). ,1997 and Jacobs, 1997). The Pelargonium oil is of 2.2.2 Material for chromatography: economic significance in food flavoring, mouth Silica gel G (60 mesh) for TLC, silica gel (70- washes, soaps, and cosmetic industries (Bailey, 1953 230 mesh) for CC, precoated TLC plates (silica gel and Kowalchik and Hylton, 1986). 60 GF254) from E. Merck (Darmstadt, Germany), Reviewing the available current literature, few sephadex LH-20 from Pharmacia (Uppsala, Sweden). Pelargonium species have been investigated for their Solvent systems: coumarin content and their biological effects (Bladt, Petroleum ether/ethyl acetate (in different ratios v/v) 1977; Baldt and Wagner, 1988; Kayser and Chloroform - methanol (in different ratios v/v). Kolodziej, 1995, El-Sherei et al., 2004; Trun et al., Ethyl acetate - methanol -water (24:4:1) 2006; Koch and Biber; 2007; Kolodziej, 2007 ; and Methanol:water (8:2) Franco and Oliveira, 2010) Spray reagents; A literature survey indicated that no data are Sulphuric acid (50%) and P-anisaldehyde were available on the presence of coumarin constituents of used (Stahl, 1969). Pelargonium fragrans Willd. Preliminary 2.3. Material for biological evaluation: phytochemical screening of the root of the title plant 2.3.1. Plant extracts: revealed the presence of coumarins in the roots. The biological evaluation was performed on the Therefore, it was deemed of interest to isolate and three isolated compounds in addition to the acetone identify these constituents; as well as to evaluate (95 %) extract of the air-dried powdered roots. The certain biological activities of the plant. extract was prepared by defatting 200 g of air-dried 2. Material and methods powdered roots with petroleum ether and the dry 2.1. Plant material defatted marc was exhaustively percolated with The roots of Pelargonium fragrans Willd. acetone: water (5:1) followed by distillation of the Were collected from plants cultivated in the solvent. The solvent-free residue was kept for Experimental Station of Medicinal and Aromatic investigation. All samples were dissolved in water by Plants, Faculty of Pharmacy, Cairo University, the aid of few drops of tween80. Giza, Egypt. The identity of the plant was kindly 2.3.2. Animals: confirmed by Prof. Dr. Mohamed El-Gebally, Prof. Male albino rats of Sprague Dawely Strain of Plant Taxonomy, NRC, Dokki, Giza. The plant (120-150g) were used. Animals were obtained from

159 Life Science Journal 2013;10(1) http://www.lifesciencesite.com the animal house, of the National Research Center, from chloroform to afford compounds C1 as a white Dokki, Giza, Egypt. The animals were fed on a powder (18 mg) & C2 as a yellow powder (15 mg). standard laboratory diet composed of vitamin mix (1 Ethyl acetate fraction (9g) was %), mineral mix (4 %), corn oil (10 %), sucrose (20 chromatographed over Sephadex LH-20 column (l x %), cellulose (0.2 %), casein (10.5 %) and starch 40cm) with methanol to give three fractions. The (54.3 %). Fresh earthworms (Allolobophora major fraction (9-15) was rechromatographed over caliginosa) were used for testing the antiwormal silica gel using ethyl acetate-methanol-water (24: 4: activity. 1) to afforded compound C3 as 19 mg, yellow 2.3.3. Fungi: powder. The following fungi available in stock culture in Structures of compounds (C1-C3) were Microbiology Department, Faculty of Pharmacy, established by a combination of physico-chemical Cairo University were used: yeast (Candida albicans, data, UV, 1H-NMR, 13C-NMR and EIMS studies Candida tropicalis and Torulepsis glabrara) and (Table 1). Mycelial fungi (Aspergillus niger, Aspergillus 2.6. Biological study: fumigatus, Penicillium vermiculatum and Rhizopus 2.6.1. Antioxidant activity: sp. (ATCC). The antioxidant activity of the tested samples 2.3.4. Reference drugs and kits: (acetone extract and isolated compounds) was Glutathione Kit (Wak, company – Germany), assessed by measuring the glutathione level in blood and Vitamin E (Pharco Pharmaceutical Co, Egypt) samples collected from alloxan-induced diabetic rats were used in the pharmacological screening as compared to Vitamin E (12 mg / kg b.wt., positive 2.3.5. Solid medium for antifungal testing: control) and adopting the procedure described by Trypticase soy agar medium. Fungal suspension Beutler (Beutler et al., 1963). The restoration of was applied as spots on the dried agar surface using blood glutathione levels (reduced due to induction of 5 diabetes) was taken as a measure of antioxidant inoculum size of nearly 10 yeast cells or fungal activity. The percentage change observed after dose spores. administration was, in each case, calculated 2.4. Apparatus: according to the following equation: % of change= UV-visible spectrophotometer, Shimadzu UV (G –G )×100/G and results are listed in (Table 2, 240 (P/N 204-58000); Mass spectrometer: Varian 90 c t c Figure 2) NMR spectrophotometer; NMR Jeol GLM, Jeol 2.6.2. Antiwormal activity: TMS Route instrument (1H-NMR, 300 MHZ, 13C, 75 The antiwormal effect of the acetone extract MHZ, Japan); Koffler,s heating stage microscope. and the isolated compounds was evaluated using 2.5. Phytochemical study: earth worms (Allolobophora caliginosa) not less than 2.5.1 Investigation of coumarin content: 10 cm long as experimental animals (Jain and Jain, 2.5.1.1. Extraction, isolation and Identification: 1972). Different dilutions viz., 0.1 and 0.2 % of each The air-dried powdered roots (2 Kg) were sample, prepared in 1 % Tween 80, were tested. A exhaustively defatted with petroleum ether and the group of three worms was dipped in 5 ml of each of dry defatted marc was exhaustively percolated with the tested solutions in addition to a negative control acetone: water (5:1). After stripping- off the solvent consisting of 1 % Tween 80. The activity of the under reduced pressure, the residue (40 g) was treated worms was observed and the time required suspended in water and successively partitioned with for complete inhibition of the response of the worms methylene chloride and ethyl acetate. The solvent of to external stimuli (indicating paralysis or death) was each fraction was evaporated, separately, under recorded and taken as a measure for antiwormal reduced pressure to give Methylene chloride fraction activity. Results are displayed in (Table 3, Figure 3). (12 g) and ethyl acetate fraction (9 g). Fractions 2.6.3. Antifungal activity: obtained were investigated on TLC using system The antifungal activity of acetone extract and Chloroform-Methanol (9:1 v/v) and the different fractions prepared thereof (methylene chromatograms were examined under UV before and chloride and ethyl acetate fractions) were tested after exposure to ammonia vapours. against representatives of yeasts (Candida albicans , Methylene chloride fraction (12 g) was Candida tropicalis and Torulepsis glabrara) and separated over a silica gel CC eluted with a mycelial fungi (Aspergillus niger, Aspergillus petroleum ether/EtOAc solvent system with fumigatus, Penicillium vermiculatum and Rhizopus increasing polarity to afford compound C1 sp) as described ( Lorian, 1980). (petroleum ether/EtOAc, 4:2) and C2 (petroleum Serial dilutions from 80 to 5 mg/mL of ether/EtOAc, 1:1). Combined fractions (10-19) were Trypticase soy agar medium were used. 20 μl of each purified on silica gel 60 CC (20 g) and re-crystallized dilution was transferred to cups preformed in

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Trypticase soy agar innoculated with suspension of 1H and 13C NMR spectra of C3, revealed the 105 /ml yeast cells or fungal spores at the surface of characteristic chemical shifts and coupling patterns agar plates and incubated at 30°C for 4-5 days. After for4-methyl-5,8-dimethoxy-coumarin-7-O-glucoside incubation, the lowest concentration producing (Murray et al., 1982), which indicated by the inhibition was recorded as the minimum effective absence of 5,7 and 8 proton signals. Long-range concentration. Results are recorded in (Table 4). coupling was observed between H-3 ( 6.05, d, J = 3. Results and Discussion 0.6 Hz), and methyl protons at C-4, which has been 3.1. Investigation of coumarin content: reported in the spectra of 4-methylcoumarin (Murray Acetone extract of the dried powdered roots et al., 1982), 5,8-dimethoxy protons appear at 4.05 was partitioned with methylene chloride and ethyl and 4.19, respectively, with integration of 3 protons acetate. Methylene chloride fraction afforded each. compounds C1 and C2, while ethyl acetate fraction Signals for five oxymethine protons in trans afforded compound C3. The structure elucidation diaxial confirmations (J = 8.4 - 9.5 Hz) and one was established on the basis of physicochemical oxymethylene protons at δ 3.60 (dd, J = 10.4/ 5.7 1 13 data, UV data, H-NMR, C-NMR and EIMS Hz, H-6a) and δ 3.74 (dd, J = 10.4/ 4.2 Hz, H-6b) (Table. 1). indicated the presence of a -D-glucopyranoside moiety. The six carbon signals observed at (δ 60.7 - Compound C1: 18 mg; white powder; soluble 99.7) in the 13C NMR spectrum also confirmed the in chloroform, The UV spectral data are similar to presence of an O-glucosyl unit in C3. The - those reported byMurray and Kayser for 7-hydroxy- anomeric configuration for the glucose moiety was 1 3 coumarin. H-NMR spectrum revealed the determined from the JH-1', H-2' coupling constant characteristic chemical shifts and coupling patterns (5.35, d, J = 8.4 Hz) corresponding to the axial for a 7-hydroxy-coumarin (Murray et al.,1982 and proton of-D-glucopyranosyl. Furthermore, a loss Kayser and Kolodziej, 1995) The molecular formula of 162 mass units from the molecular ion in the was deduced as C9H6O3 from the molecular ion peak EIMS spectrum at m/e 235 (M - H - glc)+ clearly + observed at m/z 162 (M) in the mass spectrometry, suggested the presence of O-glucose moiety in C3. ascribed to umbelliferone (Figure 1). 13C-NMR showed a signal at 166 of (C-2), due Compound C2: 15 mg; yellowish white to effect of carbonyl and hetero oxygen. Oxygenated crystals; soluble in chloroform, The UV spectral data aromatic carbons appear at 165.1 (C-5), 154.3 (C-7), are similar to those reported for 7-hydroxy-5,6- 144.4 (C-8a) and 142.3 (C-8) as well as signals at dimethoxycoumarin compounds with free OH at 104.5 (C-4a) and 100.2 (C-6) ascribed for aromatic position 7. 1H-NMR spectrum showed the signals carbons. (8-OCH3), (5-OCH3) and (CH3) appear characteristic for 7-hydroxy-5,6-dimethoxycoumarin upfield at  62.5, 61.6 and 20, respectively; (Murray et al.,1982 and Kayser and Kolodziej, From the previously mentioned 1995) , being different from C1, in protons 5 and 6, physicochemical and spectral data, as well as, where both protons were substituted by 2 methoxyl comparison with the published data (Devon. and groups and this was verified by absence of H-5 and Scott ,1975; Murray et al.,1982 and The Merck H-6 proton signals, and appearance of H-8 as a Index, 2001), compounds C1-C3 (Table 1, Figure singlet peak; in addition to the presence of, 2 sharp 1) could be identified as umbelliferone (7- singlets at  4.25 and 4.2 ppm; each integrated for 3 Hydroxycoumarin), umkalin (7-Hydroxy-5,6- protons ascribed for 2 methoxy protons. The mass dimethoxycoumarin) and 5,8-dimethoxy-4-methyl- spectrum of C2 showed a molecular ion peak at m/z 7-O-glucoside coumarin, respectively. Although = 222 compatible with the molecular formula Compounds C1 and C2 were previously reported in C1H10O5 and ascribed to umckalin (Figure 1), Other other species of Pelargonium (Bladt ,1977; Bladt and fragment ion peaks at m/z 193, indicating a loss of Wagner, 1988, Kayserand Kolodziej, 1995 and one methoxy group, and m/z 163, indicating a loss of Franco and Oliveira., 2010), they were isolated for two methoxy groups. the first time from P. fragrans Willd., while Compound C3: 19 mg; yellow powder; compound C3 was isolated for the first time from soluble in methanol. Bands characteristic for 5,8- the genus. dimethoxy-coumarin glycoside compounds were 3.2. Biological evaluation: observed by UV spectrum (Murray et al., 1982). The The different biological activities evaluated EIMS of compound C3 exhibited molecular-ion (antioxidant, antiwormal and antifungal) for extract, peaks at m/z 397 (M -H)+, and 235 (M - H - + fractions and isolated compounds, as represented in glucose) , allowed the establishment of the (Tables 2-4), revealed variable although significant molecular formula C18H22O10. efficacy & potency for all the samples when

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compared to standard and suggest their incorporation recommend the possible utility of isolates as in herbal formulations after necessary clinical trials. antiwormal agents. 3.2.1. Antioxidant effect: 3.2.3. Antifungal activity The variation in biological activities of the The antifungal activity of acetone extract and tested samples may be attributed to the difference in different fractions prepared thereof (methylene chemical nature of tested sample. As a matter of fact, chloride and ethyl acetate fractions) were tested acetone extract, C1 and C2 samples exerted high against representatives yeasts and mycelial fungi potency as antioxidant (Table 2, Figure 2); which (Table 4). may be due to synergetic effect of acetone extract The acetone extract and fractions thereof content and phenolic nature of C1 and C2 (Stephen exhibited a significant antifungal activity with MIC and Duke, 1996). between 20 - 80 mg/mL; methylene chloride and 3.2.2. Antiwormal effect: ethyl acetate fractions rich with coumarins being the On the other hand compounds C1, C2 & C3 most active. In general, yeasts were more sensitive recorded higher activity as antiwormal (Table 3, than mycelial fungi. Coumarins have been found to Figure 3); where paralysis was induced in few stimulate macrophages (Casley-Smith and J. R. minutes & dose dependent when tested in 0.1% and 1997), which could have an indirect negative effect 0.2% concentrations of tested samples. This on infections. More specifically hydroxylated response was of irreversible nature, even after derivatives of coumarins, can be presumed to have leaving the worms into recovery bath (fresh water strong antifungal activity (Hoult and Paya, 1996). bath). This observation offers a support to

Table 1. Data of compounds C1, C2 & C3 isolated from acetone extract of Pelargonium fragrans - Willd. root Data Carbon C1 C2 C3 number H C H C 1HNMR(300 MHz, DMSO- 2 - 166 d6) 3 6.2 (1 H, d, J =9.5 Hz) 6.36 (1 H, d, J= 9.5Hz) 6.05 (1 H, d, J = 0.6 Hz) 111.3 & 13CNMR (75 MHz DMSO- 4 7.93 (1H, d, J = 9.6 Hz) 7.75 (1 H, d, J= 9.5 Hz) - 152.1 d6) 5 7.65 (1 H, d, J =8.4 Hz) - - 165.1 6 6.79 (1 H, dd, J = 8.6 Hz, J = 2.6 Hz) - 6.25 (1 H, s) 100.2 7 - - 154.3 8 6.74 (1 H, d, J =2.4 Hz) 6.83 (1 H, s) - 142.3 4a - - - 104.5 8a - - - 144.4

4 CH3 - - 2.48 (3 H, d, J = 0.6 Hz) 20

5 OCH3 - 4.2 (3 H, s) 4.05 (3 H, s) 61.6

6 OCH3 - 4.25 (3 H, s) - -

8 OCH3 - - 4.19 (3 H, s) 62.5 99.7(C- 1') 73.1 (C- 2') -5.35 (1H, d, J = 8.4 Hz, H- 76.5 (C- 1'anomeric) 3') 7 glucose - - -3.15-3.73 ( remain of sugar 69.5 (C- protons) 4') 77.0 (C- 5') 60.7 (C- 6') MS (EI, 70 eV) m/z 162 (M+, 90%), 134 (100%), 105 (28%), 222 (M+, 10%), 193 (11%), 163 (12%), 111 397 (M, 95%)+, 235 ((M- glucose)+, 13%) 78 (37%) (34%), 83 (75%) UV/Vis λmax (MeOH) nm (log ε) 222, 243 sh, 259 sh, 326 220, 243, 261 sh, 322 223, 245, 261 sh, 323 physicochemical data 18 mg, white powder 15 mg, yellowish white powder 19 mg, yellow powder MP: 224-225 ºC. MP: 148-149 ºC. MP: 150-152 ºC. Rf : 0.72 (CHCl3-MeOH, 9:1) Rf : 0.83 (CHCl3-MeOH, 9:1) Rf : 0.99 (CHCl3-MeOH, 9:1)

R2 R1 R1 R2 R3 R4 R5 5 4 R 6 4a C1 H H 3 3 H H H 2 C2 H OCH3 OCH3 H H R O 7 8a O O 4 8 R C3 CH OCH H glc OCH 5 3 3 3 Figure 1: Isolated coumarins from acetone extract of Pelargonium fragrans - Willd. root

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Table 2 & Figure 2: Antioxidant effect of acetone extract and isolated coumarins obtained from the root of Pelargonium fragrans - Willd. grown in Egypt: Blood glutathione (mg%) Test Mean ± S.E.1 % of change2 Potency3 Control Saline) 36.2+0.8 - - Diabetic 24.9+0.5* 31.2 - Acetone extract 33.2+0.3 8.3 20.5 C1 32.7+0.8 9.6 17.7 C2 33.4+1.4 9.1 18.7 C3 30.6+1.1· 15.5 11.0 Diabetic + Vit E. 35.6+0.7 1.7 100.0 1: Blood glutathion level expressed in mg/kg as mean ± S.E. 2: Percentage of change as compared to control. 3: Percentage of Potency as compared to standard. * Significantly different from the control group at p < 0.01.

Table 3 and Figure 3: Antiwormal effect of acetone extract and isolated coumarins obtained from the root of Pelargonium fragrans - Willd. grown in Egypt: Time elapsed before worm death (min.) after treatment Test 0.1%1 0.2%2 Control 1% Tween80) - - Acetone extract 12 8 C1 7 4 C2 8 5 C3 9 7 1: Time elapsed before worm death in minutes after treatment by 0.1 % of tested samples. 2: Time elapsed before worm death in minutes after treatment by 0.2 % of tested samples.

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Table 4: Antifungal effect of acetone extract and fractions obtained from the root of Pelargonium fragrans - Willd. grown in Egypt: MIC(mg/mL) Tested solution Mycelia fungi Yeast Aspergillus Aspergillus Penicillium Rhizopus Candida Candida Torulepsis niger fumigatus vermiculat. sp albicans tropicalis glabrara Amphotericine B μg/mL) 2 2 1 2 1 0.5 1 Acetone extract 80 40 80 80 40 80 80 Methylene chloride fraction 80 80 40 80 40 80 80 Ethyl acetate fraction 40 40 20 40 20 40 20

Acknowledgement 11. Jacobs, J. 1997: “The Encyclopedia of Altenative The author is thankful to Dr. Amani, Sleem, Medicine”; Carlton Book Publications, Boston, Prof. of Pharmacology, NRC for her help in the Massachusetts. study of antioxidant effect and to Dr. Aly Ahmedy, 12. Jain, M.L. and Jain, S.R. 1972: Therapeutic utility of Ass. Prof of Microbiology, Microbiology Ocimum basilicum var .albumPlanta Med.; 22, 66-70. Department, Faculty of Pharmacy, Cairo University 13. Kayser, O. and Kolodziej, H. 1995: "Highly in testing the antifungal activity. oxygenated coumarins from pelargonium sidoides "Journal of Phytochemistry; 39(5), 1181-1185. Corresponding Author: 14. Koch, E. and Biber, A. 2007: "Treatment of rats with Khaled Meselhy Ibrahim the Pelargonium sidoides extract EPs 7630 has no Pharmacognosy & Medicinal Plants, Faculty of effect on blood coagulation parameters or on the Pharmacy, Misr International & Cairo Universities, pharmacokinetics of warfarinJ. Phytomedicine, 14, 1, [email protected] 40-45. 15. Kolodziej, H. 2007:" Fascinating metabolic pools of References: Pelargonium sidoides and Pelargonium reniforme, 1. Bailey, L.H. 1953: “The Standard Cyclopedia of traditional and phytomedicinal sources of the herbal Horticulture”; Vol. III, The McMillan Co., New York, medicine Umckaloabo" J. Phytomedicine, 14, 1, 9- Toronto. 17" 2. Beutler, E., Duron,O. and Kelly, BA. 1963: Improved 16. Kowalchik, C.H. and Hylton, W. 1986: “Encyclopedia method for the determination of blood glutathione. J. of Herbs”; Rdale Press, Emmaus, Pennsylvania. Lab. Clin. Med. 61, 882-888. 17. Leung, Y. A. and Foster, S. 1977: “Encyclopedia of 3. Bladt, S. 1977: Deutsche Apotheker Zeitung.; 117Oct. Common Natural Ingredients”; Wiley-Interscience, (13), 1655 -1660. John Wiley and Sons, London, Sydney, Toronto. 4. Bladt,S. and Wagner, H. 1988: Deutsche Apotheker 18. Lorian,V; 1980:"Antibiotic in Laboratory Medicine", Zeitung.; 128, Feb. (11), 292-296. 1014, Williams and Wilkins; Baltimore London. 5. Casley-Smith, J. R., and J. R. Casley-Smith. 1997: 19. Murray, R.D.H; Mendez, J. and Brown, S.A. 1982: Coumarin in the treatment of lymphoedema and other "The Natural Coumarins"; A Wiley-Interscience high-protein oedemas, p. 348. In R. O'Kennedy, and Publication; John Wiley & Sons LTD; New York, R. D. Thornes (ed.), Coumarins: biology, applications Brisbane, Toronto and Singapore. and mode of action. John Wiley & Sons, Inc., New 20. Stahl, E. 1969: “Thin Layer Chromatography”; 2nd York, N.Y Edition, Springer Verlag, Berlin, Heidelberg, New 6. Devon, T. K. and Scott, A. I. 1975 "Handbook of York. Naturally occurring Compounds" published by 21. Stephen, M. and Duke, J.A 1996: "CRC Handbook of Academic Press Inc., (London LTD.), New York. Medicinal Mints", Library of Congress; CRC Press, 7. Dweck, A.C. 1997): "African fragranced plants" Inc. Boca Raton, New York, London and Tokyo. Cosmetics and Toiletries.; 112, 47-54. 22. The Merck Index 2001: "An Encyclopedia of 8. El-Sherei, M.M., El-Sakhawy, F.S., Kassem, H. A.. , Chemicals, Drugs, and Biologicals" 13th Ed.; Meselhy, K.M. and A.A.Sleem, A.A.2004: published by Merck Research Laboratories Division "Composition and certain Bioactivities of Lipoidal of Merck & Co., Inc.; Whitehouse Station. and Coumarin Content of P. denticulatum Jacq. 23. Trun, w.; Kiderlen, A.F. and Kolodziej, H. 2006: " Cultivated in Egypt"; Egypt. Biomed. Sci 16 210-227) Nitric oxide synthase and cytokines gene expression 9. Franco, L. and Oliveira B.H., 2010 " Determination of analyses in Leishmania-infected RAW" J. umckalin in commercial tincture and Phytomedicine, 13, 8, 570-575. phytopreparations containing Pelargonium sidoides by HPLC" Talanta, (81), Issues 4–5, 1368-1372. 11/22/2012 10. Hoult, J. R. S., and Paya, M. 1996: Pharmacological and biochemical actions of simple coumarins: natural products with therapeutic potential. Gen. Pharmacol. 27:713-722

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Use of oligomer modifier to enhance the fracture characteristics of glass reinforced composites for civil and medicine

Fakhrodin Alimoradi1*, Mohammad R. Aabaadiaan2, Ali Heidari1

1 Engineering Group, Chaloos Branch, Islamic Azad University, Chaloos, Iran 2 Engineering Group, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran Corresponding author email: [email protected]

Abstract: Glass reinforced composite materials are used in civil engineering and medical applications. This material is one of the conventional constitutive materials which present good mechanical properties with low price. Herein we enhance the impact resistance of the glass-reinforced composite using oligomer modification agent. Mechanical fracture toughness tests have been conducted using standard test samples. The obtained test results show that the fracture toughness of Oligomer modified glass-reinforced composite is enhanced by addition of the Oligomer to the glass reinforced composite. Moreover the damaged surfaces of fractured samples were investigated using scanning electron microscopy (SEM) techniques. [Alimoradi F, Aabaadiaan M R, Heidari A. Use of oligomer modifier to enhance the fracture characteristics of glass reinforced composites for civil and medicine. Life Sci J 2013;10(1):165-169] (ISSN:1097-8135). http://www.lifesciencesite.com. 24

Keywords: Glass reinforced composite, Oligomer modifier, Fracture toughness properties.

1. Introduction composites. Oligomer material might be utilized as a In recent years glass reinforced polymeric safe modifier with less glass transition temperature (GRP) composite materials becomes one of the most- for GRP toughening (Kim and Mai, 1991). The applied materials in engineering, medicines and many modification of composites has been studied in branches of applied sciences. With recent previous publications. For example, Ophir et al. developments in medicine, GRP materials are used in (1995) investigated modified GRP composite for making prostheses, making human artificial parts, producing high pressure storage vessels. In another dental applications, and other medical applications. work, Zhang et al. (1995) used CTBN to modify the The GRP composites are commonly used in toughness of composite pipes. Previous research reinforcing old structures, bridges and other showed negligible increase in impact strength of applications. composites which was the result of low Despite the excellent tensile properties of toughenability of the resin used (Sanjana et al., GRP material, this material suffers from a low 1985). Abadyan et al. (2009a) observed that addition fracture roughness. This is due to the brittleness of of amine-terminated butadiene acrylonitrile (ATBN) polymeric matrix materials such as epoxy, etc. This to epoxy matrix increases impact strength of restricts its application in structural applications since modified hoop wound composite but reduces its they exhibit a low fracture resistance (Vaziri et al., compressive and interlaminar shear strength. 2011a, 2011b; Valipour et al., 2012). For example, Moreover incorporation of ATBN led to more for GRP made leg prostheses or for bridge structures favorable mechanical properties, from the viewpoint several static and dynamic loading might be of both toughness and strength, relative to CTBN happened during the operational life of the structure. (Abadyan et al., 2009b). Sobrinho et al. (2011, 2012) These loadings induce micro-cracks, defects and void applied a CTBN-modified epoxy system for nucleation that act as failure points in the matrix. development of composite pipes for riser application Considering these issues one can conclude that the in deep water. The influence of matrix toughening enhancing the fracture toughness of composite and the number of composite layers on the materials for high performance applications is very mechanical behavior of the tubes were investigated crucial. by hydrostatic and split-disk tests (Sobrinho et al., Addition of secondary polymeric phase such 2011; 2012). Moreover, exploring the energy as Oligomer oligomer to the epoxy polymer matrix is absorption of hybrid modified epoxies containing soft an appropriate method for improving impact particles has been accomplished in (Abadyan et al., resistance of these composites (Riew and Kinloch, 2011). They observed that addition of 3rd polymeric 1996; Gabr et al., 2010a; 2010b; Kim and Mai, phase (coarse waste tire particles) to a modified 1991). Other methods of improvement might not be epoxy (bi-modal epoxy) might reduce the energy as effective as addition of Oligomer to the absorption of the composite. Moreover it has been

http://www.lifesciencesite.com 165 [email protected] Life Science Journal 2013;10(1) http://www.lifesciencesite.com demonstrated that the enhancing the loading rate of techniques including measuring the impact resistance modified epoxy samples might lead to reduce the and glass transition temperature of the wound energy absorption of samples (Abadyan et al., 2010). samples. This reference shows that cavitation has a great influence on the fracture toughness of epoxy matrix. Table 1. Resin formulations made in this study Moreover, the incorporation of two types of rubber Resin Hardener Modifier Content Sample code modifier on toughness and energy absorption of bi- (phr) (phr) (phr) modal composite epoxy has been examined (Abadyan NC 100 38 0 et al., 2012a;2012b). There are several other works CV5 100 38 5 that can produce useful data for increasing the CV10 100 38 10 fracture toughness of the GRP composites. CV15 100 38 15 There is still lack of knowledge on the role CV20 100 38 20 of modifier particles in the overall performance of wound composites. Only few works has been focused 2.4 Interlaminar shear strength systematically on investigation of the impact Interlaminar shear strength (ILSS) of GRP resistance of composite materials. Therefore, the goal samples are measured using cubic test samples of the current investigation is to study the role of loaded in Three-point bending state. ASTM D2344 rubber modification in fracture toughness of GRP guideline was followed to measure the ILSS of tubes in a more systematic fashion. Fracture composite. The direction of glass fibers in the test toughness tests have been conducted in combination sample are shown in figure 1. with scanning electron microscopy (SEM). A Hycar1300×33 oligomer is used to modify the epoxy matrix and the corresponding wound composites. 2. Experimental 2.1 Materials The typical resin system used in this work for fabrication of composite samples is diglycidyle ether of bisphenol A epoxy resin (trade name of Araldite LY5052) and a polyamine hardener (trade name of HY5052). In order to fabricate glass- reinforced samples, glass fiber roving has been used for filament winding the test samples. Reinforcement used was a glass rowing with 2400 tex from Vetrotex. The reactive oligomer modifier is used was the Hycar1300×33 with 16 wt% of acrylonitrile, from Novion company. Figure 1. Direction of fibers in the ILSS test samples 2.2 Sample preparation In order to make the samples, the 2.5 Fracture toughness evaluation stoichiometric ratio of the curing agent and resin The GRP composite laminates are tested at were cured for 8h at 80 ºC. The modifier content room temperature using the double-cantilever beam (Oligomer content) was varied from 0 up to 20 phr. (DCB) specimen according to ASTM D5528 standard We added the Oligomer modifier before adding the protocol. The DCB specimens are machined from the hardener agent. After preparing the resin samples, we cured GRP laminates along the longitudinal direction fabricated the filament wound composite samples. of the fiber using a saw. Afterwards cut samples are All formulations were reinforced with glass fiber. polished with fine sand papers to reduce the First resin has been modified with Oligomer material roughness of samples. The DCB specimen is shown and then the compound has been glass reinforced in schematically in Figure 2. The loading hinges were the machine resin bath before applying on the mounted at the end of DCB specimen arms. mandrel. For this purpose we used a 3-degree of 2.6 SEM Microscopy evaluation freedom (3-DOF) filament wound automatic The surfaces of the specimens were machine. After that, we cured the samples according examined using scanning electron microscope to the datasheet instruction above. The composite (SEM). In order to produce a conductive surface the formulation used is listed in Table 1. fractured surface of samples were first coated with a 2.3 Characterization techniques thin layer of gold. After that, the surface has been In order to evaluate the behavior of investigated under a high electrical voltage Oligomer modified samples, we have used two differences.

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2.7 Optical Microscopy evaluation manufacturing high-fracture toughness GRP Reflected light optical microscopy (OM) structures. was applied to investigate the fracture surface of fracture toughness samples. An Olimpus microscope Table 2. Impact strength of composite tubes with ×70 magnification is utilized for observation Sample Interlaminar Fracture toughness CN 600 CV5 740 CV10 820 CV15 850 CV20 830

1.2 ILSS test Interlaminar shear strength (ILSS) is an important parameter in predicting the low velocity impact damage initiation of laminated or wound composites. Table 3 presents interlaminar shear data obtained from cubic test samples which show some reduction in the interlaminar shear strength (ILSS) with increasing in Oligomer content. Modification of GRP influences ILSS of modified composite as Figure 2. DCB samples for mode I fracture toughness reported by other researchers and results shows that test ILSS decreases slightly with Oligomer content.

3. Results and discussion Table 3. ILSS of GRP composite 1.1 Fracture toughness test Sample ILSS (MPa) In this work, mode I inter-laminar fracture CN 39 toughness was determined from double cantilever beam (DCB) test using ASTM D5528 standard. CV5 35 According to this standard a non-adhesive aluminum CV10 34 film was inserted in the mid-plane of the GRP CV15 35 laminate during hand layup. This thin film forms an CV20 30 initiation site for the onset of delamination at the test. The free ends of the specimen was opened by 1.3 Fractography controlling the crosshead speed with the In this paper both OM and SEM are applied recommended speed of 2 mm/min. Then, the changes for investigate the fracture surface of modified of the load, load point displacement and crack growth samples. Figure 3 (a) shows the OM fractograph of were measured during the test. the fractured samples. The white regions present the The results of the fracture toughness glass fibers that are detached from the matrix due to evaluation test of the samples are listed in table 2. As the stress at the crack tip of the samples. Note that the seen, addition of the Oligomer modifier to the epoxy OM are not able to shows the fracture mechanisms. polymeric matrix of the GRP composite can increase For investigating the fracture mechanisms SEM are the fracture toughness of the final GRP composite required. Figure 3 (b) shows the SEM photo. As samples. As seen neat epoxy has the fracture 2 mentioned before, in order to produce a conductive toughness of 600J/m . However this tale reveals that surface the fractured surface of samples was first by addition of 15 percent Oligomer, more than 40 % coated with a thin layer of gold. After that, the increase in the fracture toughness of the GRP surface has been investigated under a high electrical material is observed. This is the results of cavitation voltage differences. In figure 23we present the SEM and shear yielding of modified resin in the vicinity of images of the fracture surface of samples. In the case the crack tip of the GRP samples. However by of modified resin, cavitated rubber particles are increasing in the Oligomer content beyond 15 %, a observed in the resin surface. On the fracture surface reduction in the fracture toughness of the modified of the samples small dark spots are observed. These GRP is observed. This observation shows that there spots are the cavitated Oligomer particles that are are an optimum value of Oligomer modifier content cavitated during the stress concentration around the for fracture toughness modification. This is an crack tip. This mechanism can be schematically important issue for engineers in designing and explain as in the figure 4.

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(a)

Fig. 4: Mechanisms of Oligomer in increasing the impact resistance of composite samples: The solution of liquid oligomers in the epoxy matrix (0) precipitates into two distinct phase of epoxy and Oligomer spheres (1). Applying the external stress during impact (2) causes the matrix to cavitate (3). The cavities increase by increasing the stress during crack growth (4) and they pin the microcracks that (b) occur during the crack growth (5). Fig. 3: SEM micrographs of surface of composite fracture toughness samples: (a) TOM, (b) SEM. 4. Conclusions In this work fracture toughness of the Oligomer The mechanisms of enhancing fracture modified GRP are evaluated. Epoxy-based glass toughness resistance of composite materials by reinforced composite is one of the most common Oligomer can be illustrated in figure 4. First, the composite that is used for its good mechanical solution of Oligomer in the epoxy matrix (0) properties and low price. The optical and scanning precipitates into two distinct phase of epoxy and electron microscopy has been conducted to better Oligomer spheres (1) during the curing process. By understand the composite behavior during the applying an impact loading on the specimen, the fracture toughness test. Fracture toughness of external stress induces during growth of the crack (2) composites has been influenced by Oligomer which causes the matrix to cavitate (3) and voids modification. The obtained test results show that the nucleate. The size of these cavities increases by fracture toughness of Oligomer modified GRP increasing the stress during crack propagation (4) and composite is enhanced by addition of the Oligomer. produce a plastic deformation in the matrix. These However there is an optimum Oligomer content that plastic deformation and holes can pin and arrest the beyond it no modification can be observed by microcracks that occur during the fracture loading increasing in modifier content. As a drawback, (5). Oligomer materials reduce the ILSS of the modified GRP.

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Acknowledgement: 10. Sanjana Z N, Testa J H,. Toughened epoxy resins This work is based on a research proposal for filament winding, Proceeding of 30th founded by Islamic Azad University. National SAMPE Symposium and Exhibition.Advancing Technology in Materials Corresponding Author: and Processes, CA Covina 1985; 1221-1230 Fakhrodin Alimoradi 11. Abadyan M, Khademi V, Bagheri R, Engineering Group, Chaloos Branch, Islamic Azad Haddadpour H, Kouchakzadeh M A, Farsadi M. University, Chaloos, Iran Use of rubber modification technique to improve [email protected] fracture-resistance of hoop wound composites. Materials and Design 2009a; 30: 1976–1984. References 12. Abadyan M, Bagheri R, Haddadpour H, 1. Vaziri H S, Omaraei I A, Abadyan M, Motamedi P. Investigation of the fracture Mortezaei M, Yousefi N. Thermophysical and resistance in hoop wound composites modified Rheological Behavior of Polystyrene/silica with two different reactive oligomers. Materials Nanocomposites: investigation of nanoparticle and Design 2009b; 30(8): 3048-3055. content. Materials & Design 2011a;32: 4537– 13. Sobrinho L L, Calado V M A, Bastian F L. 4542. Development and Characterization of Composite 2. Vaziri H S, Abadyan M, Nouri M, Omaraei I Materials for Production of Composite Risers by A, Sadredini Z, Ebrahimnia M, Investigation of Filament Winding. Materials Research the fracture mechanism and mechanical 2011;14(3): 287-298. properties of polystyrene/silica nanocomposite 14. Sobrinho L L, Calado V M A, Bastian F L,. various silica contents. Journal of Materials Effects of Rubber Addition to an Epoxy Resin Science 2011b; 46(17): 5628-5638. and Its Fiber Glass-Reinforced Composite. 3. Valipour A, Moghaddam P N, Mammedov B A. Polymer Composites 2012; 33(2): 295–305. Some aspects of chemical procedures & 15. Abadyan M, Bagheri R, Kouchakzadeh M A, application trends of polyaniline as an Hosseini Kordkheili SA. Exploring the tensile intrinsically conductive polymer. Life Science strain energy absorption of hybrid modified Journal 2012; 9(4):409-421. epoxies containing soft particles. Materials and 4. Riew C K, Kinloch A J. Preformed particles as Design 2011; 32: 2900–2908. tougheners for adhesives and matrices. Adv 16. Abadyan M, Khademi V, Bagheri R, Motamedi Chem Ser. 1996; 225:112-68. P, Kouchakzadeh M A, Haddadpour H. Loading 5. Gabr M H, Elrahman M A, Okubo K, Fujii T. A rate-induced transition in toughening mechanism study on mechanical properties of bacterial of rubber-modified epoxy. Journal of cellulose/epoxy reinforced by plain woven Macromolecular Science Part B 2010; 49:602– carbon fiber modified with liquid rubber. 614. Composites: Part A, 2010a; 41: 1263–1271. 17. Abadyan M, Bagheri R, Kouchakzadeh M A. 6. Gabr M H, Elrahman M A, Okubo K, Fujii T. Study of Fracture Toughness of a Hybrid Rubber Interfacial adhesion improvement of plain woven Modified Epoxy: Part I. Synergistic Toughening, carbon fiber reinforced epoxy filled with micro- Journal of Applied Polymer Science fibrillated cellulose by addition liquid rubber. 2012a;125(3):2467-2475. Journal of Materials Science 2010b; 45: 3841– 18. Abadyan M, Kouchakzadeh M A, Bagheri R. 3850. Study of Fracture Toughness of a Hybrid Rubber 7. Kim J, Mai Y W. High strength, high fracture Modified Epoxy: Part II. Effect of Loading Rate, toughness fiber composites with interface Journal of Applied Polymer Science control- a review. Composites Science and 2012b;125(3): 2476–2483. Technology, 1991; 41: 333-378. 8. Ophir Z, Buchman A, Flashner F, Liran I, 11/22/2012 Simons H, Dodiuk H. Modified epoxy formulation for improving the fracture-resistance of filament-wound pressure vessels. Journal of Adhesion Science and Technology1995;9(2): 159-175. 9. Zhang H, Berglund L A, Ericson M. Rubber- toughening of glass fiber epoxy filament wound composites. Polymer Engineering & Science 1991; 31(14):1057-1063.

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Effect of reactive vinyl-based modifier on thermal and failure resistance of reinforced epoxy used in pipelines

Ahmad Fakhar1*, Mohammad Aabaadiaan2, Mohamad Farhat3

1 Department of mechanics, Kashan Branch, Islamic Azad University, Kashan, Iran 2 Shahrekord Branch, Islamic Azad University, Shahrekord, Iran 3 Department of engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iran Corresponding author: E-mail address: [email protected]

Abstract: In recent years, epoxy is used as the matrix for manufacturing composites that are used in medical and mechanical applications. For examples reinforced epoxy is applied in manufacturing human prostheses, scaffolds, fiberglass pipelines and other applications. In this work, we have applied a vinyl-terminated butadiene acrylonitrile reactive modifier to systematically improve the failure resistance of an epoxy resin and its reinforcing composite. Vinyl-based modifier is known as a powerful toughening agent for epoxy-based thermoset polymers. Several mechanical tests including Impact resistance test and compression test has been performed. In order to evaluate the effect of rubber toughening on thermal resistance of reinforced epoxy, the dynamical scanning calorimeter has been utilized. In addition, damaged surfaces of fractured samples were investigated using scanning electron microscopy techniques. It is found that vinyl modifier improve the failure resistance of the reinforced epoxy resin. This can be attributed to the high cavitations of oligomer particles. However this improvement is accompanied with decreasing the thermal resistance of the material. This is the result of solving vinyl-based oligomer in the matrix during the mixing. [Fakhar A, Aabaadiaan M, Farhat M. Effect of reactive vinyl-based modifier on thermal and failure resistance of reinforced epoxy used in pipelines and medicine. Life Sci J 2013;10(1):170-174] (ISSN:1097-8135). http://www.lifesciencesite.com. 25

Key words: Epoxy resin, Vinyl-terminated butadiene acrylonitrile (VTBN), Mechanical properties.

1. Introduction medicine is the use of a two- part epoxy for bonding During the past century, polymer materials end-caps to the main tube of a blood filter (Tavakoli, were utilized in production of coatings, composites, 2002). Other applications of epoxy include the medical applications and other advanced structures fabrication of fiberglass composite structures. (Tavakoli, 2002; Golestaneh et al., 2010; Makinejad Reinforced composites are used in manufacturing et al., 2012; Atef et al., 2010; Abdel- Hakim et al., large diameter pipelines that are increasingly used in 2012; Valipour et al., 2012; Vaziri et al., 2011a; conveying fluids i.e. oil, water etc. The high chemical 2011b) . Among the polymers, epoxy thermoset and moisture resistance of fiberglass pipes make polymer is one of the interesting polymeric materials them one of the most appropriate candidates for fluid used in medicines and fluid conveying applications. conveying especially in the corrosive ambient. Epoxy This resin is chemically constructed from the epoxide pipelines are resistance to acidic, basic, alkaline, sore group that is a three-membered carbon, carbon, water and other corrosive fluids. Several types of oxygen ring structure (Tavakoli, 2002);. Epoxies are reinforcement such as glass fiber, carbon fiber, one of the most widely used adhesives for structural polymeric fiber etc. are used for reinforcing the applications as well as non-structural ones (Tavakoli, epoxy matrix. Among them, glass fiber are 2002). Epoxies have been used in a number of increasingly used due to its low price and high medical devices for bonding and sealing applications strength, flexibility, impact resistance, chemical (Tavakoli, 2002). A clear, medical-grade low- resistance and other appropriate characteristics. viscosity epoxy adhesive has found application in While reinforced epoxies present good performance, fabrication of access ports that are implanted beneath they shows a brittle behavior compared to the the skin of patients requiring multiple injections. The conventional metallic materials such as steel, epoxy adhesive passed biological and toxicity tests aluminum, alloys etc. Note that thermoset material i.e. acute systemic and intracutaneous toxicity, exhibits very low impact strength and fracture implantation tests and cytotoxicity tests (Tavakoli, resistance that make them sensitive to external 2002). Use of epoxy resins in ultrasound catheters damages when they are used in structures. Therefore and pacemakers are the typical examples of its use in several methods are used by engineers and medical electronic packages (Tavakoli, 2002). manufacturers for enhancing the strength of Epoxies are also being used for joining filter fiberglass composite to prevent failure and critical components. Another application of epoxy in damages during the operational life. Previous

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researches reveal that the oligomer modification, that The model system used in this study is based is the addition of oligomer particulate phase to a on a diglycidyle ether of bisphenol A epoxy. The brittle polymer matrix, is one of the most successful trade name of the material is Araldite LY5052 from approaches for improving toughness of reinforced Hauntsman Co. The hardener part is a cycloaliphatic epoxy. Reactive oligomers can be used as rubber polyamine hardener with the trade name of HY5052 modifiers for toughening of epoxy. Rubber from Vantico. The liquid modifier is vinyl-terminated modification of epoxy has been studied by previous butadiene nitrile (VTBN) copolymer with the trade researchers. Ophir et al. (1995) investigated a name of Hycar1300×33 from Novion Co. This type modified epoxy composition for filament winding of of modifier has 16 wt% of acrylonitrile content. The pressure vessels. Their product showed an improved glass reinforcement used in this study was a glass toughness with minimal sacrifice of its thermal and rowing with 2400 tex from Vetrotex. mechanical properties. Zhang et al. (1991) used 2.2 Sample preparation carboxyl-terminated butadiene acrylonitrile (CTBN) The stoichiometric ratio of the resin and to modify the toughness of reinforced epoxy tubes. hardener were mixed and degassed at room They found that the ultimate strain was higher for the temperature for 20 min. Then it was casted in a toughened pipes (Zhang et al., 1991). Sanjana et al. mould and was cured for 8h at 80 ºC in an oven. (1985) reported that the toughness improvement of Same curing procedure was employed for all epoxy composites is affected by physical conditions toughened epoxies. The modifier content was varied as well as modifier type. Abadyan et. al. (Abadyan et up to 20 phr. The obtained mixtures were mixed al., 2009a) observed that addition of amine- under vacuum for 20 min. Table 1 presents the epoxy terminated butadiene acrylonitrile (ATBN) to epoxy formulations used. matrix increases impact strength of modified hoop All formulations made were then reinforced wound composite but reduces its compressive and with glass fiber. For this purpose a filament winding interlaminar shear strength. Moreover incorporation machine has been used to wind the cylindrical of ATBN led to more favorable mechanical composite fiberglass composite tubes. The glass properties, from the viewpoint of both toughness and roving reinforcement is first passed from a resin bath strength, relative to CTBN (Abadyan et al., 2009b). to wet the glass fiber with epoxy resin and then, the Other researchers reported that the incomplete reinforced epoxy wind around a steel made mandrel. precipitation of oligomer molecules might enhance A hoop angle was employed to provide the tube the toughness characteristics and decrease the other samples. The samples then were cured at the same mechanical characteristics of a modified resin curing temperature that was used for the resin (DeCarli et al., 2005). Moreover, in Ref. (Abadyan, samples. et al., 2010) it has been shown that the increasing in the loading rate of epoxy samples might lead to Table 1: Formulations made in this study reduce the damage zone in the vicinity of the crack Resin Hardener Modifier Content Sample code tips. This reference shows that cavitation has a great (phr) (phr) (phr) influence on the fracture toughness of epoxy matrix. E 100 38 0 In other works, the incorporation of two types of E5 100 38 5 rubber modifier on fracture toughness of epoxy has E10 100 38 10 been examined (Abadyan et al., 2011; 2012a; E15 100 38 15 2012b).The researchers reported a synergistic E20 100 38 20 toughening in fracture toughness of epoxy modified by a combination of reactive oligomer and waste tire 2.3 Characterization techniques particles (Abadyan et al., 2011; 2012a; 2012b). 2.3.1. Compressive test According to the best knowledge of authors Transverse compressive properties of hoop there is lack of knowledge on the effect of modifiers wound composite (compressive properties in the on mechanical characteristics of reinforced epoxy. direction perpendicular to the fibers direction) were Therefore, in this work we utilize a vinyl-based determined according to ASTM D5449 using tubular oligomer to modify the glass reinforced epoxy in this specimens. The tubes had dimensions of 100 mm work. Various kinds of physical and mechanical tests inner diameter × 2 mm thick × 140 mm long. and microscopic techniques are conducted in this 2.3.2. Flexural test study. The obtained results are useful for design high Flexural strength of material is an indicative toughness fiberglass composites for fluid conveying of the strength of samples in bending loadings. pipelines. Therefore the flexural tests were performed according 2. Experimental to ASTM D790 standard protocol. 2.1 Materials

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2.3.3. Impact test However flexural strength is less affected by rubber In order to evaluate the effect of modification in comparison with compressive modification on impact strength of material, special properties. test has been conducted. The impact test of the composite has been conducted according to the test Table 3: Flexural strength of samples method described in reference (Abadyan, et al., 2010; Sample Flexural Strength (MPa) 2011). E 78 2.3.4 DSC test E5 75 The glass transition temperatures of the E10 73 samples were determined by a differential scanning E15 69 calorimeter (DSC). The samples were heated from 60 E20 71 to 130 °C at the heating rate of 10 °C/min. 2.3.5. Microscopy evaluation The surfaces of the fractured specimens 3.3 Impact strength evaluation were examined using scanning electron microscope Impact strength is known as a rough (SEM). In order to produce a conductive surface the criterion for evaluating the toughness of a material. fractured surface of samples were first coated with a While it might be not accepted as a scientific data in thin layer of gold. SEM samples were coated with a scientific researches, however this produces a good thin layer of gold before examination to protect the physical interpretation of the toughness for engineers fracture surfaces from beam damage and also to and designers. The impact strength (Iimp) of VTBN- prevent charge build up. modified composite samples are measured and listed 3. Results and Discussion in Table 4. Increase in impact strength is reported in 3.1. Compressive properties samples containing VTBN oligomer. Increasing The compressive characteristic plays an modifier content leads to an increasing in impact important role in mechanical performance of strength. As seen by addition of 15 phr VTBN one reinforced epeoxy materials. Table 2 demonstrates can increase more than % in impact resistance of the the compressive strength of reinforced epoxy as a material. This is the result of inducing cavitation and function of oligomer content. The compressive massive shear yielding in the epoxy matrix due to the strength gradually slightly decreases with increasing stress concentration of VTBN particles. An vinyl-based modifier content (Table 2). This is the interesting point is that previous researches reveals result of the lower strength of oligomer in less cavitation and void grows in the case of the comparison with the epoxy. Oligomers affect impact test specimen compared to fracture toughness mechanical properties of reinforced epoxy depending test samples. Decrease in cavitation and void growth on their chemical structure and geometrical corresponds to decrease in plastic deformation and parameters of precipitated particles (Abadyan et al., energy absorption. It is the result of higher loading 2009a). Note that rubbery and elastomer materials rate of impact test in comparison with fracture usually have a lower strength and modulus than those toughness test. Previous research showed that at high of thermoset resins. loading rate, toughening mechanisms such as cavitation and void growth are suppressed. Table 2: Compressive strength of samples Table 4 Impact resistance of resin Sample Compressive Strength (MPa) 2 E 423 Sample Iimp (kJ/m ) E5 412 E 5.91 E10 388 E5 7.11 E15 392 E10 7.84 E20 380 E15 8.01 E20 7.19

3.2 Flexural test 3.4 Thermal behavior Table 6 presents the flexural strength of An indicative of the thermal strength of a composite tubes. The compressive property of final material is glass transition temperature. This composite is affected by rubber modification. As parameter shows the temperature, in which the seen, flexural strength of specimens decreases with movement of the polymer branches increases increasing rubber content similar to that of dramatically due to the heat energy increases. The compressive strength. These can be attributed to variations of glass transition temperature of the weakening effect of rubber modifiers in the matrix. samples are listed in Table 5. Decrease in glass

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transition temperature of samples is observed due to 4. Conclusions vinyl-based rubber modifiers. As seen from Table 5, Herein, a liquid vinyl-based oligomer was increase in modifier content leads to a slight decrease employed as toughening agents in composite in glass transition temperature. The reduction in glass materials. The mechanical tests including transition temperature of epoxy by addition of liquid compressive test, flexural test and impact strength rubber can be attributed to lack of complete test has been performed. Moreover, in order to precipitation of the rubber molecules from the matrix. interpret the mechanical test data, damaged surfaces Please note that rubber molecules can reduce the of fractured samples were investigated using glass transition temperature. scanning electron microscopy (SEM) techniques. It is found that the compressive and flexural strength of Table 5 Glass transition temperature of resin resin decreased by oligomer modification. Results showed Moreover the impact strength of composite Sample Tg (C) has been substantially improved. This is attributed to E 110 the high cavitations of modifier particles. However, E5 109 these improvements are accompanied with a slight E10 109 reduction in glass transition temperature. SEM E15 109 fractography reveals that oligomers precipitate from E20 108 the epoxy matrix which effectively increases epoxy

toughness. 3.4 SEM

Figure 1 shows the SEM micrographs of the Acknowledgement fracture surface of impact test samples. Hackling type This work is based on a research proposal founded by morphology is observed for unmodified matrix Islamic Azad University. remaining between the fibers and in the case of modified resin, cavitated rubber particles are Corresponding Author: observed between fibers with no hackling in resin Ahmad Fakhar surface. Department of mechanics, Kashan Branch, Islamic Azad University, Kashan, Iran Corresponding author: E-mail address: [email protected]

References 1. Tavakoli M. Adhesive Bonding of Medical and Implantable Devices - A Review of Materials, Processes and Applications. Business Briefing: Medical Device Manufacturing & Technology 2002. 2. Golestaneh M, Amini G, Najafpour G D, Beygi M A. Evaluation of Mechanical Strength of Epoxy Polymer Concrete with Silica Powder as Filler, World Applied Sciences Journal 2010;9 (2): 216-220. 3. Makinejad M D, Salit M S, Ali A, Ahmad D. Effect of the Strengthen Ribs in Hybrid Toughened Kenaf/ Glass Epoxy Composite Bumper Beam. Life Science Journal 2012;9(1):210-213. 4. Atef M, Ragab H, El-Badrawy W. Influence of resin-tags on shear-bond strength of butanol- based adhesives. Life Science Journal 2010;7(4):105-113. 5. Abdel- Hakim A A, Abdel-Salam S I, Metwally Figure 1. SEM micrographs of surface of composite M S, Begawy E S, Elshafie E S. Mechanical impact samples: (a) E, (b) E10. Properties and Morphology Studies of Nanocomposites Based on RSF/Nanoclay

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Modified /HDPE Nanocomposites. Life Science Use of rubber modiﬁcation technique to improve Journal 2012; 9(3):134-142. fracture-resistance of hoop wound composites. 6. Valipour A, Moghaddam P N, Mammedov B A. Materials and Design 2009;30: 1976–1984. Some aspects of chemical procedures & 13. Abadyan M, Bagheri R, Haddadpour H, application trends of polyaniline as an Motamedi P. Investigation of the fracture intrinsically conductive polymer. Life Science resistance in hoop wound composites modified Journal 2012;9(4):409-421. with two different reactive oligomers. Materials 7. Vaziri H S, Omaraei I A, Abadyan M, Mortezaei and Design 2009;30(8):3048-3055. M, Yousefi N. Thermophysical and Rheological 14. DeCarli M, Kozielski K, Tian W, Varley R. Behavior of Polystyrene/silica Nanocomposites: Toughening of a carbon fibre reinforced epoxy investigation of nanoparticle content. Materials anhydride composite using an epoxy terminated & Design2011;32: 4537–4542. hyperbranched modifier. Composites Science 8. Vaziri H S, Abadyan M, Nouri M, Omaraei I A, and Technology 2005;65:2156–2166. Sadredini Z, Ebrahimnia M. Investigation of the 15. Abadyan M, Khademi V, Bagheri R, Motamedi fracture mechanism and mechanical properties of P, Kouchakzadeh M A, Haddadpour H. Loading polystyrene/silica nanocomposite various silica rate-induced transition in toughening mechanism contents. Journal of Materials Science of rubber-modified epoxy. Journal of 2011;46(17): 5628-5638 Macromolecular Science Part B 2010;49:602– 9. Ophir Z, Buchman A, Flashner F, Liran I, 614. Simons H, Dodiuk H. Modified epoxy 16. Abadyan, M., R. Bagheri, M.A. Kouchakzadeh, formulation for improving the fracture-resistance S.A. Hosseini Kordkheili, 2011. Exploring the of filament-wound pressure vessels. Journal of tensile strain energy absorption of hybrid Adhesion Science and Technology 1995; 9(2): modified epoxies containing soft particles. 159-175. Materials and Design 32: 2900–2908 10. Zhang H, Berglund L A, Ericson M. Rubber- 17. Abadyan M, Bagheri R, Kouchakzadeh M A. toughening of glass fiber epoxy filament wound Study of Fracture Toughness of a Hybrid Rubber composites. Polymer Engineering & Modified Epoxy: Part I. Synergistic Toughening, Science1991; 31(14):1057-1063. Journal of Applied Polymer Science 2012; 11. Sanjana Z N, Testa J H. Toughened epoxy resins 125(3):2467-2475. for filament winding, Proceeding of 30th 18. Abadyan M, Kouchakzadeh M A, Bagheri R. National SAMPE Symposium and Exhibition. Study of Fracture Toughness of a Hybrid Rubber Advancing Technology in Materials and Modified Epoxy: Part II. Effect of Loading Rate, Processes, CA Covina 1985, 1221-1230. Journal of Applied Polymer Science 2012; 12. Abadyan M, Khademi V, Bagheri R, 125(3): 2476–2483. Haddadpour H, Kouchakzadeh M A, Farsadi M.

11/29/2012

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Comparison between methods used for manufacturing of aluminum foam

Beigi kheradmand1, S. Otroj2, Z. Soleimanpour1, M. Beigyfar1

1Faculty of Engineering, Islamic Azad University, Shahrekord branch, Iran 2Faculty of Material Engineering, Shahrekord University, Iran Corresponding author email: [email protected]

Abstract: Metal foams with porosities exceeding 50% are used for production of advanced light-weight structures. In this study, the manufacturing methods of aluminum foam were reviewed and were compared with each other.There are number of processing methods that are currently used to manufacture Al foams (Körner and Singer, 2000; Banhart et al., 1999). These methods can be categorized into foaming liquid melts, gasar, infiltration, casting, foaming of powder compacts and sintering-dissolution process (SDP). Furthermore, microstructure and properties of the final product were addressed for each manufacturing method. All these manufacturing routes have their own relative densities and cell structures. Open and closed pore morphologies and relative densities ranging from 50 to 98% can be achieved based on the applied method. [kheradmand A B, Otroj S, Soleimanpour Z, Beigyfar M. Comparison between methods used for manufacturing of aluminum foam. Life Sci J 2013;10(1):175-185] (ISSN:1097-8135). http://www.lifesciencesite.com. 26

Keywords: Aluminum foam, Manufacturing, Properties, Applications, Blowing agent

1. Introduction based on their cells morphology (Kenesei et al., In many industrial applications, new 2004; Zhu and Windle, 2002; Daxner et al., 2002; materials are required for the production of light- Ramamurty and Paul, 2004; Hai-jun et al., 2007; weight structures. Metal foams with porosities Haack et al., 2008). According to the connectivity of exceeding 50% can meet this requirement (Banhart cells, metallic foams can be categorized as either and Baumeister, 1998; Lehmhus and Banhart, 2003; closed- or open-celled. Closed cell metallic foams Kenesei et al., 2004; Zhu and Windle, 2002). These possess higher modules, strengths and impact energy materials show an interesting combination of absorbing characteristics than their open cell physical and mechanical properties that make them counterparts (Haack et al., 2008). But the particularly versatile: the low apparent density, for characteristics of which the open cell foams possess example, allows obtaining a high stiffness/ specific make the foams exploitable for multifunctional weight ratio, the presence of cavities and the essential applications. In most cases, the functions such as in-homogeneity provide them acoustic and thermal filtration, separation, heat or mass exchange, and insulation properties, besides the possibility to absorb sound or energy absorption require open celled impact loads and to damp vibrations and sound structures (Zhu and Windle, 2002; Hai-jun et al., insulation (Kenesei et al., 2004; Zhu and Windle, 2007). 2002; Daxner et al., 2002; Ramamurty and Paul, An extensive amount of research has been 2004; Hai-jun et al., 2007; Haack et al., 2008; performed on the processing and properties of Banhart et al., 1998; Nieh et al., 2000; Pcomston, lightweight metal foams since the early 1990’s (Yu et 2006; Kiratisaevee and Cantwell, 2005; Simancik, al., 2008; Banhart, 2001; Han et al., 1998). Most of 2000; Yu et al., 2008; Seitzburger et al., 1997). this work has been conducted on aluminum foams Finally, the metallic structure potentially gives those due to unique properties of these materials such good electromagnetic shielding properties, special as;low density for ultra-lightweight and buoyant electrical conductivity and inalterability in time structures,high degree of homogeneous and tailorable (Banhart and Baumeister, 1998; Lehmhus and closed-cell porosity, very high specific stiffness, Banhart, 2003; Kenesei et al., 2004; Zhu and Windle, enhanced mechanical damping compared to bulk 2002; Daxner et al., 2002; Ramamurty and Paul, aluminum, reduced thermal and electrical 2004; Hai-jun et al., 2007). The combination of these conductivity, good machinability (drilling, sawing, physical properties makes metallic foams able to be turning, milling) and joining by adhesive bonding, competitive in terms of performances and costs in soldering, active brazing, mechanical inserts, several unusual advanced applications (Hai-jun et al., advanced welding methods (Han et al., 1998; Han et 2007; Haack et al., 2008; Banhart et al., 1998; Nieh al., 2004; Rakow and Waas, 2004; Grenestedt, 1998; et al., 2000; Pcomston, 2006). Metal foams show a Kováčik et al., 2003; Simancik, 2001; Miyoshi et al., cellular structure consisting in a solid and a gaseous 2000; Akseli, 2005; Fusheng, 1999). In the last phase. In this regard, these materials can be classified decades many attempts have been made to produce

http://www.lifesciencesite.com 175 [email protected] Life Science Journal 2013;10(1) http://www.lifesciencesite.com aluminum foam structures, they were not successful, adding fine ceramic powders or alloying elementsto because of their relatively high costs. Furthermore, it the melt, which form stabilizing particles, or by other was not possible to produce metallic foam with means (Banhart, 2001; Miyoshi et al., 2000; Akseli, reproducible properties. Problems occurred with a 2005). In the past 10 years, however, a number of low foam ability of the molten metal, the varying size new developments have taken place so that nowadays of cellular structures or solidification shrinkage some improved production routes are available. (Zhaojin and Deping, 2000). However, recently Currently there aretwo known ways of foaming these issues have been solved by extensive research metallic melts: gas injecting into the liquid metal and that has lead to the development of new production gasreleasingblowing agent addition into the molten technologies (Duarte and Banhart, 2000). These new metal (Duarte and Banhart, 2000; Baumeister, 1990; foaming technologies allow the production of foam Wei-min et al., 2001). of a significant higher quality. Despite comprehensive patents and 2.1.1.1. Foaming melts by gas injection literatures have been conducted on developing new The first manufacturing method of foaming methods for production of Al foam, only few old aluminum melts is based on gas injection into molten review papers dedicated to systematically addressing metal (Figure1). Alcan N. Hydro Aluminum in the issueand comparison between the methods Norway and Cymat Corporations in Canada are the (Ashby et al., 2000). Therefore, the aim of this paper manufacturers, which apply this method to produce is to review and comparenew manufacturing methods Al foams. Hence, this process sometimes is called as which are used produce al foams. Furthermore Alcan or Cymat process (Ashby et al., 2000; Wei- microstructure and properties of the produced foam min et al., 2001; Steiner et al., 1997). During this were addressed for each manufacturing method. process, SiC, Al2O3 or MgO particles are used to enhance the viscosity of the liquid metal and adjust 2. Manufacturing methods its foaming properties because liquid metals cannot While several technologies had been easily be foamed by bubbling air through them. developed for production of aluminum foams,only Drainage of the liquid down the walls of the bubbles some of these processes are applicable for the occurs too quickly and the bubbles collapse. economically production of aluminum foams in an However, if a small percentage of these particles are industrial scale. The technologies can be divided into added to the melt, the flow of the liquid metal is that start with the liquid metal or with a metal impeded sufficiently to stabilize the bubbles. In the powder. Therefore, the manufacturing methods of next stage, the gas (air, argon or nitrogen) is injected aluminum foams can be divided in two different into molten aluminum by using special rotating categories; liquid and solid state processing. The impellers or air injection shaft or vibrating nozzles, liquid state process includes foaming Al melts and which constitute gas bubbles in the melt and the solid state process comprises of foaming of distribute them uniformly and easily through the powder compacts. Alfoamhas a composite structure melt. The resultant viscous mixture of bubbles and consisting of gaseous and solid phases, which melt floats up to the surface of the liquid where it exhibits a porosity of 70-90%. Therefore, in two turns into fairly dry liquid foam as the liquid metal ways a gas is used for producing of porosity(melt gas drains out. A conveyor belt is used to pull the foam injection (air bubbling), gas –releasing article off the liquid surface, and is then left to cool and decomposition in the melt)Almost, a foaming agent is solidify (Rakow and Waas, 2004; Grenestedt, 1998; used to generate this gas and produce of porosity. Kováčik et al., 2003; Simancik, 2001). During the process, the foaming agent must be able to generate desired amount of gas bubbles in a) Microstructure & Properties: appropriate sizes (Han et al., 1998; Akseli, 2005; Average cell size, average cell wall Friedrich et al., 2003; Leitlmeier et al., 2002). thickness and density can be adjusted by varying processing parameters including gas injection rate 2.1. Liquid state processing and parameters including gas injection rate and 2.1.1. Foaming liquid metals rotating shaft speed. The volume fraction of particles Metallic melts can be foamed directly under varies from 10% to 20%, and the mean size of the certain circumstances by creating gas bubbles in the particles ranges between 5 μm and 20 μm. This liquid. Normally, gas bubbles which are formed in a process allows the production of closed-cell foams of metallic melt tend to quickly rise to its surface due to 1 m wide to 0.2 m thick slabs with diameters ranging the high buoyancy forces in the high-density liquid, between 5 mm and 20 mm and the foam relative but this rise can be hampered by increasing the densities between 0.03-0.1. viscosity of the molten metal. This can be done by

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2.1.1.2. Foaming melts with blowing agents Adding a blowing agent into the melt is another way of foaming melts. The blowing agent decomposes under the influence of heat and releases gas, which then propels the foaming process. Shinko Wire Co. Ltd. (Osaka, Japan) has developed this method, known as the Alporas process (Banhart, 2001; Han et al., 1998; Akseli, 2005; Fusheng, 1999; Friedrich et al., 2003). The Al foams produced via Figure 1. Schematic diagram of manufacturing of Alporas–process, is the most homogeneous foams aluminum foam by melt gas injection method currently available. The method is shown (Simancik, 2001). schematically in Figure 3, in the first stage of the foam production about 1.5 wt.% calcium metal is This process allows the production of added to the Al melt at 680 °C. The melt is then closed-cell foams of 1 m wide to 0.2 m thick slabs stirred for several minutes. During the stirring , with diameters ranging between 5 mm and 20 mm viscosity of the melt continuously increases by a and the foam relative densities between 0.03-0.54 factor of up to 5. This is the result of formation of 3 g/cm . oxides, e.g. CaAl2O4, or Al4Ca intermetallics, which thicken the liquid metal. Calcium volume fraction b) Advantage & shortcomings of the method: and stirring time can influence the viscosity of Al This process is the cheapest one among all melt. Upon reaching an optimum melt viscosity, others, and the only one to have been as a continuous typically 1.6 wt.% titanium hydride (TiH2) is added production. Foam panels can be produced at rates of as the blowing agent. This material decomposes up to 900 kg/hour. according to the following reaction: A possible disadvantage of the direct TiH2 (s) →Ti (s) + H2 (g) foaming process is the eventual necessity for cutting The melt starts to expand slowly and the foam, thereby opening the cells. Also, the gradually fills the foaming vessel. The whole brittleness of the produced foam due to the foaming process can take 15 minutes for a typical reinforcing particles contained in the cell walls is in batch of about 0.6 m3 (Kováčik et al., 2003; general an unwanted side effect of the foaming Simancik, 2001; Leitlmeier et al., 2002; Stanzick et technique. Furthermore, the main disadvantage of al., 2002; Garcia-Moreno et al., 2005; Malekjafarian this process is the poor quality of the foams et al., 2011; Matijasevic and Banhar, 2011; Jufen et produced. The cell size is large and often irregular, al., 2002). After cooling the vessel below the and the foams tend to have a marked density melting point of the alloy, the liquid foam turns into gradient. Although various methods have been solid Al foam and can be taken out of the mould for developed to improve the drawing of the foam, the further processing. size distribution of the pores is still difficult to control (Figure 2) (Akseli, 2005; Ashby et al., 2000).

Figure 3. The process steps of aluminum foam forming by gas releasing agent (Degischer and Krist, 2002)

a) Microstructure & Properties The pore morphology of such foam is shown in Figure 4. It is empirically found that the melt viscosity affect the average cell diameter as well as the final foam density. Moreover it was observed that carbonate foaming ensured smaller pores but similar . densities as foaming with TiH2 (Cambronero et al., Figure 2. Al foam produced by a continuous process 2009). In the literature, zirconium hydride (ZrH2) has of gas injection to a viscous melt (Banhart, 2001) also been recommended as a blowing agent for the

http://www.lifesciencesite.com 177 [email protected] Life Science Journal 2013;10(1) http://www.lifesciencesite.com production of foams with preferred concentrations of 0.5-0.6 wt% and foaming temperatures ranging from 670 to 705 °C (Banhart, 2001). The mass of one cast foam block is approximately160 kg, corresponding to an overall density including outer skins of 0.27 g/cm3 (Banhart, 2001). Typical densities after cutting off the sides of the block are 0.18-0.24 g/cm3, with the average pore size of 2-10 mm (Friedrich et al., 2003; Leitlmeier et al., 2002; Stanzick et al., 2002; Garcia- Moreno et al., 2005).

Figure 5. Apparatus used for solid–gas eutectic solidification (Banhart, 2001)

For the porous materials formed by solid/gas eutectic solidification, the word ‘‘gasar’’ was coined meaning ‘‘gas-reinforced’’ in a Russian acronym. Removal of heat from the melt causes directional solidification. As the solidification front advances through the liquid, typically at velocities of 0.05-5

Figure 4. Pore structure of aluminum foamed by gas mm/s, the hydrogen content near the solidification plane increases and gas bubbles are formed (Duarte releasing agent (TiH2) (Duarte and Banhart, 2000) and Banhart, 2000). The process parameters have to b) Advantages & shortcomings of the method be chosen such that the bubbles do not float out into The foam manufactured via this method, has the remaining liquid and disappear but remain near very uniform pore structure and do not require the the solidification zone and are entrapped in the solid addition of ceramic particles, which makes the foam (Banhart, 2001; Friedrich et al., 2003; Leitlmeier et brittle44. However, the method is more expensive al., 2002; Stanzick et al., 2002; Garcia-Moreno et al., than foaming melts by gas injection method owing to 2005). more complex equipments are required (Ashby et al., 2000). a) Microstructure & Properties The resulting pore morphologies are largely 2.1.2. Solid–gas eutectic solidification determined by the hydrogen content, the pressure A method developed about two decade ago over the melt, the direction and rate of heat removal, exploits the fact that some liquid metals form a and the chemical composition of the melt. Generally, eutectic system with hydrogen gas. By melting one of largely elongated pores oriented in the direction of these metals in a hydrogen atmosphere under high solidification are formed. They only appear spherical pressure (typically 5–10 atmospheres of hydrogen), when observed in this direction as shown in Figure 6. one can obtains a homogeneous melt charged with hydrogen (Steiner et al., 1997; Cambronero et al., 2009; Sharpalov, 1993). Now, with a comprehensive decrease in the temperature, the melt will eventually undergo a eutectic transition to a heterogeneous two- phase solid/gas system. If the composition of the system is sufficiently close to the eutectic concentration, there will be a segregation reaction at one temperature. Because the eutectic composition depends on the system pressure, the external pressure and the hydrogen content must be coordinated. For Figure 6. Pore structure of aluminum foamed by this reason an autoclave is required in which the melt solid–gas eutectic solidification (Banhart, 2001) can be produced, charged with hydrogen and finally direction ally solidified (Figure 5) (Banhart, 2001).

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Pore diameters range from 1 mm to 10 mm, pre-form. After infiltration is complete, directional pore lengths from 100 mm to 300 mm, aspect ratios solidification is initiated from the bottom of the from 1 to 300 and porosities from 5 to 75% crucible upwards by lowering the crucible onto a cold (Friedrich et al., 2003). The pore size distribution is copper chill within the infiltration apparatus. After non-uniform because of concurrent growth of small solidification, the material is removed from the and large pores and coalescence. infiltration apparatus and machined to the desired dimensions prior to removal of the salt. The salt is b) Advantages & shortcomings of the method subsequently leached by submersion in distilled Pores may be conical or even corrugated. If water. the casting vessel is cylindrical, radial and axial pores can be made depending on how the sample is cooled. However, the homogeneity of gasars is sometimes unsatisfactory and further improvements are needed to allow for applications of the material (Banhart, 2001; Miyoshi et al., 2000; Duarte and Banhart, 2000; Friedrich et al., 2003; Leitlmeier et al., 2002; Stanzick et al., 2002; Garcia-Moreno et al., 2005).

2.1.3. Replication method The replication technique consists of three basic steps: preparation of an open-pore pattern, infiltration of the pattern with the desired metal, and subsequent removal of the pattern to produce a metal sponge (Sharpalov, 1993). Hence, this process sometimes is called as infiltration. Pure aluminum foams are produced by infiltration of porous salt patterns and subsequent dissolution of the salt as outlined in Figure 7. The pattern is prepared by sintering a packed bed of coarse chemical grade sodium chloride powder. The particles of this high- purity salt are about 500 m in diameter, and their shape is equiaxed, varying somewhat from cubic to spherical depending on the as-received lot (Goodall et al., 2005). To produce the pattern, loose salt powder is poured into alumina crucibles that have been coated with boron nitride. The crucible is then tapped until a stable density is achieved. This Figure 7. Schematic of the replication process for the crucible is placed in a cold furnace and heated to a production sintering temperature of 785 °C in air, followed by of metallic foam (Marchi and Mortensen, 2001) furnace cooling. No significant deviations in the properties of the foams have been noted for sintering times ranging from a few hours to 144 h Kennedy, a) Microstructure & Properties 2002. The temperature of the furnace is controlled to SEM micrographs of the as-cast foam reveal within ±5 °C over a length of 120 mm, such that a uniform architecture that clearly demonstrates the homogenous pre-forms with a length of about 100 detailed replication of the salt pattern structure mm and a diameter up to 38 mm can be produced (Figure 8). (Marchi and Mortensen, 2001). Facets on the salt can be replicated as well After sintering, the salt pattern is removed, as pits that apparently existed on the salt grains prior inserted into a similar alumina crucible coated with to infiltration, possibly as a consequence of thermal graphite, and a billet of high-purity (99.99%) etching during sintering (these infiltrated pits become aluminum is placed on top of the pattern. This metal protrusions after dissolution of the salt). The assembly is placed in a hot-wall gas-pressure foam consists of nonperiodic arrays of struts (or infiltration apparatus, where it is heated to 750°C beams) with an hour-glass shape that meets at large under vacuum. Once the temperature has stabilized, irregular nodes. The relative density of the foams is 3 the system is pressurized with argon to a pressure of about 0.20-0.27 gr/cm and is difficult to control 0.5 MPa, such that the liquid aluminum infiltrates the within this range (although the variation within each

http://www.lifesciencesite.com 179 [email protected] Life Science Journal 2013;10(1) http://www.lifesciencesite.com casting is small, being less than 1 vol%). The average After curing, the polymer foam is removed density is partially determined by the shape of the salt by thermal treatment and molten metal is cast into the grains, which, as mentioned, varied somewhat from resulting open voids which replicate the original lot to lot (Marchi and Mortensen, 2001). polymer foam structure. Application of pressure and heating of the mould may be necessary if no filling of the narrow cavities with the liquid metal can be achieved in simple gravity casting. After removal of the mould material (e.g. by pressurized water), a metallic structure is obtained which is an exact replicate of the original polymer foam. Such structures can be made from pre-fabricated injection moulded polymer elements in a comparably similar manner by bonding such elements together layer by layer with adhesives. The resulting structure is then used as a model for the following investment casting step which yields a metallic material that represents exactly the highly ordered polymer precursor.

a) Microstructure & Properties Figure 10 shows a micrograph of a cellular aluminum material made by this technique. Complex Figure 8. SEM micrograph of pure aluminum foam shaped parts can be fabricated by pre-forming the produced by replication (Marchi & Mortensen, 2001) polymer foam. Of course, the densities and foam morphologies of the final metal product are b) Advantages & shortcomings of the method determined by the polymer precursor. Porosities Compared with foaming techniques which typically range from 80 to 97%. require an alloy or slurry that can resist collapse of the foam structure until it solidifies, this method has the advantage that virtually any alloy can be used to produce metal sponges.

2.1.4. Casting methods 2.1.4.1. Investment casting with polymer foams Investment casting can be used to obtain ordered, highly porous metals called lattice block materials (LBMs) (Lucai et al., 1991). According to this process (Figure 9), a polymer foam, e.g. polyurethane foam, is used as a starting point. If the polymer foam has closed pores, it has to be transformed into an open porous one by a reticulation Figure 10. SEM image of Al foam parts made by treatment. The resulting polymer foam with open investment casting (Banhart, 2001) cells is then filled with a slurry of sufficiently heat resistant material, e.g. a mixture of mullite, phenolic b) Advantages & shortcomings of the method resin and calcium carbonate or simple plaster. The main advantage of these foams are their mechanical properties which are close to the theoretical optimum limit and which are easily predict able and reproducible (Banhart, 2001; Miyoshi et al., 2000; Akseli, 2005; Duarte and Banhart, 2000; Friedrich et al., 2003; Leitlmeier et al., 2002; Garcia-Moreno et al., 2005; Lucai et al., 1991). Difficulties in this process include achieving a complete filling of themould Figure 9 .

Figure 9. Production of cellular metals by investment casting (Banhart, 2001) 2.1.4.2. Casting around space holder materials Light-weight porous metals can be produced by casting liquid metal around inorganic or even

http://www.lifesciencesite.com 180 [email protected] Life Science Journal 2013;10(1) http://www.lifesciencesite.com organic granules or hollow spheres of low density, or produced by placing a pre-sintered bulk of hollow by introducing such materials into a metallic melt. spheres into a mould and casting the outer skin The granules might remain in the metallic product structure and the porous core at the same time after casting (a syntactic foam) or can be removed via (Banhart, 2001; Lucai et al., 1991). thermal treatment, leaching in suitable solvents/acids, etc.(see Figure 11). a) Microstructure & Properties The Structure of Aluminum foams produced by this methodis very uniform and the foam properties are nearly isotropic.Figure 12 shows the microstructure of these foams (Banhart, 2001).

Figure 11. Production of cellular metallic materials using space-holding fillers (Banhart, 2001)

This can be done successfully if the content of space holding fillers is so high that all the granules are interconnected. Vermiculite or fired clay pellets, Figure 12.Cellular aluminum material made by using soluble salts, loose bulks of expanded clay granules, space-holding fillers.Density1.1g/cm . sand pellets, foamed glass spheres or aluminum oxide hollow spheres can serve as inorganic filler material. b) Advantages & shortcomings of the method But, commercially available alumina hollow spheres One of the main advantages of using space of 2.1–3.6 mm diameter and 100–200 mm wall holders is the close control of the pore size thickness were used. The spheres were stacked into a distribution which is given by the distribution of steel mould and vibrated to ensure a high packing particle sizes of the filler granules. On the other hand, density. Polymer spheres can be used as organic the maximum porosities which can be achieved using space holders if the solidification of the melt is space holders are limited to values below 80%. The sufficiently fast. The latter requires high pressure filler material has been removed completely in this infiltration, e.g., in a die-casting machine. Pre-heating case and the density is as low as 1.1 g/cm3 (Banhart, of the bulk of space holders is usually necessary to 2001; Friedrich et al., 2003; Leitlmeier et al., 2002; avoid premature solidification of the melt, especially Stanzick et al., 2002). when the heat capacity of the bulk is high or infiltration pressure is low. Owing to the high surface 2.2. Solid state processing tension of liquid metals, wetting of the granules 2.2.1. Foaming of powder compacts might cause a problem, since; in general, the The process starts with the mixing of metal interstices between the granules will not be filled powders - elementary metal powders, alloy powders completely. Creating a vacuum between the granules or metal powder blends - with a powdered blowing or pressurizing the melt is therefore an important pre- agent (TiH2, ZrH2, less than 1%), after which the mix requisite for infiltration. Space-holding salts are is compacted to yield a dense, semi-finished product removed by leaching in water. Sand pellets can be (Figure13). Therefore, this method is often called easily removed if the binder decomposes under the powder metallurgical because the starting materials influence of heat, whereas the polymer spheres are are metal powders (Banhart, 2001; Akseli, 2005; brought out by thermal pyrolysis. The structure is Duarte and Banhart, 2000; Baumgartner et al., 2000; very uniform and the foam properties are nearly Kunze et al., 1993). Compaction techniques include isotropic. Sandwich panels can also be made. For uni-axial or isostatic compression, rod extrusion or this, the ceramic spheres are placed into a powder rolling, extrusion can be used to produce a prefabricated skin structure between two sheets of bar or plate and helps to break the oxide films at the metal, and are then infiltrated by the melt. The skin is surfaces of the Al powders. However, extrusion melted at its surface to be able to achieve a seems to be the most economical method at the metallurgical bond. Alternatively, the skin can be

http://www.lifesciencesite.com 181 [email protected] Life Science Journal 2013;10(1) http://www.lifesciencesite.com moment and is therefore the preferred way. Foaming temperature and heating rates (Banhart, 2001; Akseli, agent decomposes and the material expands by the 2005; Kunze et al., 1993; Rossella, 2007). released gas forces during the heating process (at 350°C-450°C) thus a highly porous structure is formed. The precursor material can be processed into sheets, rods, profiles, etc. by conventional techniques. The mixture of powders, metal powder and foaming agent, was cold compacted and extruded to give solid metal material containing a dispersion of powdered foaming agent. When this solid was heated to the metal’s melting temperature, the foaming agent decomposes to release gas into the molten metal, creatinga metal foam.

Figure 14. Expansion behavior of aluminium/TiH2 compacts when foamed at 750 °C (Banhart, 2001)

b) Advantages & shortcomings of the method The manufacturing process of the precursor has to be carried out very carefully because residual porosity or other defects will lead to poor results during further processing (Banhart, 2001). During powder compacts process, cooling the foam is a problem since after heating the precursor for foaming; the heat source could be turned off quickly. However, the metal would still be hot, and is prone to collapsing back into molten metal before it solidifies. Water-cooling or heating the foam only locally may avoid this problem; however the problem may become a significant challenge for the reliable foam production (Banhart, 2001; Akseli, 2005).

2.2.2. Sintering of metal powders and fibers Wet powder spraying consists of spraying fine powder/binder slurries containing a carrier liquid Figure 13. Foaming from powder compacts process onto a porous support, then drying and diffusion (Steiner et al., 1997) bonding this coating in a vacuum or under a hydrogen atmosphere. In this way, very fine a) Microstructure & Properties porosities down to 1 mm can be achieved. Making The foam has a closed-cell structure with porous metals from aluminum alloy powders or pore diameters in the range of 1 mm to 5 mm and the granules is more difficult than using the other metals process is called baking. Figure14 shows expansion because aluminum is usually covered by a dense curves of an aluminium/TiH2 powder compact. In this oxide layer which prevents the particles from Figure the volume of a metal sample as a function of sintering together. To circumvent this problem, one time and images of four different expansions may deform the mixture of powders or granules (including the unexpanded precursor which is 9 mm during pressing to break up the oxide films and to in height and 32 mm in diameter) are shown. An create metallic bonding between the particles. expansion maximum is shown which corresponds to Alternatively, sintering aids may be employed such fairly uniform foam morphology, after which the as copper, silicon or magnesium powders that form a foam collapses. The degree of maximum expansion, low melting eutectic alloy during sintering at 595– and therefore, the density of the solid metal foam, can 625 ºC (Banhart, 2001). For pressureless sintering be controlled by adjusting the content of blowing the aluminum granules and the sintering aids can be agent and several other foaming parameters, such as milled to initiate mechanically induced alloying processes prior to sintering. Replacing powders by

http://www.lifesciencesite.com 182 [email protected] Life Science Journal 2013;10(1) http://www.lifesciencesite.com metal fibers opens new possibilities for making range 0.20–0.75. The mixtures are compacted into porous metal structures (Banhart, 2001; Friedrich et pre-forms in steel moulds using a hydraulic press al., 2003; Leitlmeier et al., 2002; Stanzick et al., with a pressure typically of 200 MPa. The pre-forms, 2002). together with the moulds, are sintered in an electric furnace at 680°C for 180 min and are then allowed to 2.2.3. Foaming of slurries cool to the room temperature. The sintered specimens Metallic foams can also be produced by are removed from the moulds and placed into a preparing a slurry of metal powders, blowing agents warm, running water stream for 45 min to dissolve and some reactive additives. The slurry is poured into the NaCl particles embedded in the Al matrices a mould after mixing and left there at elevated (Zhao and Sun, 2001). temperatures. Under the influence of the additives and the blowing agent, the slurry turns viscous and starts to expand as gas begins to evolve. If sufficient stabilizing measures have been taken, the expanded slurry can be preserved and dried completely after which it is sintered to yield a metal foam with considerable strength. Foams have been produced from aluminum powders using orthophosphoric acid with aluminum hydroxide or hydrochloric acid as a blowing agent. Relative densities down to 7% have been achieved. Still, problems with insufficient strength and cracks in the foamed material may occur

(Banhart, 2001; Friedrich et al., 2003; Leitlmeier et Figure 15. Schematic of the sintering-dissolution al., 2002). process for manufacturing Al foams (Zhao and Sun, The two last methods are not used 2001) commercially, so details aren’t noted a) Microstructure & Properties 2.2.4. Sintering-dissolution process Figure16 shows the SEM micrograph of The sintering-dissolution process (SDP) typical Al foam with an Al weight fraction in the consists of the mixing, compacting, sintering and initial Al/NaCl pre-form of 0.2. The foam has a dissolution stages as shown schematically in homogeneous structure with open pores and pore Figure15. The raw materials are Al and NaCl sizes in the range 300-1000 mm, which represent the powders. The particle sizes of the Al powder are not particle characteristics of the original NaCl powder. critical but generally required to be smaller than 1 It is demonstrated that the morphology and sizes of mm (Surace et al., 2009). The particle sizes of the the pores in the foam can be easily controlled by NaCl powder depend on the intended pore sizes of selecting an appropriate NaCl powder. It is also the final foam. The Al powder is first mixed possible to obtain a purposely tailored distribution of thoroughly with the NaCl powder at a pre-specified pore size or relative density in the foam by using volume or weight ratio. The resultant Al/NaCl NaCl powders with different particle size ranges or powder mixture is compacted into a net-shape pre- by varying the local Al weight fraction in the form under an appropriate pressure. The pre-form is Al/NaCl pre-form (Friedrich et al., 2003; Zhao and then sintered at a temperature either above or below Sun, 2001). the melting point of Al (660 °C) but far below that of NaCl (801 °C) (Zhao and Sun, 2001). b) Advantages & shortcomings of the method After the Al in the pre-form forms a well- Foams based on powder mixes of aluminum bonded networked structure, the pre-form is cooled to alloys with sodium chloride have room temperature. The imbedded NaCl particles are successfully been made in large sections finally dissolved in water, leaving behind an open with uniform structures. The resultingcell shapes cell Al foam with the same chemical composition as differ markedly from those of foams made by other that of the original Al powder. In manufacturing the methods. In practice the method is limited to Al foam, a commercial purity gas atomised Al producing materials with relative densities between powder with particle sizes below 450 mm and a 0.3 and 0.5. The cell size is determined by the commercial purity NaCl powder with nodular powder particle sizeand lies in the range 10 µm to 10 particles and particle sizes of 300-1000 mm are used. mm (Ashby et al., 2000). The two powders are mixed thoroughly at different weight ratios with the Al weight fractions in the

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References [1] Körner C, Singer R F. Processing of metal foams challenges and opportunities. Advanced Engineering Materials 2000; 2 (4): 159-165. [2] Banhart J, Ashby M F, Fleck N A. Metal foams and porous metal structures. MIT-Verlag, Bremen, 1999. [3] Banhart J, Baumeister J. Deformation characteristics of metal foams. Journal of Materials Science 1998;33:1431–40. [4] Lehmhus D, Banhart J. Properties of heat treated aluminium foams. Materials Science and Engineering: A 2003;349:98–110. [5] Kenesei P, Kadar C, Rajkovits Z, Lendvai J. The influence of cell size distribution on the plastic deformation in metal foams. Scripta Materialia 2004; 50:295–300. [6] Zhu H X, Windle A H. Effects of cell irregularity on the Figure 16. SEM micrograph of a typical Al foam high strain compression of open-cell foams. Acta manufactured by SDP (Zhao and Sun, 2001) Materialia 2002;50:1041–52. [7] Daxner T, Bo¨hm H J, Seitzberger M, Rammerstorfer F 3. Conclusions G. Modelling of cellular metals. In: Handbook of cellular metals. H. P.Degischer andB.Kristz, Aluminum foams have received a (eds.),Weinheim, Germany: Wiley-VCH, 2002, PP: considerable amount of attention in recent years 245–280. because of their extremely low density and unique [8] Ramamurty U, Paul A. Variability in mechanical functional properties such as impacting energy properties of a metal foam. Acta Materialia 2004; absorption, sound absorption, flame resistance and 52:869–876. heat resistance. Many methods for their manufacture [9] Hai-jun Y, Guang-chun Y, Xiao-lin W. Sound insulation property of Al-Si closed-cell aluminumfoam bare board were developed. Among these methods the processes material. Transactions of nonferrous metals society are of great importance for the production of 2007;17:93–98. aluminum foams, which are basing on the use of a [10] Haack D P, Butcher K R, Kim T, Lu T J. Novel foaming agent. Instead of foaming agents inert gases Lightweight Metal Foam Heat Exchangers. 2008. can be directly entrapped in the precursor. All these available online: http://www.bitlib.net [11] Banhart J, Schmoll C, Neumann U. Light-Weight manufacturing routes have their own relative Aluminum Foam Strectures for Ships. Proc. Conf. densities and cell structures. Open and closed pore Materials in Oceanic Environment (Euromat ’98) 1998, morphologies and relative densities ranging from 50 pp: 55-63. to 98% can be achieved. There are a variety of ways [12] Nieh T G, Higashi K, Wadsworth J. Effect of cell to produce open-celled aluminum foams, in which morphology on the compressive properties of open-cell investment casting, pre-form infiltration and SDP are aluminum foams. Materials Science and Engineering: A 2000; 283:105–110. the most widely used routes. Other methods such as; [13] Comston P. Low Energy Impact Damage Modes in Alcan, Alporas and gasar are the most widely used to Aluminum Foam and Polymer Foam Sandwich produce closed-celled aluminum foams.With a Structures. Journal of Sandwich Structures and rapidly increasing demand for high quality Al foams, Materials 2006;8(5): 365-379. there has been a growing need for developing other [14] Kiratisaevee H, Cantwell Wmj. Low-velocity Impact cost effective manufacture technologies. There are Response of High-performance Aluminum Foam Sandwich Structures. Journal of Reinforced Plastics and many factors influencing aluminum foam properties. Composite 2005;24(10): 1057-1072. A key point is the use of an appropriate blowing [15] Simancik F. Alulight - Aluminum Foam for Lightweight agent which releases gas at the right temperature to Construction. SAE 2000 World Congress, Detroit, ensure high expansion and the formation of a uniform Michigan, 2000. porosity. [16] Yu J, Wang E, Li J, Zheng Z. Static and low-velocity impact behavior of sandwich beams closed-cell Acknowledgement aluminum-foam core in three-point bending. This work is based on a research proposal International Journal of Impact Engineering 2008;35: founded by Islamic Azad University. 885–894. Corresponding Author: [17] Seitzburger M, Rammerstorfer F G, Degischer H P, A. Beigi kheradmand Gradinger R. Crushing of Axially Compressed Steel Faculty of Engineering, Islamic Azad University, Tubes Filled with Aluminium Foam. Acta Mechanica 1997;125: 93-105

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[18] Banhart, J., 2001. Manufacture, characterization and [36] Stanzick H, Wichmann M, Weise J, Banhart J, Helfen L, application of cellular metals and metal foams. Progress Baumbach T. Process control in aluminum foam in Materials Science, 46: 559–632. production using real-time X-ray radioscopy. Advanced [19] Han F, Zhuand Z, Gao J. Compressive Deformation and Engineering Materials 2002; 4:814–823. Energy Absorbing Characteristic of Foamed Aluminum. [37] Garcia-Moreno F, Babcsan N, Banhart J. X-ray Metallurgical And Materials Transactions A 1998; 29: radioscopy of liquid metal foams: influence of heating 2497-2502. profile, atmosphere and pressure.Colloids Surf A: [20] Han F, Cheng H, Wang J, Wang Q. Effect of pore Physicochem Aspects 2005;263:290–294. combination on the mechanical properties of an open [38] Malekjafarian M, Sadrnezhaad S K, Abravi M S, cell aluminum foam. Scripta Materialia 2004; 50:13–17. Golestanipour M, AminiMashhadi H. Manufacturing [21] Rakow J F, Waas A M. Size effects and the sheer Aluminum Foams by Melt Gas Injection Process. 7th response of aluminum foam. Mechanics of Materials International Conference on Porous Metals and Metallic 2004; 37:69–82. Foams, MetFoam, 2011, pp:195-202 [22] Grenestedt J. Influence of wavy imperfections in cell [39] Matijasevic B, Banhar J. Improvement of aluminium walls on elastic stiffness of cellular solids. Journal of the foam technology by tailoring of blowing agent, 2011. Mechanics and Physics of Solids, 1998; 46:29–50. available online: http://www.helmholtz-berlin.de [23] Kováčik J, Simančík F, Jerz J, Tobolka P. Reinforced [40] Jufen L, Rongpei L, Qingnan S, Xiaoqing Z. Study of Aluminium Foams. International Conference in Fabricating Process for New Type Aluminum Foam. Advanced Metallic Materials, Smolenice, Slovakia Materials review, 2002. 2003, pp: 154-159. [41] Cambronero L E G, Ruiz-Roman J M, Corpas F A, Ruiz [24] Simancik F. Metallic foams – ultra light materials for Prieto J M. Manufacturing of Al–Mg–Si alloy foam structural Applications. Inzynieria Materialowa 2001, 5: using calcium carbonate as foaming agent. journal of 823-828. materials processing technology 2009;209: 1803–1809. [25] Miyoshi T, Itoh M, Akiyama S, Kitahara A. Alporas [42] Sharpalov Y. Method for manufacturing porous articles. Aluminum Foam: Production Process, Properties, and US Patent No. 5,181,549, 1993. Applications. Advanced Engineering Materials [43] Degischer H P, Krist Z. Handbook of cellular 2000;2(4): 179-183 Metals:Production,Processing, Applications. Willy- [26] Akseli I. The application of aluminum foam for the heat VCH VerlagGmbH&Co. KGaA, 2002. and noise reduction in automobiles. M. S. Thesis, [44] Goodall R, Marmottant A, Despois J F, Salvo L, Engineering and Sciences of İzmir Institute of Mortensen A. in: H. Nakajima, N. Kanetake (Eds.). Technology, 2005. Proceedings of the Fourth Interna-Tiona Conference on [27] Fusheng H, Zhengang Z. The mechanical behavior of Porous Metals and Metal Foaming Technology, Japan foamed aluminum. Journal of Materials Science 2005,pp: 207–210. 1999;34: 291-299. [45] Kennedy A R. The effect of TiH2 heat treatment on gas [28] Zhaojin W, Deping H. Changes in porosity of foamed release and foaming in Al–TiH2. preforms. Scripta aluminum during Solidification. Chinese Science Materiali 2002;47:763–767. Bulletin 2000; 45(18): 1667-1672. [46] Marchi C S, Mortensen A. Deformation of Open-Cell [29] Duarte I, Banhart J. A study of aluminium foam Aluminum Foam. Acta Mechanica 2001; 49: 3959- formation - kinetics and microstructure. Acta Mechanica 3969. 2000;48: 2349-2362 [47] Lucai W, Xin C, Yuesheng C, Yan Z. Study on [30] Ashby M F, Evans A, Fleck N A, Gibson L J, Preparing Techmque of the Open Pores Foam Hutchinson J W, Wadley H N G. Metal foams-a design Aluminum Using Investment Casting Process. Foundry, guide. Butterworth-Heinemann, London, 2000. 1991. [31] Friedrich B, Jessen K, GRombach. Aluminum foam – [48] Surace R, Filippis L A C, Ludovicoand A D, Boghetich Production, Properties and Recycling Possibilities. G. Influence of processing parameters on aluminium Erzmetall 2003; 56(11): 656-660. foam produced by space holder technique. Materials and [32] Leitlmeier D, Degischer H P, Flankl H J,. Development Design 2009;30: 1878–1885. of a foaming process for particulate-reinforced [49] Baumgartner F, Duarte I, Banhart J. Industrialization of aluminium metals. Advanced Engineering Materials P/M foaming process. Advanced Engineering Materials 2002; 4:735–740. 2000; 2: 168–174. [33] Baumeister J. 11 Method for Producing Porous Metal [50] Kunze H D, Baumeister J, Banhart J, Weber M. P/M Bodies. IJ German Patent DE 4018.36. 1990. technology for the production of metal foams. Powder [34] Wei-min Z, Yan-dong M, Shu- H.Ping, Zhong-wei H. Metallurgy International 1993;25:182–185. Development Resesrch and Application about Foam [51] Rossella S, Filippis L A C D, Ludovico A D, Boghetich Metals.Hebei University of Technology, 2001 G. Experimental analysis of the effect of control [35] Steiner K, Banhart V J, Baumeister J, Weber M. factors on aluminium foam produced by powder Production and Properties of Ultra-Lightweight metallurgy. Proceedings of the Estonian Academy of Aluminum Foams for Industrial Applications. Sciences and Engineering 2007;13(2): 156–167. Proceeding from the 4th International Conference on [52] Zhao Y Y, Sun D X. A Novel Sintering-Dissolution Composites Engineering, 1997, pp: 943-944 Process for Manufacturing Al Foams. Scripta Materialia 2001;44: 105–110.

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Simulating the buckling deflection of carbon nanotube-made detectors used in medical detections by applying a continuum mechanics model

Alireza Vahdati*1, Mehdi Vahdati1,2, R. A. Mahdavinejad2

1Mechanical Engineering Department, Naein Branch, Islamic Azad University, Naein, Iran 2Department of Mechanical engineering, Faculty of Engineering, University of Tehran, Tehran, Iran Corresponding author: [email protected]

Abstract: Carbon nanotubes are increasingly used in fabrication of nano-detectors and other nano devices. Herein, the buckling of a carbon nano-tube made detector is simulated. In order to obtain nonlinear constitutive equation of the detectors we assume the distributed electrostatic and Van der Waals attractions along the CNT length. By considering the nano forces in a continuum mechanics model we were able to achieve the differential equation of the CNT. In the next step by applying Adomian series solution, we provided an analytical closed-form solution of CNTs. The deflection and the buckling parameters are determined and discussed in detail. The analytical obtained results are compared with those of literature as well as numerical solution. The obtained results revealed that the presented continuum based model is in good agreement with experimental result. Moreover it is found that the analytical solution can be considered as a reliable approach to study the buckling stability of detectors in nanoscales where the presence of van der Waals force is important. [Vahdati A, Vahdati M, Mahdavinejad R A. Simulating the buckling deflection of carbon nanotube-made detectors used in medical detections by applying a continuum mechanics model. Life Sci J 2013;10(1):186-191] (ISSN:1097-8135). http://www.lifesciencesite.com. 27

Key words: Carbon nanotube (CNT), Buckling, Continuum model, Nano-detector, Electrostatic, Modified Adomian method (MAD).

1. Introduction While there are several forces such as casimir, After discovering carbons nano-tubes (CNTs), capillary and van der Waals force that are acting in these materials are increasingly used in sub-micro distances. As the gap decreases from manufacturing small-scale structures. In recent micro to nano-scale, the van der Waals interaction decades these materials are specially for occurs. The prediction of the molecular force- constructing nano-detectors, nanotweezers, induced instability of CNTs nano-detector is a nanoelectromechanical switches, etc. (Paradise et critical subject in design nano-detector: A nano- al., 2007; Baughman et al., 1999; Ke et al., 2005). detector might adhere to its substrate with an It has been demonstrated that the elastic modulus, applied voltage less than buckling voltage or even bending flexibility and tensile strength of carbon without an applied voltage as a result of molecular nano-tubes are much higher than the conventional force, if the minimum gap between the nano- metallic materials (Esawi and Farag, 2007). These detector and substrate is not considered (Lin and materials have a great potential for medical Zhao, 2005;Abadyan et al., 2010;Koochi et al., applications in manufacturing medical detectors, 2011; Tsai and Tu, 2010; Tserpes, 2007; Desquenes biological sensors etc. Assume a typical cantilever et al., 2002; Batra and Sears, 2007; Lin and Zhao, CNT detector suspended near electrode surface 2005; Hayt and Buck, 2001). Therefore predicting with a small gap in between. By applying a voltage the effect of nano-scale forces on performance of difference between the nano-components causes the nano-detector is very important issue for design the CNT to deflect and be attracted toward the reliable detectors. electrode surface due to the presence of There are several approaches for investigate electrostatic forces. Once this voltage exceeds a the nano-world. In order to simulate the critical value, an increase in the electrostatic force nanomaterials, several theoretical techniques might becomes greater than the corresponding increase in be employed by researchers. The most famous the restoring force, resulting in the unstable molecular dynamics (MD) and molecular collapsing of the CNT to the ground position. This mechanics (MM) simulations could be used to behavior is known as the buckling instability and study the mechanical behavior of carbon-based the critical voltage is called the buckling voltage. nano-materials (Tsai and Tu, 2010; Tserpes, 2007; Predicting this voltage is very important for Desquenes et al., 2002; Batra and Sears,2007). engineers. However these methods are very time-consuming and might not be easily used in complex structures.

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Although continuum models are more time-saving  V 2 than MM and MD, their approach often leads to f  0 (3) elec DU nonlinear equations that might not be worked out (DU )arccosh2 ( ) by analytical methods, accurately (Desquenes et RW al., 2002; Lin and Zhao, 2005). Therefore It’s must be noted in this equation we analytical approaches are used to solve the assumed that: constitutive equations of the nano-system. Due to the importance of the instability of DRD  (4) CNT nano-detectors this work is dedicated to w Equation (3) can be simplified by using simulating the instability of the nano-detectors. In the following assumption: this paper, the buckling instability of cantilever 1 CNT detector has been studied. Modified Adomian  DU decomposition (MAD) is employed to solve the (DU )arccosh2 ( ) nonlinear governing equation of the system. The RW obtained results are verified by comparing with 2   those from literature as well as numerical solution.   Results will be useful for design the nano- 1 1    (5) detectors. DU DUDU   2  ln[( ) ( )  1]  2. Theoretical Model RRWW  2.1. Electrostatic interaction 1 1 Let us consider a freestanding multi-  DU 2 DU ln [2 ] walled CNT above a ground plane consisted of R graphene layers, with interlayer distance d = 3.35 W Å, as illustrated in Fig. 1. When a conductive nano- Therefore 2 tube is placed over an electrode substrate in the 0V presence of an applied potential difference between f elec  (6) 2 DU the tube and the electrode, the electrostatic charge (DU )ln (2 ) would be induced both on the tube and the RW substrate. To calculate the electrical forces acting on the tube, a capacitance model may be used. For 2.2. van der Waals interactions infinitely long metallic cylinders, the capacitance Lennard-Jones potential is a suitable per unit length is given by (Hayt and Buck, 2001): model to describe van der Waals interaction 2 (Lennard-Jones, 1930). It defines the potential C() q  0 (1) between atoms i and j by D arccosh(1 ) C 12 C 6 Rw    (7) ij r12 r 6 Where D is the initial distance between ij ij -12 C 2 whre rij is the distance between atoms i the tube and ground plate, 0=8.854×10 2 is Nm and j while C6 and C12 are the attractive and the permittivity of vacuum so the electrostatic force repulsive constants, respectively. For distances per unit length is given by: higher than 3.4 Å, such as in this paper, the 1 repulsive term decays extremely fast and can be d(()) C D V 2 2 neglected (Tserpes, 2007). For the carbon-carbon f  6 elec d() D interaction, C6=15.2 eVÅ (Girifalco et al., 2000). (2) A reliable continuum model has been established to  V 2  0 compute the van der Waals energy by double- 2 D volume integral of Lennard-Jones potential (Ke and DDR( 2w ) arccosh (1  ) Rw Espinosa, 2006), that is

Where Rw the radius of CNT and V the C 6 EvdW  n1 n 2()  dυ 1 dυ 2 (8) applied voltage. υ  υ 6 1 2 r (,)υ1 υ 2 By applying external voltage the nano- where  and  represent the two tube deflected to ground and the distance between 1 2 domains of integration, and n and n are the the nano-tube and ground plate reduce to D-U 1 2 densities of atoms in these domains, respectively. therefore the electrostatic force per unit length of deflected detector can be rewrite as:

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The distance between any two points on  and  E 1 2 d()vdW is r( , ). 1 2 f L  Eq. (8) provides acceptable results for vdW dr explaining the CNT-graphene attraction compared Ro D( N  1) d 4 3 2 2 3 4 2 2 R(8 r 32 r R  72 r R  80 rR  35 R ) to that of direct pair wise summation through C6    2r4.5 ( r 2 R ) 4.5 molecular dynamics in Eq. (7). For a (15,0) single R Ri r  D walled carbon nanotube (SWCNT) over a graphene (10) surface and for distances larger than 5 Å, the In most applications it is practically difference between EvdW specified by Eq. (8) and assumed that the diameter of tubes is much smaller molecular dynamics, is less than 1% (Tserpes, than the distance between nanotube and the 2007). graphene surface, i.e. (2R)<

2.3. Governing equations In order to develop the governing equation of the beams, the constitutive material of the nanotube is assumed to be linear elastic, and only the static deflection of the nano-tube is considered. The minimum energy principle was applied, which implies equilibrium when the free energy reaches a minimum value. By applied the Hamilton principle the governing equilibrium equation can be Figure1. Schematic representation of Cantilever determined as:

CNT nano-detector WWWW elas   elec   vdW

L d2 U d 2 U E Ro D(N1)d R(Rr)[3R2 2(Rr)2 ] ()E I  f  U  f  U dX vdW  C σ2π2  effdX2 dX 2 elec vdW L 6   2[(Rr)2 R2 ]3.5 0 RRi rD LL 2 3 (9) d U dU d U  EIEIUeff2 eff 3  where Ri and Ro are the inner and outer dXdX dX 0 0 radii of CNT, N is the number of graphene sheets L -2 d4 U and  38nm is the graphene surface density. (E I  f  f ) UdX  0  eff4 elec vdW Once the van der Waals energy is computed, the 0 dX corresponding energy terms are employed to derive (14) the component of the intermolecular force per unit Where denotes the variation symbol, X is length, fvdW, along r-direction as below: the position along the nano-tube measured from the clamped end, U is the beam deflection, Eeff, is the effective Young’s modulus of CNT which is

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Life Science Journal 2013;10(1) http://www.lifesciencesite.com typically 0.9-1.2 TPa (Gupta and Batra, 2008) and I 1 1 1   u()() x  C x2  C x 3   x 4 is the cross-sectional moment of inertia, equal to 1 2 2 4 4 2! 3! 4!  ln (2 ) (Ro - Ri )/4. By integrating Eq.(14), the governing 1 4  2  6 ()   C1 x equation of cantilever nano-tube detector is 6!  ln2 (2 ) ln 3 (2  ) derived as: 1 4  2  7 4 ()   C x d U 2 3 2 EI f  f (15.a) 7!  ln (2 ) ln (2  ) dX 4 elec vdW 1  4   2  With the B.C. of: {(  )(   )C 8! 2  2 3 2 dU ln (2 ) ln (2  ) ln (2  ) U (0) (0)  0 dX 60 6  3 3 8 [  (1   )]C1 } x (Geometrical B.C. at fixed end), (15.b) ln2 (2 )ln(2  ) ln 2 (2  ) and 1 200 20 3 3 9 2 3  [ (1   )]C1 C 2 x d U d U 9!  ln2 (2 )ln(2 ) ln 2 (2  ) 2(LL ) 3 ( )  0 dX dX ... (Natural B.C. at free end). (15.c) (18) Eqs. (15a-c) can be made dimensionless using the following substitutions, Where the constants C1 and C2 can be U determined by solving the resulting algebraic u  , D equation from the B.C at x=1 i.e. using equation X (17-c). x  , L For any given , β and , equation (31) can be used to obtain the buckling parameters of CNL2  2 4   6 W , (16) the nano-tube detector. The instability in 4 dEeff ID equation (18) occurs when dβ(x=1)/du→0. The D buckling voltage of the system can be determined  , via plotting the u vs. β. RW 2 4 3.2 Numerical Solution 0VL   In order to verify the analytical results, the E ID2 eff cantilever beam-type nano-detector is numerically These transformations yield simulated and the results are compared with those obtained via MAD and LPM. The nonlinear d4 u     (17-a) governing differential equation (Eq. (17)) is solved dx4(1 u ( x )) 4 (1  u )ln 2 [2  (1  u )] with the boundary value problem solver of MAPLE commercial software. The step size of the With the B.C. : parameter variation is chosen based on the du sensitivity of the parameter to the tip deflection. u (0) (0)  0 (17-b) The buckling parameters of the system can be dX determined via the slope of the u-β graphs. (Geometrical B.C. at fixed end), and 4. Results and Discussion d2 u d 3 u (LL ) ( )  0 (17-c) 4.1. Verification dx2 dx 3 First we verify the solution with (Natural B.C. at free end). experiments. In order to verify the obtained results, the buckling voltage of a typical cantilever CNT 3. Solution base nano-detector with the following parameters In this section two solving methods has was compared to experimental data in Table. 1. been applied for solving the governing equation. The length of the nanotube, L= 6:8 m; initial gap First is MAD and the second is Nemerical solution: between nanotube and electrode, D = 3 m; 3.1 Adomian series solution Rw=5nm; E = 1 TPa. As seen the MAD results are The detail of the MAD can be find in in good agreement with experimental results. (Adomian, 1983). The analytical MAD solution of 4.2. Simulation of deflection equation (17) can be obtained as the following:

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0.6 u u 0 PI Numerical 0.5 MAD =0 0.1 0.4

0.2 12 0.3

0.2 0.3

18(Pull-in) 0.1 0.4 0 0.2 0.4 0.6 0.8 1 x 0 0 10 20 30 40  50 (a)

Figure 4. Effect of van der Waals force (f) on pull-

u in deflection (=50) 0 After validating the solution by =0 experiments, we simulate the deflection of the 0.1 nano-detector. Figure 2 shows the centerline deflection of a typical nano-tube detector under

0.2 12 intermolecular force and external voltage obtained using MAD, and numerical methods. This figure * 17.4(Pull-in) utip increases from zero to utip , when  increases 0.3 from zero to *. This figure reveals that the CNT nano-detector has initial deflection without 0.4 applying voltage difference. This is the result of the 0 0.2 0.4 0.6 0.8 1 x presence of vdW attraction. (b) Figure 2. Deflection of the cantilever CNT for 4.3 Effect of van der Waals force on deflection of different values of  when =25 and =10. (a) CNT analytical, (b) Numerical The buckling deflection is an important parameter for design the detectors. The relation between buckling deflection and van der Waals force () is presented if figure 3 and 4 for 2 0.6 different values of radios to initial gap ratio (=10 uPI Numerical 0.5 MAD and =50) as seen increasing the intermolecular force the buckling voltage decrease. When no voltage applied (β=0) the CNT buckling if the van 0.4 der Waals force exceeds from its critical values * 0.3 ( ) the critical values of  can find from the horizontal axes of figure 3 and 4. By comparing

0.2 Figure 3 and 4 reveal that by increasing the values of , the critical values of  increase as well as the

0.1 buckling voltage.

0 Table 1. Buckling voltage obtained from different 0 2 4 6 8 10  method Experimental Figure 3. Effect of van der Waals force (f) on pull- Method MAD Numerical (Ke et al.,2005 ) in deflection (=10) Buckling 48 50.39 48.79 Voltage

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5. Conclusions Nano-actuators. International Journal of The buckling behavior of a cantilever Modern Physics: B 2011; 25(29): 3965–3976. CNT nano-detector has been studied. The obtained 8. Tsai JL, Tu JF. Characterizing mechanical results show that: properties of graphite using molecular The Adomian series solution is a very dynamics simulation. Materials & Design power full method for study the buckling behavior 2010;31(1):194-199. of CNT based nano-detectors. 9. Tserpes KI. Role of intertube spacing in the In the absence of electrical loading on pullout forces of double-walled carbon CNT based detector, it can buckling to ground if nanotubes. Materials & Design the van der Waals force exceeds from its critical 2007;28(7):2197-2201. values and this critical values increase if the  10. Desquenes M, Rotkin SV, Alaru NR. increase. Calculation of Pull-in Voltages for Carbon- The van der Waals force reduces the Nanotube-Based Nanoelectromechanical instability deflection of the CNT detector. Switches. Nanotechnology 2002;13:120-131. 11. Batra RC, Sears A. Continuum models of Acknowledgement multi-walled carbon nanotubes. International This work has been founded as a research Journal of Solids and Structures 2007;44:7577- project and supported by Islamic Azad University, 7596. Naein Branch. 12. Lin WH, Zhao YP. Nonlinear behavior for nanoscale electrostatic actuators with Casimir Corresponding Author: force. Chaos Solitons and Fractals Alireza Vahdati 2005;23:1777–1785. Mechanical Engineering Department, 13. Hayt WH, Buck JA. Engineering Naein Branch, Islamic Azad University, electromagnetics. 6th ed.. New York: Naein, Iran McGrawHill; 2001. E-mail: [email protected] 14. Lennard-Jones J E. Perturbation Problems in Quantum Mechanics. Proc. R. Soc. A 1930; References 129: 598-615. 1. Paradise M, Goswami T. Carbon nanotubes– 15. Girifalco L A, Hodak M, Lee R S. Carbon Production and industrial applications. nanotubes, buckyballs, ropes, and a universal Materials & Design 2007;28(5):1477-1489. graphitic potential. Physical Review B 2. Baughman RH, Cui C, Zakhidov AA, Iqbal Z, 2000;62(19): 13104-13110. Barisci JN, Spinks GM, Wallace GG, 16. Ke C. and Espinosa H. D., Handbook of Mazzoldi A, Rossi DD, Rinzler AG, Jaschinski Theoretical and Computational O, Roth S, Kertesz M. Carbon nanotube Nanotechnology (American Scientific actuators. Science 1999; 284:1340-1344. Publishers, Valencia CA, 2006. 3. Ke CH, Pugno N, Peng B, Espinosa HD. 17. Gupta S S, Batra R C. Continuum structures Experiments and modeling of carbon equivalent in normal mode vibrations to single- nanotube-based NEMS devices. Journal of walled carbon nanotubes. Computational Mechanics and Physics of Solids Materials Science 2008;43:715-723 (2008). 2005;53:1314-1333. 18. Adomian G, Stochastic Systems, Academic 4. Esawi AMK, Farag MM. Carbon nanotube Press, London, 1983. reinforced composites: Potential and current challenges. Materials & Design 2007;28(9):2394-2401. 11/29/2012 5. Lin WH, Zhao YP. Casimir effect on the pull- in parameters of nanometer switches. Microsystem Technologies 2005;11:80-85. 6. Abadyan M, Novinzadeh A, Kazemi AS. Approximating the effect of Casimir force on the instability of electrostatic nano-cantilevers. Physica Scripta 2010;81:015891 (10pp) 7. Koochi A, Noghrehabadi A, Abadyan M, Roohi E. Investigation of the effect of van der Waals force on the instability of electrostatic

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Protective effects of (-)-epigallocatechin gallate on blue light-induced damage in retinoblastoma Y79 cells by activating estrogen receptor pathway

Mei-Ling Peng1, 2#, Ching-Ju Lee3#, Chung-Liang Chien4, Chun-Lan Liu5, Cheng-Yu Tsai6, Yang-Cheng Wen6, Kuang-Wen Tseng1, 6*

1 Department of Ophthalmology, Chung Shan Medical University Hospital, Taichung, Taiwan 2 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC 3Internal Medicine, Taipei Hospital, Department of Health, Taipei, Taiwan, ROC 4 Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 5Department of Anatomy, Chung Shan Medical University, Taichung, Taiwan, ROC 6School of Optometry, College of Medical Sciences and Technology, Chung Shan Medical University, Taichung, Taiwan #Contributed equally. [email protected] Abstract: Light-induced photoreceptor cell death can be caused by a variety of cellular mechanisms that involve oxidative stress. Therefore, the eye depends on the presence of antioxidants to protect the retina from light-induced damage. Visible light is generated by the sun as well as by a wide variety of artificial illumination sources such as light emitting diodes (LED). Excessive exposure to light would be damaging to the eye. The short-wavelength visible light between 430 nm to 500 nm (blue light) is especially associated with retina damage as evidenced by photoreceptor degeneration. Recent investigations demonstrated that estrogen receptors (ERs) have antioxidant and antiinflammatory effects on neuronal cells in brain. However, estrogen receptor (ER)-mediated effects of the (-)- epigallocatechin gallate (EGCG), extracted from green tea, have not been examined extensively in photoreceptors of the eyeball. EGCG were examined for the ability to elicit ERs and ER-mediated gene expression in vitro. Our studies were demonstrated that the cell degeneration of retinoblastoma Y79 cells was observed after blue light exposure. Apoptosis related proteins, p53 and caspase-3, increased the expression after blue light illumination. After EGCG treatment, increased ER proteins production and inhibited the blue light-induced retinoblastoma Y79 cells death were investigated. These results indicated the short-wavelength visible light, such as white LED exposure, leads to retinoblastoma Y79 damage. EGCG regulates the expression of neuroprotective proteins, ER, and modulates degeneration responses in human retinoblastoma Y79 cells. [Mei-Ling Peng, Ching-Ju Lee, Chung-Liang Chien, Chun-Lan Liu, Cheng-Yu Tsai, Yang-Cheng Wen, Kuang- Wen Tseng. Protective effects of (-)-epigallocatechin gallate on blue light-induced damage in retinoblastoma Y79 cells by activating estrogen receptor pathway. Life Sci J 2013;10(1):192-198] (ISSN:1097-8135). http://www.lifesciencesite.com. 28

Keywords: Green tea, Estrogen receptor, Photoreceptor, Retinoblastoma Y79, Blue light

1. Introduction Besides UV light, the visible blue light can Photodegeneration resulted in extensive cell cause damage to the eye [6]. Short-wave length blue death in the retina, mainly in the outer nuclear layer radiation is believed to cause retinal damage or to (ONL), where nuclei of the rod and cone contribute to the development of age-related macular photoreceptors were contained. Bright light and degeneration [7-8]. Animal experiments indicate continuous exposure to moderately intense light photochemical damage of photoreceptor and retinal interfere with the visual cycle and induced pigment epithelial cells after eye exposure to excessive photoreceptor damage [1-2]. In addition, light levels of short-wave length visible blue light [9-10]. exposure has a role in the progression of age-related The high-energy photons generate reactive oxygen macular degeneration (AMD) [3] as determined in a species, which are deleterious to DNA and to a variety human population based study [4]. Photoreceptor cell of cellular organelles, predominantly the mitochondria loss is an irreversible injury and constriction of the [11-12]. Shorter wavelength light is the most visual field, leading to the loss of central vision. hazardous component of the visible spectrum, and is Light-induced photoreceptor cell death can be caused known to generate reactive oxygen species in the retina by different of intracellular mechanisms that involve [13-14]. oxidative stress, reactive oxygen species, and AMD is the most common cause of lost activation of caspase-relative proteins [5]. central vision in elderly people [15]. Human public- health studies signify that women with early

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menopause appear at hazard for worse AMD [16] and the treatment and prevention of neurological estrogen absence may contribute to the onset or disorders. Nevertheless, the exact role and underlying severity of AMD in these patients [17]. These molecular mechanisms of ERs in photoreceptor statistical studies have provided the evidences for a protective roles are largely unknown. In this study, great clinical trial to evaluate the effectiveness in we propose that EGCG can protect retinoblastoma preventing the onset or progression of AMD [18]. Y79 from blue light-induced cell death through its Recent findings of an association between use of antioxidative effect. We investigated the protective postmenopausal exogenous estrogen and a minor effects of EGCG against blue light-induced hazard of macular degeneration also suggest a role retinoblastoma Y79 cell death and the possible for estrogen in the pathogenesis of the disease [19]. mechanisms involved in retinoblastoma Y79 cell Estrogens are steroid hormones long known survival. for their profound effects on both male and female . reproductive systems. Estrogens regulate growth, 2. Material and Methods differentiation, and function of diverse tissues both Human cell line culture within and outside the reproductive system. The Human Retinoblastoma Y79 cells, revealed effects of estrogens are mediated by specific nuclear with photoreceptor-specific nuclear receptor receptors, which act as hormone-inducible localized expression in retinal photoreceptor cells, transcription factors. Relatively recent findings were obtained from the American Type Culture demonstrate important roles of the steroid hormones Collection (Manassas, VA). Media and fetal bovine in the cardiovascular system, in specific brain regions, serum (FBS) were purchased from Gibco-BRL in the liver, and in the maintenance of bone tissue. (Rockville, MD). Y79 was cultured in RPMI (Gibco- Despite the wide range of tissues influenced by BRL) 1640, supplemented with 10% heat-inactivated estrogens and the broad transcriptional regulatory fetal calf serum, 0.1% ciprofloxacin, 2 mM L- properties of the ligand-activated ERs, the literature glutamine, 1 mM sodium pyruvate, and 4.5% contains limited evidence of these receptors in dextrose. Another control neuro2A was propagated in normal ocular tissues [20-22]. DMEM (Gibco-BRL), supplemented with 7.5% heat- Green tea has been shown to have inactivated FBS, 7.5% heat-inactivated horse serum. antiinflammatory and antioxidant properties on Cultured cell lines were grown at 37°C in a 5% CO2- different types of cells [23-24]. The green tea extracts humidified incubator. contain (-)-epigallocatechin gallate (EGCG), (-)- epigallocatechin (EGC), (-)-epicatechin gallate Blue light exposure on cultured cells (ECG), (-)-epicatechin (EC), and catechin. Among Spectrum distribution from LED lamps and these constituents, EGCG is the most abundant and white floursent lamps (China electric MFC, Taiwan) the most active component in green tea. Transcription was analyzed using an UV-visible spectrophotometer factors play a crucial role in regulating gene (Ultrospec 3000, Pharmacia Biotech, Cambridge, expression by interacting with epigenetic modulators. UK) from 300 nm to 800 nm. Peak wavelengths of In breast cells, EGCG can affect chromatin the LED lighting are 440 nm and 470 nm within the modifications as well as the binding alteration of a blue light wavelengths. To create a constant level of transcription repressor complex, Rb/p130-E2F4/5- illumination, we placed some cells into the same cell HDAC1-DNMT1-SUV39H1, to the ERs promoter, culture incubator, with identical ambient conditions resulting in ER reactivation [25]. HDAC/DNMT1 to normal cultures except that light was housed 15 cm involves a series of gene silencing through recruiting directly above culture plates to insure that all cultures transcriptional repressors to the gene promoter. received the same lighting levels. Cultured cells were Moreover, histone methyltransferase, SUV39H1, is exposed to diffused light from LED lamps ranged another important epigenetic factor for transcriptional from 10 minutes. The core temperatures were silencing. A recent study has shown that a measured and were found not to increase by more multimolecular complex, pRb2/p130-E2F4/5- than 1°C during illumination. Cells were further HDAC1-DNMT1-SUV39H1, binding to the ER incubated for 12 to 24 hours under normal conditions promoter, is associated with ER transcriptional (culture medium, 37°C, 5% CO2, 95% air) before repression [26]. analysis. Studies demonstrate that activation of ER EGCG treatment on cultured cells enhance the viability of neuronal cells exposed to a EGCG was purchased from Sigma Chemical variety of toxic conditions. The protective effects of Co. (St. Louis, MO). Human retinoblastoma Y79 activation of ER in models of acute neuronal stress cells were treated with various concentrations of and neurodegeneration suggest that these female sex EGCG for 3 days. The medium with EGCG was hormones might represent an effective approach for replaced every 24 h for the duration of the

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experiment. Control cells received equal amounts of 3. Results DMSO (Sigma) in the medium. For the combination 3.1 The spectral irradiance profile of LED source study, cells were treated with an optimal The emission spectrum of UV/visible lighting concentration of EGCG based on our following ranging from 300 to 800 nm was recorded. The results and ER antagonist (ICI 182,780) (Sigma) wavelength within the UV range was not detectable alone or together for a total 3 days as the common from LED source. The spectrum distribution of recommended doses of these compounds. lighting began to increase at 300 nm and reached sustained maximum transmission at 450 nm. The Immunocytochemistry for cultured cells chief peak of the spectrum distributes was about 0.35 Cultured cells were washed in PBS, fixed in mW/cm2 within the visible wavelength of the blue methanol at 20°C for 10 minutes, equilibrated in light. Spectral irradiance profile of LED source PBS, blocked for 30 minutes with 5% normal goat showed that the main optical radiation ranging from serum, incubated with the primary antibodies 430nm to 500 nm as our previous study [6]. overnight, and washed extensively with PBS. Cells were than incubated for 1 hour with the secondary 3.2 Effects of Human Retinoblastoma Y79 cells antibodies and again washed with PBS. damage induced by short-term blue light LED Subsequently, cells were mounted and observed light exposure under a Leica TCS SP2 confocal spectral microscope. No available studies had investigated on the cytotoxic effect of short-wave length blue light Cell fractionation and Western Blot radiation on human retinoblastoma cells. To illustrate Samples were rinsed once with ice-cold PBS the relationship between short-wave length blue light and then lysed with PBS containing 1% Triton X- radiation and cell death, the immunostaining of active 100, 0.1% SDS, 0.5% sodium deoxycholate, 1 μg/ml caspase-3 was applied. Apoptosis-related protein, leupeptin, 10 μg/ml aprotinin and 1 mM caspase-3, was determined at 30 min and then for 12 phenylmethylsulfonyl fluoride on ice for 10 minute. and 24 incubated time after blue light LED After sonication using microprobe sonicator, crude irradiation. As we expected, Human Y79 cells extracts were subjected to centrifugation at 4 °C. The underwent apoptosis cell death after blue light LED supernatants were collected as cell lysates. All exposure in our system. The increase percentage of protein concentrations were determined by a protein caspase-3 positive cells after light exposure was assay (Bio-Rad laboratories, Richmond, California). observed (Fig. 1). After 12 and 24 hr, very few Aliquots (50 μg) of cell lysates or nuclear extracts immunopositive cells with active caspase could be were separated electrophoresed on 8 or 12% SDS- detected in the Y79 cells from control and visible- polyacrylamide gel and then transblotted onto the light exposure groups, whereas a number of neurons ImmobilonTM-P membrane (Millipore). After being were labeled with active caspase-3 and their nuclei blocked with 10% skim milk in Tween-20/PBS, blots were in an eccentric position after blue light-exposed. were incubated with various primary antibodies and With a statistical analysis, the percentage of then incubated with HRP-conjugated secondary active caspase-3 immuno-positive neurons was antibodies. The protein bands in the blots were significantly increased in the cultured neurons blue detected using enhanced chemiluminescence kit light irradiation compared with that from control and (ECL; PerkinElmer Life Sciences, Inc. Boston). visible-light exposure groups. These results implied that the cell death was associated with short-wave Morphological evaluation length blue light radiation. Cultured cells were fixed in methanol for 30 min at 4°C. Hematoxyline was applied to stain 3.3 Effects of EGCG on activating ER expression nuclei. Subsequently, culture cells were mounted and To elucidate the effects of the green tea examined under a Zeiss Axiophot microscope (Carl polyphenol, EGCG, on cellular viability and ER Zeiss, Oberkochen, Germany). expression in human retinoblastoma Y79 cells, we initiated to determine the optimal dose that will Statistical Analysis induce ER transcriptional activation without causing Data are presented as the mean ± SEM. toxicity to cells. We performed western-blot assays to Statistical comparisons were made using a one-way detect the protein level of ER expression in human analysis of variance (ANOVA) followed by a retinoblastoma Y79 cells (Fig. 2A). More strikingly, Student’s t test. The statically significance was studies demonstrated that EGCG treatment can assessed at p < 0.05. induce significant effects on cellular expression when chose to use the concentration of 25 μM EGCG (Fig. 2B). In summary, these results suggest that EGCGs

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may play an important role in ER expression in retinoblastoma Y79 cells.

Fig. 2: EGCG induced ER proteins expression in human retinoblastoma Y79 cells The retinoblastoma cells were treated either 10 μM or 25 μM EGCG for 1, 2, and 3 days. EGCG induced ER proteins expression in retinoblastoma cells (A). EGCG Fig. 1: Effects of Human Retinoblastoma Y79 cells induced maximal ER expression at a concentration of damage induced by short-term blue light 10 μM for 3 day treatment. β-Actin was as the loading irradiation control (B). Data are expressed as means (±S.D.); * Cultured cells were exposed to light for 10 minutes, and then for 12 to 24 hr incubated time. Human Retinoblastoma Y79 cells, revealed with photoreceptor-specific nuclear receptor localized expression in retinal photoreceptor cells, showed a significantly increased percentage of caspase-3 positive cells after light exposure with blue light, but not with visible light irradiation. Data are expressed as means (±S.D.); * indicate a value statistically different (p < 0.05) from the control.

3.4 Neuroprotection of ER expression in EGCG- treated human retinoblastoma cells The morphological evaluation was performed to determine whether ER can inhibit apoptotic cell Fig. 3: EGCG modulate degeneration response in death induced by short-wavelength visible blue light. human retinoblastoma Y79 cells The cytotoxic responses were measured by the Few positive staining cells were noted in control morphological evaluation. Cultured Y79 cells were cultured cells; whereas cultures treated with short- pretreated with 5 μM ER antagonist 30 min prior to 25 wavelength visible blue light h had large numbers of μM EGCG treatments. The cell number of apoptosis cells undergoing apoptosis. However, pretreatment of cells with chromatin condensation (arrows) was Y79 cells with 25μM EGCG for 3 day prior to light significantly increased after blue light irradiation or exposure led to a dramatic decrease in the numbers of light irradiation combined with ER antagonist (ICI apoptotic cells. Moreover, pretreatment with ER 182,780) treatment. Pretreatment of retinoblastoma antagonist (ICI 182,780) prior to the addition of Y79 cells with EGCG for 3 day prior to blue light EGCG significantly block the neuroprotective effect exposure caused a significant decrease in the numbers of apoptotic cells of EGCG in this study (Fig. 3)

4. Discussion In the present study, we demonstrated that EGCG has a neuroprotective effect in the retina which is mediated via the activating estrogen receptor pathway. It has been known that EGCG promotes

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viability and survival of other primary neuronal results demonstrated that EGCG inhibits blue light- culture systems, such as cortex, hippocampus, and induced caspase-related proteins expression. hypothalamus [27-30]. EGCG is also known as a Moreover, EGCG increases human retinoblastoma scavenger of ROS in extracellular environments and cell survival after exposure to visible blue light for its protective effect on many cell types against the radiation. This suggests that EGCG is valuable in damage of UV radiation in retinal pigment epithelium preventing retinal cells from short-wave length [31]. This is the first observation demonstrating that visible light-induced damage and may be appropriate EGCG inhibits blue light-induced human to be further developed as a prophylactic health food retinoblastoma Y79 cell apoptosis, and activates ER for the prevention of retinal diseases. In addition, our expression. results imply that green tea catechins can have a As excessive light exposure is known to positive effect and can play an important role in the induce retinal dysfunction [1-2, 6], we investigated prevention of short-wave length visible light -induced the effect of human retinoblastoma cells on light- visual disorders. induced cell degeneration in mice by Immunocytochemistry staining. Previous studies 6. Acknowledgment demonstrated that light exposure caused apoptosis in This work was supported by a grant (CSMU- the retinal tissue [31] and that apoptosis was INT-101-19) from Chung Shan Medical University, prevented by antioxidants such as ascorbic acid and Taiwan, ROC. dimethylthiourea [32-33]. Main effects of light- induced cell death have been reported to depend on Corresponding Author: the apoptotic pathway and also on ROS production Kuang-Wen Tseng, Ph.D. [34]. In some tissues, the neuroprotection may be School of Optometry, College of Medical Sciences mediated by the estrogen receptor and relative protein and Technology, Chung Shan Medical University, synthesis, because estradiol can bind intracellular No. 110, Jianguo N. Rd., Taichung 402, Taiwan; specific estrogen receptors, and the complex binds to Phone: +886-4-24730022, ext. 12137 specific sites on genomic DNA and control mRNA E-mail: [email protected] transcription. Definitely, estradiol was reported to provide neuroprotection mediated by estrogen References receptors in cultured neurons including neurones 1. Noell WK, Walker VS, Kang BS, Berman S. (1966) from the ipothalamus, amygadala, cortex midbrain Retinal damage by light in rats. Invest and hippocampus [35]. We observed that a Ophthalmol, 5 (5): 450-473. competitive estrogen receptor antagonist, ICI 2. Reme’ CE, Grimm C, Hafezi F, Marti A, Wenzel A. 182,780, significantly attenuate the protection (1998) Apoptotic cell death in retinal provided by ER. The absence of neuroprotective degenerations. Prog Retin Eye Res, 17 (4): 443- effects by ICI 182,780 completely impair the 464. possibility that the neuroprotective functions that 3. Hirakawa M, Tanaka M, Tanaka Y, Okubo A, were mediated by estrogen receptors. Koriyama C, Tsuji M, Akiba S, Miyamoto K, It is well identified that age is a risk factor for Hillebrand G, Yamashita T, Sakamoto T. (2008) loss of visual function in many diseases, such as Age-related maculopathy and sunlight exposure AMD [17]. Symptoms observed in menopausal evaluated by objective measurement. Br J women have been considered related to the abrupt Ophthalmol, 92(5): 630-4. decline in estrogen activity after the reproductive 4. Krishnamoorthy RR, Crawford MJ, Chaturvedi years. Several human public-health studies suggest MM, Jain SK, Aggarwal BB, Al-Ubaidi MR, potential participations of estrogen and its receptors Agarwal N. (1998) Photo-oxidative stress down- in the homeostasis of the eye, but the mechanisms modulates the activity of nuclear factor-κB via involved remain unclear. In our studies, these results involvement of caspase-1, leading to apoptosis of indicated that EGCGs may play an important role in photoreceptor cells. J. Biol. Chem, 274 (6): 3734- ER expression in retinoblastoma Y79 and ER 3743. expression modulates degeneration responses in 5. Wood JP, Lascaratos G, Bron AJ, Osborne NN. vitro. (2007) The influence of visible light exposure on cultured RGC-5 cells. Mol. Vis, 14: 334-344 5. Conclusion 6. Peng ML, Tsai CY, g Chien CL, Hsiao CJ, Huang In summary, our study provides the first SY, Lee CJ, Lin HY, Wen YC, Tseng KW. evidence that EGCG is a protective agent for human (2012) The Influence of Low-powered Family retinoblastoma Y79 cells after exposure to blue light LED Lighting on Eyes in Mice Experimental radiation by activating ER expression pathway. Our Model. Life Sci J, 9(1): 477-482.

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7. Taylor HR, West S, Munoz B, Rosenthal FS, 19. Marin-Castano ME, Elliot SJ, Potier MK, Striker Bressler SB, Bressler NM. (1992) The long-term LJ, Striker GE, Csaky KG. Cousins1 SW. (2003) effects of visible light on the eye. Arch. Regulation of estrogen receptors and MMP-2 Ophthalmol, 110(1): 99-104. expression by estrogens in human retinal pigment 8. Braunstein RE, Sparrow JR. (2005) A blue- epithelium. Invest. Ophthalmol. Vis. Sci, 44 (1): blocking intraocular lens should be used in 50-59. cataract surgery. Arch. Ophthalmol, 123(4): 547- 20. Kumar V, Green S, Stack G, Berry M, Jin JR, 549. Chambon P. (1987) Functional domains of the 9. Suter M, Reme C, Grimm C, Wenzel A, Jaattela human estrogen receptor. Cell, 51 (6): 941-951. M, Esser P, Kociok N, Leist M, Richter C. (2000) 21. Evans RM. (1988) The steroid and thyroid Age-related macular degeneration. The lipofusion hormone receptor superfamily. Science, 240 component Nretinyl- N-retinylidene ethanolamine (4854): 889-895. detaches proapoptotic proteins from mitochondria 22. King WJ, Greene GL. (1994) Monoclonal and induces apoptosis in mammalian retinal antibodies localize oestrogen receptor in the pigment epithelial cells. J. Biol. Chem, 275(50): nuclei of target cells. Nature, 307 (5953): 745-747. 39625-30. 23. Tipoe GL, Leung TM, Hung MW, Fung ML. 10. Boulton M, Dontsov A, Jarvis-Evans J, Ostrovsky (2007) Green tea polyphenols as an anti-oxidant M, Svistunenko D. (1993) Lipofuscin is a and anti-inflammatory agent for cardiovascular photoinducible free radical generator. J. protection. Cardiovasc Hematol Disord Drug Photochem. Photobiol, 19(3): 201-204. Targets, 7 (2): 135-144. 11. Boulton M, Dontsov A, Jarvis-Evans J, Ostrovsky 24. Farris P. (2007) Idebenone, green tea, and M, Svistunenko D. (1993) Lipofuscin is a coffeeberry extract: new and innovative photoinducible free radical generator. J. antioxidants. Dermatol Ther, 20 (5):322-329. Photochem. Photobiol. B, 19 (3): 201-204. 25. Macaluso M, Cinti C, Russo G, Russo A, 12. Rozanowska M, Pawlak A, Rozanowski B, Giordano A. (2003) pRb2/p130-E2F4/5- HDAC1- Skumatz C, Zareba M, Boulton ME, Burke JM, SUV39H1-p300 and pRb2/p130-E2F4/5- Sarna T, Simon JD. (2004) Age-related changes HDAC1-SUV39H1-DNMT1 multimolecular in the photoreactivity of retinal lipofuscin complexes mediate the transcription of estrogen granules: role of chloroform-insoluble receptor-alpha in breast cancer. Oncogene, 22 components. Invest. Ophthalmol. Vis. Sci, 45 (4): (23): 3511-3517. 1052-1060. 26. Vaute O, Nicolas E, Vandel L, Trouche D. (2002) 13. Crockett RS, Lawwill T. (1984) Oxygen Functional and physical interaction between the dependence of damage by 435 nm light in histone methyl transferase Suv39H1 and histone cultured retinal epithelium, Curr. Eye Res, 3 (1): deacetylases. Nucleic Acids Res, 30 (2): 475-481. 209-215. 27. Cambiasso, M. J., and Carrer, H. F. (2001) 14. Ruffolo JJ, Ham WT, Mueller HA, Millen JE. Nongenomic mechanism mediates estradiol (1984) Photochemical lesions in the primate stimulation of axon growth in male rat retina under conditions of elevated blood oxygen. hypothalamic neurons in vitro. J. Neurosci. Res, Invest. Ophthalmol. Vis. Sci, 25(8): 893-898. 66(3): 475-481. 15. Fine SL, Berger JW, Maguire MG, Ho AC. (2000) 28. Gallaher, B. W., Hille, R., Raile, K., and Kiess, Age-related macular degeneration. N. Engl. J. W. (2001) Apoptosis: live or die--hard work Med, 342 (7): 483-492. either way! Horm. Metab. Res, 33 (9):511-519 16. Vingerling JR, Dielemans I, Witteman JC, 29. Honda K, Shimohama S, Sawada H, Kihara T, Hofman A, Grobbee, DE, de Jong PT. (1995) Nakamizo T, Shibasaki H, Akaike A. (2001) Macular degeneration and early menopause: a Nongenomic antiapoptotic signal transduction by case-control study. BMJ 310 (6994): 1570-1571. estrogen in cultured cortical neurons. J. Neurosci. 17. Mitchell P, Smith W, Attebo K, Wang JJ. (1995) Res, 64 (5): 466-475 Prevalance of age related maculopathy in 30. Yang C, Landau J, Huang M, Newmark H. (2001) Australia. The Blue Mountains Eye Study. Inhibition of carcinogenesis by dietary Opthalmology, 102 (10): 1450-1460. polyphenolic compounds. Annu Rev Nutr, 21: 18. Smith W, Mitchell P, Wang JJ (1997). Gender, 381-406. oestrogen, hormone replacement and age-related 31. Chan CM, Huang JH, Lin HH, Chiang HS, Chen macular degeneration: results from the Blue BH, Hong JY, Hung CF. (2008) Protective effects Mountains Eye Study. Aust. N. Z. J. Ophthalmol. of (-)-epigallocatechin gallate on UVA-induced 25 (1): S13-S15. damage in ARPE19 cells. Mol Vis, 14: 2528-34. 31. Abler AS, Chang CJ, Ful J, Tso, MO, Lam TT.

http://www.sciencepub.net/life 197 [email protected] Life Science Journal, 2013;10(1) http://www.lifesciencesite.com

(1996) Photic injury triggers apoptosis of 34. Krishnamoorthy RR, Crawford MJ, Chaturvedi photoreceptor cells. Res. Commun. Mol. Pathol. MM, Jain SK, Aggarwal BB, Al-Ubaidi MR, Pharmacol, 92 (2):177-189. Agarwal N. (1999) Photo-oxidative stress down- 32. Organisciak DT, Wang HM, Li ZY, Tso MO. modulates the activity of nuclear factor-κB via (1985) The protective effect of ascorbate in involvement of caspase-1, leading to apoptosis of retinal light damage of rats. Invest. Ophthalmol. photoreceptorcells. J. Biol. Chem, , 274 (6): 3734- Vis. Sci, 26 (11): 1580-1588. 3743. 33. Organisciak DT, Darrow RM, Jiang Y I, Marak G. 35. Amantea D, Russo R, Bagetta G, Corasaniti MT. E, Blanks JC. (1992) Protection by (2005) From clinical evidence to molecular dimethylthiourea against retinal light damage in mechanisms underlying neuroprotection afforded rats. Invest. Ophthalmol. Vis. Sci, 33 (5): 1599- by estrogens. Pharmacol Res, 52(2):119-132. 1609.

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Biomass-density relationships and self-thinning of shell and tissue in marine intertidal barnacles

Claver Sibomana and Gen-Xuan Wang

College of Life Sciences, Zhejiang University, Hangzhou 310058, China [email protected]

Abstract: Although self-thinning exponent in barnacles was assumed to be close to -3/2 widely acknowledged for space-limited plants, steeper slopes have been observed as a result of allometric growth. There is a trade-off between shell and tissue growth in barnacles and self-thinning lines for these individual parts would differ as a result of different biomass allocation. We quantified biomass-density relationships for shell and tissue in self-thinning population of Tetraclita squamosa in two intertidal levels to determine the corresponding scaling exponents to see whether they are close to each other and to the “-3/2 power law”. The self-thinning slope for tissue in low interidal was steeper than the shell one in mid-intertidal and all slopes were higher than -3/2. In higher heights, individuals are subject to more desiccation stress and would allocate more biomass to shell than their counterparts in lower heights which allocate more biomass to tissue, thus self-thinning slope for tissue in low intertidal would be steeper. Our results suggested that self-thinning exponent for barnacle individual parts is not invariant and these different exponents would be explained by different biomass allocation to shell and tissue in response to environmental conditions. [Sibomana C, Wang GX. Biomass-density relationships and self-thinning of shell and tissue in marine intertidal barnacles. Life Sci J 2013;10(1):199-203] (ISSN:1097-8135). http://www.lifesciencesite.com. 29

Key-words: barnacle, biomass-density relationship, biomass allocation, self-thinning, Tetraclita squamosa

1. Introduction slope (Sibomana et al. in press). Growth of sessile intertidal invertebrates is Although the “-3/2 power law” of self- limited by space availability. As a result, suppression thinning has been considered as an empirical of weaker individuals due to density-dependent generalization (Yoda et al. 1963; White & Harper mortality is accompanied by an increase in biomass 1970), the self-thinning exponent has been of survivors during growth in the so-called process of demonstrated to vary in plant communities (Weller self-thinning (Yoda et al. 1963; Hughes & Griffiths 1987; Deng et al. 2006; Dai et al. 2009; Bai et al. 1988; Fréchette & Lefaivre 1995). Self-thinning can 2010; Zhang et al. 2011). Recent results indicated be quantified by the equation: that the exponent depends on species (Pretzsch 2006) or environmental conditions (Morris 2002; Deng et al. W=kNα or logW=logk+αlogN 2006; Bai et al. 2010; Zhang et al. 2012). Self- thinning in barnacles is still poorly understood and Where W is mean individual biomass, N is density; k the generality of the scaling exponent may be and α are intercept and exponent of the self-thinning questionable (Sibomana et al. in press), thus studies line respectively (Yoda et al. 1963; Morris 2003). are needed for a better understanding of the self- Self-thinning in sessile invertebrates has been thinning process and the underlying mechanisms in widely studied especially in the cultured mussels sessile barnacle populations. Moreover, previous (Fréchette et al. 2010; review by Guiñez 2005). In studies have considered whole individual biomass barnacles, self-thinning has drawn less attention without taking different biomass components (e.g. despite their importance in marine intertidal shell, tissue) into account (Hogarth 1985; Hughes & community dynamics (Little & Kitchings 1996) and Griffiths 1988; Sibomana et al. in press). their potential in aquaculture (Lopez et al. 2010). Self-thinning of individual parts has been Early studies suggested that the slope (α) would be well studied in plant communities. Biomass-density close to -3/2, the exponent that has been widely relationship lines for roots are steeper than the shoot acknowledged for plants since barnacles are space- ones in arid nutrient-limited environments where limited (Hughes & Griffiths 1988). However, steeper plants allocate more biomass to belowground parts to slopes have been found and were accounted for by maximize uptake of water and nutrients while in allometric growth and the nature of barnacle food light-limited benign environments, the allometric different from the evenly distributed light used by exponent is higher for shoot since additional biomass plants (Hogarth 1985). The difference of competition is allocated to aboveground part in response to light mechanisms between plants and barnacles has competition during growth (Deng et al. 2006; Bai et recently been suggested for interpreting this steeper al. 2010; Zhang et al. 2011). Likewise, there is a

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trade-off between shell and tissue growth in barnacles sampled barnacles were separated before they were (Lively 1986) and individuals subject to desiccation dried in oven at 70°C for 72 hours. stress tend to allocate more biomass to shell while those spending longer period submerged allocate Mass-density relationships of barnacle parts more biomass to tissue (Bertness et al. 1998; Lopez In LI and MI, 40 10 cm x 10 cm, 5 cm x 5 cm, & Gonzalez 2003). Biomass-density relationships of 2.5 cm x 2.5 cm quadrats were set along 20-40 m barnacle shell and tissue are related to those of whole transects parallel to the coastline randomly, according individuals by pattern of biomass allocation, therefore to the density and the individual size (Sibomana et al. analyzing them is important for understanding the in press). However, plots with empty shells or barnacle strategy for survival, reproduction and markedly heterogeneous regarding individual size population dynamics. were not sampled. In laboratory, individuals from In higher intertidal levels, barnacles are each quadrat were counted and dried shell and tissue subject to higher desiccation stress, thus self-thinning weighed to determine density and mean individual lines for shell should be steeper if individuals tend to biomass for the two individual parts. allocate more biomass to shell during growth while in low heights the corresponding lines for soft parts Density and tidal level effects on biomass would be steeper if individuals allocate more biomass allocation to tissue. In this study, we quantify biomass-density For density and tidal level effects on biomass relationships of shell and tissue in self-thinning allocation to shell and tissue, individuals were also population of Tetraclita squamosa Bruguière, 1789 to sampled in LI and MI. Three density levels, solitary determine the corresponding scaling exponents to see (SOL) (with no contact with neighbors-at least 1 cm whether they are close to each other and to the “-3/2 from the nearest conspecifics), low density (LD) (4-5 power law”. Density and tidal height effects on ind/5cm²) and high density (HD) (>5 ind/5cm²) were biomass allocation were also determined. considered at each tidal height. 40 individuals were randomly chosen for each density level within each 2. Material and Methods tidal height. We took adult barnacles into account, The samples were collected in Zhujiajian, an measuring at least 5 mm of opercular rostro-carino island of the Zhoushan archipelago in East China diameter (density-independent parameter) (29°55’12’’ North and 122°25’05’’ East) from corresponding to 22 mm of basal rostro-carinal October 2011 to July 2012 during low tides. The site diameter in size for non-crowded individuals (Chan is a wave exposed rocky shore mainly covered by two & Williams 2004). Nevertheless, only 26 solitary acorn barnacle species, Tetraclita squamosa individuals could be found in LI and broken Bruguière, 1789 and Tetraclita japonica Pilsbry, 1916 individuals were not considered in the analysis. Dry and 3 seaweed species, Ulva lactuca, Grateloupia shell and tissue weights were determined for filicina and Corallina officinalis covering the lowest collected individuals and mean tissue:shell ratio levels. T. squamosa is the dominant barnacle (Cai et estimated for each density level within the two tidal al. 1991) and occupies the space between 0.4 and 2.6 heights. m above mean lower low water where it settles twice a year (Chen et al. 1987). Post-recruitment mortality Statistical analysis of barnacles due to predation and interspecific The self-thinning exponents were estimated competition has been assumed not to be an important by OLS (ordinary least square) of log-transformed factor as compared with intraspecific density- data. Significant differences between exponents were dependent mortality since the densities of the most tested using a permutation test in SMATR version 2.0 conspicuous mobile organisms are low in the (Falster et al. 2006) and the regression lines were community (Sibomana et al. in press). In the lowest plotted using OriginPro 8 SR3 package for LI and MI heights, low-intertidal (LI) (0.4 m-0.7 m) and mid- respectively. Mean individual tissue biomass and intertidal (MI) (0.7 m-1.8 m), the population of T. mean individual shell biomass were used as the squamosa undergoes self-thinning (Sibomana et al. in dependent variable and density as the independent press), thus we considered this tidal heights for our variable. investigation on self-thinning of barnacle individual The means of tissue:shell ratio for different parts. density levels were compared pairwise within and Sampled barnacles were carefully scraped off between tidal levels by one-way ANOVA using SPSS the rock and transferred into airtight plastic bags. statistical package. When the samples could not be taken back to the laboratory the same day, they were chilled in a 3. Results refrigerator. In laboratory, shell and tissue from all the The results showed that self-thinning

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exponent in the studied barnacle individual parts is one for shell in MI (p=0.016) (fig. 1, table 1). In not invariant. In fact, although mass-density scaling addition, all the barnacle components thinning lines exponent did not significantly differ from each other were steeper than the theoretical “-3/2 power law” within tidal levels (p>0.05), tissue thinning exponent (table 1). in LI was significantly higher than the corresponding

Figure 1. Shell (□) and tissue (○) mass-density relationships of Tetraclita squamosa in the low intertidal (a) and mid-intertidal (b).

Figure 2. Comparison of tissue:shell ratio between density levels and tidal heights. Means with dissimilar letters were significantly different (ANOVA 1; p<0.05). The bars represent mean +/-2 SE of 26-40 individuals.

Biomass allocation in T. squamosa differed in solitary and crowded individuals. High density and low density individuals had higher tissue:shell ratio than solitary individuals (fig. 2). In addition, high density individuals in LI had a tissue:shell ratio higher than their counterparts in MI ((F=8.765, p=0.004) whereas there was no significant difference in other density levels between tidal levels (F=0.546, p=0.462 for Sol; F=0.182, p=0.671 for LD) (fig. 2).

Table 1: Slopes and intercepts of linear relationships between mean shell and tissue biomass and density in the two tidal levels. Tidal level Individual Slope (α) (α) 95% CL Intercept (log k) 95% CL R² n part (log k) LI Shell -2.166 -2.563; -1.769 -0.936 -1.120; -0.752 0.810 30 LI Tissue -2.332 -2.816; -1.848 -1.983 -2.397; -1.922 0.759 30 MI Shell -1.776 -1.995; -1.558 -1.035 -1.169; -0.901 0.880 38 MI Tissue -1.811 -2.058; -1.564 -2.093 -2.245; -1.941 0.856 38

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4. Discussion studied site, barnacles are subject to desiccation stress It is well known that crowded barnacles undergo during low tides especially on summer days when self-thinning (Wethey 1983; Hogarth 1985; Hughes & rocky substratum temperatures can reach 45°c Griffiths 1988; Lopez and Gonzalez 2003; Sibomana et (personal obs.). Individuals attached in higher heights al. in press) and barnacle population structure varies spend longer period emerged, thus need a thicker shell with tidal levels (Liu & Morton 1994; Bertness et al. to enable them to lower their water loss rates (Lopez & 1998; Chan et al. 2006; Macpherson & Scrosati 2008). Gonzalez 2003). As a result, the surviving barnacles in Although the studied T. squamosa shell and tissue self- MI should allocate more biomass to shell than their thinning exponents are close to each other within tidal counterparts in LI to minimize the desiccation stress levels, tissue regression line in the LI is significantly effects whereas tissue biomass in LI increase at a faster steeper than the shell line in the MI (fig. 1). All the rate than in MI in response to decreased desiccation exponents are significantly higher than the classical “- stress. Thus, the self-thinning exponent is higher for 3/2 power law”. These results suggested that there is no tissue in LI. common scaling exponent for barnacle individual parts, Most studies on biomass allocation in barnacles thus the data support the hypothesis of variability of the have focused on crowded vs. solitary individuals (Wu self-thinning exponent in barnacles. Different thinning et al. 1977; Wu 1980; Wethey 1984; Lopez & Gonzalez exponents were also found in previous studies on 2003); our results suggested that difference in biomass barnacles (Hogarth 1985; Hughes & Griffiths 1988; allocation in response to variation in environmental Sibomana et al. in press). In plant communities, factors occurs in crowded barnacles. The present study empirical studies reported that self-thinning of below- has shown that self-thinning exponent of shell and ground and above-ground parts varied with drought tissue in Tetraclita squamosa is not invariant, stress (Deng et al. 2006; Bai et al. 2010), nutrient confirming that a general self-thinning law in barnacles availability (Morris 2002) and forest type (Zhang et al. may be unwarranted. Different biomass allocation to 2011). shell and tissue in response to variation in Our results indicated that the tissue biomass- environmental conditions would explain the difference density exponent in LI is significantly higher compared in biomass-density exponents of these individual parts to the corresponding one for shell in MI (fig 1; Table 1), in thinning barnacles between low and higher tidal suggesting that individuals in the LI allocate more heights. biomass to their soft parts than their conspecifics in higher tidal heights during the course of growth. Our Acknowledgments: results on density and tidal height effects on biomass We thank Chao Liu and Zhong-Ji Shen for their help allocation showed that crowded individuals allocate during field investigations and Dr. Benny K.K. Chan more biomass to tissue compared to solitary ones in for his helpful comments on the manuscript. This study each tidal height (fig 2). On the other hand, high was supported by the Natural Science Foundation of density individuals in LI allocate more biomass to China (30730020) and the Hi-Tech Research and tissue than their counterparts in MI, thus the tendency Development (863) Program of China to allocate more biomass to tissue is more pronounced (2011AA100503). in the lower heights in crowded barnacles. Therefore, the studied thinning T. squamosa allocate more Corresponding author: biomass to shell in the higher intertidal heights than the Dr. Gen-Xuan Wang specimens attached in the lower tidal levels. In College of Life Sciences crowded individuals of the acorn barnacle Semibalanus Zhejiang University balanoides, it has been shown that specimens between Hangzhou 310058, China “hummocks” and on “hummock” tops presented Tel/Fax: +86 571 88206590 different biomass allocation to shell and tissue Email: [email protected] (Bertness et al. 1998). Our results are consistent with previous studies which indicated that more resource is References allocated to shell in higher intertidal level species 1. Bai YY, Zhang WP, Jia X, Wang N, Zhou L, Xu SS, (Lopez & Gonzalez 2003) and specimens (Bertness et Wang GX. Variation in root:shoot ratios induced the al. 1998). differences between above and belowground mass- Energy allocation to different barnacle density relationships along an aridity gradient. Acta Oecologica 2010; 36: 393-395. individual parts in response to variation in tidal level 2. Bertness MD, Gaines SD, Bermudez D, Sanford E. may be considered an adaptive trait to maximize Extreme spatial variation in the growth and reproductive survivorship and enable individual growth and output of the acorn barnacle Semibalanus balanoides. reproduction in the harsh intertidal environment Marine Ecology Progress Series 1991; 75: 91-100. (Bertness et al. 1998; Lopez & Gonzalez 2003). In the 3. Cai RX, Zheng F, Wang YH, Ding YP, Yu ZM, Zhou YZ,

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Jiang LH, Shao WH. Studies on ecology of Zhoushan Marine Ecology 2003; 24: 289-302. intertidal zone II．Quantities and distributions. Donghai 19. Lopez DA, Lopez BA, Pham CK, Isidro EJ, Girolamo Marine Science 1991; 9: 58-72 (in Chinese with English MD. Barnacle culture: background, potential and abstract). challenges. Aquaculture Research 2010; 41: 367-375. 4. Chan BBK, Williams GA. Population dynamics of the 20. Macpherson AE, Scrosati R. Population structure of the acorn barnacles, Tetraclita squamosa and Tetraclita barnacle, Semibalanus balanoides (cirripedia, thoracica), japonica (Cirripedia: Balanomorpha), in Hong Kong. across intertidal environmental stress gradients in Marine Biology 2004; 146: 149-160. northern Sova Scotia, Canada. Crustaceana 2008; 81: 5. Chan BKK, Morritt DM, Pirro MD, Leung KMY, 725-736. Williams GA. Summer mortality: effects on the 21. Morris EC. Self-thinning lines differ with fertility level. distribution and abundance of the acorn barnacle Ecological Research 2002; 17: 17–28. Tetraclita japonica on tropical shores. Marine Ecology 22. Morris EC. How does fertility of the substrate affect Progress Series 2006; 328: 195-204. intraspecific competition? Evidence and synthesis from 6. Chen K, Dong L, Cai R. Biology and ecology of self-thinning. Ecological Research 2003; 18: 287-305. Tetraclita squamosa squamosa and Tetraclita japonica 23. Pretzsch H. Species-specific allometric scaling under in Zhoushan waters. Acta Oeceanologica Sinica 1987; 9: self-thinning: evidence from long-term plots in forest 93-103 (In Chinese). stands. Oecologia 2006; 146: 572–583. 7. Dai XF, Jia X, Zhang WP et al.. Plant height–crown 24. Sibomana C, Jia X, Qiu YP, Wang GX. Self-thinning in radius and canopy coverage–density relationships a space-limited rocky intertidal barnacle system. determine above-ground biomass–density relationship in Annales Zoologici Fennici (in press). Preprint available stressful environments. Biology Letters; 2009; 5: 571- from URL: 573. http://www.annzool.net/anzf_forthcoming.htm 8. Deng JM, Wang GX, Morris EC et al.. Plant mass- 25. Weller DE. Self-thinning exponent correlated with density relationship along a moisture gradient in north- allometric measures of plant geometry. Ecology 1987; west China. Journal of Ecology 2006; 94: 953-958. 68: 813-821. 9. Falster DS, Warton DI, Wright IJ (2006). User’s guide to 26. Wethey D. Intrapopulation variation in growth of sessile SMATR: Standardised major axis tests and routines, ver organisms: natural population of the intertidal barnacle 2.0., http://www.bio.mq.edu.au/ecology/SMATR/. Balanus balanoides. Oikos 1983; 40: 14-23. 10. Fréchette M, Lefaivre D. On self-thinning in Animals. 27. Wethey D. Effects of crowding on fecundity in barnacles: Oikos 1995; 73: 425-428. Semibalanus balanoides, Balanus balanoides, Balanus 11. Fréchette M, Lachance-Bernard M, Daigle G. Body size, glandula, and Chthamalus dali. Canadian Journal of population density and factors regulating suspension- Zoology 1984; 62: 1788-1795. cultured blue mussel (Mytilus spp.) populations. Aquatic 28. White J, Harper JL. Correlated changes in plant size and Living Resources 2010; 23: 247-254. number in plant populations. Journal of Ecology 1970; 12. Guiñez R. A review on self-thinning in mussels. Revista 58: 467-485. de Biologia Marina y Oceanografia 2005; 40: 1-6. 29. Wu RSS, Levings CD, Randall DJ (). Differences in 13. Hogarth PJ. Population density, mean weight, and the energy partitioning between crowded and uncrowded nature of the “thinning line” in Semibalanus balanoides. individual barnacles. Canadian Journal of Zoology 1977; Crustaceana 1985; 49: 215-218 55: 643-647. 14. Hughes R N, Griffiths CL. Self-thinning in barnacles 30. Wu RSS. Effects of crowding on the energetics of the and mussels: the geometry of packing. American barnacle Balanus glandula (Darwin). Canadian Journal Naturalist 1988; 132: 484-491. of Zoology 1980; 58: 559-566. 15. Little C, Kitching JA. The biology of rocky shores. 31. Yoda K, Kira T, Ogawa H, Hozumi K. Intraspecific Oxford University Press. Oxford. 1996. competition among higher plants. XI. Self-thinning in 16. Lively CM. Competition, comparative life histories, and overcrowded pure stands under cultivated and natural maintenance of shell dimorphism in a barnacle. Ecology conditions. Journal of Biology Osaka City University 1986; 4: 858-864. 1963; 14: 107-129. 17. Liu JH and Morton B. The temperature tolerances of 32. Zhang WP, Jia X, Bai YY, Wang GX. The difference Tetraclita squamosa (Crustacea: Cirripedia) and Septifer between above- and below-ground self-thinning lines in virgatus (Bivalvia: Mytilidae) on a sub-tropical rocky forest communities. Ecological Research 2011; 26: 819- shore in Hong Kong. Journal of Zoology 1994; 234: 825. 325-339. 33. Zhang WP, Jia X, Morris EC, Bai YY, Wang GX. Stem, 18. Lopez DA, Gonzalez ML. Density-dependent effects in branch and leaf biomass-density relationships in forest Jehlius cirratus (Darwin, 1854) Cirripedia: communities. Ecological Research 2012; 27: 819-825. Chthamalidae) under different growth conditions.

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Assessment of Zinc and Copper Contents in the Hair and Serum and Also Superoxide Dismutase, Glutathion Peroxidase and Malondi Aldehyde in Serum in Androgenetic Alopecia and Alopecia Areata

Mehdi Amirnia1, Sima Sinafar2, Hassan Sinafar3, Mohammad Nuri4, Abdoreza Taban Sadeghi5

1. Associated professor of Dermatology, Department of Dermatology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 2. General physician, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 3. Dermatologist, Department of Dermatology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 4. Associated professor of Biochemistry, Department of Biochemistry, Tabriz University of Medical Sciences, Iran 5- Pathologist, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran [email protected]

Abstract: Alopecia areata (AA) is a recurrent nonscarring type of hair loss that can affect any hair-bearing area. Androgenetic alopecia (AGA) or male pattern hair loss is a very common condition that has a significant psychosocial impact for patients. There are claims that imbalance of trace elements may trigger the onset of alopecia. The aim of this research was to assess the level of zinc, copper contents in the hair and level of lipid peroxidation as super oxide dismutase, Glutathion peroxidase and malon di aldehyde in serum in androgenic and areata alpecia. The prospective study was conducted in department of Dermatology of Sina Hospital and Biochemistry department of Tabriz University of medical science. The study population consists of 27 patients with alopecia areata and 27 patients with androgenetic alopecia. 27 age and sex matched healthy control subjects were studied. The level of zinc, copper contents in the hair and serum and level of lipid peroxidation as super oxide dismutase (SOD), Glutathion peroxidase(GPX-Px) and malon di aldehyde (MDA) in serum in androgenetic, areata alpecia and healthy control subjects were measured in all samples. The collected data were statistically analyzed, using SPSS. The duration of hair loss varied between 1-240 month among patients with alopecia areata and 6-180 month among patients with androgenetic alopecia. The mean of hair zinc level in AA patients, AGA patients and controls were 98.33 µg/dl, 105.35 µg/dl and 129.52 µg/dl. The mean level of hair copper level in study and control groups were, 7.91, 7.25 and 10.34. The mean of serum level of SOD, MDA and GPX-Px in study and control groups were (1945.25, 1861.57 and 2296.77), (3.64, 3.49 and 1.62) and (129.11, 118.84 and 138.74). Zinc and copper contents of hair and serum were significantly lower among patients with alopecia areata and androgenetic alopecia compared to controls (P<0.05). The serum level of SOD, GPX-Px were significantly lower and level of MDA were higher among patients with alopecia areata and androgenetic alopecia compared to controls (P<0.05). These results suggest that low levels of zinc and copper of hair and serum and lipid peroxidation and alterations in the oxidant- antioxidant enzymatic system (SOD, GPX-Px) with high level of MDA at serum may play a role in the pathogenesis of AA and AGA. [Amirnia M, Sinafar S, Sinafar H, Nuri M, Taban Sadeghi A. Assessment of Zinc and Copper Contents in the Hair and Serum and Also Superoxide Dismutase, Glutathion Peroxidase and Malondi Aldehyde in Serum in Androgenetic Alopecia and Alopecia Areata. Life Sci J 2013;10(1):204-209] (ISSN:1097-8135). http://www.lifesciencesite.com. 30

Keywords: Alopecia; Zinc; Copper

1. Introduction pathophysiology of the disease it is said that T-cells Alopecia is one of the most common are responsible for this autoimmune phenomenon and problems amongst the youth and it is responsible for increase the incidence of probability in the people numerous psychosocial problems especially in the who are genetically susceptible. (Ruiz-Doblado et al., youth. The patients in whom the disease emerges in 2003, Garcia-Hernandez et al., 1999, Gulec et al., younger ages may experience more discomfort. 2004, Gulec et al., 2005). Alopecia areata can be devastating in the people who Androgenic alopecia (boldness in men) is care much about their appearance as they try to cover also seen in the men who are genetically susceptible. the hairless areas with long hairs. These patients Regarding the possible causes for areata and gradually become reserved and experience androgenic alopecia, diverse hypotheses have been psychological disorders (Ruiz-Doblado et al., 2003). introduced however major causes of these two Etiology of this disease is not yet known but diseases have not been discussed in any of these autoimmune events are to be blamed. Regarding the hypotheses. Finding the probable causes of this

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disease could lead to making preventive and distance between the clinic and the laboratory. therapeutic decisions. Different hypotheses such as Therefore only patients with complete information lack of some elements in the blood and hair have are brought in the findings section. been discussed. On the other hand, some researchers As above mentioned, the groups of the have blamed the increase in antioxidant enzymes in patients with areata and androgenic alopecia the hair as an initiative factor of the disease. Our consisted of 27 people each and to be compared with hypothesis is that the decrease in copper and zinc normal population, 27 normal people were also serum levels causes hair loss in these patients. The entered that study. It should be mentioned that these concentration of these elements in the hair is also is two groups and the control group were matched of importance. Prior to our study, no similar study regarding age and gender. The researcher was present was previously done in Iran regarding measuring in the above mentioned clinics and offices and copper and zinc content in the hair. On the other hand completed the provided questionnaires and then regarding the existing articles, the hypothesis that the guided the patients for having the necessary tests deficiency of these elements in the hair could cause done after offering them the necessary explanations. and develop the disease was considered by the After obtaining written consent from the patients, researcher and defined as one of the primary goals of almost 1 g (100 to 200 hairs) fallen hairs were the current study. The role of serum and hair fat collected and the patients were introduced to the peroxidation in the patients with alopecia has been laboratory. discussed in previous studies. The serum levels of The hair sample consists of the fallen hairs super oxide dismutase, glutathione peroxidase, which have been collected by the patient and sent to malondialdehyde were compared in the patients and the laboratory. Blood samples were also taken in the regular people. We hope that the present research laboratory. The primary questionnaire consisted of could help us discover alopecia etiology so that we questions regarding demographic information, can provide preventive and therapeutic measures. marital status, pregnancy and delivery items, history 2. Material and Methods of recent surgery or disease, physical or mental The present study is a descriptive-analytic stresses and hair fall duration and severity. study in which 27 patients with areata alopecia and Laboratory findings were also included in 27 patients with androgenic alopecia referred to this questionnaire including serum levels of copper, dermatology clinic of Tabriz Sina Hospital were zinc, SOD, malon di aldehyde (MDA) and GPX and randomly chosen and their findings were compared also copper and zinc levels in the hair. with 27 normal people. The patients having symptoms for areata and The referred patients with areata and androgenic alopecia were interred the study. androgenic alopecia were examined by physicians in Areata alopecia is defined as completely hair their offices and selected using Hamilton’s fall in a particular part of the scalp skin with a clear classification. Patients with areata alopecia were margin which is usually round. Androgenic alopecia chosen considering their clinical signs including clear is defined as unusual thinning of the hair in the patches without single or multiple scales in head and frontotemporal region followed by circular hair fall in face in different age ranges. one part of the vertex. Regarding the role of antioxidant enzymes, Exclusion criteria from this study were as it could be said that in normal conditions there is following: any factor interfering deteriorating or equilibrium between oxidative destruction and anti- amending patient status such as extrinsic factors like oxidative protective reactions in cells. Super oxide the history of consuming zinc, multivitamin, dismutase (SOD) and Glutathion peroxidase(GPX) medications (allopurinol, cytotoxic medications), have been proved to play roles in stress oxidative history of radiotherapy, thyroid dysfunction, reactions and skin diseases. psychological stress, local trauma, severe weakening Based on the results obtained from the diseases such as tuberculosis, any accompanying studies carried out on total antioxidant SOD and GPX disease affecting hair fall like hirsutism, diabetes and levels in patient and control groups, using the age limitations. On the other hand, other interfering formula for defining sample volume, the sample factors such as gastrointestinal disorders, stress, number were calculated as 40 which was considered diet… that can impair copper and zinc absorption 50 after a 20% increase for each of the groups. were borne in mind and patients with above As previously mentioned, one of the mentioned diseases were excluded from the study. objectives of this study was to measure copper and After collecting the samples, copper and zinc levels of the hair. However in spite of the zinc levels were measured using the following explanations of the researcher not all of the selected technique: patients were eager to go to the laboratory due to the

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Hair samples should be collected from occipital normal group was 27.11 ± 5.55 with a median of 47, region and 1 cm above the scalp skin. Each sample they youngest patient was 15 and the oldest was 38 should be placed in separate plastic bags and stored years old. in a desiccator in the dark. The amount of the metals Independent-test revealed no meaningful in the hair is significantly influenced by external difference between the mean age of the patients with pollutants such as: dust, sweat, epidermal scaling, alopecia areata or androgenic alopecia with normal and especially detergents and cosmetics. Therefore it people (P=.435). is essential for the samples to be washed and cleaned In the group of patients with alopecia areata, from any extrinsic pollutants so that we can have there were 24 females(88.9%) and three appropriate samples. Washing procedure is carried males(11.1%) and in the group of patients with out in three steps: androgenic alopecia there were 21 females(77.8%) 1. Washing and shaking for 3consequent times and six males(18.5%)(P=0.546). for 10 minutes in a shaker with a 3 to 1 ethyl 13 Females with alopecia areata and 13 ether-acetone solution to eliminate the fat females with androgenic alopecia were housewives. layer around the hair. 7 people with alopecia areata and 5 people 2. Soaking and immersing along with one-hour with androgenic alopecia were students. There were shaking in an EDTA 5% solution to 13 single (48.14%) and 14 married (51.85%) people neutralize chemical elements on the surface in the group of patients with androgenic alopecia of the hair. .There were also 12 single (4 4.44%) and 15 married 3. Frequent washing with the deionized water (55.55%) in the group of patients with androgenic with high purity degree alopecia. After drying the hair samples, almost 0.25 16 females from the group of the patients grams of the washed hair is transferred to a tube with alopecia areata (59.3%) and nine people from filled with tetrafluoroethylene. Later we add HNO3 patients with androgenic alopecia (39.1%) had no solution and oxygenated water with a 3 to 1 ratio. history of delivery. None of the patients with alopecia After 24 hours a mega-microwave mortar MLS1200 areata had an underlying disease. Only one patient is used to dissolve the minerals in the hair samples as with androgenic alopecia had a history of diabetes, following: hypertension and cardiac disease. Three minutes in 250 W. A background of emotional stress was found Six minutes in 0 W in 2 patients with alopecia areata that and 2 patients Five minutes in 250 W. with androgenic alopecia. There was one case of Six minutes in 0W family member death and divorce in the patients with Two minute in 450 W. alopecia areata. There was also a case of family death Two minute and 500 W. and examination stress in the patients with After cooling the sample, we increase the volume of androgenic alopecia. the samples to 10 ml using DW. All of the above Nine people from the patients with alopecia mentioned steps in the procedures should be done in areata (33.3%) had not been examined by a physician a clean room. (Mustafa and Ibrahim 1999) regarding their hair fall and not given any After data being collected by the researcher, all data medication.5 people (18.51%) of the patients with were interred SPSS software and analyzed. In this androgenic alopecia had not received any medication. research, statistical and descriptive analyses, and There was no family history of hair fall in the statistical indices like mean, median and… and K2 patients with alopecia areata. and independent t-test were used to compare the Family history was positive for six patients means. with androgenic alopecia. The result of K2 test It should be mentioned that P less than 0.05 is revealed a meaningful relation between a positive considered to be meaningful. family history and the incidence of androgenic 3. Results alopecia in our study (P=0.011). In our study 27 patients with alopecia areata The mean duration of hair fall in the patients and 27 patients with androgenic alopecia were with alopecia areata was 34.11 ± 58.33 months with a compared with 27 healthy people. The mean age for minimum of one month and a maximum of 240 the patients with alopecia areata was 66.27 ± 9.90, months (20 years).The mean duration of hair fall was with a median of 25; the youngest patient was 2 and also 35.80 ± 51.48 months for the patients with at the oldest was three years old. The mean age of the androgenic alopecia with a minimum of six months patients with androgenic alopecia was 88.28 and a maximum of 180 months (15 years). ± 9.85 with a median of 28, they youngest was 17 The results of the independent t-test revealed and the oldest 62 years old. The mean age for the that there was no meaningful difference between the

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duration of hair fall before referring to the center serum levels can be observed in these between the two groups of the patients with alopecia patients(P<0.005). areata and androgenic alopecia(P=0.914). The mean GPX-Px serum level in the In our study the hair fall of almost 50 to 100 patients with alopecia areata was 129.11 ± 12.77, in hairs in a day was considered as mild hair fall and the patients with androgenic alopecia 118.84 ± 8.09 more than 100 hairs a day as severe hair fall. µg/dl, and in normal people 138.74 ± 7.18 µg/dl. Based on the above mentioned definition, in Independent t-test revealed that there was a the group of the patients with alopecia areata 11 meaningful difference between the mean GPX-PX people (40.74 %) had mild hair fall and 16 people serum level in the patients with alopecia areata (59.25 %) severe hair fall. In the group of the patients (P=0.001), androgenic alopecia (P<0.005) and with androgenic alopecia 10 people had mild hair fall normal people and lower serum levels can be (37.03%) and 17 people (62.96%) severe hair fall observed in these patients. (P=0.50).

Table 1.A comparison of lab tests in patients with alopecia areata, androgenic alopecia and normal people

Serum level Hair content Mean Hair content Serum level Zinc Copper SOD MDA GPX Zinc Copper alopecia areata 98.33 ± 24.25 7.91 ± 72 1945.25 ± 105.41 3.64 ± 0.53 129.11 ± 12.77 64.25 ± 19.40 79.03 ± 28.22 60.08 ± 28.16 65.23 ± 29.65 118.84 ± 8.09 3.49 ± 0.45 1861.57 ± 98.79 7.25 ± 2.5 105.35 ± 32.43 androgenic alopecia 105.35 ± 32.43 7.25 ± 2.5 1861.57 ± 98.79 3.49 ± 0.45 118.84 ± 8.09 65.23 ± 29.65 60.08 ± 28.16 Normal people 129.51 ± 29.61 10.34 ± 2.3 2296.77 ± 286.89 1.62 ± 0.49 138.74 ± 7.18 82.77 ± 5.77 96.77 ± 6.48 P_ Value 1 <0.005 0.001 <0.005 <0.005 0.001 <0.005 0.002 P_ Value 2 0.007 <0.005 <0.005 <0.005 <0.005 0.004 <0.005

The mean zinc level in the hair of the 4. Discussions patients with alopecia areata was 98.33 ± 24.25 In different studies, the prevalence of µg/dl, 105.35 ± 32.43 µg/dl in the patients with alopecia areata has been reported to be 1 to 2%. The androgenic alopecia, and 129.51 ± 29.61 µg/d in onset age is also varied in different studies ranging normal people from 0 to 80 years. In a study Yasmeen et al reported Independent t-test revealed that there was a that the prevalence of alopecia areata is more in meaningful difference between the zinc content of the males compared to females with a ratio of 36 to 16. hair in the patients with alopecia areata (P<0.005), The mean age of the patients in their study was 27.3 androgenic alopecia (P=0.007) and normal people. years and most patients were under 40 (Yasmeen et The mean copper content of the hair in the patients al., 2009). with alopecia areata was 7.91 ± 2.72 µg/dl, in the This research was similar to the results of patients with androgenic alopecia 7.25 ± 2.5 µg/dl, the study of Sharma et al. The age of the patients and in normal people 10.34 ± 2.3 µg/dl. with alopecia has been reported to be 15 to 29 years Independent t-test revealed that there was a in another study (Sharma et al., 1996). meaningful difference between the copper content of In our study also the mean age of the the hair in the patients with alopecia areata patients with alopecia areata was 27.6 69.90 (P=0.001), androgenic alopecia (P=0.005) and (between two and 53 years) and the mean age for the normal people. patients with alopecia androgenic was The mean SOD serum level in the patients 28.889.85(between 17 to 62 year) which is in with alopecia areata was 1945.25 ± 105.41 µg/dl, in accordance with their results of other studies. In a the patients with androgenic alopecia 1861.57 ± study carried out on 736 people with alopecia areata, 98.79 µg/dl, and in normal people 2296.77 ± 286.89 the male to female ratio was reported to be 1 to 1. In µg/dl. a similar study their ratio of females was a little Independent t-test revealed that there was a higher. (Tobin 2006). in our study both in the group meaningful difference between the mean SOD serum of the patients with androgenic alopecia and alopecia level in the patients with alopecia areata (P<0.005), areata, considering the randomized sampling, the androgenic alopecia (P<0.005) and normal people. ratio of females was higher than males. Although this The mean MDA serum level in the patients result is in contrary to the results obtained from the with alopecia areata was 3.64 ± 0.53 µg/dl, in the study of Yasmeen et al (2009)and other studies, patients with androgenic alopecia 3.49 ± 0.45 µg/dl, cultural characteristics and authors insight should and in normal people 1.64 ± 0.49 µg/dl. also be borne in mind. (Yasmeen et al., 2009).It is Independent t-test revealed that there was a obvious that the ratio of the female to male in our meaningful difference between the mean MDA study is not the prevalence ratio of the disease in both serum level in the patients with alopecia areata, genders whereas it shows the referring ratio of androgenic alopecia and normal people and higher females to males. Therefore it seems that men pay

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less attention to their hair fall than females do and in both groups (P<0.005) whereas the difference they consider it as the natural cycle of their lives or between serum copper levels was not meaningful an incurable disease and avoid referring to (Yasmeen et al., 2009). physicians. This issue should also be studied more In this study like the similar studies, comprehensively. meaningful results were obtained comparing zinc Regarding the relation between serum levels in patients with alopecia areata and psychological or physical stress and chronic androgenic alopecia and normal people. Serum levels psychological disease with alopecia incidence, of this element was meaningfully lower than normal expanded studies have been carried out. The studies people in both groups (P= 0.004 and P<0.005). revealed a high incidence of psychological disorders Regarding the copper serum level, a in these patients including anxiety, depression, meaningful difference also was observed between the phobia, paranoia and mood disorders which are more patients with areata and androgenic alopecia. The frequent in patients with alopecia areata compared to serum levels of this element also were meaningfully the normal people. These studies emphasize on recent lower than normal people. Inspite of not being in stress such as the death of a family member, divorce, accordance with the findings of Yasmeen et al., 2009, difficult exams, or severe physical stresses in the these findings are in accordance with most studies patients with alopecia (Garcia-Hernandez et al., 1999, (Yasmeen et al., 2009). Koo et al., 1994, Kalafi et al., 1993). For the first time in Iran, the copper and zinc In the study of Van der Steen P, the contents of the hair of the patients with alopecia were possibility of having a positive family background in compared to normal people. The results obtained the patients with alopecia areata is mentioned to be from this study are in accordance with the similar 10 to 20% which is considered to be 1 to 7% in studies and show meaningful lowers zinc contents in normal people. (Chantal and Harvey, 2009) A the hair of the patients with areata and androgenic positive family history has been reported to be as a alopecia compared to the normal people risk factor in the incidence of androgenic alopecia. In (P= 0.007 and P<0.005). In a series of experiments, this study none of the patients with alopecia areata the copper contents of the hair of the patients with had a positive family history whereas 22.22% of the areata and androgenic alopecia were also patients with androgenic alopecia had a positive meaningfully lower compared to normal people (P= family history. As it was mentioned, the relation 0.001 and P<0.005). between the positive family history and the type of Numerous studies have also been carried out alopecia was meaningful (P= 0.011) regarding the relation between serum level of In different studies carried out on the antioxidants and alopecia. In the research carried out relation between the etiologies of different types of in 2002 by Ecar et al, it was said that the level of alopecia so far, diverse results have been obtained super oxidase dismutase, glutathione peroxidase and regarding the relation between serum levels of copper malondialdehyde are higher in the scalp of the and zinc and also copper and zinc contents of hair patients with alopecia areata compared to normal with alopecia. people. The number of the patients and normal In his research, AF Alexis mentions that people was 10 in this research. Experiments revealed zinc and iron are two major elements in the growth of significant differences in the enzyme levels of hair. (Alexis 2004) antioxidants (Akar et al., 2002). On the other hand Musaaloa Rauhama, unlike Alexis, found no other studies have shown higher levels of lipid meaningful difference in the serum levels of zinc, peroxidase and antioxidant enzymes in the scalp of iron and copper in the patients with alopecia the patients with alopecia. In Mustafa Naziroglu compared to the normal people. study, blood levels of vitamin E., beta carotenes and He reported that only in the universal the activity level of glutathione peroxidase in 37 alopecia the urine levels of copper was meaningfully patients with alopecia were compared with plasma lower (Mussalo-Rauhamaa et al., 1986) and RBC levels in 34 normal people. In this study the In a study titled as “serum levels of elements activity level for glutathione peroxidase was in alopecia areata” Jasmeen J Bhat et al compared meaningfully lower than normal people (Mustafa and serum levels of copper and zinc in the patients with Ibrahim 1999). alopecia areata and normal people. In the study the In Koca R study on the comparison of serum mean serum copper level was reported as 787.45 in levels of antioxidants in the patients with alopecia the patients and 8878.8 in normal people. The mean and normal people following results were obtained: serum copper levels also were reported as 11417.5 serum level of malondialdehyde was meaningfully in the patients and 11717.02 in the normal people. higher in the patients compared to the normal people. The difference in copper serum level was meaningful The activity level of super oxidase dismutase enzyme

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was also meaningfully lower compared to the normal psychiatric disorders. J. Dermatol 1999; 26(10): people. This study suggests that the increase in 625-632. peroxidation of the fats in the patients with areata 3-Gulec AT, Tannverdi N, Cagay D, Yasemin S, alopecia is in relation with degrees in the activity of Cenk A. The role of psychological factors in super oxidase enzyme and therefore fat peroxidation alopecia areata and the impact of the disease on is a key factor in areata alopecia pathogenesis (Koca the quality of life. Int. J. Dermatol 2004; 43(5): et al., 2005). In this study the serum levels of the 352-356. enzymes (SOD, MDA, and GPX) were evaluated. As 4-Gulec, A.T., N. Tanriverd, C. Duru, Y. Caray and was mentioned in the results section, the serum levels C. Akcali. The role of psychological factors in of all three enzymes were meaningfully different alopecia areata and the impact of the disease on from normal people. Serum level of SOD enzyme the quality of life. Int Dermatol 2005; 46(7): 332- was meaningfully lower in the patients with areata 336. and androgenic alopecia compared to normal people 5-Yasmeen, J.B., M. Sheikh, A.R. Khan and Q. (P<0.005 and P<0.005). These findings are similar to Seema. Trace element levels in alopecia areata. the results obtained from the study of Koca et al., Indian Journal of dermatology 2009; 75(1): 29- 2005 whereas Kara reported opposite results in his 31. studies. Comparing serum levels of this enzyme in 6-Sharma, V.K., G. Dawna nd B. Kumar. Profile of two groups with areata and androgenic alopecia also alopecia areata in Northern India. Int J Dermatol revealed meaningful differences. The serum levels of 1996; 35(1): 22-7. this enzyme were meaningfully lower in patients with 7-Tobin, DJ. The genetically programmed hair androgenic alopecia (P= 0.004). growth cycle and alopecia: What is there to Like the results obtained from Koca et al., know?. Expert Rev Dermatol 2006; 1(3): 413- 2005 study, our study also revealed a meaningful 428. difference in the serum levels of MDA in patients 8-Koo, J.Y., W.V. Shellow, C.P. Hallman and J.E. with alopecia and normal people. The serum levels of Edward. Alopecia areata and increased prevalence this enzyme was meaningfully more in the patients of psychiatric disorders. Int. J. Dermatol 1994; with areata and androgenic alopecia compared to 32(12):849-850. normal people (P<0.005 and P<0.005). 9-Kalafi, Y., M. Mousavi Nassab, S. Tobaei and G. No meaningful difference was found Nateghi. The role of psychiatric disorders in comparing the serum levels of this enzyme in two patients with Alopecia Areata. Iranian J. Med. Sci groups of areata and androgenic alopecia. 1993; 3-4(18): 138-142. In our study, like the study of Mustafa 10-Chantal, B. and L. Harvey. Alopecia areata. Naziroglu, serum levels of glutathione peroxidase Dermatology J 2009; 41(7): 36-42. was lower in the patients with areata and androgenic 11-Alexis, AF. Alopecia areata: Autoimmune basis alopecia compared to normal people (P=0.001 and of hair loss. Eur J Dermatol 2004; 14(6): 364-70. P<0.005). 12-Mussalo-Rauhamaa, H., E.L. Lakomaa, U. Kianto Conclusion: and J. Lehto. Element concentrations in serum, The results of this study confirm the erythrocytes, hair and urine of alopecia patients. hypothesis that some elements in the blood and hair Acta Derm Venereal 1986; 66(2): 103-9. could affect hair loss and on the other hand the 13-Akar, A., E. Arca, H. Erbil, C. Akay, A. Sayal and increase in the level of antioxidant enzymes in hair A.R. Gur. Antioxidant enzymes and lipid has been blamed as an initiating factor in these peroxidation in the scalp of patients with alopecia patients which our study also confirms this areata. J. Dermatol. Sci 2002, 29(2): 85-90. hypothesis. 14-Mustafa, N. and K. Ibrahim. Antioxidants and Corresponding Author: lipid peroxidation status in the blood of patients Dr. Mehdi Amirnia with alopecia. Cell Biochem Function 2002, Department of Dermatology, Faculty of Medicine, 18(3): 169-73. Tabriz University of Medical Sciences, Tabriz, Iran 15-Koca, R., F. Armutcu, C. Altinyazar and A. Gürel. E-mail: [email protected] Evaluation of lipid peroxidation, References oxidant/antioxidant status, and serum nitric oxide 1-Ruiz-Doblado S, Carrizosa A, Garcia-Hernandez levels in alopecia areata. Med Sci Monit 2005; MJ. Alopecia areata: Psychiatric co morbidity and 11(6): 296-299. adjustment to illness. Int. J. Dermato 2003; 42(6): 434- 437. 9/26/2012 2-Garcia-Hernandez M, Ruiz-Doblado S, Rodriguez- Pichardo A, Alopecia-Areata CF. Stress and

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Correlation between body mass index (BMI) and immunologic Response to Hepatitis B Vaccine

Mojtaba Varshochi and Sepehr Taghizadeh

Infectious Diseases and Tropical Medicine Research Center, Faculty of medicine, Tabriz University of Medical Sciences, Tabriz, Iran. [email protected]

Abstract: Problem statement: To evaluation the Correlation between body mass index (BMI) and immunologic Response to Hepatitis B Vaccine. Methods: In a cross sectional descriptive analytic study carried out on the health care Workers (HCWs) of Tabriz Shahid Madani Hospital in 20010-2011, we evaluated the immunologic response of the staff to vaccination against Hepatitis B and correlation by body mass index (BMI). Results and Conclusion: Results: 122 people (36.8%) from the studied HCWs were male and 209 people (63.2%) female. The mean Primary and secondary antibody titer in the studied HCWs were 304.07±199.98 and 369.13 ± 169.78 IU/L. From 331 studied people, 31 (9.4%) showed no response to vaccination, 52 (15.7%) low response and 248 (74.9%) good response and after one booster dose, 7 (2.1%) showed no response to vaccination, 5 (1.5%) low response and 319 (96.4%) good response. In our study, the response rate to the vaccine has been reduced by increased weight and BMI, although this reduction was not statistically significant. Mean of Primary response antibody titer in male was significantly higher than female HCWs(P=0.011).Significant liner correlation was not found between HBsAb level and HCWs weight and BMI. [Varshochi M, Taghizadeh S. Correlation between body mass index (BMI) and immunologic Response to Hepatitis B Vaccine. Life Sci J 2013;10(1):210-213] (ISSN:1097-8135). http://www.lifesciencesite.com. 31

Keywords: Hepatitis B Vaccines; Occupational Diseases; BMI; Immunologic Response

1. Introduction Zhuang et al showed that three-dose In recent decades, hepatitis is among the intramuscular revaccination did play an important diseases considered as serious threats to healthcare immune protection for non-responder children to workers and patients (Mast, 1998). primary HB vaccination, but its efficacy could not Infection with hepatitis B virus, as one of the most reach the level of primary vaccination in common infections in the world, is one of the major responders(Zhuang, 2006). health problems in our country as well. Over 2 billion Testing of blood for anti-HBs one month people worldwide have been infected with this virus, after vaccination is recommended to recognize non- of which 400 million are chronic carriers of the virus responders as a booster dose will be beneficial in the and approximately 53,000 new cases of infection are majority of them (Perera, 2002). reported every year, 8,000 of which are job-related, Hofmann et al demonstrated that and recommendations have been made for Estimations of post-vaccination-anti Hbs showed that vaccination of at-risk groups, including healthcare women, younger persons, persons with a body mass providers working in hospitals and health centers index < 25 and non-smokers had higher anti-HBs against hepatitis B virus (Lemon, 1997; Lavanchy, concentrations than men, elderly person's overweight 2008). persons and smokers (Hofmann, 1997). Vaccine-preventable viral hepatitis Chlíbek et al show that body weight was continues to be a cause of considerable morbidity and also observed, with the obese subjects showing the mortality: on worldwide basis, approximately 1.4 lowest immune response (Chlíbek, 2007). million cases of hepatitis A are reported every year. In this study, we were to review the results The true incidence, however, has been estimated to of vaccination of healthcare workers and evaluate the be 3-10 times higher. Regarding hepatitis B, more effect of BMI on its response rate. than a third of the world's population has been infected (Van Damme and Van Herck, 2006). 2. Material and Methods Hepatitis B vaccination is recommended for In a cross sectional descriptive analytic study all healthcare workers (HCW) at risk of exposure to carried out on the HCWs of Tabriz Shahid Madani infectious body fluids. However, the absolute Hospital in 2010-2011, we evaluated the duration of protection from immunization is immunologic response of the HCWs to vaccination unknown. The purpose of this randomized against Hepatitis B and correlation by body mass comparison trial was to determine how previously index (BMI). immunized HCW respond to different booster doses of hepatitis B vaccine(Williams, 2001).

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HCWs are routinely vaccinated against hepatitis next step, antigen S conjugated with peroxidase B and therefore antibody titration is essential to enzyme is added which attaches to the part of the insure appropriate immunity. antibodies not bound to antigens. Later, adding The studied HCWs are vaccinated by hepatitis B chromogene and substrate dies the solution whose vaccine with fallowing properties (entitled EUVAX color can be read by ELISA reader. B, met the WHO requirements, made in Korea, LG After performing tests and life sciences company) for three doses (0, 1 and 6 reading the plates by ELISA reader, standard curves months) and antibody was titrated three months after were provided using standard samples. Later using vaccination. All utilized vaccines were from one these curves, the concentration of antibodies in the brand and all tests were performed in one laboratory. tested sample were calculated. Based on the ELISA test was used to evaluate the samples used kit instructions of the kit manufacturer, antibody level less in this study was the Anti-HBs kit, made by ROCHE than 10 units per mL was considered negative and Company entitled “COBAS”. Methods of this kit was amounts higher as positive. ELISA, in which ELISA micro plates in this method In this study, HCWs vaccinated on schedule, ELISA micro plates are covered by antigen S so that routinely 3 times during the past 5 years (6-1-0), and after adding serum containing antibodies against had a negative HBcAb and HBsAg, were examined antigen S, antigens would bind with antibodies. In the in terms of HBsAb titer, height, weight, and BMI.

Table 1. Evaluation of Age, Height, Weight and BMI based on Primary and Secondary response. Primary Response Secondary response (after one booster dose) Good Non Good Low Non P P Responders Low Responders Responders Responders Responders Responders Age 31.32 ± 7.02 32.65 ± 7.96 33.71 ± 8.51 0.146 31.40 ± 7.08 37.20 ± 7.46 43.86 ± 8.30 <0.001 Height 1.64 ± .08 1.64 ± .07 1.65 ± .08 0.908 1.64 ± .08 1.71 ± .09 1.65 ± .08 0.129 weight 66.28 ± 12.79 67.75 ± 14.02 69.48 ± 11.96 0.365 66.41 ± 12.71 82.40 ± 12.05 74.00 ± 15.73 0.007 BMI 24.56 ± 4.02 24.94 ±4.10 25.52 ± 3.33 0.408 24.61 ± 3.95 28.08 ± 3.71 26.87 ± 4.16 0.053

Table 2. Evaluation of primary and secondary HBsAb titers of HCWs based on BMI groups. BMI Groups <20 20-25 25-30 30-35 > 35 P Primary Response 272.48 ± 177.27 313.86 ± 197.01 314.59 ± 212.81 225.11 ± 174.85 403.00 ± 125.37 0.173 Secondary response 336.10 ± 149.55 377.71 ± 165.40 377.86 ± 178.08 313.70 ± 165.11 403.00 ± 125.37 0.114

331 HCWs were studied and those without response” group and 248 people (74.9%) in “Good ideal HBsAb titer (below 100) were selected. These response” group and Secondary Response: 7 people employees received a booster dose of hepatitis B (2.1%) were in “No response” group, 5 people vaccine, and were evaluated again in terms of HBsAb (1.5%) in “Low response” group and 319 people titer 3 to 6 months later in the same laboratory. (96.4%) in “Good response” group(Chart I). Finally, the response to vaccination was Mean age of male HCWs was 34.05 ± 8.58 evaluated in three groups of proper response and in female HCWs was 30.43 ± 6.15 year (HBsAb>100), acceptable response (P<0.001).Mean of Primary response antibody titer in (100>HBsAb>10) and inadequate response male HCWs was 340.59 ± 205.15 IU/L and in female (HBsAb<10). HCWs were 282.75 ± 194.22 IU/L. Mean of Primary response antibody titer in 3. Results male was significantly higher than female HCWs This study was carried out on 331 HCWs. (P=0.011).Significant liner correlation was not found The mean Primary and secondary antibody titer in the between HBsAb level and HCWs weight and BMI. studied HCWs were 304.07±199.98 IU/L and 369.13 Mean and STD deviation of age, height, weight and ± 169.78 IU/L. 122 people (36.8%) from the studied BMI of HCWs based on Primary and secondary HCWs were male and 209 people (63.2%) female. response to vaccination was shown in table 1. Dividing the antibody titer into three groups of “no Evaluation of primary and secondary response” (Titer < 10 IU/L), “Low response” HBsAb titers of HCWs based on BMI groups were (Titer10-100 IU/L) and “Good response” (Titer shown in table 2. Frequency of Primary and >100 IU/L) revealed that from 331 studied HCWs, Secondary Responders based on BMI Groups were at Primary Response: 31 people (9.4%) were in shown in table 3. “No response” group, 52 people (15.7%) in “Low

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Primary Response were associated with lacking anti-HBs only for 100 Secondary Response Recombivax HB recipients; and gender (P = .03) was associated with lacking anti-HBs only for Engerix-B 80 recipients (Wood, 1993). Roome et al demonstrated that the frequency of 60 inadequate level of antibody increased significantly relative to age, from 2.8% among those younger than 40 30 years to 42.1% among those older than 60 years (P < .0001). Smoking (odds ratio [OR], 3.6; 95% 20 confidence interval [CI], 2.0 to 6.4), extreme obesity (OR, 13.3; 95% CI, 3.8 to 49.1) (Roome, 1993). 0 Good response Low response No response In the study of Cockcroft et al, the responders were Char 1. Primary and Secondary response to Vaccinations in significantly younger than the non-responders and HCWs had significantly lower values of body mass index (wt/ht2) (Cockcroft, 1990). 4. Discussions Chlíbek et al demonstrated that body weight was also Several factors affect the rate of response to observed, with the obese subjects showing the lowest hepatitis B vaccination of healthcare workers immune response (Chlíbek, 2007). including obesity and high BMI, the effect of which Although body mass index affected the response to is evaluated on the response to vaccination of health the first hepatitis B booster, when full compliance to care workers that there was no significant linear regular revaccination was ensured, all non- and low- relationship between the HBsAb level, weight and responders eventually reached sufficient anti-HBs BMI of health care workers under study; however, levels (Clemens, 1997). the response rate to the vaccine has been reduced by Horowitz and et al show that, low levels HBsAB increased weight and BMI, although this reduction were associated with smoking, older age, and higher was not statistically significant. body-mass index (Horowitz, 1998). Martínez Pérez et al showed that Obesity is a factor As well, in two other studies by Averhoff et al in providing a prior indication of a minor response to 1998 and Rendi-Wagner et al in 2000 there was no the same (Martínez Pérez, 1998). negative relationship between high weight and Anti- Wood et al demonstrated that age (P = .01), body HBs titer (Averhoff, 1998; Rendi-Wagner, 2000). mass index (P < .01), and smoking status (P < .01)

Table 3. Frequency of Primary and Secondary Responders based on BMI Groups BMI Group <20 20-25 25-30 30-35 > 35 P Non Responders 2 13 13 3 0 Primary Response Low Responders 6 23 16 7 0 <0.001 Good Responders 21 118 89 17 3 Non Responders 1 1 3 2 0 Secondary response Low Responders 0 1 3 1 0 <0.001 Good Responders 28 152 112 24 3

In the study of Averhoff et al conducted significantly associated with anti-HBs levels (Perera, double blinded and prospective, obesity, being male, 2002). chronic disease, and smoking were the factors Cardell K et al showed that Sex or body effective on reduced Anti-HBs titer (Averhoff, 1998). mass index had no influence on response rate of In a similar research by Sezer et al on 50 HCWs to Hepatitis B vaccination (Cardell, 1999). hemodialysis patients, being male was one of the Like the results of the above mentioned factors effective on reduction of Anti-HBs titer studies, in our study as well there was no significant (Sezer, 2000). relationship between high weight and BMI and The above mentioned studies suggest that reduced response to vaccination. high weight and BMI are among the factors effective on reduction of the response to Hepatitis B Conclusions vaccination. From 331 studied people, 31 (9.4%) showed Perera J et al demonstrated that duration of no response to vaccination, 52 (15.7%) low response vaccination, sex and body mass index were not and 248 (74.9%) good response and after one booster

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dose, 7 (2.1%) showed no response to vaccination, 5 C, García Pasan MJ. Coverage and immune (1.5%) low response and 319 (96.4%) good response. response to hepatitis B vaccine in adolescents of In our study, the response rate to the vaccine has been the Guadalajara province. Rev Esp Salud Publica reduced by increased weight and BMI, although this 1998;72(2):119-26. reduction was not statistically significant. 11-Wood RC, MacDonald KL, White KE, Hedberg CW, Hanson M, Osterholm MT. Risk factors for Corresponding Author: lack of detectable antibody following hepatitis B Dr. Mojtaba Varshochi, vaccination of Minnesota health care workers. Infectious Diseases and Tropical medicine research JAMA 1993;270(24):2935-9. center, Tabriz University of Medical Sciences, 12-Roome AJ, Walsh SJ, Cartter ML, Hadler JL. Tabriz, Iran. Hepatitis B vaccine responsiveness in E-mail: [email protected] Connecticut public safety personnel. JAMA 1993; 270(24):2931-4. References: 13-Cockcroft A, Soper P, Insall C, Kennard Y, 1-Mast EE, Williams JT, Alter MJ, Margolis HS. Chapman S, Gooch C, Griffiths P. Antibody Hepatitis B vaccination of adolescent and adult response after hepatitis B immunisation in a highrisk groups in the United States. Vaccine group of health care workers. Br J Ind Med 1998; 16: 27-9. 1990;47(3):199-202. 2-Lemon SM, Thomas DL .Vaccines to prevent viral 14-Clemens R, Sänger R, Kruppenbacher J, Höbel W, hepatitis .N Engl J Med 1997;336:196-204. Stanbury W, Bock HL, Jilg W. Booster 3- Lavanchy D. Chronic viral hepatitis as a public immunization of low- and non-responders after a health issue in the world. Best Pract Res Clin standard three dose hepatitis B vaccine schedule-- Gastroenterol 2008;22:991-1008. results of a post-marketing surveillance. Vaccine 4- Van Damme P, Van Herck K. A review of the 1997;15(4):349-52. long-term protection after hepatitis A and B 15-Horowitz MM, Ershler WB, McKinney WP, Battiola vaccination. Travel Med Infect Dis 2007; 5(2):79- RJ. Duration of immunity after hepatitis B 84. Epub 2006 Jun 19. vaccination: efficacy of low-dose booster vaccine. 5-Williams JL, Christensen CJ, McMahon BJ, Bulkow LR, Ann Intern Med 1988;108(2):185-9. Cagle HH, Mayers JS, Zanis CL, Parkinson 16- Averhoff F, Mahoney F, Coleman P, Schatz G, AJ, Margolis HS. Evaluation of the response to a Hurwitz E, Margolis H, et al. Immunogenicity of booster dose of hepatitis B vaccine in previously hepatitis B vaccines implications for persons at immunized healthcare workers. Vaccine occupational risk of hepatitis B virus infection. 2001;19(28-29):4081-5. Am J Prev Med 1998; 15(1): 1-8. 6- Zhuang GH, Yan H, Wang XL, Wu Q, Wang LR, Gao 17- Rendi-Wagner P, Kundi M, Stemberger H, HY. A five-year observation on efficacy of Wiedemann G, Holzman M, Hofer M, et al. revaccination in children of non-response to Antibody-Response to three recombinant hepatitis hepatitis B vaccine. Zhonghua Yu Fang Yi Xue Za B vaccines: comparative evaluation of multicenter Zhi 2006;40(1):8-12. travel-clinic based experience. Vaccine 2000; 7-Perera J, Perera B, Gamage S. Seroconversion after 19:1055-60. hepatitis B vaccination in healthy young adults, 18- Sezer S, Ozdemir FN, Guz G, Arat Z, Colak T, and the effect of a booster dose. Ceylon Med J Sengul S. Factors influencing response to 2002;47(1):6-8. hepatitis B virus vaccination in hemodialysis 8-Hofmann F, Kralj N, Hasselhorn HM, Nübling M, patients. Transplant Proc 2000; 32: 607-8. Michaelis M, Berthold H. Efficacy of hepatitis B 19-Cardell K, Frydén A, Normann B. Intradermal preventive vaccination. Gesundheitswesen 1997; hepatitis B vaccination in health care workers. 59(8-9):527-34. Response rate and experiences from vaccination 9-Chlíbek R, Smetana J, Sindelár R, Cecetková B, Prymula in clinical practise. Scand J Infect Dis R, Kohl I. Immunogenicity of vaccines against 1999;31(2):197-200. viral hepatitis A and B in the population above 40 years of age -- impact of risk factors. Epidemiol 11/13/2012 Mikrobiol Imunol 2007;56(3):119-28. 10-Martínez Pérez JA, Belmonte Santamaría JE, Gascueña Luengo M, Caballero Moreno L, Sabroso Alcázar

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Isolation of Cryptococcus neoformans from pigeon excreta in Qazvin

Fatemeh Samiee Rad

Assistant professor of Pathology, Qazvin Metabolic Disease Research Center, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran. [email protected]

Abstract: Background: Cryptococcus neoformans is a major pathogen in patient's immunocompromised, ubiquitous fungi that can live free from dust, bird droppings and plant materials are separated. Objective: purpose of this study was isolation of Cryptococcus neoformans from pigeon excreta in Qazvin. Methods: The total of 50 pigeon excreta were collected and cultured on the brain heart infusion agar and Saboured dextrose agar with Chloramphenicol media, to isolate Cryptococcus neoformans at the 25 degrees centigrade and 37 degrees centigrade. Identification was performed by direct examination and by means of the urease test and Carbohydrate assimilation. Finding: From 50 pigeon excreta 2 cases (4%) Cryptococcus neoformans were detected. Besides the Cryptococcus neoformans were isolated 10 species of Cryptococcus Candida unigattulatus 4 cases (5.72%), Candida laurentii 3 cases (4.28%), Candida albidus 2 cases (2.86%) and Candida humicola 1 cases (1.43). Conclusion: Immunosuppressed individuals, especially HIV infected persons (AIDS patients) should avoid contact with pigeons and AIDS patients should avoid contact with pigeons and pet birds. [Samiee Rad F. Isolation of Cryptococcus neoformans from pigeon excreta in Qazvin. Life Sci J 2013;10(1):214-219] (ISSN:1097-8135). http://www.lifesciencesite.com. 32

Keywords: Cryptococcus neoformans; Pigeons excreta; AIDS

1. Introduction excreta have been isolated (Gugnani, 2005). Tame Cryptococcus neoformans is opportunistic pigeons and wild pigeon healthy, can be used as both and basidiomycete yeast that cause meningitis in carrier types neoformans function (Blaschke- immunocompromised patients. People having Hellmessen, 2000). 5-10% of patients with advanced cryptococcus are usually AIDS patients. Origin of HIV infection with disseminated cryptococcus have Cryptococcus is mostly pigeon feces, but it has been been reported. This infection is the most common isolated from feces of other birds like parrots and lethal fungal infection in these population (Mitchell, sparrows too. 1995), and almost in 40% of cases, this is the first Pet bird's excreta and those around us can be presenting sign of getting infected with AIDS a reservoir for this pathogen. Causative agents of (Mitchell, 1995; Powderly, 1995). Always CD4 Cryptococcus neoformans are neoformans and gattii fraction of T lymphocytes are less than 100 in 1cc, varieties, and infections spread to human through but usually it is less than 50 at the time of diagnosis environmental sources. Cryptococcal fungal (Shankar, 2007). Cryptococcus neoformans are an infection is transmitted by soil and air contaminated, encapsulated yeast and causative agent of therefore immune-deficient people and people who cryptococcosis in man and animal. The are HIV-infected should avoid of contact with feces environmental source of Cryptococcus neoformans is of tamed birds (Lugarini, 2008). pigeon's feces and it is a cause of death in immune Cryptococcus neoformans causes deficient patients (Shankar, 2007). cryptococcus probably rarely is at the people with Levits, (1991) stated that Cryptococcus natural immunity (Blaschke-Hellmessen, 2000). neoformans rarely produces cryptococcosis in people Infections with gattii variety mostly have with normal immunity and the infection is mostly been reported from Australia, Brazil, Hawaii, limited to AIDS patients (Levitz, 1991). Southeastern California, Mexico, Thailand, Vietnam, Faggi, (1993) mentioned that domestic Nepal and central African countries, or generally animals like dogs and cats also are infected with speaking, tropical and sub-tropical areas while cryptococcosis. He has reported of cryptococcosis neoformans variety have been reported from infection at the nervous system in three dogs and two Denmark, Belgium, France, Germany, Netherland, cats (Faggi, 1993). Italy, Switzerland, and Japan(Kwon-Chung and Mitchell, conducted a study that prognosis Bennett, 1984). of cryptococcosis with gattii variety of Cryptococcus In 2005, Gugnani has reported segregation neoformans is worse than neoformans variety in gattii of Clemens flowers and eucalyptus trees in human. Gattii variety usually affects on the people India, but the variety neoformans from pigeon residing in rural area or those having occupation in

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rural areas. He studied 118 cases of nervous system in 15% of AIDS patients having disseminated cryptococcosis from 1985 to 1992 and stated that cryptococcosis (Dharmshale, 2006). cryptococcosis with gattii variety is several times In the year 2006, Seo presented a report of more prevalent than neoformans variety in Australia prostatitis with Cryptococcus neoformans. He (Mitchell, 1995). reported a case of prostatitis with Cryptococcus Bava also stated in 1997 that AIDS is a neoformans in an immune deficient alcoholic patient predisposing factor for acquisition of cryptococcosis. having cirrhosis, where the diagnosis had been He noted that men were at a higher risk of getting established with sonography and biopsy (Seo, 2006). infection, and this was due to higher prevalence of In the year 2006, Andreola reported a case AIDS in men during the years 1981 through 1993 of ophthalmic choroiditis involvement in a 27 year (Bava, 1997 ). old woman having HIV with disseminated Arteaga, (1998) has reported 211 cases of cryptococcosis infection. In histological examination autopsies of patients with AIDS that 29% of them of the eyes after her death, presence of Cryptococcus had nervous system cryptococcosis, 9.6% neoformans yeast was established. Detecting this histoplasmosis and 3.2% pulmonary condition by the ophthalmologist can influence aspergillosis(Arteaga Hernandez, 1998). treatment and prognosis of the disease (Andreola, In the year 2000, Blaschke stated that 2006). immune deficient people and those having AIDS or In the year 2007, Umemura reported a case HIV should avoid contact with feces of domestic of cryptococcal meningoencephalitis in a male AIDS birds and pigeons, since they may have Cryptococcus patient after autopsy. In autopsy of his brain, multiple neoformans and cause infection of these patients with cysts caused from free from Cryptococcus fungus in cryptococcosis. He described that wild and tamed these patients, physicians should apply a greater care birds and pigeons that are healthy carriers of in diagnosis (Umemura, 2007). Cryptococcus neoformans and this yeast is mixed In the year 2007, Shankar spoke of having with feces and produces infection through inhalation conditions like AIDS, cancer, organ transplant, of contaminated soil by susceptible people immune deficiency and … as a way of getting (Blaschke-Hellmessen, 2000). infection with Cryptococcus (Shankar, 2007). In 2001, Pitisuttithum stated that high doses In the year 2008, Yoshida put forth of Amphotericin B were not as effective as it was measurement of glucuronoxy lomannan, a capsule thought in preventing mortality of AIDS patients structural antigen of Cryptococcus fungus for having cryptococcosis. He provided this conclusion diagnosing cryptococcosis, and suggested using after one year of studying on 106 AIDS patients with drugs like fluconazol, itraconazol, and Amphotericin cryptococcal meningitis being treated with B together with flucytosin (Yoshida, 2008). Amphotericin B and regular culturing of spinal fluids Cabanes, (1995) stated that cryptococcosis of these patients and evaluation of Cryptococcus in domestic animals is an uncommon event and it is yeasts in spinal fluid samples (Pitisuttithum, 2001). seen as a sporadic infection. These infections often In the year 2002, Iyer stated that PCR can be used as take place with neoformans variety, but infections a rapid tool for diagnosing cryptococcal meningitis. with gattii variety are also seen in different species of All samples becoming positive in culture were animals. Among domestic animals, the highest confirmed as positive by PCR too (Iyer, 2002). prevalence of cryptococcosis belongs to cats In the year 2003, Malik presented a report of (Cabañes, 1995). cryptococcosis of Australian parrots and stated that In the year 2008, Kwon-Chung stated that cryptococcosis in parrots involves nasal cavity, upper most isolates of grubii variety of Cryptococcus respiratory tract, beak, sinuses and facial surfaces. neoformans (serotype A) are not lethal for mice. Causative agent of four parrots was gattii variety of Most cryptococcosis in man is caused from grubii Cryptococcus neoformans. It seems that sitting of variety of Cryptococcus neoformans, which is parrots on eucalyptus tree containing gattii variety of ubiquitous in the nature and spreads with pigeon’s Cryptococcus neoformans yeast has acted as a feces. He used mice nasal injection model of clinical predisposing factor for getting cryptococcosis. Malik lethality for testing pathologic strength of also reported two cases of infection with grubii environmental strains of serotype A Cryptococcus variety of Cryptococcus neoformans in parrots due to neoformans and from eleven strains tested, only one skin trauma from America (Malik, 2003). strain produced illness in mice in 60 days after In the year 2006, Dharmshale reported wide injection (Chen, 2008). spread skin involvement with Cryptococcus In the year 2010, Liaw evaluated 100 cases neoformans in AIDS patients having cryptococcosis. of cryptococcal isolates with serotype, molecular and He stated that skin infection with this fungus is seen pathologic factors methods. Eight isolates were from

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pigeons feces and the remaining was clinical. 99 spore in each ml from its new culture, and injected in isolates of the whole 100 were grubii variety of amount of 0.3 ml through caudal vein, and 0.8 ml Cryptococcus neoformans, and the other one was through peritoneum. Brain, liver and spleen tissues gattii variety of Cryptococcus neoformans, serotype were taken from dead mice and stained with Indian B. all isolates were tested for production of urease, ink, gimsa, and methylen blue. Preparing blocks and phospholipase, capsule and melanin, and the amounts staining the slices with hematoxilen eosine were also of production were different in isolates. Melanin done. For preparing suspensions to be injected to production has a direct influence in meaningful mice, counting spores on neobar slides was also used. protection of Cryptococcus against being killed by Feces of parrots, love bird, canary and poultry were Amphotericin B in isolates (Liaw, 2010). Since also cultured in Sabouraud dextrose Agar for finding Cryptococcus neoformans is found in places where Cryptococcus. For analyzing data, descriptive pigeons haunt, haunting of pigeons in urban places statistics methods were exploited. where immune deficient patients are kept can be dangerous for their health. 3. Results This study was done in 6 months (February 2. Material and Methods through August 2010) in Qazvin, Iran. In this study, This is an analytical descriptive study 70 types of colonies of different yeasts and carried out in Qazvin from 2009 to 2010. The study saprophytes were isolated from 50 samples of pigeon population consists of pigeons feces at the Qazvin. feces (table 1). Qazvin was divided into 25 areas in accordance with Colonies suspected to be Cryptococcus postal map of the city. During this period, two produced a brownish color after being transferred to saplings were planted in each area. Sampling was chrome agar media, and produced blastoconidia after classified as a Census study was done while we were being transferred to corn meal agar media. Injection trying to check all the points. In each postal area, of 10 to power 6 Cryptococcus neoformans in ml from houses where pigeons were kept, some feces of suspension to Souris mice led to death after 5 to 12 pigeon were taken from cages. Feces from under days. In evaluating the brain, liver and spleen of these roofs of houses, holy shrines, and pigeon selling mice, encapsulated yeast cells were seen with Indian shops were also collected. In later stages, suspensions ink. Urea test after 10 minutes to 48 hours to was were made from the collected feces and some positive. Cryptococcus neoformans isolated from antibiotics like penicillin or streptomycin were added pigeons feces grew in Sabouraud dextrose Agar at to them in order to reduce bacterial population. both temperatures of 25 and 37 degrees centigrade. Before preparing suspensions, samples were placed in nylon bags and mixed thoroughly, and then, one Table 1. Frequency of isolated subtypes gram of it was put in a tube and 9 cc of physiologic Fungus type Number of percent serum was added to it. Then, tubes were shaken for 5 isolates Cryptococcus ioni gotulatus 4 5.72 minutes and left stationary for 30 minutes, and then, Cryptococcus larneti 3 4.28 cultures were made from the supernatant fluid part, Cryptococcus neoformans 2 2.86 with a sterile device and in Sabouraud dextrose Agar Cryptococcus albidus 2 2.86 containing chloramphenicol. Two culture plates were Cryptococcus homicula 1 1.43 Candida crusei 6 8.57 prepared from each sample, one to be taken to a 37 Candida albicans 4 5.72 degree incubator and the other to be kept at 25 Rhodotorula glotinis 2 2.86 degrees centigrade. After 48 hours, routine Geotrichum capitatum 3 4.28 mycological examinations like observing the fungus Trichosporon asahi 2 2.86 Aspergillus spp. 15 21.43 with Indian ink, using urea culture media, corn meal Rhizopus spp. 12 17.14 agar , chrome culture media and sugar kit like api Mucor spp. 6 8.57 20C- Aux were carried out on colonies appeared. For Penicillium spp. 5 7.14 transferring the fungus to sugar media, first, the Fusarium spp. 3 4.28 fungus was transferred to a Brain Heart Infusion Total 70 100

Agar (BHI) media to clarify the fungus off from Culture taken from feces of domestic birds sugar. Then, a suspension with turbidity of 2 Mc like canary, love bird, parrot and poultry were all Farland was prepared from the fungal colonies grown negative. The result of sugar absorption with api 20 c in this suspension and the instructions of the related AUX kits was 12 instances of different Cryptococcus sugar kit were followed. For injecting to the lab species from the whole 50 pigeon feces samples animal (souris), a sample, approved to be (24%) and isolation of two Cryptococcus neoformans Cryptococcus neoformans via all tests carried out, yeasts (4%). Isolated cryptococcuses were obtained was taken and turned into a suspension of one million

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from pigeon feces collected from dark and shadow feces of pigeons and domestic birds through PCR places with no access to sun light. In samples method. 50 percent of isolates belonged to collected from different geographical regions of Cryptococcus albidus (Kielstein, 2000). Qazvin, meaning north, south, east, west and center, In 2002, Horta reported isolation of 17 no difference was observed from view point of clinical and 10 environmental Cryptococcus different Cryptococcus species. From the two cases neoformans cases from pigeon feces in Rio de of Cryptococcus neoformans, one was obtained from Janeiro state of Brazil (Horta, 2002). east and the other from west. No difference was Tay (2005) also reported 20 isolates of observed from viewpoint of presence of Cryptococcus neoformans from feces of zoo birds, Cryptococcus divided by place of collection bird selling shops and public places of Clang city in including cage floor, under roof, or on the walls. Malaysia. All isolated strains of Cryptococcus Saprophyte fungi like Aspergillus, Mucor, Rhizopus, neoformans were of a serotype and identified as and Pencillium, and yeasts like Rhodotorula, grubii variety of Cryptococcus neoformans. All Trichosporon, Geotrichum and Candida and bacteria isolates were sensitive to Amphotericin B, also appeared on cultures of pigeon feces. Itraconazol (Tay, 2005). Gugnani (2005) reported isolation of two 4. Discussions varieties of gattii and grubii of Cryptococcus In this study, two isolates of Cryptococcus neoformans from eucalyptus trees in India. He neoformans were obtained from samples of pigeon collected 233 samples from eucalyptus tree parts feces of Qazvin. including flowers, barks, and leaves of the tree. Two Chung (1984) has stated that more than 85 cases of Cryptococcus gattii were isolated from percent of cryptococcuses isolated in Argentina, flowers and two other grubii varieties of Canada, England, America ( except south –east Cryptococcus neoformans from barks of the tree California ) belong to neoformans variety of (Gugnani, 2005). Cryptococcus neoformans and the remaining 15 In 2005, Rosario isolated Cryptococcus percent belong to gattii variety of Cryptococcus neoformans from cloak of pigeons in Spain. He neoformans. High prevalence of gattii variety of prepared 331 samples from cloak of pigeons with a Cryptococcus neoformans (35 to 100 percent) is seen swab, of which, 26 cases had Cryptococcus spp (7, in Australia, Brazil, and south –east California, 58%), 11 (3.32%) had Cryptococcus Uniguttulatus, 6 Mexico, Paraguay, Vietnam, Nepal, and central (1.81%) had Cryptococcus Laurentii, 6 (1.81%) had African countries. He states that gattii variety of neoformans variety of Cryptococcus neoformans, and Cryptococcus neoformans is prevalent only in 3 (0.91%) had Cryptococcus albidus. The results tropical and subtropical areas (Kwon-Chung and showed that pigeons act as a reservoir for Bennett, 1984). Serotype studies in these areas cryptococcosis and as a carrier for neoformans showed that 70 percent of isolates of neoformans variety of Cryptococcus neoformans (Rosario, 2005). variety of Cryptococcus neoformans were of a In the year 2005, Chee reported isolation of serotype, and from the two serotypes of gattii variety serotype a grubii variety of Cryptococcus neoformans of Cryptococcus neoformans, B serotype was 4.5 from feces of pigeons in Seoul, Korea. 72 samples of times more than C serotype (Chen, 2008). pigeon feces sere collected from 25 points of Seoul In 1993, Li isolated Cryptococcus city, 17 cases of which from 8 points contained neoformans from pigeon feces in China and stated Cryptococcus neoformans. All isolates were obtained that 78 percent of isolates were of a serotype and 22 from dry feces of pigeons, and all went under percent of AD serotype. He found only neoformans identification and serotyping through DNA variety of Cryptococcus neoformans from pigeon fingerprinting, where all turned to be A serotype feces in China, although gattii variety of grubii variety of Cryptococcus neoformans (Chee, Cryptococcus neoformans has also been isolated 2005). from clinical samples in China and this fungus has a Cermeno(2006) isolated Cryptococcus special place in the nature and is specific to tropical neoformans from doves in BolivGorganar province and subtropical areas (Li, 1993). of Venezuela (Cermeño, 2006). In 1998, Yildriran isolated 29 cases of Duncan (2006) obtained Cryptococcus gattii Cryptococcus neoformans from 634 samples pigeon from nasal culture of a gray squirrel in Vancouver feces in Turkey. In this isolation, air humidity of air Canada, and stated that wild animals of Vancouver and being away from sun light were influential can become a reservoir for this fungus, like domestic factors (Yildiran, 1998). animals of this region (Duncan, 2006). In 2000, Kielstein proved presence of In the year 2006, Abegg from Brazil, reported neoformans variety of Cryptococcus neoformans in isolation of Cryptococcus neoformans and

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Cryptococcus gattii from bird’s garden of Rio de more attention be paid to possible infliction of AIDS Janeiro. He obtained 32 isolates belonging to a patients of Qazvin with this fungus. serotype grubii variety and 5 isolates belonging to B serotype Cryptococcus gattii. All isolates were Corresponding Author: evaluated for virulence factors and quantitative Dr. Fatemeh Samiee Rad, Qazvin Metabolic Disease production of urease, and the urease amount of gattii Research Center, Faculty of Medicine, Qazvin variety was similar to the isolates obtained from University of Medical Sciences, Qazvin, Iran. clinical cases (Abegg, 2006). E-mail: [email protected] Rosario (2008) reported from Spain that pigeons were not the only reservoir of Cryptococcus, References: and other birds could act as reservoir for it too 1- Lugarini C, Goebel CS, Condas LA, Muro MD, de (Rosario, 2008). Farias MR, Ferreira FM, Vainstein MH. Cryptococcus In 2008, Lugarini from Brazil reported neoformans Isolated from Passerine and Psittacine isolation of Cryptococcus neoformans from feces of bird excreta in the state of Paraná, Brazil. parrots and sparrows. He stated that feces of domestic Mycopathologia 2008;166(2):61-9. 2- Blaschke-Hellmessen R. Cryptococcus species- birds and those around us, act as reservoir for etiological agents of zoonoses or sapronosis? Mycoses Cryptococcus neoformans (Lugarini, 2008). 2000; 43 (1):48-60. In 2008, Baltazar from Spritosanto state of 3- Kwon-Chung KJ, Bennett JE. Epidemiologic Brazil reported an isolation of Cryptococcus gattii differences between the two varieties of Cryptococcus from trees of the region. In addition to Cryptococcus neoformans. Am J Epidemiol 1984; 120 (1): 123-130. gattii, he isolated Cryptococcus laronti and 4- Gugnani HC, Mitchell TG, Litvintseva AP, Lengeler Cryptococcus neoformans from woods trees too. He KB, Heitman J, Kumar A, et al. Isolation of stated that environment is a source of infection with Cryptococcus gattii and Cryptococcus neoformans this fungus (Baltazar Lde, 2008). var. grubii from the flowers and bark of Eucalyptus trees in India. Med Mycol 2005;43(6):565-9. In 2010, Liaw reported isolation of 5- Mitchell DH, Sorrell TC, Allworth AM, Heath CH, Cryptococcus neoformans from feces of pigeons and McGregor AR, Papanaoum K, Richards MJ, Gottlieb stated that 99 percent of Cryptococcus neoformans T. Cryptococcal disease of the CNS in isolates he evaluated in his study were a serotype of immunocompetent hosts: influence of cryptococcal grubii variety (Liaw, 2010). variety on clinical manifestations and outcome. Clin In 2010, Centers from America reported Infect Dis 1995;20(3):611-6. isolation of Cryptococcus gattii from woods trees of 6- Powderly WG, Finkelstein D, Feinberg J, Frame P, He four states of Iowa, Oregon, Washington, and Idaho. W, van der Horst C, et al. A randomized trial He states that Cryptococcus neoformans mostly comparing fluconazole with clotrimazole troches for the prevention of fungal infections in patients with inflicts people having HIV, while Cryptococcus gattii advanced human immunodeficiency virus infection. mostly contaminates people not having HIV in NIAID AIDS Clinical Trials Group. N Engl J Med tropical and subtropical areas (Centers for Disease 1995;332(11):700-5. Control and Prevention, 2010). 7- Shankar SK, Mahadevan A, Sundaram C, Sarkar C, In 2009, Souza isolated Cryptococcus gattii; Chacko G, Lanjewar DN, et al. Pathobiology of fungal serotype B from eucalyptus trees (Souza, 2010). infections of the central nervous system with special We used Sabouraud dextrose Agar for reference to the Indian scenario. Neurol India culturing Cryptococcus, and Menezes (2002) used the 2007;55(3):198-215. same as culture media. We used direct observation 8- Levitz SM. The ecology of Cryptococcus neoformans and the epidemiology of cryptococcosis. Rev Infect method with Indian ink for seeing capsules of Dis 1991; 13(6):1163-1169. Cryptococcus fungus grown on culture media, again, 9- Faggi E, Gargani G, Pizzirani C, Pizzirani S, as how Menezes had done (Menezes, 2002). For Saponetto N. Cryptococcosis in domestic mammals. identification of species, urease test and absorption of Mycoses 1993;36(5-6):165-70. different sugars were used, like what Li had done (Li, 10- Bava AJ, Negroni R, Arechavala A, Robles AM, 1993). And finally, we used PCR method for Bianchi M. Cryptococcosis associated with AIDS in identification of isolates, and Kielstein used the same the Muñiz Hospital of Buenos Aires. Mycopathologia PCR method for identification of isolates (Kielstein, 1997;140(1):13-7. 11- Arteaga Hernandez E, Capo de Paz V, Perez 2000). Since Cryptococcus neoformans is a Fernandez-Teran ML. Opportunistic invasive mycoses basidiomycete opportunistic yeast, mostly infeccting in AIDS. An autopsy study of 211 cases. Rev Iberoam immune deficient and specially AIDS patient Micol 1998; 15(1):33-35. (Lugarini, 2008), and also since this yeast was found 12- Pitisuttithum P, Tansuphasawadikul S, Simpson AJ, in feces of pigeons of Qazvin city, it is necessary that Howe PA, White NJ. A prospective study of AIDS- associated cryptococcal meningitis in Thailand treated

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with high-dose amphotericin B. J Infect neoformans isolates in the Brazilian state Rio Grande 2001;43(4):226-33. do Sul. Med Mycol 2002;40(6):565-71. 13- Iyer RS, Pal RB, Patel RY, Banker DD. Polymerase 27- Tay ST, Chai HC, Na SL, Hamimah H, Rohani MY, chain reaction based diagnosis of systemic fungal Soo-Hoo TS. The isolation, characterization and infections and sensitivity testing of the fungal isolates. antifungal susceptibilities of Cryptococcus Indian J Med Microbiol 2002;20(3):132-6. neoformans from bird excreta in Klang Valley, 14- Malik R, Krockenberger MB, Cross G, Doneley R, Malaysia. Mycopathologia 2005;159(4):509-13. Madill DN, Black D, et al. Avian cryptococcosis. Med 28- Rosario I, Hermoso de Mendoza M, Deniz S, Soro G, Mycol 2003;41(2):115-24. Alamo I, Acosta B. Isolation of Cryptococcus species 15- Dharmshale SN, Patil SA, Gohil A, Chowdhary A, including C. Neoformans from cloacaof pigeons. Oberoi C. Disseminated crytococcosis with extensive Mycoses 2005; 48 (6): 421-424. cutaneous involvement in AIDS. Indian J Med 29- Chee HY, Lee KB. Isolation of Cryptococcus Microbiol 2006;24(3):228-30. neoformans var. grubii (serotype A) from pigeon 16- Seo IY, Jeong HJ, Yun KJ, Rim JS. Granulomatous droppings in Seoul, Korea. J Microbiol 2005; 43(5): cryptococcal prostatitis diagnosed by transrectal 469-72. biopsy. Int J Urol 2006;13(5):638-9. 30- Cermeño JR, Hernández I, Cabello I, Orellán Y, 17- Andreola c, Carli CRS , Gouvea ALF. Multifocal Cermeño JJ, Albornoz R, Padrón E, Godoy G. choroiditis in disseminated Cryptococcus neoformans Cryptococcus neoformans and Histoplasma Infection.Am J Ophthalmol 2006; 142:346–348. capsulatum in dove's (Columbia livia) excreta in 18- Umemura T, Hirayama M, Niimi Y, Matsui K, Bolívar state, Venezuela. Rev Latinoam Microbiol Hashizume Y. An autopsy case of cryptococcal 2006;48(1):6-9. meningoencephalitis with AIDS: correspondence 31- Duncan C, Schwantje H, Stephen C, Campbell J, between MRI and pathological findings of basal Bartlett K. Cryptococcus gattii in wildlife of ganglia and cerebellum. Brain Nerve 2007;59(6):623- Vancouver Island, British Columbia, Canada. J Wildl 7. Dis 2006 ;42(1):175-8. 19- Yoshida K, Niki Y. Cryptococcosis. Jap J Clin Med 32- Abegg MA, Cella FL, Faganello J, Valente P, Schrank 2008; 66(12):2350-2355. A, Vainstein MH. Cryptococcus neoformans and 20- Cabañes FJ, Abarca ML, Bonavia R, Bragulat MR, Cryptococcus gattii isolated from the excreta of Castellá G, Ferrer L. Cryptococcosis in a cat psittaciformes in a southern Brazilian zoological seropositive for feline immunodeficiency virus. garden. Mycopathologia 2006 ;161(2):83-91. Mycoses 1995;38(3-4):131-3. 33- Rosario I, Acosta B, Colom MF. Pigeons and other 21- Chen J, Varma A, Diaz MR, Litvintseva AP, birds as a reservoir for Cryptoccus spp. Rev Iberoam Wollenberg KK, Kwon-Chung KJ. Cryptococcus Micol 2008; 25 (1): 513-518. neoformans strains and infection in apparently 34- Lugarini C, Goebel CS, Condas LA, Muro MD, de immunocompetent patients, China. Emerg Infect Dis Farias MR, Ferreira FM, Vainstein MH. Cryptococcus 2008;14(5):755-62. neoformans Isolated from Passerine and Psittacine 22- Liaw SJ, Wu HC, Hsueh PR. Microbiological bird excreta in the state of Paraná, Brazil. characteristics of clinical isolates of Cryptococcus Mycopathologia 2008;166(2):61-9. neoformans in Taiwan: serotypes, mating types, 35- Baltazar Lde M, Riberio MA. First isolation of molecular types, virulence factors, and antifungal Cryptococcus gattii from the environment in the State susceptibility. Clin Microbiol and Infection 2010; 16 of Espirito Santo. Rev Soc Bras Med Trop 2008 (6), 696–703. .41(5): 449-453. 23- Li A, Nishimura K, Taguchi H, Tanaka R, Wu S, 36- Centers for Disease Control and Prevention (CDC). Miyaji M. The isolation of Cryptococcus neoformans Emergence of Cryptococcus gattii - Pacific Northwest, from pigeon droppings and serotyping of naturally and 2004-2010. MMWR Morb Mortal Wkly Rep 2010; clinically sourced isolates in China. Mycopathologia 59(28): 865-8. 1993;124(1):1-5. 37- Souza LK, Souza Junior AH, Costa CR, Faganello J, 24- Yildiran ST, Saracli MA, Gönlüm A, Gün H. Isolation Vainstein MH, Chagas AL, et al. Molecular typing of Cryptococcus neoformans var. neoformans from and antifungal susceptibility of clinical and pigeon droppings collected throughout Turkey. Med environmental Cryptococcus neoformans species Mycol 1998;36(6):391-4. complex isolates in Goiania, Brazil. Mycoses 25- Kielstein P, Hotzel H, Schmalreck A, Khaschabi D, 2010;53(1):62-7. Glawischnig W. Occurrence of Cryptococcus spp. in 38- Menezes EA, Monteiro MN, Angelo MR, Santos CD, excreta of pigeons and pet birds. Mycoses 2000;43(1- Freire CC, Cunha FA. Cryptococcus neoformans 2):7-15. causing meningitis in AIDS patients. Rev Soc Bras 26- Horta JA, Staats CC, Casali AK, Ribeiro AM, Schrank Med Trop 2002;35(5):537-9. IS, Schrank A, Vainstein MH. Epidemiological aspects of clinical and environmental Cryptococcus

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Hypodense area within epidural hematoma in brain CT scan; Prediction of active bleeding in epidural hematoma

Mahboub Pouraghaei1, Payman Moharamzadeh1, Sima Dibazar2, Ali Taghizadieh1, Alireza Ala1, Parham Maroufi3, Reza Rikhtegar Ghiasi4

1- Department of Emergency Medicine, Faculty of Medicine, Tabriz University of medical sciences, Tabriz, Iran 2- General Medicine, Faculty of Medicine, Tabriz University of medical sciences, Tabriz, Iran. 3- Department of Orthopedic, Faculty of Medicine, Tabriz University of medical sciences, Tabriz, Iran. 4- Neuroscience research center (NSRC), Department of Neurology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. [email protected]

Abstract: Background: One of the most important factors in management of patients with epidural hematoma is the existence of hypodense area within the hematoma (SWIRL sign) in brain CT scan. This finding can be the sign of active bleeding and developing the hematoma. Purpose: Study the value of brain CT scan of the patients with epidural hematoma in order to predict the possibility of expansion of the hematoma because of active bleeding. Method: 49 patients with traumatic epidural hematoma entered the study and divided in to 2 groups. Group A was those with epidural hematoma who had indications for immediate craniotomy such as significant midline shift, large size of hematoma, epidural hematoma due to hydrocephalus and etc, and group B was that small or moderate size of epidural hematomas that were kept under close observation without surgery. [Pouraghaei M, Moharamzadeh P, Dibazar S, Taghizadieh A, Ala A, Maroufi P, Rikhtegar Ghiasi R. Hypodense area within epidural hematoma in brain CT scan; Prediction of active bleeding in epidural hematoma. Life Sci J 2013;10(1):220-222] (ISSN:1097-8135). http://www.lifesciencesite.com. 33

Keywords: Hypodense Area; Epidural Hematoma; Brain CT Scan

1. Introduction Nakshabandi, 2001). Active bleeding and fresh blood Epidural hematoma, one of the most life in the clotted area in brain CT (Swirl Sign) will threatening events, presents in 1_2% of the head manifest as a lower density area regarding to the trauma patients (Mishra and Mobanty, 2001). density of the clotted hematoma (Picture 1) (Nizar Bleeding is usually from vessels near the fractures, and Al-Nakshabandi, 2001). This formation is especially the arteries, which an appropriate predictor of continuous bleeding, the possibility of homeostasis will result in a good prognosis for the expansion of hematoma and high pressure over patient. Over all, epidural hematoma has a fatal rate parenchyma of the brain. The goal of this article is to of 15-20 %( Sandres and Mckenna, 2001). One of the see how much important is to care carefully about major reasons of mortality following the epidural this sign in hematomas who had no indications of hematoma is its development and speed of expansion surgical evacuation; Also, we would like to estimate (Singh and Stack, 2006). Expanding of the epidural the existence of active bleeding vessel during surgery hematoma is reported in the first 24 hrs in 90% of the and the possibility of hematoma expansion in these 2 patients (Hardman, 1979). groups. To estimate the amount of expanding of hematoma there are some factors including: source of 2. Material and Methods hematoma (artery or vein), spasm in the ruptured a. Study Design vessel, Walled off vessel by packed bloods, Trauma patients with epidural hematoma in their decompression of the hematoma by the fractured brain CT scan, who referred to Emergency Room, bone (Zimmerman and Bilanuk, 1982). were studied. Patients with the past history of All the clinical and para-clinical findings coagulopathy were excluded. Finally, forty-nine which help to understand, the possibility of patients were classified in two groups, A and B. expanding of the hematoma earlier would be a Group A, included all the patients transferred to OR benefit in the management of the patients. Computed and group B, included all the patients underwent Tomographic CT scan is a valuable imaging method close observation in the neurosurgical ICU. This in order to evaluate the head trauma patients with selection was based on the patients Glasco Coma epidural hematoma. CT results vary depending on the Scale (GCS), hematoma size, midline shift, source of bleeding (artery or vein), time interval hydrocephalus or indication of immediate operation between injuries and doing CT, bleeding’s rate, in order to evacuate the hematoma and making formation of clots and active bleeding (Nizar and Al- homeostasis. All the patients in the operating room

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were evaluated for any bleeding vessel following considerable correlation between transferring to the craniotomy. In group A patients with evidence of OR and existence of swirl sign. Patients that did not hypodense area in hyperdense epidural hematoma transferred to the OR underwent close observation in (SWIRL sign; indicating active intra hematoma the ICU (38.8%; 19 patients) (Group B), and delayed bleeding) in primary brain CT Scan were compared CT scan was taken within 24 hrs. In group B with 19 to the patients without SWIRL sign in terms of patients, 21% had SWIRL sign. All the patients with presence or absence of active bleeding vessel positive swirl sign (4patients) had increasing size of following craniotomy. hematoma in their second brain CT scan and 3 In group B, the increase in epidural patients of them transferred to the OR within 24 hrs hematoma size in the second brain CT scan within 24 of admission. 79% of patients in group B had hours of patients with positive SWIRL sign was epidural hematoma without SWIRL sign in primary compared to that of the patients with negative brain CT scan. Of those with negative swirl sign, SWIRL sign. only 20% (3patients) had increasing size of b. Outcome Measurements and Statistical hematoma in their second controlled brain CT scan analysis (P<0.05). The significance level was set at 0.05. Statistical analyses were conducted using SPSS15.0 (SPSS Inc, 4. Discussions Illinois, and USA). Statistical analysis was conducted Epidural hematoma is one of the most consisting of student’s t-test. Correlation between important and significant emergencies which is bleeding mark in the CT and the need for surgery and treatable in the rapid diagnostic settings. Regarding other qualitative predictions was conducted using to the arterial source of the bleeding under the skull binary logistic regression. bone, the continuous of bleeding leads to the high pressure over the parenchyma and ends in death. Estimation of the continuous or active bleeding in this group of patients is a key point to undergo surgery and make homeostasis. In the patients with epidural hematoma less than 15 mm and lack of any indication for immediate surgery, any sign of active bleeding in the hematoma and no packing sign of the bleeding vessel by the hematoma will necessitate evacuation of the hematoma and make homeostasis. Also, physicians should take care of the hospitalized patients in the ER or ICU precisely. The hypodense area in the hyperdense epidural hematoma (swirl sign) can be a predictive of active bleeding in the hematoma. Helmet et al. showed that active bleeding in the epidural hematoma makes an aneurismal sac or swirl sign. Similar to our study in which 56.5% of the hematomas had swirl sign, in Helmer and Zimmerman study, 58% and 57.7% of the hematomas had this sign, respectively (Zimmerman and Bilanuk, Picture 1 1982; Nizar and Al-Nakshabandi, 2001). All the patients with the immediate surgical indication who 3. Results transferred to the ER had the positive swirl sign. Of Totally forty-nine patients were included in these patients, 90% had the obvious bleeding during this study. The mean age was 31.43±15.79 years old. craniotomy; this is in agreement with Al- The mean of maximum diameter of hematoma in Nakshabandi et al. who reported 84.6% in their study brain CT scan of patients with negative swirl sign over 13 patients with epidural hematoma with was 11.95 (SD=8.40) and was 21.80 (SD=8.40) in the hypodense area in the hyperdense clot (Nizar and Al- patients with positive swirl sign (p<0.05). Nakshabandi, 2001). Among the patients, 61.2%(30 patients) Transferring the patients to the ER and take transferred to the OR for emergent craniotomy this image, meanwhile, the primary bleeding will be (Group A) and all of them had positive swirl sign in organized and the clot will be formed. Any their brain CT scan and of these patients 90% (27 hypodense area in the organized clot would be the patients) had obvious active bleeding artery within mark of active bleeding or possibly, a bleeding the hematoma during craniotomy. There was vessel.

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In patients with coagulopathy, there is the References: risk of developing hematoma and active bleeding. 1- Mishra A, Mobanty S. Contre-coup extra dural haematoma: a short report. Neorology India Conclusion 2001;49(94):94-99. In the CT of the patients with head trauma, 2- Sandres MJ, Mckenna K. Mosby’s para medic text hypodense area in the epidural hematoma is a serious book Dovromin, 2nd revised Ed. chapter 22, Head caution of developing bleeding and existence of an and Facial Trauma Mosby, WB Sanders, active site of bleeding in the hematoma. Also, swirl Philadelphia, 2001;1124-1132. sign can be placed as an immediate indication for 3- Singh J, Stack A. Head Trauma. Emedicine,2006; surgery of epidural hematoma. 239(65):117-139. 4- Hardman JM. The pathology of traumatic brain Corresponding Author: injuries. Adv Neurol 1979;22:15-50. Dr. Payman Moharamzadeh 5- Zimmerman RA, Bilaniuk LT. Computed Emergency Department, Faculty of Medicine, Tabriz tomographic staging of traumatic epidural University of medical sciences, Tabriz, Iran. bleeding. Radiology 1982;144(4):809-12. E-mail: [email protected] 6- Nizar A, Al-Nakshabandi. The Swirl Sign. Radiology 2001;32(43): 218- 443.

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Time Frequency of Guillain-Barre Syndrome in Northwest of Iran

Mehdi Farhoudi 1, Hormoz Ayromlou 1, Amir Mohammad Bazzazi 2, Farrokh Bakht Shadi 3, Samad EJ Golzari 4,5, Kamyar Ghabili 6,7, Marjan Dehdilani 8

1. Neurosciences Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 2. Department of Neurosurgery, Urmia University of Medical Sciences, Urmia, Iran 3. Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 4. Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 5. Students’ Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran 6. Physical Medicine and Rehabilitation Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 7. Young Researchers Club, Tabriz Branch, Islamic Azad University, Tabriz, Iran 8. Medical Education Research Center, Tabriz University of Medical Science, Tabriz, Iran [email protected]

Abstract: Guillain-Barre syndrome (GBS) is an immune mediated polyradiculoneuropathy usually proceeded with respiratory and gastrointestinal infections 3 to 5 days prior to neurologic signs appearance. It seems that GBS seasonally outbreaks in our region. This study was aimed to evaluate seasonal relationship of clinical and epidemiological findings of GBS in hospitalized patients in Imam Khomeini hospital in the period of 1992-2004. One hundred and seventy five cases of hospitalized patients diagnosed with GBS were studied considering following factors: age, sex, events prior to the onset, seasonal incidence, clinical patterns, and cerebrospinal fluid (CSF) protein, electrodiagnosis and mortality rate in patients. The age distribution was in the range of 2-83 years with the mean age of 38 years and male to female ratio was 1 to 2.3. Seasonal onset was spring in 21.1% of the cases, summer in 26.6%, fall in 28% and winter in 24.6%. There was no significant epidemic relation with the seasons of the year. The mortality rate was 8.6% during admission time. There was no significant relation between the season of onset of the disease and mortality rate. [Farhoudi M, Ayromlou H, Bazzazi AM, Shadi FB, Golzari SE, Ghabili K, Dehdilani M. Time Frequency of Guillain-Barre Syndrome in Northwest of Iran. Life Sci J 2013;10(1):223-225] (ISSN:1097-8135). http://www.lifesciencesite.com. 34

Keywords: Guillain-Barre syndrome; seasonal incidence; electrodiagnosis

1. Introduction admitted periodically. As seasonal prevalence is In 1916, Guillain-Barre and Stroll reported a confirmed in some of the articles that we reviewed French soldier who had experienced motion (Cheng et al., 2003) and not confirmed in some weakness, areflexia and changes in CSF (Cerebro (Hughes and Rees, 1997; Alter, 1990). Considering Spinal Fluid) protein. Later, they reported further the controversy over this issue, we decided to study cases with the same manifestations and these clinical this subject in hospitalized patients with GBS in conditions were called Guillain-Barre syndrome or previous years and relation of it to short-term GBS (Trojaborg, 1998; Seneviratne, 2000). GBS prognosis. affects people from every race or age. This disease is non epidemic and females are more vulnerable. The 2. Material and Methods age spectrum for being affected by this disease is In an analytic-descriptive and retrospective from 8 months to 81 years. The most probable ages to study, all the patients with GBS admitted to Imam experience the onset was 50-74 years. The annual Khomeini hospital from 1992-2004 were studied. incidence is 0.4 -1.7 in one hundred thousand (Victor This hospital was the biggest referral university and Roper, 2001; Aminoff, 2001). The main cause of medical center in north-west of Iran in that period. GBS remains unknown and it is believed to have an The following variables were studied: age, sex, immunologic etiology. Most of the cases have a season of onset, recent infection history, clinical and history of an infection before the clinical onset .It is paraclinical findings, duration of admission and respiratory infection in 40% and gastroenteritis in 14- discharge situation. The collected data were analyzed 20% of cases (Jacobs et al., 1998; Hughes and Rees, using SPSS statistics software and the statistical 1997). analyses of the relation between variables were Considering our experiences with referred evaluated using chi square and T-test. A P value patients with GBS at neurology ward of Tabriz Imam <0.05 was considered statistically significant. Khomeini hospital, it seemed that the patients were

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3. Results 4. Discussions One hundred and seventy five patients This study reveals that there are no (male:female ratio of 2.3:1) with GBS were admitted significantly seasonal outbreaks of GBS and no in the period of 1992 to 2004. The age range was 2- relation of it to preceding infections, GBS type and 83 years with mean age of 68. Considering the onset mortality. Regarding sex prevalence, Rowland (2005) season, 21.1% experienced the disease in spring, and Aminoff (2001) reported an equal prevalence, 26.3% in summer, 28% in fall and 24.6% in winter. whereas Victor and Roper (2001) and Alter (1990) The events prior to the weakness onset happened 2 to reported a higher prevalence in females and other 4 weeks earlier including upper respiratory infections studies suggest higher prevalence in males (Hughes (59.4%), gastrointestinal infections (5.7%), and both and Rees, 1997; Yuan et al., 2002; Rocha et al., (6%). Nearly one third of the cases had facial nerve 2004) which is in accordance with our study. These palsies and 8.6% had other cranial nerves palsies. statistical differences are indicative of the inequality Considering clinical pattern, almost 97.8% in sex distribution in different parts of the world. of the cases suffered from weakness in extremities The literature shows controversial results including paraparesia (6.9%), quadriparesia and about seasonal distribution of GBS. Similar to our numbness (90.9%), and respiratory complaints study, some researchers suggested no significant (13.4%). Regarding loss of deep tendon reflexes seasonal difference (Victor and Roper, 2001; (DTRs), 46.6% of the cases experienced loss of Aminoff, 2001; Rowland, 2005; Hughes and Rees, DTRs in lower extremities and 51.4% in both upper 1997; Alter, 1990; Dyck, 1994; Walton, 1993; Hui et and lower extremities and decrease in reflexes was al., 2005; Soffer et al., 1978; Chroni et al., 2004; reported in 4% of the cases. Regarding sensory Sedano et al., 1994). In contrast, other studies impairment in examination, 23.4% of the cases had showed a significant prevalence in spring (Olivé et pain and temperature impairment and 9.7% had deep al., 1997; Lyu et al., 1997), a significant prevalence sensory dysfunction. Autonomic dysfunction was in summer (Rocha et al., 2004; Van Koningsveld et present in 15.9% of cases, 5.7% of the cases had al., 2000), and a higher prevalence in winter (Cheng cardiovascular involvement, 9.1% bladder et al., 2003; Cheng et al., 2000; Bogliun et al., 2004; involvement and 1.1% had sweating dysfunction. Bahou et al., 1996). These statistics suggest that Mechanical ventilation was required in 22.9% of the climatic diversity has been probably effective on cases due to the weakness in respiratory muscles. chronological prevalence in different parts of the Electrodiagnostic tests showed GBS types as world. Regarding the preceding events in GBS, in our follows: acute inflammatory demyelinating study respiratory infections were present in 59.4% of polyneuropathy (AIDP) (67%), acute motor axonal the cases and gastrointestinal infections in 5.6% and neuropathy (17%), M. Fisher syndrome (4%), acute there was no seasonal relation. Pioneer events have motor sensory axonal neuropathy (2%), and been reported in 50-60% of the cases in the literature unclassified (10%). Hospitalization period lasted (Aminoff, 2001; Goldman and Bennett, 2004; from 1 to 90 days. The mean admission days were 16 Walton, 1993). In a Greek study, respiratory days. Mortality rate was 8.6% in admitted patients infections (28.8%) and gastrointestinal infections which was mostly due to the cardiopulmonary arrest. (7.5%) were reported as 50% of all pioneer events Regarding the distribution of GBS in and there has also been a report of a significant different seasons of the year, there was no significant relation between different seasons and affecting relation between the disease and different seasons of infections incidence (Chroni et al., 2004). In a the year (P=0.61). Regarding the epidemiologic Taiwanese study, there was a relation between relation in different seasons of the year (e.g., 2002, respiratory infections incidence in spring with GBS 2003 and 2004), there was no significant relation. (Cheng et al., 2003). Regarding the mortality rates during admission Regarding the different types of period in different seasons of the year, 8.1% of the electrodiagnosis in our study, AIDP type was the affected cases expired in spring (P=0.6), 2.2 % in most common (67%) and the incidence of any types summer (P=0.57), 12.2% in fall (P=0.21), and 11.6% did not have relation with different seasons. In the in winter (P=0.25). Regarding the seasonal pattern, previous studies, AIDP was also the most common AIDP type was more common in winter but the type (Bogliun et al., 2004; Kuwabara, 2004; Alam et relation was not significant (P=1.00). There was no al., 1998). However, AMAN type was more common significant relation between history of respiratory in studies of northern China and Japan (Hiraga et al., infections and GBS in seasons of year (P=0.08) and 2003; Walton, 1993). Furthermore, Cheng et al. between mortality in GBS and previous respiratory (2003) reported that AIDP was more common in infections (P=0.59). spring compared to other seasons. In addition, mortality rate was 8.6% in our study and it had no

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relation with seasons of the year and pioneer events. 14. Hui AC, Chow KM, Tang AS, Fu M, Kay R, Wong Mortality had been reported differently from 2-12% KS. Electrophysiological, clinical and epidemiological in the literature (Seneviratne, 2000; Victor and study of Guillain-Barre Syndrome in Hong Kong Roper, 2001; Cample, 1999). Chinese. J Clin Neurosci. 2005;12:134-136. 15. Soffer D, Feldman S, Alter M. Epidemiology of In conclusion, in our region, there is no Guillain-Barré syndrome. Neurology. 1978;28:686- seasonal increase in incidence of GBS. Also, there is 690. no significant relation between season of GBS 16. Chroni E, Papapetropoulos S, Gioldasis G, Ellul J, occurrence with preceding infection, GBS pattern and Diamadopoulos N, Papapetropoulos T. Guillain-Barré mortality during a 12-year study period. syndrome in Greece: seasonality and other clinico- epidemiological features. Eur J Neurol. 2004;11:383- Corresponding Author: 388. Dr. Amir Mohammad Bazzazi 17. Sedano MJ, Calleja J, Canga E, Berciano J. Guillain- Department of Neurosurgery, Barré syndrome in Cantabria, Spain. An Urmia University of Medical Sciences, epidemiological and clinical study. Acta Neurol Scand. 1994;89:287-292. Urmia, Iran 18. Olivé JM, Castillo C, Castro RG, de Quadros CA. E-mail: [email protected] Epidemiologic study of Guillain-Barré syndrome in children <15 years of age in Latin America. J Infect References Dis. 1997;175 Suppl 1:S160-S164. 1. Trojaborg W. Acute and chronic neuropathies: new 19. Lyu RK, Tang LM, Cheng SY, Hsu WC, Chen ST. aspects of Guillain-Barré syndrome and chronic Guillain-Barré syndrome in Taiwan: a clinical study of inflammatory demyelinating polyneuropathy, an 167 patients. J Neurol Neurosurg Psychiatry. overview and an update. Electroencephalogr Clin 1997;63:494-500. Neurophysiol. 1998;107:303-316. 20. Van Koningsveld R, Van Doorn PA, Schmitz PI, Ang 2. Seneviratne U. Guillain-Barré syndrome. Postgrad CW, Van der Meché FG. Mild forms of Guillain-Barré Med J. 2000;76:774-782. syndrome in an epidemiologic survey in The 3. Victor M, Roper A. Adams and Victor’s Principle of Netherlands. Neurology. 2000;54:620-625. Neurology, 7th ed. McGraw Hill, New York, 21. Cheng Q, Jiang GX, Press R, Andersson M, Ekstedt B, 2001;1380-1387. Vrethem M, Liedholm LJ, Lindsten H, Brattström L, 4. Aminoff M. Neurology and General Medicine, 3rd ed. Fredrikson S, Link H, de Pedro-Cuesta J. Clinical Churchill Livingston, USA, 2001;1011-1012. epidemiology of Guillain-Barré syndrome in adults in 5. Jacobs BC, Rothbarth PH, van der Meché FG, Sweden 1996-97: a prospective study. Eur J Neurol. Herbrink P, Schmitz PI, de Klerk MA, van Doorn PA. 2000;7:685-692. The spectrum of antecedent infections in Guillain- 22. Bogliun G, Beghi E; Italian GBS Registry Study Barré syndrome: a case-control study. Neurology. Group. Incidence and clinical features of acute 1998;51:1110-1115. inflammatory polyradiculoneuropathy in Lombardy, 6. Hughes RA, Rees JH. Clinical and epidemiologic Italy, 1996. Acta Neurol Scand. 2004;110:100-106. features of Guillain-Barré syndrome. J Infect Dis. 23. Bahou YG, Biary N, al Deeb S. Guillain-Barre 1997;176 Suppl 2:S92-S98. syndrome: a series observed at Riyadh Armed Forces 7. Cheng BC, Chang WN, Chang CS, Chee CY, Huang Hospital January 1984--January 1994. J Neurol. CR, Chen JB, Chang CJ, Hung PL, Wang KW, Chang 1996;243:147-152. HW, Lu CH. Guillain-Barré syndrome in southern 24. Goldman L, Bennett C. Textbook of Cecil internal Taiwan: clinical features, prognostic factors and medicine, Vol 2, 21st Ed. Saunders, New York, therapeutic outcomes. Eur J Neurol. 2003;10:655-662. 2004;2193-2194. 8. Alter M. The epidemiology of Guillain-Barré 25. Kuwabara S. Guillain-Barré syndrome: epidemiology, syndrome. Ann Neurol. 1990;27 Suppl:S7-S12. pathophysiology and management. Drugs. 9. Rowland L. Merritt’s Neurology, 11th ed. Lippincott 2004;64:597-610. Williams and Wilkins, USA, 2005;613-615. 26. Alam TA, Chaudhry V, Cornblath DR. 10. Yuan CL, Tsou HK, Wang YJ, Tsai CP. Guillain- Electrophysiological studies in the Guillain-Barré Barré syndrome: a retrospective, hospital-based study. syndrome: distinguishing subtypes by published Zhonghua Yi Xue Za Zhi (Taipei). 2002;65:540-547. criteria. Muscle Nerve. 1998;21:1275-1279. 11. Rocha MS, Brucki SM, Carvalho AA, Lima UW. 27. Hiraga A, Mori M, Ogawara K, Hattori T, Kuwabara Epidemiologic features of Guillain-Barré syndrome in S. Differences in patterns of progression in São Paulo, Brazil. Arq Neuropsiquiatr. 2004;62:33-37. demyelinating and axonal Guillain-Barré syndromes. 12. Dyck PJ. Peripheral Neuropathy, 2nd ed. Saunders, Neurology. 2003;61:471-474. Philadelphia, 1994;2050-2057. 28. Cample WI. Essential of electro diagnostic medicine. 13. Walton J. Brain disease of the nervous system. Oxford Lippincott Williams and Wilkins, USA, 1999;238- University Press, Oxford, 1993;599-603. 239.

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Analysis of Various Anti-Windup Schemes used to Control PMDC Motors employed in Orthopedic Surgical Simulators

G. Murugananth1, S. Vijayan 2, S. Muthukrishnan3

1 Department of EEE, Pollachi Institute of Engineering and Technology, Pollachi, India. E-mail: [email protected] 2 Principal, Surya Engineering College, Erode, India 3 Department of ECE, Sri Eshwar College of Engineering, Coimbatore, India

Abstract: Orthopedic surgical simulators are used by the trainee surgeons to drill the bones and place the screws. These simulators use PMDC motors for bone drilling. In this paper a closed loop chopper controlled drive is proposed and evaluated. The chopper controlled drive has an inner current control loop and an outer speed control loop. The outer control loop employs a conventional PI controller for the speed control of the PMDC motor. The anti-windup PI controller based system is proposed in order to enhance the performance of the system. The system is simulated using Matlab / Simulink and the performances of various anti-windup schemes are analysed. The properties of these controllers were measured and tabulated. The simulation results inferred that the proposed closed loop system with tracking anti-windup schemes can be used for the effective control of the PMDC motor in orthopedic surgeries. [G. Murugananth, S. Vijayan, S. Muthukrishnan. Analysis of Various Anti-Windup Schemes used to Control PMDC Motors employed in Orthopedic Surgical Simulators Life Sci J 2013;10(1):226-230] (ISSN:1097-8135). http://www.lifesciencesite.com. 35

Keywords: Anti-windup, chopper, maximum peak overshoot, orthopedic surgical simulator, PI controller, PMDC Motor

1. Introduction current control loop uses a hysteresis controller. The Novice surgeons practice drilling and screw outer speed loop is employed with a PI controller placements in cadaver bones rather than live patients. and is simulated using Matlab/Simulink. The various The use of orthopedic surgical simulators for parameters are measured and tabulated. practicing drilling and screwing can help for such The anti-windup phenomenon improves the practices (Ann Majewicz et al, Chantelle et al, R. transient state performance. The various types of Thomas et al). The drilling and screwing of bones anti-windup mechanisms are studied (R. Hanus et al, depends on the resistive force offered by the bones Youbin Peng et al, G. Murugananth et al). These and the screw geometry respectively (R. Thomas et anti-windup mechanisms are simulated using al, Ming-Dar Tsai et al, Olga Sourina et al and Matlab/Simulink and their transient state Robert V. O et al) . The resistive force offered by the performances were analysed and compared with un-fractured bones will be more while that of the conventional PI controller based system. fractured will be less. The screwing of the bones is 2. Mathematical Model of PMDC Motor done in three phases namely insertion, tightening and The advantages of PMDC motor include stripping (Ann Majewicz et al, Chantelle and Robert linear speed – torque characteristics with high V. O et al). Different torque and speed combinations stalling torque and reduced power loss. Due to these are needed for optimal placement of screws. advantages the PMDC motors are widely used in In the surgical simulators PMDC motors are orthopedic surgical simulators. The mathematical used, because of their linear speed torque model of the motor is derived from the following characteristics. The mathematical model of the motor equations. is derived (R. Sankar, S. Ramareddy & N. dIa Chandrasekar, K. Thiyagarajah). The speed control V E  IaRa  La (1) of the motor is employed with conventional PI dt (2) controller for various applications which include EK 1 rock drilling and robotics (Robert V. O et al, R. d Sankar, S. Ramareddy & N. Chandrasekar, K. TTBJ   (3) Thiyagarajah, Michel E Fisher et al and Nitai Pal et EL dt al). In this paper a closed loop chopper controlled T K Ia (4) system with an inner current control loop and an E 2 Where, outer speed control loop is attempted. The inner

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Ra = Armature Resistance in Ohms current and compared with the set value. The La = Armature Inductance in H difference between the set value and the present Ia = Armature Current in A values drives the hysteresis controller and the E = Back EMF in Volts controller controls switch S2. During drilling or K1 = Voltage Constant in volts sec/rads screwing when the drill bit reaches or touches the ω = Angular Speed in rads/sec un-fractured bone the torque required increases. TE = Electromagnetic torque developed This increase in torque is sensed and compared TL = Load torque in Nm with the set value and the error is processed by the J = Moment of Inertia in kg.m2/s2 hysteresis controller. As the torque value is B = Damping Coefficient in Nms increased, the controller generates appropriate K2 = Torque Constant in Nm/A pulse to switch off the switch S2. Figure 1 illustrates the mathematical model of the 3.2. Outer Speed Control Loop PMDC motor derived from the above equations. The three phases of screwing such as insertion, tightening and stripping needs three different levels of speed in the motor. Based on the operation the value of speed is set. The current speed of the motor is sensed and is compared with the set value. The error is processed by the PI controller, which in-turn generates the required PWM signal for the switch S1. The switch S1 generates the required voltage for the motor and Figure1. Mathematical Model of PMDC Motor thus the speed of the motor is controlled. 3.3. Conventional PI Controller 3. Proposed System The schematic model of the PI controller is The proposed closed loop control system shown in Figure 3. The speed error is calculated consists of two power electronics switches S1 and from the current speed and set speed and is given S2. Switch S1 is used to regulate the speed of the to the controller. The controller processes the motor in the outer speed control loop and S2 is speed error and generates the required pwm signal used for On/Off control. An inner current control for the chopper. loop and an outer speed control loop as shown in Figure 2. The current control loop employs a hysteresis control system and the outer speed control loop employs a PI control system.

Figure 3 - Schematic Model of Conventional PI Controller

4. Anti-Windup PI Controller PMDC Motors consists of a permanent magnet field system. When a linear control system Figure 2 - Block Diagram of the Proposed is designed for the control of the these motors, System employing an integral action and a limiter, then the integrator will integrate the error signal such 3. 1. Inner Current Control Loop that the integral term may become very large if The inner current control loop is meant for integration lasts for a long time and saturation ON/OFF control of the switch S2. The torque occurs. This is termed as windup problem. required for drilling and the screw placements The windup phenomenon can be avoided by differs for fractured and un-fractured bones. The keeping the integrator output value within limits is un-fractured bones have good strength and so the called Anti-Windup control. The anti-windup resistive force required by them increases. This in- schemes are used to limit the over value in the turn increase the torque required to drill them. integrator and reduces the integration time and During surgery the un-fractured bones should not hence, the overshoot and steady state error. be drilled. In PMDC motors torque is a function of In PMDC motors, the relationship current. Here torque is measured in terms of between speed and voltage is linear. To improve

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the dynamic state of the PMDC motor the Anti- 4. 3. Anti-Windup with Tracking Wind up PI controller is employed. The schematic The tracking scheme calculates the model of the anti-windup PI controller is shown in difference between the input and output saturation Figure 4. block and reduces the integrator’s value.

Figure 4 - Schematic Model of Anti-Windup PI Controller Figure 7 - Anti-Windup with Tracking

There are various anti-windup PI schemes 5. Simulation Results which include back calculation, conditioned, anti- The proposed closed loop chopper controlled windup with dead zone, anti-windup with tracking, system is simulated using Matlab/Simulink. The anti-windup tracking with gain etc. In this paper we simulation model of the system is shown in Fig. 8. attempted with back calculation, anti-windup with The torque error determined from the current and set dead zone and tracking schemes. torque values is processed by the hysteresis 4.1. Back Calculation Anti-Windup Scheme controller and inturn controls the ON/OFF condition The model of back calculation anti-windup of switch S2. The speed error is determined form the scheme is shown in Figure 5. In this scheme the current speed and set speed values and is porcessed integral limit is set form the feedback of the output by the PI controller and it generates the approporiate signal. In back-calculation technique the integral PWM signal needed for siwtch S1. The Zeigler – term is calculated based on the saturation of the Nichols method of tuning is used to fix the values of output. The schematic model of back calculation proportional and integral gains. The response of the scheme is shown in Figure 5. system with PI controller is shown in Figure 9.

Figure 5 - Back Calculation Anti-Windup Scheme

Figure 8 - Matlab / Simulink Model of the 4.2. Anti-Windup with Dead Zone Proposed System Anti-Windup with dead zone scheme utilizes a dead zone element to control the integral limit as Table 1 – Motor Specifications shown in Fig. 6. The integral value remains linear Output Power 52 W and unchanged until it achieves the dead zone limit. Voltage 9V Once it becomes higher than the dead zone limit, Rated Speed 4990 rpm Armature Resistance 1 ohms then the total integral value is reduced. Armature Inductance 0.13mH

Figure 6 - Anti-Windup with Dead Zone

Figure 9 - Response of Conventional PI Controller

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The comparative analysis of the conventional PI and the three schemes of anti-windup PI controller are shown in Figure 10. The results infer that there is a drastic change in the maximum peak overshoot compared to the conventional PI controller and the other anti-windup schemes.

Figure 11 - Steady State Response of Conventional and Anti-Windup PI Controllers

The comparative result implies that in the anti-windup scheme with dead zone, the integral limit is not adjusted, which results in large peak overshoot. The gain of the tracking system is the Figure 10 - Comparative Analysis of cause for reduced peak overshoot in the tracking Conventional and Anti-Windup PI Controllers anti-windup scheme. The transient and steady state responses of the systems are tabulated from the simulation results as shown in Table 2 and Table 3 for various speed values.

Table – 2 – Simulated Parameters for Set Speed = 1000 RPM Anti-Windup Scheme Parameter Conventional PI Dead Zone Back Calculation Tracking Maximum Peak Overshoot 9.5 % 9.9 % 9.1 % 8.5 % Steady State Error 1% 0.65% 0.2% 0.1% Rise Time (ms) 0.063 0.063 0.062 0.062 Settling Time (ms) 0.55 0.35 0.31 0.3

Table – 3 – Simulated parameters for Set Speed = 2500 RPM Anti-Windup Scheme Parameter Conventional PI Dead Zone Back Calculation Tracking Maximum Peak Overshoot 10.2 % 10.6% 9.5 % 9.1 % Steady State Error 1.6% 0.7% 0.4% 0.16% Rise Time (ms) 0.07 0.07 0.07 0.068 Settling Time (ms) 0.6 0.45 0.4 0.4

6. Conclusions E-mail: [email protected] A closed loop chopper controlled drive system is proposed for orthopedic surgical References simulators. The simulation results of the proposed 1. Ann Majewicz, Jason Glasser, Rosemary system shows that the speed becomes zero when Bauer, Stephen M. Belkoff, Simon C. Mears, there is a sudden increase in the torque value. The Allison M. Okamur, “Design of a Haptic transient and steady state analyses show that by Simulator for Osteosynthesis Screw Insertion”, using the anti-windup techniques the performance IEEE Haptics Symposium 2010, 25 - 26 March of the system can be improved. Finally it is 2010, Waltham, Massachusetts, USA, pp 497- concluded that the system with tracking anti- 500. windup scheme can give better performance by 2. Chantelle Saegenschnitter, Tania Walker, reducing the peak overshoot and with low rise and “Orthopedic surgical simulator”, S.M.B.E. (SA) settling time. By the way this proposed system can Inc. Newsletter Vol. X1 No 1, 2003 pp1-2. be used in orthopedic surgical simulators. 3. R. Thomas, et al., “Automated surgical screwdriver: Automated screw placement,” Corresponding Author: Proceedings of the Institution of Mechanical Prof. G. Murugananth Engineers, vol. 222, no. 5, June 2008, pp. 451– Department of EEE 454. Pollachi Institute of Engineering and Technology 4. Ming-Dar Tsai, Ming-Shium Hsieh, Chiung- Pollachi, Tamilnadu, India Hsin Tsai, “Bone drilling haptic interaction for

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orthopedic surgical simulator”, ELSEVIER - Multi conference of Engineers and Computer Computers in Biology and Medicine, volume Scientists, Volume 2, March 2012, pp 14-16. 3,2007, 1 pp1709 – 1718. 11. R. Hanus, M. Kinnaert and J.L. Henrotte 5. Olga Sourina, Alexei Sourin, Howe Tet “Conditioning Technique, a General Anti- Sen,”Orthopedic Surgery Training Simulation”, windup and Bumpless Transfer Method”, Journal of Mechanics in Medicine and Biology, Automatica, Vol. 23, No. 6, pp729-739, 1987 Volume 7, No. 1 2007, pp 37–53. 12. Youbin Peng, Damir Vrancic, and Raymond 6. Robert V. O’ Toole, Branklajva Ramaz, Hanus, “Anti-Windup, Bumpless, and Anthony M, DrrGlora III, Chrsitopher. D, Conditioned Transfer Techniques for PID Visnica and Robert H. Reid, “Biomechanics for Controllers”, IEEE Control Systems Mag., Preoperative Planning and Surgical Simulations 16(4), 48-57, 1996. in Orthopedics”, ELSEVIER - Computers in 13. Hayk Markaroglu Mujde Guzelkaya Ibrahim Biology and Medicine, volume 25, No. 2, 1995, Eksin Engin Yesil, “Tracking Time Adjustment pp183 – 191. in Back Calculation Anti-Windup Scheme”, 7. R. Sankar, S. Ramareddy, “A Novel Control Proceedings 20th European Conference on Strategy Using Neuro-Fuzzy Controller for Modelling and Simulation, Wolfgang Borutzky, PMDC Drive”, European Journal of Scientific Alessandra Orsoni, Richard Zobel, 2006. Research, Volume 54 No.1, 2011, pp.29-45. 14. J. Espina, A. Arias, J. Balcells, C. Ortega & S. 8. N. Chandrasekaran, K. Thiyagarajah, Galceran, “Speed Anti-Windup PI strategies “Modeling and MATLAB Simulation of review for Field Oriented Control of Permanent Pumping System using PMDC Motor Powered Magnet Synchronous Machines Servo Drives by Solar System”, European Journal of with Matrix Converters”. Scientific Research, Volume 59 No.1, 2011, 15. G. Murugananth and S. Vijayan, “Performance pp.6-13. Evaluation of PMDC Motors using Anti - 9. Michael E. Fisher Arindam Ghosh Adel M. Windup PI Controller”, European Journal of Sharaf, “Intelligent Control Strategies for Scientific Research, Volume 85 No.2 Permanent Magnet DC Motor Drives”, September 2012, pp 218-224. Proceedings of the 1996 International 16. G. Murugananth and S. Vijayan, S. John Alexis Conference on Power Electronics, Drives and and S. Muthukrishnan, “A Comparative Energy Systems for Industrial Growth, Volume Analysis of PI, PID and Anti-Windup PI No. 1, 8-11 Jan 1996, pp 360-366. Schemes for PMDC Motors”, International 10. Nitai Pal, Pradip Kumar Sadhu and R. Journal of Computer Applications (0975 – Swaroop, “Closed Loop Speed Control of DC 8887) Volume 57– No.2, November 2012, pp Motors used in Rock Drilling and Mud Pump 21-25. Application”, Proceedings of the International

12/15/2012

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Knowledge and Practice of Female Employee About Premenstrual Syndrome and its Effect on Daily Life Activities in EL-Minia University

Mona A.Abd EL-Hamid.¹ , ² Diaa Ahmed El Moghazy, ³ Manal F..Moustafa and. 4Ekbal A. Emam.

¹,4 Women Health & Obstetrics Nursing Faculty of Nursing, El-Minia University, Egypt ² Obstetrics and Gynecological Faculty of Medicine El-Minia University, Egypt ³Obstetrics& Gynecological Nursing Faculty of Nursing Assuit University, Egypt

Abstract: Premenstrual syndrome is a common health problem affecting females and because of its cyclic occurrence it is postulated to have different effects on quality of life. Aim of this study was to assess the female employee knowledge and practice about premenstrual syndrome and its effect on daily life activities. Method and materials: A cross sectional descriptive study was conducted on one hundred and thirteen women. A specially designed self-administered questionnaire was developed which included socio-demographic data, obstetrical history, symptoms of premenstrual syndrome (PMS), source of information about PMS, its effect on daily life activities and work and management of premenstrual syndrome. Results: The study revealed that the mean age ± SD of the studied sample was (31.7 ± 9) years,(%84.96) of the studied samples were normal age of menarche, (61.95%) of the females were taken information about PMS from mothers, (63.72%) of the studied samples were had an effect on work. The most common reported physical symptom of PMS (79.64%) was backache while the most common reported Psychiatric symptom (76.99%) was worry. The measures practiced by the studied sample to overcome symptoms of PMS were (warm drinks, warm bathing, sports and activities, comfortable and rest period and medications).PMS symptoms was significantly more intense in the single female participants. Conclusion, PMS had an effect on work and daily life activities of female employee working in El- Minia University. Health education, appropriate medical treatment and counseling services, as part and parcel of the overall health service, should be provided to the affected female. [Mona A.Abd EL-Hamid;.Diaa Ahmed El Moghazy; Manal F.Moustafa and. Ekbal A. Emam. Knowledge and Practice of Female Employee About Premenstrual Syndrome and its Effect on Daily Life Activities in EL- Minia University. Life Sci J 2013;10(1):231-243] (ISSN:1097-8135). http://www.lifesciencesite.com. 36

Keywords: premenstrual syndrome, physical, psychological, daily life activities.

1. Introduction decreased appetite, and crave food), and emotional Premenstrual syndrome(PMS) refers to a symptoms are (depressed mood, sad, lonely, anxious, group of menstrually related disorders is nervous, mood swings, trouble with relationships, characterized by mental and physical symptoms that irritable, angry, impatient , difficulty concentrating , vary with different phases of the menstrual cycle. feel out of control, cannot cope, less productive in job These symptoms result in the deterioration of or home and avoid social activity) (4). interpersonal relationships, personal health and The different types of PMS according to function. Symptoms start shortly after ovulation, WHO, (5) Primary PMS, Secondary PMS, Mild increase in severity, and reach a maximum during the PMS, Moderate PMS, Severe PMS (treatment last five premenstrual days. After the onset of resistant) Mood alteration and psychological effects , menstrual bleeding, the symptoms rapidly disappear Gastrointestinal symptoms, Skin problems, and are usually gone within three to four days (1) Respiratory problems, Eye complaints & PMS is related to ovulatory cycles and resolves at Neurological and vascular symptoms (6) menopause. PMS may have an onset at any time PMS is known to have a great impact on during the reproductive years, and once symptoms are daily life activities and social functions, and might established they tend to remain fairly constant until result in significantly decreased quality of life (QOL). menopause (2). These symptoms sufficient to impair Women with PMS had the worse QOL score in daily activities, A woman's experience of almost all domains of QOL except for spiritual health premenstrual symptoms has been found to reduce (physical function, psychological health, social work efficiency, increase absenteeism, and negatively function, pain, vitality, role limitation, health status impact on family (3). perception, and health status change (7). The most common physical symptoms are Treatment goals for PMS are to eliminate (headaches, breast tenderness, swelling, abdominal symptoms, reduce their impact on activities and bloating, heaviness, low energy, tired and weak, back interpersonal relationships, and minimize adverse and muscle pain, sleep more, stay in bed increased / effects of treatment. The current treatment options for

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PMS vary considerably and reflect the multiple be fun. As well as exercising, make sure you get etiology theories and the complexity of hormonal enough rest each day (14&15). interactions likely involved in PMS. (8) Herbal remedies and other supplements: Once an accurate diagnosis is made, The following is a description of some commonly appropriate interventions should be based on two used herbal preparations used to treat PMS. St. principles; first, PMS is a chronic problem that John's wort (Hypericum perforatum): is an herbal typically does not resolve until menopause, making remedy that may help some patients with mild-to- both cost and side effects important components of moderate depression. ex Ginger tea. , help to reduce the treatment choice. Second, women experience mild nausea and other minor symptoms of PMS Seed different degrees of symptom severity, and the oil , for treatment of PMS Dong Quai, used for have intensity of the treatment approach should matched to vasodilating, antispasmodic, and anti platelet. the symptoms. Most women seek treatment as a result Dandelion promoting diures treating the fluid of problems with mood (irritability, mood liability, retention Vitamin B6 It’s help reduce sugar cravings, etc.)(9) Non-pharmacologic interventions for PMS irritability, and bloating Calcium Take daily calcium include patient education, supportive therapy, and it helps prevent water retention, mood swings, and behavioral changes. Women who have been educated cramps(16-18). about the biological basis and prevalence of PMS Complementary and Alternative Medicine report an increased sense of control and relief of (CAM): Many remedies for PMS have been listed symptoms. Psychological interventions such as such as (chiropractic, acupuncture, homoeopathy, relaxation therapy and cognitive behavioral therapy light, yoga and meditation therapy reduce negative show some benefit. Behavioral measures include emotions through cognitive restructuring, enhanced keeping a symptom diary, getting adequate rest and problem solving skills in personal.(19). exercise, making dietary modifications and lifestyle Pharmacologic therapy of PMS: pharmacologic changes.(10). treatment should be considered and ranged of Dietary modifications & life style changes: hormone treatments and medications available to help Dietary restrictions are often recommended to help you manage your symptoms. Medications are given to alleviate the physical and psychological symptoms of treat specific symptoms or alter the menstrual cycle. PMS. The most common dietary recommendations Treatment should be individualized to target the most are to restrict sugar, don’t eat food high in salt reduce troublesome symptoms in each patient Hormonal intake (junk food –fast foods, fatty foods, fatty foods, treatments The aim of hormone treatment is to sweeter, burgers, crisps, salts, tea and coffee and suppress ovulation and reduce the hormones of the intake of cigarettes and alcohol and increase premenstrual phase.(20). consumption of complex carbohydrates. Try to Medications: group of medicines are mood change your eating pattern. Eat frequently and never stabilizers and antidepressants and can improve PMS go without food for more than 5 hours. Increase the symptoms significantly. Anti-prostaglandin and anti- amount of (water, green vegetables and salad, fruit inflammatory effective for breast pain and bloating and nuts, pasta, brown rice, and dried beans, chicken Non-steroidal anti-inflammatory drugs help to reduce and fish,(11) breast tenderness and pain(21). Also Carbohydrates intake are said to raise Surgery the use of surgery should be the last the positive effect on mood and cognition. Fiber-rich treatment option for women with PMS. It’s usually foods are particularly important in maintenance or reserved for individuals with sever symptoms that restoration of healthy estrogen levels. (12) have not respond to medical therapy and after failure Lifestyle Changes : Lifestyle changes to of all conservative measures. Bilateral oophorectomy decrease premenstrual symptoms include physical with hysterectomy is rarely indicated for the treatment exercise and stress reduction technique. The Dietary of PMS alone. Clearly hysterectomy and bilateral Guidelines for Americans, 2005 recommends that oophorectomy is extremely effective in the treatment people perform at least 30 minutes of moderate- of PMS.(22) The nursing role in PMS involves intensity physical activity on most, preferably all, helping the women and her family to understand the days of the week.(13) possible causes of the syndrome, specific symptoms Stress: reduction of stress is a great help in and the rationale for any planned treatments. eliminating the PMS symptoms Incidence of PMS ; Depending on how it is Exercise will reduce physical and emotional assessed, between 5% and 97% of women have been symptoms .It releases built up stress and enhances reported as suffering from PMS. More accurate your sense of well being. Do some gentle exercise estimates point to around 35% of women having such as going for a short walk. . Try to exercise for moderate to severe symptoms which disrupt social 20-30 min 3 times a week. Remember exercise should life, work, and family life and may cause a woman to

232 Life Science Journal 2013;10(1) http://www.lifesciencesite.com seek treatment, whilst 5-10% will experience severely disease, bleeding, and any thing affect on menstrual debilitating symptoms causing major disruption in all cycle ex. Family planning method (I U D). aspects of life (23) Tools: self-administered questionnaire as research The prevalence in Egypt was also found to instrument completed by researcher for collecting this be variable in a study by Mekhail,(24) PMS was data found to be high prevalence (47.2%) among the 1-Socio-demographic characteristics of the studied studied females in El-salam district at Ismailia city. sample such as: This information included the age of Of theses, 39.1% had moderate, and 8.1% had sever the female employee as well as the education, marital PMS, another study by El-Defrawi, (25) in Suez status, place of work and residence. Canal area has reported prevalence rate of( 69.6% ) 2-Data about their obstetrical history: This Significance of the Study includes the age of menarche, menstrual duration, Premenstrual syndrome (PMS) refers to a interval, menstrual regularity, duration and source of group of menstrual related disorders that occur in up information about premenstrual syndrome. to 40 percent of women of reproductive age, with 5 3- Knowledge of female employee about percent of these women having severe impairment. premenstrual syndrome: This include female Which affect their ability to work, work achievement, employee knowledge about premenstrual syndrome affect the quality of their lives and their activities (physical symptoms such as back pain, breast Knaapen, (26). Data generated from this study will tenderness, headache, fatigue…etc and psychological help in identifying the female employee knowledge symptoms such as anxiety, irritability, depression, and practice about premenstrual syndrome and its mood swings…etc). effect on their daily life activity 4- The effect of premenstrual syndrome on daily life activity: 2. Subjects and Methods: This includes data about: Aim of the Study a- The effect of premenstrual syndrome on daily life The present study was conducted to assess activities. the female employee knowledge and practice about - Mild effect with good participation premenstrual syndrome and its effect on daily life - Moderate effect with satisfactory participation activities. - Sever effect with no participation Research hypothesis b- The effect of premenstrual syndrome on the work 1- Identifying the female employee knowledge and in forms of: practice about premenstrual syndrome and its - Absenteeism- Coming late to work - Excusing early effect on their daily life activity. - Low productivity Delaying work for after end of Technical designs syndrome - Research design:- 5- Management of premenstrual syndrome: A cross sectional descriptive research design This include female employee knowledge was utilized in this study. and practice about how to manage premenstrual Subjects: syndrome such as drinks taken or avoided, Setting medication, sports and activities, rest period and This study was carried out at all faculties bathing. (N=16) (Nursing, Medicine, Arts, Fine arts, Administrative Design: Education, Specific education, Science, pharmacy, Procedure: Computers &information, tourism & hotels,  Before conducting the study an official Language, Dar Al Uloom, Engineering, Dental permission was obtained from the Deans of the medicine, Agriculture and physical education) in El- faculties to proceed with study. Minia University.  The researcher introduced herself to the eligible Sample female employee and briefly explained the nature The sample of the present study included 113 of the study. female employees. The data collection started from  Formal consent was obtained from female 3/2010 to 6/2010 which is collected through two days employee orally before being involved in the per week. study after explanation of the nature and purpose Inclusion criteria: All of female employee at of the study and there are no risks or cost in faculties in El- Minia University in reproductive age participation, and there are voluntary participation from 18 to 48 years old. and confidentiality of each subject who agrees to Exclusion criteria: Women have gynecological participate and to fill the questionnaire. diseases, cardiac disease, hypertension disease, liver  The researcher collected the sample through two days, one faculty per week and given sample self

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administered questionnaire to fill data and Part V: Distribution of the studied sample according explained any question from them about the to the effect of symptoms of premenstrual syndrome questionnaire. on daily life activities and work.  The filling of questionnaire took 10-15 minutes Part VI: Distribution of the studied sample according by participants. to their habits to mange premenstrual syndrome  After filling the questionnaire, the researcher symptoms. provided health education about hygienic Part VII: effect of symptoms on work and daily life practices during menstruation and premenstrual activities and the female practice to overcome the syndrome and recommendations for dietary syndrome modification and prescription of regular moderate aerobic exercise within the context for her life & Table (1): Distribution of the studied sample responsibilities according to their socio demographic Operational Design characteristics. Socio- demographic Pilot study: % Pilot study was done on 10% of the women characteristics (N=113) to evaluate the clarity and understanding of the tools. Age It also helped in the estimation of the time needed to 18 to < 28 years 62 54.87 fill the form. According to the results of the pilot, 28 to < 38 years 28 24.78 tools modifications were done. The women who were 38 ≤ 48 years 23 20.35 tested in the pilot study were included in the main Mean age ± SD 31.7 ± 9 study sample. Marital status Fieldwork Single 47 41.59 A Clear explanation of the nature and the Married 59 52.21 aim of the study were given to the women to obtain Divorce 5 4.42 their informed verbal consent which includes the Widow 2 1.77 rights for privacy and confidentiality. Educational level Statistical analysis Secondary or (Diploma degree) 55 48.67 Data were analyzed using the statistical University 49 43.36 package for social science (SPSS) version 11.5 Post graduate 9 7.96 (Windows Microsoft). Continuous data were Place of residence expressed as frequency, percentage, mean and SD. Urban 77 68.14 discrete data were expressed as frequency and Rural 36 31.86 percentage. Comparison between variables was done Place of work using chi-square test. Probability (p-value) less than Faculty of Nursing 13 11.50 0.05 was considered significant and less than 0.001 Faculty of Fine Arts 8 7.1 was considered highly significant. Faculty of Physical Education 14 12.38 Limitation of the study: Faculty of Language 6 5.3 There were some of the limitations facing Faculty of Medicine 9 7.96 the researcher during the period of data collection:- Faculty of Specific Education 2 1.77 1. Some women can’t tolerate to Faculty of Computer and 6 5.3 complete the all questionnaire sheet with the Information researcher because of the overload of the work. Faculty of Dar Al uloom 8 7.1 2. As the information was collected about Faculty of Science 5 4.42 previous practices, recall bias was a possibility. Faculty of Pharmacy 5 4.42 Faculty of Agriculture 11 9.73 3. Results Faculty of Dental Medicine 7 6.19 The results of this study will be described Faculty of Arts 6 5.3 according to the following parts: Faculty of Engineering 5 4.42 Part І: Socio- demographic characteristics of the Faculty of Education 6 5.3 studied sample. Faculty of Hotel and Tourism 2 1.77 Part ІІ: Distribution of the studied sample according to

their obstetrical history. Table (1): Illustrated distribution of the Part IІІ: Distribution of the studied sample according studied sample according to their socio- demographic to their knowledge about source of information. characteristics. It was noticed that the mean age ± SD Part ІV: Distribution of the studied sample according of the studied sample was (31.7 ± 9) years. As regards to their symptoms of premenstrual syndrome. marital status, it was observed that around half

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(52.21%) of the studied sample were married, &only (%84.96) of the studied sample were normal age of (1.77%) were widow. Concerning the educational menarche. As regards duration of menstruation, it was level, it was observed that secondary or diploma noticed that the vast majority (81.42%) Concerning degree have the nearly half percentage in our studied regularity and interval of menstruation, it was sample (48.67%), (43.36%) of them were universal observed that the entire studied sample (100%) was education respectively normal.

Table (2): Distribution of the studied sample Table (3): Distribution of the studied sample according to their obstetrical history according to their knowledge about source of information. Obstetrical data % (N=113) Source of information about % Age of menarche: mean premenstrual syndrome# (N=113) age=(13 ±.8) 96 84.96 Mother 70 61.95 Normal (11-16) 17 15.04 Sisters 21 18.58 Abnormal (<11->16) Grand mother 7 6.19 Duration of menstruation Beers 53 46.90 Normal (3_7) 92 81.42 Books and news paper 49 43.36 Abnormal (<3->7) 21 18.58 Mass media 31 27.43 Regularity of menstruation More than one 58 51.33 Yes 113 100.00 All of them 1 0.88 Others 17 15.04 ـــ ـــ No Interval of menstruation N.B: More than one answer # Normal (21_35) 113 100.00 Table (3): Show distribution of the studied ـــ ـــ (Abnormal (<21->35 Duration of premenstrual sample according to their knowledge about source of syndrome 54 47.78 information about premenstrual syndrome. As regards 3days before 24 21.23 source of information about premenstrual syndrome. 5days before 35 30.97 It revealed that more than half(61.95%) of the studied 7days before sample were taken information from mothers, and (15.04%) was other sources (eg. studying of obstetric Shows distribution of the studied sample subject, internet ,quraan, religious lessons and according to their obstetrical history and age of working in clinical faculties as medicine and nursing menarche it was observed that the vast majority ).

90 79.64 80 75.22 70 65.49 61.95 60

40 33.63 32.74 30 21.24 15.93 20

0 backache fatigue bloating breast headache Nausea weight gain vomiting tenderness

Figure (1): Shows the most common reported physical symptoms of PMS by the studied sample.

Figure (1): Shows that the most common 15.93%) respectively were; backache, fatigue, reported physical symptoms of premenstrual bloating, breast tenderness, headache, Nassau, weight syndrome by the studied sample (79.64%, 75.22%, gain and vomiting. 65.49%, 61.95%, 33.63%, 32.74%, 21.24% and

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90.00 76.99 80.00 75.22 68.14 70.00 61.95 60.00 46.90 50.00 37.17 40.00 32.74 30.00 23.01 20.00 10.00 0.00 worry nervous mood tension depression insomnia social crying swings withdrawal attack Figure (2): Shows the most common reported psychiatric symptoms of PMS by the studied sample.

Figure (2): Displays that the most common 23.01%) respectively were; worry, nervous, mood reported Psychiatric symptoms of premenstrual swings, tension, depression, insomnia, social syndrome by the studied sample(76.99%, 75.22%, withdrawal and crying attack. 68.14%, 61.95%, 46.90%, 37.17%, 32.74% and

26.55% 29.20%

44.25% Mild effect with good participation Moderate effect with satisfactory participation Sever effect with no participation

Figure (3): Effect of premenstrual syndrome on daily life activities of the studied sample.

Figure (3) revealed the effect of participation. While nearly one third (29.20%) of premenstrual syndrome on daily life activities. It them were had sever effect with no participation and was observed that nearly half (44.25%) of the studied more than quarter (26.55%) of them were had mild sample were had moderate effect with satisfactory effect with good participation.

90.00 79.16 80.00

70.00 61.11

60.00

50.00

40.00 33.33

25.00 30.00 22.22 20.00 9.72 10.00 0.00 difficulty low delaying work excusing early absenteeism coming late to concentration at productivity work work

Figure (4): Shows forms of premenstrual syndrome effects on work of the studied sample.

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Figure (4): Displays the form of the effect was (difficulty concentration at work, low of symptoms of PMS on work of the studied productivity, delaying work for after end of sample. It was observed that studied sample were had syndrome, excusing early, absenteeism and coming an effect on work in form of (79.16% ,61.11%, late to work). 33.33%, 25.00%, 22.22% and 9.72%) respectively

77.88% 80.00% 75.22% 73.75% yes no 70.00% 60.18% 60.00% 51.33% 48.67% 50.00% 39.82% 40.00%

26.55% 30.00% 24.78% 22.12% 20.00%

10.00%

0.00% warmdrinks medictions sports and comfortable and warm bathing activities rest period

Figure (5): Displays the measures used by the studied sample to overcome premenstrual syndrome symptoms

Figure (5): Shows the measures used by the used measure by the studied sample to overcome the studied sample to overcome premenstrual syndrome premenstrual syndrome symptoms were: warm symptoms. It displays that (77.88%, 60.18%, 51.33%, bathing, warm drinks, sports and activities, 26.55% and 24.78%) respectively the most common comfortable and rest period and medications).

Table (4): Frequency difference between the rural and the urban female of studied sample regarding source of information about premenstrual syndrome.

Urban Rural Source of information # ( n = 77 ) ( n = 36 ) X2 P-value No % No % Mother 46 59.74 24 66.67 0.50 0.480 Sisters 14 18.18 7 19.44 0.03 0.872 Grand mother 2 2.60 5 13.89 5.38 0.020* Beers 33 42.86 20 55.56 1.59 0.208 Books and news paper 37 48.05 12 33.33 2.16 0.141 Mass media (Television and internet) 29 37.66 2 5.56 12.70 0.000* NB1: More than one answer # NB2: Statistical significant when P value ≤ 0.05

Table (4) illustrated that there no statistical from the grand mother and the significant is for the significant difference between rural and urban female rural female (p= 0.020). While there is a highly of the studied sample regarding source of information statistical significant difference between rural and about premenstrual syndrome taken from the mother, urban female of the studied sample regarding source sisters, beers, books and news paper. But there is of information about premenstrual syndrome taken statistical significant difference between rural and from(mass media) and the significant is for the urban urban female of the studied sample regarding source female (p= 0.000). of information about premenstrual syndrome taken

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Table (5): Frequency difference between the studied sample marital status in symptoms of premenstrual syndrome and effect of symptoms on work and daily life activities. Single Married Symptoms of premenstrual syndrome ( n = 47 ) ( n = 59 ) X2 P-value No % No % a) Physical symptoms # Backache 36 76.60 47 79.66 0.70 0.872 Breast tenderness 31 65.96 34 57.63 1.60 0.656 Headache 12 25.53 25 42.37 6.18 0.103 Fatigue 32 68.09 47 79.66 2.63 0.453 Nausea 24 51.06 10 16.95 8.92 0.050* Vomiting 26 55.32 9 15.25 8.97 0.050* Wait gain 10 21.28 13 22.03 0.57 0.904 Bloating 30 63.83 40 67.80 0.48 0.924 b) Psychiatric symptoms# Worry 34 72.34 46 77.97 1.67 0.645 Nervous 34 72.34 45 76.27 0.96 0.810 Crying attack 9 19.15 16 27.12 1.58 0.664 Depression 19 40.43 30 50.85 5.13 0.163 Fell lonely 11 23.40 17 28.81 3.86 0.277 Mood swings 29 61.70 44 74.58 2.48 0.479 Insomnia 16 34.04 26 44.07 5.54 0.136 Social withdrawal 15 31.91 26 44.07 5.52 0.138 Tension 20 42.55 35 59.32 10.09 0.018* Effect of premenstrual syndrome on daily life activities 15 31.91 35 59.32 10.09 0.017* Effect of symptoms of premenstrual syndrome on work 25 53.19 48 81.35 2.54 0.030* Absenteeism 4 16.00 20 41.66 5.15 0.021* Coming late to work 2 8.00 11 22.91 7.27 0.054* Excusing early 6 24.00 11 22.91 3.36 0.080* Low productivity 9 36.00 40 83.33 5.52 0.010 Difficulty concentration at work 20 80.00 35 72.91 10.09 0.018 Delaying work for after end of syndrome 7 28.00 15 31.25 2.59 0.459 NB1: More than one answer # NB2: Statistical significant when P value ≤ 0.05

Table (5) illustrated that there is statistical 4.Discussion significant difference between the single and married Premenstrual Syndrome (PMS) is a disorder participants regarding physical symptoms (vomiting that characterized by the cyclic recurrence of and nausea) with p value (0.050) the significant is symptoms during the luteal phase of the menstrual for the single female participants. In addition, there is cycle.1-3 Symptoms typically begin between the ages statistical significant difference between them of 25 and 35 years. Women who have severe affective regarding the psychological symptoms in only symptoms may also meet criteria for premenstrual (tension) with p value (0.018) and the significant is dysphoric disorder (PMDD). In both PMS and for the married female participants. PMDD, symptoms diminish rapidly with the onset of Concerning the effect of PMS on the daily menses (27). life activities of the single and married participants, it The aim of this study is to assess the was found that there is statistical significant female employee knowledge and practice about difference between them with p value (0.017) and the premenstrual syndrome and its effect on daily life significant is for the married female participants. Also activities. there is statistical significant difference between them The results of the current study will be regarding the effects of PMS on work with p value discussed and compared them with other related (0.030) and the significant is for the married female studies, literature, as well as representing the participants and this effect in forms of (absenteeism, researcher interpretation of the current results. coming late to work and excusing early).

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As regarding socio-demographic sample. Similar results were found by Ghonamy (33) characteristics of the studied sample, the findings of who studied premenstrual syndrome among Egyptian the present study showed that the mean age of the Cairo university females and reported the most studied sample was (31.7 ± 9) common somatic symptoms were backache, fatigue, The results of table (2) Shows distribution of headache, abdominal cramps and breast tenderness. the studied sample according to their obstetrical On the other hand, the present study found history, concerning the age of menarche it was that (79.64%) backache was the most reported observed that the vast majority (84.96%) of the complaint among the studied sample. These results studied sample were normal age and the mean age of are accordance with the finding of Aly & menarche was (13 ±.8) and this supported by Aly,(8) amasha,and Tenkir et al.,(28,34,35)who mentioned who studied premenstrual syndrome among Assuit that backache was the most reported complaint among university students and the mean age of menarche of the studied sample. But this result in accordance with hid studied sample was (13.5 ±1.2). the study of Mekhail, (24) who reported that As regards duration of menstruation, it was abdominal bloating was the most common complaint noticed that the vast majority (81.42%) of the studied among the studied group. sample had normal duration of menstruation. Also regarding psychiatric symptoms Concerning regularity and interval of menstruation, it Figure (2) revealed that (76.99%, 75.22%, 68.14%, was observed that the entire studied sample (100%) 61.95% and 46.90%) respectively the most common was normal regularity and interval of menstruation reported psychiatric symptoms were (worry, nervous, and this is agreed with the results of Diaz ,(29) who mood swings, tension, depression). This result is studied menstruation in girls and adolescents and supported by the results of Atwood(36),Lee et found that all studied participants (100%) was normal al.,(37) who found that the most observed regularity and interval of menstruation. psychological symptoms were: worry, mood swings, Also results of table (3) showed that the depression, tension, and nervous. These findings were most common source of information about in line withAngst etal (Angst et al.(38) Alters & premenstrual syndrome. It revealed that more than Schiff (39) Hylan, et al.,(40) who mentioned that up half (61.95%) of the studied sample were taken to 60% of American women with PMS reported information from mothers (6.19%) was from grand Psychiatric symptoms such as worry, depression, mother, and (15.04%)was other sources eg. studding tension, nervousness and mood swings. of obstetric subject, internet, quraan, religious lessons On the other hand, the present study found and working in clinical faculties as Medicine and that most common reported psychiatric symptom of Nursing). This result disagreed with the results of premenstrual syndrome by the studied sample (Marván, and Claudia,(30) who studied the role of (76.99%) was (worry). This result is accordance with Prior knowledge about premenstrual syndrome and the finding of Aly,(28); Amasha(34) who mentioned found that the most common source of information that worry was that most common reported about premenstrual syndrome are from videotape and psychiatric symptoms of PMS by the studied sample, films describing PMS with percent of (52.80%). Also also is accordance with the study of Jill and Elissa our results disagreed with Choi (31) who studied the (41) who reported that worry was the most common impact of premenstrual symptoms on activities of complaint among the studied group. But the result daily life in Korean women and found that Korean disagreed with Mohammed (42) who studied the women have no or little knowledge about PMS and prevalence and severity of premenstrual syndrome only infrequently consult their physicians. among adolescent Iranian Girls and found that the In the present study Figure(1): showed that most common complaints were; crying attack (84%) (79.64%, 75.22%, 65.49%, 61.95% and 33.63%) and sudden feeling of sadness (72.3%). respectively the most common reported physical Concerning the effect of premenstrual symptoms of premenstrual syndrome by the studied syndrome on daily life activities, results of Figure sample were; backache, fatigue, bloating, breast (3) showed that nearly half (44.25%) of the studied tenderness and headache. This result is agreed with sample were had moderate effect with satisfactory the results of Aly, (28) who found that the most participation. While nearly one third (29.20%) of frequently physical symptoms reported by the studied them were had sever effect with no participation and students were backache, fatigue, headache, bloating more than quarter (26.55%) of them were had mild and breast tenderness. Also the result is agreed with effect with good participation. This result in the findings of Alvir and Thys-Jacobs, (32) who consistent with the results of Dennerstein (43) who mentioned that backache, fatigue, bloating, breast studied the effect of PMS on activities of daily life tenderness and headache were the most common and found that activities of daily life were reported physical symptoms of PMS by the studied predominantly mild, moderate and sever affection

239 Life Science Journal 2013;10(1) http://www.lifesciencesite.com with percentages of ( 26.73%, 48.15% and 25.22%) work and personality and found that the most respectively. Similar results were found by (Balaha common effects is (85.21%) difficulty concentration (44)who studied the effect of premenstrual syndrome at work. in female medical students and his results were Results showed the practices used by founded his studied participants affected from; 45% studied sample to overcome the syndrome and there mild, 32.6% moderate to 22.4% severe affection on were various types of measures mentioned in this their daily life activities. Also the result agreed with research which were performed by the female the finding of Ova, (45) who studied premenstrual employee. Their aim was to alleviate PMS symptoms syndrome (PMS) and premenstrual dysphoric and the measures (77.88%, 60.18%, 51.33%, 26.55% disorder (PMDD) in Indonesian women and these and 24.78%) respectively were: warm bathing warm women affected form moderate to sever affection on drinks, , sports and activities, comfortable and rest their daily life activities with a percentage of (3.9% period and medications). This result supported by and 1.1%) respectively. But the results disagreed with Stevinson, & Ernst, (51) who observed that the most Choi et al, (31) who studied the impact of practiced measures were warm drinks and sports and premenstrual symptoms on activities of daily life in observed that women practiced exercise were had Korean women and found that premenstrual high pain tolerance and helps in accepting PMS as a syndrome have no effect on Korean women daily life normal experience. Also the results agreed with activities. Amasha, (34) who found that the majority of the In addition the results of Figure (4) studied sample drink warm fluids (51.2%), (26.7) Concerning the effect of symptoms of taking medications, (48%) practice exercise, (25%) premenstrual syndrome on work. It was observed taking rest period and (89.4%) warm bath to reduce that about two thirds (63.72%) of the studied sample PMS symptoms. In the same line the results were had an effect on work in form of (79.16%) was supported by George, (52) who studied a review of difficulty concentration at work, (61.11%) was low treatment approaches to pre-menstrual syndrome and productivity, (33.33%) was delaying work for after found that the most practice measures were high end of syndrome and (22.22%) was absenteeism. proportion of these women perceived lifestyle while more than one third (36.28%) of the studied adjustments- rest, exercise, less caffeine/ alcohol/ sample had no effect at all. This result is in agreement sugar, increase fluid intake and taking warm bathing. with Ichino and Moretti,(46) who studied the On the other hand, the present study found biological gender differences, absenteeism and the that most common reported practice by the studied earning gap and found that women with PMS had an sample to overcome the symptoms of premenstrual effect on work in form of absenteeism, low syndrome was; warm drinks and warm bathing this productivity, delaying work and difficulty disagreed with the finding of Ismail, (53) who concentration at work with a percentage of (24.55, observed that most common reported practice by the 60.95, 25.35 and 73.02) respectively. studied sample to overcome the symptoms of Also the results supported by Ekholm, (47) premenstrual syndrome was nutritional approaches to who studied influence of premenstrual syndrome on overcome the syndrome. Also the results disagreed family, social life, and work performance and relieved with Bakr, & Ez-Elarab, (54) who mentioned that that the most reported effect were low productivity, sleep was the commonest remedy used to overcome absenteeism and delaying work for a later time or the symptoms of PMS reported by the studied female missed work. In the same line our finding supported participants’. by (Borenstein, et al, (48) who studied premenstrual There is statistical significant difference syndrome carries high costs for employers the women between rural and urban female of the studied sample recorded missed work time and estimates of lost regarding source of information about premenstrual productivity related to PMS. Similar results were syndrome taken from the grand mother and the found by Pharma (49) who studied PMS/ in real life: significant is for the rural female (p= 0.020) and this its impact to women's quality of life and observed that supported by the results of Abou Seeeda, and Abdel the physical and emotional manifestations of PMS Hafez, (55)who studied the PMS in rural and urban causes increased absenteeism and reduced work Egyptians and found that the most common source of productivity. information about premenstrual syndrome taken from On the other hand, the present study found the grand mother and the significant is for the rural that most common reported effect of symptoms of female. premenstrual syndrome on work by the studied While there is a highly statistical significant sample was; (79.16%) difficulty concentration at difference between rural and urban female of the work this is agreed with finding of Davydov (50) studied sample regarding source of information about who studied the effects of premenstrual syndrome on premenstrual syndrome taken from mass media and

240 Life Science Journal 2013;10(1) http://www.lifesciencesite.com the significant is for the urban female (p= 0.000) this 5. Conclusion result agreed with the finding of Portella (56) who The following were concluded from this study: studied the association between the source of  PMS is an intermittent problem that cannot be information about PMS and the place of residence resolved until menopause. However, appropriate and mention that internet and mass media were the management can help to alleviate the disturbing most common source about PMS among the studied symptoms of PMS. Findings, of this study, could sample. be used to guide health care providers, who Table (5) illustrated that there is statistical work with those experiencing PMS, regarding significant difference between the single and married what symptoms tend to occur most often, which participants regarding physical symptoms (nausea and symptoms tend to have the highest intensity and vomiting) with p value (0.050) the significant is for what strategies to use to effectively deal with the the single female participants. This result disapproved most disturbing symptoms. by the finding of Dennerstein.,(43) who had a study  The results of this explorative study provide about the effects of premenstrual symptoms on sufficient evidence that PMS have significant activities of daily life and found that married women impact on quality of life, assessed as ADL. reported more symptoms of premenstrual syndrome. (26.55%) of studied sample were had mild effect These findings inconsistent with Ghanbari (57) who with good participation, (44.25%) moderate found that there is no statistical significant difference effect with satisfactory participation, (29.20%) between his studied sample group regarding marital severe effect with no participation. PMS is status and the symptoms of the syndrome .Also associated with difficulty concentration at work, disagreed with (Bakhshani et al) (58) who stated that reduced work productivity, increased work no significant difference was found between married absenteeism and coming late to work. These and unmarried women in his studied sample regarding results suggest that severe premenstrual disorder symptoms of premenstrual syndrome. results in an economic burden both for women Concerning the effect of PMS on the daily as well as for society. life activities of the single and married participants, Recommendations it was found that there is statistical significant In the light of the findings of the present study, the difference between them with p value (0.017) and following recommendations are suggested: the significant is for the married female counseling about premenstrual syndrome and participants. This supported with the finding of  menstrual disorders”& hygienic practices should Dennerstein. (43) who had a study about effects of be given by nurses to help the female employee premenstrual symptoms on activities of daily life understanding what her symptoms most likely and found that married women reported more represent, their causes, to enhance self- efficiency impact of symptoms on activities of daily life and self-management also the goals and (ADL). components of treatment, recommendations for Also there is statistical significant dietary modification and prescription of regular difference between them regarding the effects of moderate aerobic exercise within the context for PMS on work with p value (0.030) and the her life & responsibilities. significant is for the married participants and this effect is in forms of (absenteeism, coming late to  The mass media should be used more effectively work and excusing early), this is supported by Lee to improve awareness of women about the PMS. (37) who study the effect of premenstrual  Further researches should be carried out to syndrome and its economical burden in employed investigate other factors and their association women and mention that there is significant with prevalence of PMS. difference between married and unmarried women in his studied sample regarding the effects of PMS References symptoms on work and the married women 1. Brown, J., O' Brien, P.M., Marjoribanks, J., and reported effects in forms of (missed work, high Wyatt K., (2009): Selective serotonin reuptake level of tension at work and absenteeism) . Also inhibitors for premenstrual syndrome.Cochrane approved with the finding of Chang (59)who Database Syst Rev. 15 ;( 2):CD001396. studied premenstrual syndrome in employed 2. Women’s Health Center., (2007): Premenstrual Chinese women in Hong Kong and found that Syndrome (PMS). Retrieved March 10, 2007, absence and low achievement was significantly fromhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi more frequent among married employed women as ?cmd=retrieve&db=pubmed&list_uids58909&dop t=Abstract they had more responsibilities and concerns than 3. Kleijnen,J., Ter, R. G., and Knipschild, P., (2006): unmarried women. Vitamin B6 in the treatment of the premenstrual

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syndrome--a review. Br Journal Obstet & Gynaecol; calcium and manganese effects on premenstrual 97:847-52 published erratum in Br Journal Obstet & syndrome symptoms. Am J Obstet Gynecol; 168 : Gynaecol; 98:329-30. 1417 – 23. 4. Mitchell, E., Woods, N., and Lentz, M., (2005): 19. Christensen, H., (2010): the potential for dietary Recognizing PMS when you see it: Criteria for PMS supplements to reduce premenstrual syndrome., J. of sample selection. In D. Taylor & F. Woods (Eds.), the American college of Nutrition;19pp:3-12. Menstruation, health and illness (pp. 89–118). New 20. Jarvis, C.I., Lynch, A.M., and Morin, A.K., (2009): York: Hemisphere. Management strategies for premenstrual 5. World Health Organisation (WHO) (2008): syndrome/premenstrual dysphoric disorder. Ann International Classification of Diseases, 10th revision. Pharmacotherapy; 42(7): 967-78 Geneva, Available at: www.who.int/ nut/ 21. Beigon, A., Fischette, C.G., Rainbow ,T.C., and publications.htm# McEwen, B.S. (2003): Serotonin receptor modulation 6. Behrman, S. J., & Buxton, C.L., (2006): by estrogen in discrete brain nuclei. Premenstrual tension. American Journal of Obstetrics Neuroendocrinology; 35:287–291[Medline]. and Gynecology, 81, 606–609. Evidence-based 22. Hart, C., Amieva, H., Goff, M. L., and Portet, F., recommendations for managing the premenstrual (2008): Clinical trials for treatments of premenstrual syndrome. Women’s Health in Primary Care, 3, 735– syndrome In: Gynecology by ten teachers.,16th ed., 738. London, USA, Arnold,pp:177-179. 7. Rizk, D., Mosallam, M., Alyan, S., and 23. O’Brien, P. M. S., (2007): Premenstrual Syndrome, Nagelkerke, N., (2010): Prevalence and impact of Blackwell Scientific Publications. Vol.307, pp.1471- premenstrual syndrome in adolescent school girls in 5. the United Arab Emirates. Acta Obstet Gynecol 24. Mekhail, N.R., (2006): The problem of premenstrual Scand.;85(5):589-98. syndrome in general practice in EL-Salam district, 8. Plouffe, T., Backstrom, A, B., and Macgibbon,T. R., Ismailia. Thesis MSC.,Suez canal university, faculty (2010): Diagnosis of premenstrual tension syndrome: of Medicine. description and evaluation of a procedure for 25. EL –Defrawi, M.H., Lotfi, G., and Mahfouz, R., diagnosis and treatment. Journal Psychosom Obstet (2007): Late luteal phase dysphoric disorder. Do we Gynecol; 10(5): 25– need another psychiatric category?. Egyptian Journal 9. Mira, M., McNeil, D., Fraser, I. S., Vizzard, J., of Psychiatry; 13:205-212. and Abraham S., (2006): New approaches in the 26. Knaapen,L., (2010): Recommendations for treatment of premenstrual syndrome. Obstet& managing the premenstrual syndrome. Women’s Gynecol; 68:395-8. Health in Primary Care;3,735–38. 10. Pearlstein, T. B., (2006): Nonpharmacologic http://www.womenshealthpc.com/10_00/pdf/735WNI treatment of the premenstrual syndrome. ManagingPMS. df; accessed 14 July 2005). Psychiatric Annals; 26: 590–594 27. Freeman, E., Phillip, D., and Rickels K., (2009): 11. Caan, S.B., Walter, S.D., Brazil, K., and Loeb, Relationships between age and symptom severity M.B., (2009): Diet and sex Hormones Binding among women seeking medical treatment for globulin, Dysmenorrhea , and Premenstrual premenstrual symptoms. Psychological Medicine; 25: symptoms., J. of Obstet & Gyenecol; 95(2) :395-8. 309-315. 12. Chuong, A., and Dawson, R. C., (2009): Critical 28. Aly, S. A., (2001): Study of premenstrual syndrome evaluation of nutritional factors in the patho- among Assuit university students. pp: 63-64. physiology and treatment of premenstrual syndrome. 29. Diaz, A., Laufer, M.R., and Breech, L.L., (2009): Clin. Obstet. & Gynecol., Vol; 35, pp : 679 – 88. Menstruation in girls and adolescents: using the 13. Daugherty, M., (2007): Treatment strategies for menstrual cycle as a vital sign. Pediatrics; premenstrual syndrome. Am Family Physician; 118(5):2245-50. 58:183- 92,197-8. 30. Marvan, M.L., Diaz-Erosa, M., and Montesinos, 14. Friedman, D., and Jaffe, A., (2007): Influence of A., (2007): Premenstrual symptoms in Mexican life-style on the premenstrual syndrome, Analysis of a women with different educational levels. J Psychol; questionnaire survey. J Reprod Med; 30:715-9. 132:517-26. This article on PubMed. 15. Byrne, A., and Byrne, D.G., (2010): The effect of 31. Choi, C.R., Wong, L.P., and Khoo, E.M., (2007): exercise on depression, anxiety, and other mood the impact of premenstrual symptoms on activities of states: a review. J Psychosom Res; 37:565-74. daily life in Korean women; 58(4):147-152. 16. Goodale, P.W., Wyatt, K.M., Jones, P.W., and 32. Alvir, J.M., and Thys-Jacobs,S., (2005): O’Brien, P.M., (2007): Premenstrual syndrome and premenstrual and menstrual symptoms cluster and premenstrual dysphoric disorder: guidelines for herbal response to calcium treatment. Psychopharmacology- management. Journal of Nutrition; 25(5): 459–468. Bull; 29(6), pp: 145-148. 17. Kleijnen,J., Ter, R. G., and Knipschild, P., (2006): 33. Ghonamy, G. E., (2004): The Menstrual Symptoms, Vitamin B6 in the treatment of the premenstrual Self-care and Attitude toward Menstruation, among syndrome--a review. Br Journal Obstet & Gynaecol; Cairo university students. Thesis MSC.,Cairo 97:847-52 published erratum in Br Journal Obstet & University, Faculty of Nursing. pp: 55-7. Gynaecol; 98:329-30. 34. Amasha, H. A. R., (2005): Prevalence of 18. Penland, J.G., and Johnson, P.E., (2006): Dietary premenstrual tension syndrome among student Nurses

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And its relationship with Environmental factors. 47. Ekholm, D., (2009): Influence of premenstrual Thesis MSC. Suez canal University, High Institute of syndrome on family, social life, and work Nursing. performance. Clinical Obstetrics and Gynecology; 35 35. Tenkir, A., Fisseha, N., and Ayele, B., (2008): (3): 587-600. Premenstrual syndrome. Prevalence and effect on 48. Borenstein, Y., Kelly, T., and Lubitz, J., (2008): academic and social performances of students in Selective serotonin Serotonin re-uptake inhibitors for Jimma University, Ethiopia, Ethiop J Health Dev; the treatment of premenstrual syndrome. International 17:181-8. Clinical Psychopharmacology; 14:27–3. 36. Atwood, J. D., (2005): Effect of premenstrual mood 49. Pharma, M., Hammarback, S., Lindhe, B.A., change on the couple relationship. Journal Backstrom ,T., (2005): PMS/ in real life : its impact Annoucement, CIJ;7(3):17-19. on women's quality of life The Internet Journal of 37. Lee, M., Wallenstein, G., Hagan, M., and Guo, A., Obstetrics and Gynecology; 3 (1): 85-93.. (2009): Burden of premenstrual syndrome on health- 50. Davydov, D. M., Shapiro, D., and Goldstein, I. B., related quality of life. J Womens Health (Larchmt); (2006): Moods in everyday situations: Effects of 17(1):113-21. Premenstrual syndrome on work and personality. 38. Angst, J., Sellaro,R., Merikangas,K.R., and Journal of Psychosomatic Research; 56:27-33. Endicott,J.,(2007): Prevalence of Premenstrual 51. Stevinson, C., Ernst, E., (2009): syndrome , epidemiology and treatment., J. of Complementary/alternative therapies or premenstrual Reproductive Med;32,pp: 405-8 syndrome: a systematic review of randomized 39. Alters, S., and Schiff, W., (2009): Premenstrual controlled trials. Am J Obstet Gynecol;185: 227-35. syndrome and Menstrual cycle. In: Essential concepts 52. George, J., (2007): A Review of Treatment for Healthy living. 2nded., London, Jones approaches to Pre-Menstrual Syndrome- What do &Bartlett;pp:111-113. British women perceive to be effective for their 40. Hylan, T. R., Sundell, K., and Judge, R., (2010): symptoms?. Human Reproduction; 9 (6): 1058-1063. The impact of premenstrual symptomatology on 53. Ismail, M. K., (2009): The Management of Symptoms functioning and treatment-seeking behavior: Associated with Premenstrual Syndrome. Journal of Experience from the United States, United Kingdom, Manipulative and Physiological Therapeutics. 14(3): and France. Journal of Women’s Health & Gender- 209-216. based Medicine, 8,1043–1052. 54. Bakr, I., Ez-Elarab, H., (2010): Prevalence of 41. Jill, R., and Elissa, K., (2009): Depressive premenstrual syndrome and the effect of its severity Symptomatology Among Early Maturing Girls. on the quality of life among medical students. The journal of youth & adolescence, Vol. 20 (4) pp : 451- Egyptian Journal of Community Medicine, Vol. 28 455 No. 2, pp: 19-30. 42. Mohammad, A., Ganga, M., and Abbas, M., 55. Abou Seeeda, M., and Abdel Hafez,S., (2005): An (2008): Prevalence and severity of premenstrual epidemiological study of premenstrual syndrome in symptoms in adolescent Iranian Girls; 23, pp: 235-8. urban and rural Egyptian J. of Obstet. & Gynecol; 43. Dennerstein, SR., Reinken, JA., and Sparrow, 21(1) pp:7-12. MJ., (2010): Styding the effect of premenstrual 56. Portella ,A.T., Haaga, D.A., and Rohan, K.J., (2010): syndrome on activities of daily life. The Journal of The association between the source of information Reproductive Medicine; 42 (10): 637-645. about PMS and the place of residence and premenstrual 44. Balaha, M. H., Mostafa, A. M., Mohammed, S, A., symptoms is continuous and is not fully accounted by (2010): The phenomenology of premenstrual depressive symptoms. J Nerv Ment Dis; 194(11):833-7. syndrome in female medical students: a cross 57. Ghanbari, M. D., Farideh, D. M., and Mina, J. M., sectional study The Pan African Medical Journal; 5:4- (2008): Practice of women with PMS at different age 8. group to overcome the symptoms of PMS; 2(4), pp: 45. Ova, E., (2008): Premenstrual syndrome (PMS) and 169-172. premenstrual dysphoric disorder (PMDD) in 58. Bakhshani, N. M., Mahnaz, N. M., and Golbahar, Indonesian women. Journal of Women’s Health; 8 Kh., (2009): Severity and effect of Premenstrual (1): 75-85. Syndrome on Quality of Life of Iranian female 46. Ichino, D.R., & Moretti, A., (2008): The biological university students Iranian Girls J Psychiatry; 3:105- gender differences, absenteeism and the earning 109. gap for women with Premenstrual Syndrome. 59. Chang, A.M., Holroyd, E., and Chau, J.P., (2010): Academic Science; 26: 576-580 Premenstrual syndrome in employed Chinese women in Hong Kong. Health Care Women; 16:551-61.

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An Outlier Based Bi-Level Neural Network Classification System for Improved Classification of Cardiotocogram Data

Sundar Chinnasamy1’*, Chitradevi Muthusamy2 and Geetharamani Gopal3

1Christian College of Engineering and Technology, Oddanchatram – 624619, Tamil Nadu, India. 2PRIST University, Trichy Campus, Trichiy – 620009, Tamil Nadu, India. 3Anna University Chennai, BITS Campus, Trichy – 620024, Tamil Nadu, India. *Corresponding Author. E-Mail: [email protected]

Abstract: Cardiotocography (CTG), consisting of fetal heart rate (FHR) and tocographic (TOCO) measurements, is used to evaluate fetal well-being. It is one of the most common diagnostic techniques to evaluate maternal and fetal well-being during pregnancy and before delivery. By observing the Cardiotocography trace patterns doctors can understand the state of the fetus. Even few decades after the introduction of cardiotocography into clinical practice, the predictive capacity of the existing methods remains inaccurate. In a previous work (Sundar.C and et al, 2012), we showed that a model based CTG data classification system using a supervised artificial neural network (ANN) can classify the CTG data better than most of the other methods. But, the performance of the normal neural network based classifier was limited because of the presence of potential outliers in the training data. The presence of outliers in training data affects the neural network training as well as testing. In this work, we present improved classification models which will consider outliers in the data and eliminate them from training phase of the classification process. We used Precision, Recall, F-Score and Rand Index as the metric to evaluate the performance. The proposed idea considerably improved the performance in classifying Normal, Suspicious and Pathologic CTG patterns. It was found that, the improved classifier was capable of identifying Normal, Suspicious and Pathologic condition with very good accuracy than normal methods. [Sundar C, Chitradevi M, Geetharamani G. An Outlier Based Bi-Level Neural Network Classification System for Improved Classification of Cardiotocogram Data. Life Sci J 2013; 10(1):244-251] (ISSN: 1097-8135). http://www.lifesciencesite.com. 37

Keywords: Outlier Detection; CTG; BPN; Dimensionality Reduction; RBF

1. Introduction al, 2012). Even then death and long term disablement One of the major challenges in medical occurs due to hypoxia during delivery. More than domain is the extraction of comprehensible 50% of these deaths were caused by not recognizing knowledge from medical diagnosis data such as CTG the abnormal FHR pattern, even after recognizing not data. In this information era, the use of machine communicating the same without knowing the learning tools in medical diagnosis is increasing seriousness and the delay in taking appropriate action gradually. This is mainly because the effectiveness of (Antonia Costa and et al, 2009). Computation and classification and recognition systems has improved other datamining (C.Domeniconi and et al, 2007), in a great deal to help medical experts in diagnosing (K.Cios and et al, 1998), (J. Han and M.Kamber, diseases. 2000) techniques can be used to analyze and classify Cardiotocography (CTG) the CTG data to avoid human mistakes and to assist Cardiotocography (CTG) is a technical doctors to take a decision. means of recording the fetal heart rate (FHR) and the In a recent work (Shomona and at el, 2012) uterine contractions (UC) during pregnancy, typically they evaluated the performance of the ten in the third trimester to evaluate maternal and fetal classification algorithms with CTG -Morphology well-being (Diogo Ayres-de-Camposa and et al, Pattern dataset. The algorithms C-RT, CS-CRT, 2005). FHR patterns are observed manually by NBC, PLS-DA and RBF show improved accuracy obstetricians during the process of CTG analysis after outlier detection. However the algorithms C4.5, (Stirrat, Mills and Draycott, 2003). In the recent past CS_MC4, ID3, PLS-LDA and Random Tree show fetal heart rate baseline and its frequency analysis has decrease in performance after outlier removal. been taken in to research on many aspects (Sundar.C 2. Material and Methods and et al, 2012). Cardiotocography (CTG), consisting of fetal Fetal heart rate (FHR) monitoring is mainly heart rate (FHR) and tocographic (TOCO) used to find out the amount of oxygen a fetus is measurements, is used to evaluate fetal well-being acquiring during the time of labor (Sundar.C and et during the delivery. Since 1970 many researchers

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have employed different methods to help the doctors we also have a test data set that is used to determine to interpret the CTG trace pattern (Philip A Warrick the effectiveness of a classification. In general, and Emily F Hamilton, 2011) from the field of signal commonly used and popular neural networks can be processing and computer programming (Shahad trained to recognize the data directly, whereas in Nidhal and et al, 2010), (Chen CY and et al, 2009). simple networks there is a chance of the system being They have supported doctors with interpretations in complex and training may be difficult. The time order to reach a satisfactory level of reliability so as taken and the accuracy of classification depend on to act as a decision support system in obstetrics the dimension of the input given and also on the (Onisko, A and Druzdzel, M. J, 2011). Up to now, dimension in the training data. For input data with predictive capacity of the method remains high dimension, the process will take a longer time controversial. The scope of this work is to improve (Sundar.C and et al, 2012). the performance of a neural network based classification system for CTG data classification. In(Shomona and at el, 2012), Among the evaluated algorithms, the algorithms C4.5, CS_MC4, ID3, PLS- LDA produced improved accuracy but, the accuracy was reduces after removing the outlier. In other words, the algorithms which give high accuracy were very much affected by the outliers. This confirms that all the outliers in the data are actually not noise. Even the rarest of occurrences of a peculiar record in a dataset may provide novel insights into new patterns corresponding to a disease identification and diagnosis (Shomona and at el, 2012). Even the best performing tree based Figure 1. Feed forward Network algorithm like C4.5 will get effect by an abnormal change in individual attribute of the input data. In Feed forward networks often have one or other words, a tree based algorithms will work good more hidden layers of sigmoid neurons followed by if the data is a categorical data but it cannot an output layer of linear neurons (Sundar.C and et al, approximate a continuous variable better manner. So, 2012). Multiple layers of neurons with nonlinear according to our understudying, we cannot improve transfer functions allow the network to learn the accuracy just by removing all the outliners in the nonlinear and linear relationships between input and data. Because all the outliers in the data need not output vectors. The linear output layer lets the necessarily be a noise (Bin Jiang and Jian Pei, 2011). network produce values outside the range -1 to +1. Those outliers like abnormal data also should be On the other hand, if you want to constrain the considered during classification of the data. outputs of a network (such as between 0 and 1), then In this work, we are detecting outliers or the output layer should use a sigmoid transfer abnormal records in the training data during the first function (Sundar.C and et al, 2012). stage of training and testing of the back propagation The following diagram shows the standard neural network (BPN). After detecting outliers, those way of classifying the CTG data using a neural outliers will be removed from the training data, and network. again the same network will be trained with the The Training The Testing Data outlier removed data to improve the training Data with Class performance of the neural network and all the outliers Labels will be included in the classification process. So, in this work, we are going to address some of the Train ANN using Classify the machine learning based hybrid datamining techniques Training Data Test Data using for the better classification of CTG data. the Trained Network Standard Neural Network Based Classification Here in this classification (Ben Kao and et al, 2010), we use supervised learning by using a set Predicted Class of training data which is accompanied by class labels Labels (Klimesova A and Ocelikova E, 2010). When a new data arrive, then classification of that data will be Figure 2. The Standard BPN based CTG Data done based on the training set by generating Classifier descriptions of the classes. In addition to training set

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The Proposed Outlier based Bi-level BPN The Training Data The Testing Approach (BL-BPN) with Class Labels Data Outliers In statistics, an outlier (Xiaojun Chen and et al, 2012) is an observation that is numerically distant from the rest of the data. Outliers can occur by Train Network using chance in any distribution, but they are often Training Data indicative either of measurement error or that the population has a heavy-tailed distribution (Barnett. V and Lewis.T, 1994). In the former case one wishes to Classify the Training discard them or use statistics that are robust to Data using the outliers, while in the latter case they indicate that the Trained Network distribution has high kurtosis and that one should be very cautious in using tools or intuitions that assume a normal distribution (Barnett. V and Lewis.T, 1994). Separate the Outliers In a neural network based classification Based on the Log- system, the presence of outliers in training data will Sigmoid Transfer have significant impact on classification performance Function output of the because, the network will not get optimum training output layer due to the presence of outliers in training data. In this proposed classification model the outliers from the training CTG data is removed after training the network with the training data. After that, the Train Network using Classify the Test network is again trained with the outlier removed Outlier Removed Data using the Trained Network data to get better classification performance. Data Outlier Separation Using Log-Sigmoid Transfer Function Transfer functions of the neural network Predicted Class calculate a layer's output from its net input. During Labels the unsupervised competitive learning process of the neural network, the nodes compete for the right to Figure 4. The Outlier Removed Training and respond to a subset of the input data. We used Log- Classification Model Sigmoid Transfer function in the layers of the neural network. The Log-Sigmoid Transfer function will try The above block diagram shows the to produce output between 0 and 1 (Han and et al, proposed BL-NN system. We can consider this 1995). model as virtual cascade of two Neural Networks in serial (but we use only one network to simulate this virtual cascade). The first level network is removing outliers and the second level network is trained to classify the normal CTG data in a better manner. Bi-level BPN Classification Algorithm Inputs: Training Data: DL= {d1, d2…,dn} Training Targets: CL= {c1, c2…, cn} Figure 3. Log-Sigmoid Transfer Function n = Total Number of Training records Testing Data: DT= {t1, t2…, tm} So, we can predict outliers in the training m = Total Number of Testing Data data based on the Log-Sigmoid Transfer function Outputs: output in the output layer. The value of near 1 value Predicted Class labels of Test Data will signify that the input is classifiable. The near CT = {l1, l2… lm} zero values signifies that the input belongs to a Procedure BL-BPN { potential outlier. In our implementation, we consider 1. Read training data DL and targets CL an input as outlier if it produces the Log-Sigmoid and test data DT Transfer Function outputs of value less than 0.5 at the 2. Create Network N1 to learn DL and output layer. The following algorithm explains the map it to the original output class CL proposed classification model.

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3. Classify DL using the trained network The Rand index has a value between 0 and 1 N1. with 0 indicating that the two set of data clusters do 4. Separate the Outliers OL from DL not agree on any pair of points and 1 indicating that Based on the Log-Sigmoid output of the the two data clusters are exactly similar. output layer of N1 Precision 5. Train Network N1 only using data Precision is calculated as the fraction of without Outliers OL correct objects among those that the algorithm 6. Classify the DT using the trained believes belonging to the relevant class. The network N1 and find the Predicted Precision is calculated as (Sundar.C and et al, 2012): Class labels. P(Lr, Si) = nri/ni } for Advantages class Lr of size nr Since the outliers are removed from the cluster Si of size ni training, the trained network will get optimum nri data points in Si from class Lr training for the normal data and so the classification Recall will get improved in the case of normal data in the Recall is the fraction of actual objects that test data set. were correctly identified. The recall is calculated as Still the system will not classify the (Sundar.C and et al, 2012) : potential outliers in the testing dataset in a accurate R(Lr, Si) = nri /nr manner since the network is not trained to handle F-Score abnormalities in the input data. F-Score or F-Measure is the harmonic mean The Metrics Used for the Evaluation of Precision and Recall and will tries to give a good Precision, recall and F-Score are computed combination of the two. It is calculated with the for every (class, cluster) pair. But Rand index is a equation (Sundar.C and et al, 2012): metric which will consider all the classes and the 2 R(L , S ) P(L , S ) F(L , S )  r i r i clusters as the whole (Sundar.C and et al, 2012). r i R(L , S )  P(L , S ) Rand Index r i r i The Rand index or Rand measure is a In a classification task, a precision score of commonly used technique for measure of such 1.0 for a class C means that every item labeled as similarity between two data clusters. belonging to class C does indeed belong to class C Given a set of n objects S = {O1, …, On} and two (but says nothing about the number of items from data clusters of S which we want to compare: X = class C that were not labeled correctly) whereas a {x1, ..., xR} and Y = {y1, ..., yS} where the different recall of 1.0 means that every item from class C was partitions of X and Y are disjoint and their union is labeled as belonging to class C (but says nothing equal to S; we can compute the following values about how many other items were incorrectly also (Sundar.C and et al, 2012): labeled as belonging to class C). Often, there is an a is the number of elements in S that are in the inverse relationship between precision and recall, same partition in X and in the same partition in where it is possible to increase one at the cost of Y, reducing the other. Usually, precision and recall b is the number of elements in S that are not in the scores are not discussed in isolation. Instead, either same partition in X and not in the same value for one measure are compared for a fixed level partition in Y, at the other measure (e.g. precision at a recall level of c is the number of elements in S that are in the 0.75) or both are combined into a single measure, same partition in X and not in the same such as their harmonic mean the F-measure, which is partition in Y, the weighted harmonic mean of precision and recall d is the number of elements in S that are not in the (Sundar.C and et al, 2012). same partition in X but are in the same partition Validating the Performance of the Classification in Y. Classifier performance depends on the Intuitively, one can think of a + b as the characteristics of the data to be classified. number of agreements between X and Y and c + d the Performance of the selected algorithms is measured number of disagreements between X and Y. The for Rand Index, Precision, Recall and F-Measure. Rand index, R, then becomes (Sundar.C and et al, Various empirical tests can be performed to compare 2012), the classifier like holdout, random sub-sampling, k- fold cross validation and bootstrap method. Here we a  d RI  did Holdout Cross validation for evaluating the a  b  c  d proposed classification models.

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Holdout Cross validation (It is equal to k-Fold 3. FM - # of fetal movements per second Validation with k=2) 4. UC - # of uterine contractions per second The holdout method is the simplest kind of 5. DL - # of light decelerations per second cross validation. This 2-fold cross validation is the 6. DS - # of severe decelerations per second simplest variation of k-fold cross-validation. For each 7. DP - # of prolongued decelerations per second fold, we randomly assign data points to two sets d0 8. ASTV - percentage of time with abnormal short and d1; so that both sets are equal size (this is usually term variability implemented by shuffling the data array and then 9. MSTV - mean value of short term variability splitting it in two). We then train on d0 and test on 10. ALTV - percentage of time with abnormal long d1, followed by training on d1 and testing on d0. The term variability advantage of this method is that it is usually 11. MLTV - mean value of long term variability preferable to the residual method and takes no longer 12. Width - width of FHR histogram to compute. However, its evaluation can have a high 13. Min - minimum of FHR histogram variance. The evaluation may depend heavily on 14. Max - Maximum of FHR histogram which data points end up in the training set and 15. Nmax - # of histogram peaks which end up in the test set, and thus the evaluation 16. Nzeros - # of histogram zeros may be significantly different depending on how the 17. Mode - histogram mode division is made. 18. Mean - histogram mean This has the advantage that our training and 19. Median - histogram median test sets are both large, and each data point is used for 20. Variance - histogram variance both training and validation on each fold. 21. Tendency - histogram tendency We used Holdout Cross validation (or k- 22. CLASS - FHR pattern class code (1 to 10) Fold Validation with k=2) because, the dataset 23. NSP - fetal state class code (Normal=1; contains sufficient amount of samples which can be Suspect=2; Pathologic=3) separated and used for training and testing (50%, Class Information 50%). We used the data for a three class Further, instead of doing holdout cross classification problem. The descriptions for the three validation for one time, the data set is randomly classes are permuted and the training and testing records were Normal: A CTG where all three features fall into the randomly taken for 10 times and the average result of reassuring category 10 such holdout cross validations were only Suspicious: A CTG whose features fall into one of considered. the non-reassuring categories and the reassuring 3. Implementation and Evaluation category and the remainder of features are reassuring For implementing and evaluating the Pathological: A CTG whose features fall into two or proposed improved neural network based more of the Non-reassuring the reassuring category classification system, and normal BPM and SVM or two or more abnormal categories. based classifier, we used Matlab 7. The RBF method The Visualization of Data Space is implemented and evaluated using Weka data The image (Figure 5) shows the projection mining tool. of this 21 attribute (dimension) data in to a virtual Data Set Information three dimensional data space. We used three principal For evaluating the algorithms under components of the data for this projection. In this consideration, we used cardiotocograms data from plot, the normal CTG data points are shown in black UCI Machine Learning Repository. dots, the suspicious data points are shown as blue This data set contains 2126 fetal dots and the Pathologic data points are shown as red cardiotocograms belonging to different classes. The ‘x’ mark. This figure roughly shows the distribution data contains 21 attributes and two class labels. The of the data in the virtual space. CTGs were classified by three expert obstetricians 4. Results and a consensus classification label assigned to each The following table shows the performance of them. Classification was both with respect to a of RBF Networks. morphologic pattern (A, B, C. ...) and to a fetal state (N, S, and P). Therefore the dataset can be used either Table 1. Classification Performance of RBF Network for 10-class or 3-class experiments. Here we use this Class Precision Recall F-Measure data set for these evaluations. Normal 0.952 0.897 0.924 Attribute Information Suspicious 0.512 0.729 0.601 1. LB - FHR baseline (beats per minute) Pathological 0.822 0.682 0.745 2. AC - # of accelerations per second

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in terms of Precision is good while classifying the Normal and Pathologic records, it was not good in identifying the suspicious cases. Particularly, the proposed method significantly improved the performance in the case of suspicious class.

Performance in terms of Precision

RBF Network SVM BPN BL-BPN

0.95 0.98 1.00 0.920.93 0.90 0.90 0.84 0.82 0.75 0.80 0.71 0.70 0.63 0.60 0.510.52 0.50

Score . Score 0.40 0.30 0.20 0.10 0.00 Figure 5. The 3D projection of CTG data shows Normal Suspicious Pathological Potential Outliers Classes

The following tables show the performance Figure 6. Performance in terms of Precision of SVM algorithm. The following chart shows the Comparison Table 2. Classification Performance of SVM of Recall under four different methods. The ANN Class Precision Recall F-Measure based classifier provided good Recall in all the cases. Normal 0.84 1.00 0.91 In terms of recall, SVM was not good in identifying Suspicious 0.52 0.20 0.29 the suspicious cases. Pathological 0.98 0.30 0.46 Performance in terms of Recall The following tables show the performance RBF Network SVM BPN BL-BPN of BPN algorithm. 1.000.970.96 1.00 0.90 0.90 Table 3. Classification Performance of BPN 0.80 0.73 0.67 0.68 0.70 Class Precision Recall F-Measure 0.70 0.62 0.62 Normal 0.9238 0.9697 0.9452 0.60 0.50

Suspicious 0.6292 0.6176 0.6220 . Score 0.40 0.30 Pathological 0.7482 0.6238 0.6780 0.30 0.20 0.20 0.10 The following tables show the performance 0.00 of the proposed BL-BPN algorithm. Normal Suspicious Pathological Classes Table 4. Classification Performance of BL-BPN Figure 7. Performance in terms of Recall Class Precision Recall F-Measure Normal 0.9345 0.9637 0.9488 The following chart shows the Comparison Suspicious 0.7110 0.6723 0.6905 of F-Score under four different methods. The Pathological 0.9021 0.6978 0.7584 proposed BL-BPN based classifier provided good F- Score in all the cases (Normal, Suspicious and

pathological). Even though the performance of SVM 5. Discussions in terms of recall is good while classifying the The following chart shows the Comparison Normal and Pathologic records, it was not good in of Precision under four different methods. The identifying the suspicious records. proposed BL-BPN based classifier provided good

Precision in all the cases (Normal, Suspicious and pathological). Even though the performance of SVM

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Performance in terms of F-Score The derived results obviously show that the proposed bi-level training improved the classification RBF Network SVM BPN BL-BPN performance of system. The BL-BPN approach 0.95 1.00 0.920.910.95 provided good performance in all cases than 0.90 0.76 compared other methods. 0.80 0.75 0.69 0.68 0.70 0.60 0.62 0.60 0.46 6. Conclusion 0.50 We have evaluated the performance of the Score. 0.40 0.29 0.30 four methods with respect to three different metrics. 0.20 The performance of standard neural network based 0.10 0.00 classification model, RBF, and SVM were has been Normal Suspicious Pathological compared with proposed BL-BPN Model. According Classes to the derived results, the performance of the proposed supervised machine learning based Figure 8. Performance in terms of F-Score classification approach provided significant performance than other compared methods. The following chart shows the performance of BPN It was found that, the proposed BL-BPN algorithm. In general, the algorithm gives good based classifier was capable of identifying Normal, performance for normal records and poor Suspicious and Pathologic condition, from the nature performance in all other cases. of CTG data with very good accuracy. If we see the Classification Performance of BPN performance of BL-BPN with respect to all the

Normal Suspicious Pathological metrics, then we can say that it almost provided

0.97 double the performance of the other three compared 1.00 0.92 0.95 0.90 methods. 0.80 0.75 0.68 So, future works may address the way to 0.70 0.63 0.62 0.62 0.62 0.60 improve the system to recognize the suspicious CTG 0.50 patterns and treat them separately while training and

Score. 0.40 0.30 testing. One may address the way to improve the 0.20 system for getting proper training with different 0.10 0.00 classes of CTG patterns. Future works may address Precision Recall F-Measure hybrid models using statistical and machine learning Metric techniques for improved classification accuracy.

Figure 9. Performance of BPN Corresponding Author: SUNDAR.C The following chart shows the performance Associate Professor, of BL-BPN algorithm. In general, the algorithm gives Department of Computer Science and Engineering, good performance for normal and pathological Christian College of Engineering and Technology, records and poor performance in suspicious records. Oddanchatram – 624619, Tamil Nadu, India. Classification Performance of BL-BPN E-Mail : [email protected] Normal Suspicious Pathological

0.93 0.96 0.95 References 1.00 0.90 0.90 Xiaojun Chen, Yunming Ye, Xiaofei Xu, Joshua 0.76 1. 0.80 0.71 0.67 0.70 0.69 0.70 Zhexue Huang , “A feature group weighting 0.60 method for subspace clustering of high- 0.50 dimensional data”, Pattern Recognition 45 Score. 0.40 (2012) 434-446, Elsevier 0.30 0.20 2. Shahad Nidhal, M. A. Mohd. Ali1 and Hind 0.10 Najah, “A novel Cardiotocography fetal heart 0.00 Precision Recall F-Measure rate baseline estimation algorithm”, Scientific Metric Research and Essays Vol. 5(24), pp. 4002-4010, 18 December, 2010 3. Ana. Klimesova, Eva Ocelikova Figure 10. Performance of BL-BPN algorithm Multidimensional Data Classification,

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Proceedings of the 10th WSEAS International 14. http://www.iasri.res.in/ebook/win_school_aa/no Conference on Automation & Information, tes/Evaluation_Measures.pdf ISSN: 1790-5117, ISBN: 978-960-474-064-2 15. Sundar Chinnasamy, Chitradevi Muthusamy 4. Stirrat, Mills and Draycott, "Notes on Obstetrics and Geetha Ramani G “ An Analysis on the and Gynaecology for the MRCOG, 5th Edition", Performance of Fuzzy C -Means Clustering 04 Aug 2003, ISBN: 9780443072239 Algorithm for Cardiotocogram Data Clustering” 5. Diogo Ayres-de-Camposa, Cristina Costa- Caspian Journal of Applied Sciences Research, Santosb, Joa Bernardesa, "Prediction of ISSN 2251 – 9114 Vol. 1 no. 13 pp. 35-42, neonatal state by computer analysis of fetal 2012 heart rate tracings: the antepartum arm of the 16. C. Domeniconi, D. Gunopulos, S. Ma, B. Yan, SisPorto1 multicentre validation study”, M. Al-Razgan, D. Papadopoulos, 2007.Locally European Journal of Obstetrics & Gynecology adaptive metrics for clustering high dimensional and Reproductive Biology 118 (2005) 52-60. data, Data Mining and Knowledge Discovery 14 6. Shomona Gracia Jacob and R. Geetha Ramani, (1) 63–97. "Evolving Efficient Classification Rules from 17. K. Cios, W. Pedrycz, and R. Swiniarsk, 1998. Cardiotocography Data through Data Mining Data Mining Methods for Knowledge Methods and Techniques", European Journal of Discovery. Boston: Kluwer Academic Scientific Research, ISSN 1450-216X Vol.78 Publishers. No.3 (2012), pp.468-480 18. J. Han and M. Kamber, 2000. Data Mining; 7. Sundar.C, M.Chitradevi and Dr.G.Geetharamani Concepts and Techniques, Morgan Kaufmann “Classification of Cardiotocogram Data using Publishers Neural Network based Machine Learning 19. Ben Kao, Sau Dan Lee, Foris K.F.Lee, David Technique” International Journal of Computer W. Cheung, Wai-Shing Ho,” Clustering Applications, ISSN 0975 – 888 Vol. 47 No. 14 Uncertain Data using Voronoi Diagrams and R- (2012) pp. 19-25. Tree Index” IEEE Transactions on Knowledge 8. Han, Jun; Morag, Claudio (1995). "The and Data Engineering, Vol. 22(9), pp. 1219 – influence of the sigmoid function parameters on 1233, sep 2010. the speed of backpropagation learning". In Mira, 20. Bin Jiang; Jian Pei, 2011, Outlier detection on José; Sandoval, Francisco. From Natural to uncertain data: Objects, instances, and Artificial Neural Computation. p. 195-201 inferences, IEEE 27th International Conference 9. Sundar.C, M.Chitradevi and G.Geetharamani, on Data Engineering, ,doi: “An Analysis on the Performance of k-means 10.1109/ICDE.2011.5767850 Clustering algorithm for Cardiotocogram Data 21. Onisko, A., & Druzdzel, M. J. 2011. Impact of clustering” International Journal on quality of Bayesian network parameters on Computational Sciences & Applications, ISSN accuracy of medical diagnostic systems. In 2200 – 0011, Vol. 2 No. 5 (2012) pp. 11-20. Working Notes of the 2011 AIME’11 10. http://www.academicjournals.org/SRE, ISSN Workshop on Probabilistic Problem Solving in 1992 – 2248 , 2010 Academic Journals Biomedicine. Bled, Slovenia. 11. Antonia Costa, MD; Diogo Ayres-de-Campos, 22. Philip A Warrick, Emily F Hamilton, 2011. PhD; Fernada Costa, MD; Cristina Santos, MS; Subspace detection of the impulse response Joao Bernardes, PhD, “Prediction of neonatal function from intra-partum Cardiotocography, academia by Computer analysis of fetal heart Conf Proc IEEE Eng Med Biol Soc. 2011 Aug, rate and ST event sibnals” 2009 AJOG – pp5678-81 American Journal of Obstetrics and 23. Chen CY, Chen JC, Yu C, Lin CW.2009. A Gynecology. comparative study of a new Cardiotocography 12. http://www-h.eng.cam.ac.uk/help/tpl/ programs/ analysis program. Conf Proc IEEE Eng Med matlab.html. Biol Soc. 2567-70. 13. Barnett, V. and Lewis, T.: 1994, Outliers in Statistical Data. John Wiley & Sons., 3rd edition.

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The impact of descriptive evaluation on knowledge and achievement of Meta cognitive of the elementary students of Boushehr city

Fatemeh Mahzounzadeh

E-mail: [email protected]

Abstract: The current research has been done with the purpose of impact of reviewing of descriptive evaluation on met cognitive knowledge of the elementary students. The process of this research is over- comparative. Its statistic society includes the students of four ranks of elementary students of Bousheshr city in 2009-2010. Due to the fact that the evaluation is done only in two schools and includes 180 students, due to being small society, the entire students were used as sample. The statistic sample includes girl students and boy students of fourth rank. In this direction there was used of the Panaura & Filipoo met cognitive knowledge questionnaires (2005) of students. The conclusion of the regression analysis test implies of significant impact of descriptive evaluation into the traditional evaluation on the level of the Meta cognitive knowledge. [Fatemeh Mahzounzadeh. The impact of descriptive evaluation on knowledge and achievement of Meta cognitive of the elementary students of Boushehr city. Life Sci J 2013;10(1):252-255] (ISSN:1097-8135). http://www.lifesciencesite.com. 38

Keywords: Descriptive evaluation, Meta cognitive knowledge, achievement, Boushehr, elementary course

1. Introduction problem solving skills, supervision on learning itself Education system needs to use education and its control… evaluation needs to use evaluation in order to determine the measure of untraditional strategies (Gary Natriello. 1987). One success of the programs and or implementation of of the reduction ways of the current problems in the education purposes. (Shymansky James A. Larry D. current evaluation system is replacing of the Yore, John O. 2004) defines the education descriptive evaluation with traditional evaluation. achievement evaluation the measurement of learner Descriptive evaluation plan is a pattern that by using functions and comparison of the results with of genetic evaluation, functional evaluation and education purposes, in order to make decision in this giving feedback tries to guide the teaching-learning issue that whether the educational activities of the process of noting to the preserves and mental teacher and learning efforts of the students, how accumulations of the students, refer to the much cause to favorable results. Evaluation affects comprehensive profundity and quality of them and the contents and education quality. (Hill Heather C., or by using of the different equipment’s like working et.al. 2005). Some define the evaluation impact in the directory, check list, view validity registration, education with the word outcome of the evaluation. functional tests …,provide a description of their (Wall & Alderson, 1993:52), This word means that learning situation and cause to the improvements and evaluation effects on teaching and learning, developments of the skills, knowledge and student education system and all of the participants in the attitudes and at the end cause to their educational education process (Andrews,2004:46 ).The main achievements. The most researches dedicates that purpose of the evaluation is facilitate of student changing the evaluation approach of traditional to the learning and improve the education and learning descriptive cause to the improvements of the students environment.Teachers use of obtained information of learning (Darling-Hammond, L. (2000), (Hanushek, all kinds of measurement strategies and evaluation in Eric A. (1992), ( Kane, Thomas J., Douglas O. order to judge about students, this information helps Staiger (2008).The reason of this act is increase in the teachers to plan learning experiences and activities in interaction between teacher and students that cause to the classroom for efficient and effective learning and frequent feedbacks between them and at the end or change it. Also evaluation is used to report the cause to the learning improvement, in this process achievements to the students and parents and help in teacher is the supporter of the students in the learning education decisions and promotion of the students environment, Dreeben, R. (2000) saw that interaction too. Evaluation was focused on the content and of the students with the teacher and classmates in the keeping in the past and present and student classes subject to descriptive evaluation plan is more. achievement is done by using of the traditional Humiliation, vituperation and extremist blames of the strategies like pencil tests-measured papers. Noting students is due to the undesirable education function the new approaches in the content of the curriculums of the current events in the schools that can consider and necessity of orientation to the creative thought, it of the emotional child abuse indicates that exam

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anxiety fear of assertive, lack of self-confident, knowledge in order to make clear the impact of the physiologic reactions in the schools under the descriptive evaluation on the meta cognitive descriptive evaluation plan is significantly less. knowledge. If the level of the Meta cognitive Whereof exam anxiety cause to the physical and knowledge of the students that are evaluated by psychological symptoms (Elaine M. Justice, Teresa descriptive process will make clear that effectiveness M.2001), then descriptive evaluation changes the and level of the descriptive evaluation on the Meta emotional space of the class with its methods and cognitive knowledge more than students that are policies and so make an environment without stress evaluated traditionally. In this direction the main and pressure. This will cause to reduction in the problem is that the level of the impact of descriptive trauma to the children (DOUGLAS F. KAUFFMAN. evaluation on the Meta cognitive knowledge of the 2004). By implementing the descriptive evaluation in students will be determined. Thus the current the elementary schools, needed knowledge and research follows up the implementation of the teachings such as Meta cognitive knowledge is following purpose. provided to the students that if be provided in 1.1. Reviewing of the level of the impact of appropriate expertise of its learning, can turn the descriptive evaluation on the Meta cognitive students to the most efficient learners. This is done knowledge of the student by informing the student by teacher. In fact this This issue consist the logical infrastructure of activity is a kind of Meta cognitive feedback that is the questions of the current research, there are raised provided by teaches to the student. Accessibility to two main questions in this research: 1- How much is the child Meta cognitive needs attention in the the impact of the level of the impact of descriptive learning process and analysis of the mistakes and evaluation on the Meta cognitive knowledge of the students achievements that cause to a kind of student? 2- How much is the impact of the traditional feedback that is famous to the Meta cognitive evaluation on the level Meta cognitive knowledge of feedback. Black & William (1998:57) too believe the student? that one of the main reasons in the education 2. Material and Methods promotion through class measurement is effective 1.2. Research process feedback to the students. In fact the feedback effect is Considering that the current research is so strong that some of the pundits called it a main reviewing the of the impact of descriptive evaluation principle in the learning. By involving the students in on the meta cognitive knowledge of the student that the descriptive evaluation can increase the power of are involved in the descriptive evaluation plan against self-measurement and similar-measurement in them. the students under the traditional evaluation and Research studies also indicated the positive effects of comparing them in two group so the used research self-measurement and similar-measurement on the process s of the kind of over-comparative. student’s educational achievement and function in Statistic society: Statistic society of this different age groups and lessen (Fotana & Fernandes, research includes students of the fourth rank of the 1994:410). Perkins & Salomon (1992 ) emphasize on elementary school of Boushehr city in the 2009-2010. the necessity of the awareness of the student of its 2.2. The sampling process: educational status and involving in a meta cognitive Generally the plan of descriptive evaluation process and consider its notion awareness of one in in the fourth rank was done only in toe schools that its thought that can control it in this way whenever he provided some questionnaire researches to all of the wants (Elaine M. Justice and Teresa M. Dornan. girl and boy students’ f these classes that included 50 2001). Black & Harrison & Lee & Marshall & girl student and 40 boy students and their meta William (2004:12) And Berry (2005:227) believe that cognitive knowledge was evaluated.From all of the students learn by two process of self-measurement elementary schools of the Boushehr, that were and similar-measurement that how to supervise on evaluated traditionally, two school was chosen that own and others learning and manage and control it. were similar to the schools that were involved in the For this reason can expect that students under the descriptive evaluation plan and from point of quantity cover of the descriptive evaluation plan, by using of of the students and education area provided some these methods will have high Meta cognitive questionnaire researches to the fourth rank students. knowledge. In this research the researcher tries to Information compilation tools in the students: From review the level of the impact of descriptive Panaura & Filipoo questionnaire (2005), that was evaluation on the Meta cognitive knowledge of the composed to measure the Meta cognitive knowledge elementary students. In It means he plans to compare of the elementary students, was used. This the impact of the descriptive evaluation and questionnaire includes 30 inserted phrase and of the traditional and the students that were evaluated by likert spectrum. The selections are (always, often, these two methods, from the level of meta cognitive sometimes, and rarely) and grading them is

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considered for positive statements since 4 to 1 and for 4. Discussions negative statements since 1 to 4. Panaura & Filipoo The subject of this research is reviewing of (2005) reported the stability of this test 78%. It the impact of the descriptive evaluation on the Meta considered this test with high justifiability. In the cognitive knowledge of the elementary students. The current research too once again the coefficient of purpose of the implementation of this research, is stability of the test was calculated by method of determining the level of the effectiveness of the Alpha-Chronbach and the value of it was 82% that is descriptive evaluation on increasing the Meta significant in the level of the (p<0.001). cognitive knowledge of the students of the 3.2. Statistic methods of data analysis elementary school. For this purpose to determine the There was used of SPSS software with 16 impact of the descriptive evaluation on the Meta versions in this research in order to analysis of the cognitive evaluation the test of the regression data. In the perceptive level there was used of the analysis was calculated. The result of this test simple regression analysis. dedicated that descriptive evaluation on the meta 3. Results cognitive knowledge has significant and positive 1.3. The impact of the descriptive evaluation on effect and is one of the main reason and factors in the Meta cognitive knowledge prediction of the meta cognitive knowledge.The There was used of the regression analysis in obtained results had coordination with the similar order to determine the impact of the descriptive researches such as Mouhen &Stalling (1972), Gracc evaluation on the Meta cognitive knowledge, the (1992) Fontana & Fernandes (1994), Mouya & Mali obtained results of this test are mentioned in the (1994), Camp & Petrov (1998), Fucoz And following table. colleagues (1999), Yong(2000), Van ovara (2004). Relevant to the main purpose of the research that is Table (1): coefficients of standardized and the same reviewing of the impact of the descriptive unstandardized regression evaluation on the Meta cognitive knowledge, the Significant level R2 R2Adjusted B β relation between these two variant is calculated too. This relation is obtained (r=0.48) that was significant Descriptive evaluation 0.234 0.226 5.298 0.484 0.000 in the (p<0.000) level. Generally all of the results The results indicated that the impact of the indicate of this main principle that the Meta cognitive descriptive evaluation on the Meta cognitive knowledge is under the impact of the descriptive knowledge is significant in the (p<0.01) level. The evaluation. In the interpretation of these results can results indicated that 22.6% of variance of the Meta refer to the theory of the Bendoura (1986). He cognitive knowledge is due to the descriptive believes that one of the dimensions of the meta evaluation Prediction of the Meta cognitive cognitive knowledge, is the supervision and knowledge. evaluation of the function of oneself that this is due 3.2. The impact of the traditional evaluation on to the internal evaluations and outer feedbacks. the Meta cognitive knowledge Whereas in the descriptive evaluation system, the There was used of the regression analysis in teacher gives feedbacks to the students during order to determine the impact of the traditional learning process continuously, it seems obvious that evaluation on the Meta cognitive knowledge. The descriptive evaluation cause to increase the meta following table shows the relation and the impact of cognitive knowledge. This test was calculated about the traditional evaluation on the Meta cognitive the impact of the traditional evaluation on the meta knowledge: cognitive knowledge but the different result was obtained, in this way that traditional evaluation has Table (2) coefficients of standardized and not significant impact on the meta cognitive unstandardized regression knowledge and only the student with the grade-point Significant level average more than 19 have high meta cognitive 2 2 R R Adjusted B β knowledge. The result of the reviewing of the

Descriptive evaluation 0.039 0.028 3.527 0.197 0.063 traditional evaluation on the meta cognitive knowledge is not conform with the researches of the above mentioned researchers. Can interpret the The results indicated that the impact of the results in this way that in the evaluation in the traditional evaluation on the Meta cognitive traditional method that is mostly turn on a final knowledge is not significant in the (p<0.05) level. evaluation, teachers evaluates only instead of The level of the (β=0.197) indicates that the teaching some thing, the this that is to evaluate they traditional evaluation has not the power of the teach.. For this reason the teacher’s feedback to the prediction of the Meta cognitive knowledge. students is too simple and clear. Thus that teacher

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tests and the student receive feedback through grade, Instructional Tools Designed To Facilitate degree or a general judgment.The relied reasons to Cognitive Strategy Use, Metacognitive these researches show that this kind of feedback Processing, And Motivational Beliefs. Journal of method does not help to the student that be aware of Educational Computing Research. Volume 30. his thought process and control it and as a result has 7. Dreeben, R. (2000). Structural Effects in not any role in the educational promotion and Education: A History of an Idea. In M. T. achievement of meta cognitive knowledge. Generally Hallinan (Ed.), Handbook of the Sociology of these calculations, somehow shows the impact of the Education (pp. 107-135). New York: Kluwer descriptive evaluation on the Meta cognitive Academic. knowledge of the students. From the interpretation of 8. Elaine M. Justice, Teresa M.2001. Dornan. the results of the done researches and this research it Metacognitive Differences between Traditional- is clear that descriptive evaluation system due to the Age and Nontraditional-Age College Students. having special properties such as being performance- Adult Education Quarterly May vol. 51 no. 3 oriented, process-oriented, qualitative feedback, 236-249. dynamism and being comprehensive cause that the 9. Fontana, D, & Fernandez, M.(1994). student be active and dynamic in the class and Improvements in mathematics performance as a continuously ask question about the taught issues and consequence self assessment in Portuguese the issues of the next session, so the student increase primary school pupils.British journad of the participation in learning and subversions on his Educational psychology, 64, 407-417. affords and in fact obtains his own adjusting and self- 10. Gary Natriello. 1987. The Impact of Evaluation control, also make assessment of effectiveness of his Processes on Students. Educational Psychologist learning level. This cause to increase the Meta Volume 22, Issue 2. cognitive knowledge among the students included in 11. Hanushek, Eric A. (1992). The Trade-Off the descriptive evaluation plan into the other ordinary between Child Quantity and Quality. Journal of school ‘students. Political Economy 100 (1), pp. 84–117. Acknowledgements: 12. Hill Heather C., Brian Rowan, Deborah Author is grateful to persons for support to Loewenberg Ball. 2005. Effects of Teachers’ carry out this work. Mathematical Knowledge for Teaching on Corresponding Author: Student Achievement. Are Educ Res J June 20, Fatemeh Mahzounzadeh vol. 42 no. 2 371-406? E-mail: [email protected] 13. Kane, Thomas J., Douglas O. Staiger (2008). References Estimating Teacher Impacts on Student 1. Berry, R. (2005). Entwining feedback, self, and Achievement: An Experimental Evaluation. peer assessment. Academic Exchange Quarterly, NBER Working Paper 14607. Cambridge, MA: 9, 225-230. National Bureau of Economic Research. 2. Black, p., Harrison, C. Lee, C, Marshall, B.& 14. Perkins, D. & Salomon, G (1992). The Science William, D.(2004).Working inside the black and Art of Transfer. box: Assessment for learning in the 15. Richard T. Lapan, Norman C. Gysbers., classroom.phi Delta kappan. 86,8-21. Yongmin Sun.1997. The Impact of More Fully 3. Black P.I & Wiliam, D.(1998).Assess ment and Implemented Guidance Programs on the School classroom learning. Experiences of High School Students: A 4. Bransford, J. D., Brown, A. L., & Cocking, R. R. Statewide Evaluation Study. Journal of (Eds.). (1999). How People Learn:Brain, Mind, Counseling & Development Volume 75, Issue 4, Experience, and School. Washington, D.C.: pages 292–302. National Academy Press. 16. Shymansky James A. Larry D. Yore, John O. 5. Darling-Hammond, L. (2000). Teacher Quality 2004.Anderson. Impact of a school district's and Student Achievement: A Review of State science reform effort on the achievement and Policy Evidence. Education Policy Analysis attitudes of third- and fourth-grade students. Archives, 8(1), 50. Journal of Research in Science Teaching, 6. Douglas F. Kauffman. 2004. Self-Regulated Volume 41, Issue 8, pages 771–790. Learning In Web-Based Environments:

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Consistency of linguistic domain of the Quran of Quds with Dehei dialect in central plateau of Iran

Ali Akbar Nargesian

Natanz branch, Islamic Azad University,Natanz, Iran

Abstract: Translation of Quds Quran is of special value from the perspective of Persian language history. This valuable work has also allocated a special position to itself from the view of translating Quran to Persian language, since some existing documents indicate that the creation of this valuable work belongs to the time of language separation between Middle Persian and New Persian. The history of Persian language during the early centuries after Islam is an aura of mystery. And the Persian written inheritance in Arabic script which has remained from the language separation era or silence period is very little. Dr. Ravaghi, the hardworking editor and introducer of Quds Quran says: “Quds Quran is worth of profound historical social research ; and as a historical document of Persian language, it must be studied and researched by Persian language researchers and linguists.” In this paper, the author has analyzed Quds Quran by studying the language domain of translation of Quds Quran and its similarities with Dehei dialect (Boreh and Bashe. [Ali Akbar Nargesian. Consistency of linguistic domain of the Quran of Quds with Dehei dialect in central plateau of Iran. Life Sci J 2013;10(1):256-261] (ISSN:1097-8135). http://www.lifesciencesite.com. 39

Keywords: Species richness; beta-diversity; taxonomic diversity; forest

1. Introduction of Astan-E Quds-E Razavi as number 54. The date of In the Sassanid era, education and knowledge its compilation is unknown, but Quranic and literary was devoted to certain classes of the society and it researchers consider it as one of the most precious was impossible for all to pick clusters of the and ancient and perfect translations of Quran in knowledge harvest. Moreover, “it was unjust to teach Persian language. Respected researcher Dr. Ali people reading and writing.” (Tamimdary, 2009 : 98) Ravaghi after several years of doing research and Therefore at that time, most people were familiar attempting hard published this translation in 1985 and with their own native language just through introduced it to Quranic society and Persian literature conversation in society or in family and ordinary and from then it became known as “Quds Quran” or people didn’t know how to read and write. But after “Quran of Quds translator”. “Quran of Quds” is both the emergence of Islam in Iran and its acceptance by an example of an accurate, perfect and conscious Iranian scientists specially people of Transoxiana translation of Quran and an indicator of people’s (Big Khorasan), they used Quranic or Arabic script need and their attention to translating Quran to and this way they opened the doors of learning and Persian language.” (Mahootian Shahrzad, (1997).). education to public and ended the domination of The manuscript translation contains 205 large pages Pahlavi written language. This event led to the bigger than that of Vaziri with external size of gradual training of scientists and scholars in first (29*30) cm. Each line is as long as 24cm. Its pages century who had good command of both oral and are fawn-colored and thick but they are Vera written Persian language. Then they began to spread damaged in edge. There is no date of its writing or its theological and Quranic science in Persian language translation or the name of writer or translator in text and on the other hand they published Persian culture or in the margin of the book. The existing manuscript and sciences in Arabic language in Islamic countries lacks any ornament or illumination like what exists among which Persian translations of Quran such as today, but in the margin of some pages there are translation of the Quds Quran, Arabic books such as several spiral, concentric, radial bearing circles or Tabari History in Arabic and its translation in Persian semicircles which have made it really attractive. entitled Tabari Interpretation, Bal’ami history, Arabic writing of the book goes back to the time science evolution (Ehya’ol Oloom) by Imam when Quranic verses and suras were not numbered Mohammad Ghazali and its translation in Persian by yet. Each surah begins with “In the name of God” in one bold line except the “surah (ﺑﺴﻢ ﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﯿﻢ) -the same author entitled Prosperity Chemical(kimiya ye Sa’adat) can be referred to( Mahootian Shahrzad, of repentance” and after the name of each prophet the (1997). expression “peace be upon her” is used. Arabic and 1.1. Quran of Quds Persian texts lack any punctuations, symbols or letter The precious Quran in manuscript translated by signs or any division to smaller sections (verse, “Quds translator” is an ancient translation by an chapter, unit, part,). There are no Quranic signs such ٍ◌ )/unknown translator which is maintained in the library as / œ /( ◌َ ),/ e / ( ◌ِ ),/ ō / ( ◌ُ ), or /an/( ◌ً ), /en

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on/ ( ◌ٌ ) like what exists today, but on stressed expressive one like ancient translations, and the/,( letter the sign of stress( ◌ّ ) is seen. Some translator has tried to make a translation which on punctuations related to the first century which were one hand expresses Quranic meanings and on the invented by Abolasvad Dueli (death : 69 solar year) other hand will be a tool for knowing and are seen on letters such as hollow or full circles, not understanding Quran. Therefore it seems that less as bold as the background color, which are used as attention is paid to the structure of sentences. vowels /œ /, / e /, / ō /, above, below, or in front of Translation is in the form of simple and fluent prose letters. Also Tanvin (diphthongs) are places as two without any rhyme and for ordinary people in an area vertical dots above, below, and in front of words with special language and is free from any technical respectively. Most similar letters with or without dots verbal, scientific or artistic expressions. Moreover, and letters with tooth are written like the past style, there is no explanation about the contents in the text that is without dots or tooth both some similar letters or in the margins. It seems that the translator has in one word are distinguished by placing dots such as mainly tried to offer a perfect pure translation and / s/ without any extra information and in this style he has (ث)/θ/ ,(ت)/t/ ,(ب)/and /b ( ذ) /and /z (د) /d/ Arabic text is translated the words and expressions very carefully .(خ)/x/ ,(ح)/h/ ,(ج)/and /j ,(ش)/ ŝ/,(س) written in changed legible Kufic script – in and cautiously by finding proper equivalence and or comparison to old Kufic script- and with black ink. making equivalence and by using borrowed words Persian text is written in middle Naskh which is not from Arabic language or local terms. In examining very ancient. This balanced translation is considered translation, many cases are found in which the as one of the rich sources of studying and knowing translator himself has created new terms through Persian language and local-regional dialects. combination and derivation by means of prefixes, “Although the historical social value of most ancient infixes, and suffixes such as ( ﺗﻨﮭﺎ ﮐﺎن =translations overweigh their linguistic value, this ( Tanhakan ( راﺳﺖ ﮐﺮﮔﺮﻓﺘﻦ=precious translation is important not only from the (rast kargereftan ( وراورورزﻧﺪ = view of the history of translation to Persian language (vera varvarzand ( ﺳﺘﻢ ﮐﻨﺎران = but also from the perspective of the history of (setamkenaran (ﺗﺮﺳﺎﮐﺎن =language and is considered as one of the most (Tarsnakan ( در ﮔﺮﻓﺘﺎر =precious and unique literary works.” (Lazard, Gilbert, (dar gereftar ( ﭘﺪﯾﺪه آﻣﺪاران =Translation of Quran began since the (padideh umdaran .(1971 ( ﻧﯿﮑﺒﺨﺘﺸﺪاران =beginning of Islam with the efforts of Salman Farsi (Nikbakhtashdaran .and etc ( ﻧﯿﺸﺘﺎواﻧﯿﺪﯾﺪ = and has continued until today (Windfuhr, Gernot. (nishtavanidid 2009). Lots of essays and books have been written about the history of translation and translations of Deriving local-regional terms is another feature of Quran. Translation of the Quran of Quds is a literal -- this style which is found a lot in the text such as: word-for-word – translation written between lines. gorse : gorosneh)= hungry )ﮔﺮﺳﮫ: ﮔﺮﺳﻨﮫ The translator has tried to write Persian words one by givekan : Bive gan)= widows) ﮔﯿﻮه ﮔﺎن: ﺑﯿﻮه ﮔﺎن one under the Quranic terms. This method is as old as kang : ankaboot) : spider) ﮐﻨﮏ: ﻋﻨﮑﺒﻮت one thousand years and many of independent dojak : gereh, oghde) = knot) دﺟﮏ: ﮔﺮه،ﻋﻘﺪه ’translations or the translations of predecessors kolunak : cheraghdan) =light stick) ﮐﻠﻮﻧﮏ: ﭼﺮاغ دان interpretations, like the translation of Cambridge copy, translation of pure Quran, and translation of Another feature of this translation is using a few some part of ancient interpretation, Tabari Arabic terms with little changes in Persian text: interpretation, and Surabadi interpretation are of this In verses 66 and 73 of Surah Ya Sin: (ﺑﺴﻢ ﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﯿﻢ) kind. For example in translating ( َو ﻟَﻮ َﻧ ُﺸﺎء ﻟَ َﻄ َﻤﺴﻨﺎ َﻋﻠﯽ اَﻋﯿُ ِﻨ ِﮭﻢ...): ,it has come, In the name of the God of Kindness ار ﺧﻮاھﯿﻢ ﻣﮭﺎ ﮐﻨﯿﻢ ور ﭼﺸﻤﮭﺎ اﯾﺸﺎن... merciful along. Also in translating verses 4, 5, and 12 of Safat Surah it has come: If we want to cover their eyes َو ﻟَﮭُﻢ ﻓﯿﮭﺎ َﻣ ِﻨﺎﻓﻊُ و َﻣ ِﺸﺎر ُب َا َﻓﻼ َﯾ ُﺸﮑﺮون واﯾﺸﺎن را در آﻧﺠﺎ ﻣﻨﻔﻌﺖ ھﺎﯾﯽ و ﺷﻤﯿﺪن ﺟﺎی ھﺎﯾﯽ ای ( ِا ﱠن ِا َﻟﮭ ُﮑﻢ ﻟَ ِﻮاﺣ ٌﺪ ) ﺷﮑﺮ ﻧﻤﯽ ﮔﺰارﻧﺪ .Your God is one ( َر ﱡب اﻟﺴﻤﻮات َو َاﻻرض َو َﺑﯿﻨﮭُﻤﺎ َو َر ﱡب اﻟﻤﺸﺎرِق) The God of heavens and the Earth and that of And there are benefits and drinks for them between them and the God of the East. there, but they do not thank. ( َو اِذا ُذ ﱢﮐﺮوا ﻻ ﯾَ ﱠﺬ ﱠﮐﺮون) They were given advice by us, they won’t accept Another characteristic is applying words any advice. Translation style of Quds Quran is an which are derived from Arabic text without making

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any changes in them. Although these borrowed terms equivalent to 8th or 9th century A.D.” (Rypka, Jan. are not a lot in comparison to those of 6th and 7th solar 1968). Various gusses made about the writing time of centuries translations (12th and 13th C), whenever the the text could be studied based on the script and the translator has not been able to find a Persian linguistic signs used in the text but considering the equivalence for a word, or to make an equivalence script of Quran which is developed Kufic script and himself, he has exactly entered some Arabic terms in the black ink which is used and the translation which Persian language which is somehow remarkable. is written in elementary Naskh script and under the Some examples are as follows: lines, it seems that both Quran and its translation had been written in the same time due to the similar color ( ُد ًﺣﻮرا َو َﻟﮭُﻢ َﻋ ٌﺬاب ِواﺻ ٌﺐ ) (ﺻﺎﻓﺎت/9): of the ink used in them as in translator’s manuscripts ﺑﮫ دور ﮐﺮدن و اﯾﺸﺎن را ﻋﺬاﺑﯽ داﺋﻢ ﺑﮭﺪ Avoid them and for them there is an everlasting it is assumed that both the original text and its punishment (Safat: 9) translation belong to the same period of time. Based on this assumption there are some script signs which .can direct us toward the estimated time of its writing ( َو ﯾَ َﺮی َاﻟﺬﯾﻦ ِاوﺗﻮااﻟﻌﻠﻢ...) (ﺳﺒﺎ/6): Quran’s manuscripts attributed to the companions of ﮔﯿﻨﻨﺪ اﯾﺸﺎن ﮐﺪاده ﺷﺪﻧﺪ ﻋﻠﻢ... And they see those who were given knowledge (Saba: the prophet (PBUH) or Imams (AS) which have been 6) written after the death of the holy prophet (PBUH) lack any punctuation or writing signs or shapes and ( ُﮐﻼٌ َﻧ ٍﻔﺲ ذاﺋِ َﻘﺖ اﻟﻤﻮت...) (اﻧﺒﯿﺎ/35): similar letters are free from dots. The script used by ھﺮ ﻧﻔﺲ ﭼﺸﯿﺪار ﻣﺮﮔﯽ اﺳﺖ... Each soul will taste death (Prophets: 35) Arabs in early periods of Islam for showing the sounds of vowels or voiceless sounds and recognizing similar letters was very simple and (و اِذ ﯾَ ُﻘﻮل ُاﻟﻤ َﻨﺎﻓﻘﻮن و َاﻟﺬﯾﻦ ﻓﯽ ﻗُﻠﻮﺑِ ِﮭﻢ َﻣ َﺮ ٌض ﻣﺎ َو َﻋﺪﻧﺎ elementary and free from symbols or signs of today’s ﷲُ َو َرﺳﻮﻟﮫ اِﻻ ًﻏﺮورا ) scripts. At first Arabs were reading Quran loudly and accurately without any mistakes due to their own (اﺣﺰاب/12):ﮐﮫ ﻣﯽ ﮔﻔﺘﻨﺪ ﻣﻨﺎﻓﻘﺎن و اﯾﺸﺎن ﮐﺪر دﻟﮭﺎ اﯾﺸﺎن intelligence and their interest and with the help of ﺑﯿﻤﺎری:وﻋﺪه ﻧﮑﺮد اﯾﻤﺎ را ﺧﺪای و ﭘﯿﻐﺎﻣﺒﺮ اوی ﺑﯽ ﻓﺮﯾﻮ. And when he says to hypocrites and those who have their strong memory and there were rarely mistakes unhealthy hearts, they wound not accept the promise in their readings. But after the expansion of of God and his prophet but proudly. geographical territory of the Moslems to other Arab and non-Arab lands and linguistic exchanges of The Quran of Quds has been translated by words and accents and dialects, arrangements of an unknown translator. There is no sign of the changes in accents and eloquent language were translator in this book. The date of its writing is provided. Arab scholars set out the writing rules and unknown and it can’t be surely said when or where or grammar to modify eloquent accent for reading and in what language domain it has been formed. But memorizing Quran. In the book of the history of holy some guesses have been made by researchers based Quran it is said: on the linguistic evidences and witnesses. Dr. First, simple and complete grammar was Ravaghi, the respected editor of holy Quran of Quds made for Arabic language in first half of first century considers this translation as the most ancient under the command of Amir Al-Mo’menin Imam Ali translation to Persian believes that it is closer to (PBUH) and with the attempts of Abolasvad Dueli. middle Persian due to the morphological and the He began to punctuate and use phonetic symbols for conjugation forms and the words used in it. He vowels in Quranic letters and words for the first time. argues that this Quran belongs to the second half of (Hojati, 456 : 1985). third solar century or the first half of fourth The signs made by Abolasvad, were very century.(Ravaghi, 1985: 10). Considering the time of simple and elementary like these: writing the Quran of Quds, Professor Gilbert Lazar 01. /æ/ (above) : a hollow circle on the letter believes that with regard to the use of verb making 02. /e / (under) : a hollow circle under the letter prefix “mi-” instead of “hami-” and the preposition 03. /o/ (next to) : a hollow circle in front of the letter “dar” instead of “andar” in translated text, this This signs are used in the Quran of Quds instead of translation is not older than the fifth solar century in forms, signs and symbols of vowels. spite of all its ancient signs and preferably belongs to From this viewpoint, the book has been written the second half og 5th century. (Lazar, 2005: 158). earlier than 4th or 5th centuries (10th and 11th Another researcher has stated about the time of centuries. A.D.). translation that: “This ancient precious work has been From linguistic viewpoint, the ancient language of written in old Kufic script and its writing seems to translation and using very rare or obsolete words are belong to the late first solar century which is remarkable:

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texts. And with referring to lexical and structural khish) : mud characteristics, he finally proposed this theory in) ﺧﯿﺶ kank): spider terms of geographical terms of the translation which)ﮐﻨﮏ .barkhari): ignorance and unawareness belongs to Sistan)ﺑﺎرﺧﻮاری setadi): shelter Professor Lazar, believes that this work is)ﺳﺘﺎدی kinva) :trunk close to ancient Persian-Jews while showing some)ﮐﯿﻨﻮا azan): mate dialect features and based on the presence of some)ازن bar mou girid ): wait obsolete and archaic words in the Quran of Quds, and)ﺑﺮﻣﻮﮔﯿﺮﯾﺪ barkhari kardam) : I exceeded he guesses that it was written in an area near the)ﺑﺎرﺧﻮاری ﮐﺮدم :dajak bastid): you promised south east of Iran, that is Baloochestan. (Lazar, 2005)دﺟﮏ ﺑﺴﺘﯿﺪ .(rast kar gereft) : he believed 159-162) راﺳﺖ ﮐﺮﮔﺮﻓﺖ dandan sepid kard): smiled, laughed Although Dr Ravaghi believes that the)دﻧﺪان ﺳﭙﯿﺪﮐﺮد Also some linguistic signs of Middle Persian are used translation of this Quran belongs to Sistan, he has va): and also posed this question that : since every text)«و» gu) instead of)«ﮔﻮ» such as including the translations of Quran has been created ﮔﻮرده (gurde)= ورده( varde)= ﺑﺮده:(has taken : (borde in response to social needs, have been people who ﮔﻮاﻓﺘﮫ(Guofte)=واﻓﺘﮫ(vafte)=ﺑﺎﻓﺘﮫ(has woven:(bafte vah)wow were familiar with the language of this translation)=وه(guh)ﮔﻮه living in other places except Sistan, too? (Ravaghi, b/ of Middle Persian in this 1985:71). In response to this main question it should/ «ب» The beginning g/ : be noted that “in villages and small towns in central/ «گ» translation is used as part of Iran and in the rural areas scattered along the ﮔﺘﺮ(gatar) = ﺑﺘﺮ(better:(batar deserts there are still numerous dialects including the ﮔﯿﻮه(give)ﺑﯿﻮه(widow:(bive ,dialects in areas between Kashan and Isfahan ﮔﮭﺸﺖ(gehesht)=ﺑﮭﺸﺖ(put:(behesht .dialects of Natanz and Naeen” (Clawson, Patrick ﮔﯿﻨﺪ(ginad )= ﺑﯿﻨﺪ(will see:(binad 2005). In relation to the dialects of central part of Iran Second person pronoun is used as which are generally called Raji, Madi dialects, some Ima)(we) …. and research has been done. Geokovski, Aranski, La)اﯾﻤﺎ toa)(you) and first person as) ﺗﻮا and Cook, and Yar Shater have done some valuable"ﺑﻲ" from Middle Persian as "اﺑﻲ" using the word .ela) (to), research in this area and have raised some arguments)، اﻟﻲ ، (ala)(Ay)اﻻ with the same meanings laken) (but) and special script of certain terms Some of these linguists have classified all the dialects)ﻟﻜﻦ Khorshid) of this area under a series of signs in a general)ﺧﺮﺷﯿﺪ:ﺧﻮرﺷﯿﺪ ,(aan) (that)اان:آن such as framework. Among the central dialects of the deserts ھﻤﺘﺎان:ھﻤﺘﺎﯾﺎن ,(Jahan)(world)ﺟﮭﻦ:ﺟﮭﺎن ,(Sun) Mah) of Kashan and Naeen, is Dehei dialect (Boreh and) ﻣﺎه : (mayegan)ﻣﺎﯾﮕﺎن ,(hamtayan) (the same) Basheh) in central plateau of Iran which is a part of ”آن“ Moon) and making plural nouns by suffixes) ha), in spite of other translated Qurans, the town of Natanz and is similar to the ancient)”ھﺎ“ aan) and) not using Arabic irregular plural form in the dialect of this town which is known as Raji dialect. æ) such as As mentioned before, dialect of Dehei is now very)"ا" translated text and using initiating .common in some districts and villages of Natanz ﯾﺎد =(ayad)اﯾﺎد shotor):camel, With an area of about 3400 km2 and population of)ﺷﺘﺮ=(Oshtor)اﺷﺘﺮ,yad):memory) shekam): belly 50,000 people, Natanz is one of the 20 towns of)ﺷﻜﻢ=(eshkam)اﺷﻜﻢ Are some other features of this style of translation? Isfahan province which is located in an area between the central mountains of Iran ( Karkas mountain) and 2.1. Language domain of the Quran of Quds with the desert plain. This town includes a central part and Dehey dialect in central plateau of Iran a shrine. Shrine in desert area includes two small A number of words of the Quran of Quds towns Badrood, Khaledabad, and several villages, have certain similarities with the dictionary of among which Arisman that is 7 km far from Badrood “Mohazabol-Asma” (purified names) “The History of is an ancient historical village. In 1966, archeologists Sistan” and “Masaderologhat”(word infinitive), and have estimated that ancient Arisman is as old as third certainly all three books have been written in Sistan. or fourth millennium B.C. Aryanpur, Manoochehr, This similarity of the terms of the Quran of Quds believes that: “Arismani-Badroodi dialect or Dehei with these texts indicates that the translator of Quran dialect is the ancient heritage of our ancestors and it must be probably from Sistan –Kerman. (Ravaghi, is believed that this dialect has been originated from 1985:35). This is the first claim about the region of Ancient Farsi and Pahlavi” (Aryanpur, Manoochehr. writing the Persian text of Quds Quran. In order to 1973). know the geographical region of this translation, Dr.Ravaghi has studied common words in ancient

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Table 1: compatibility between this dialect of central During the research and his studies, the area and the dialect of the Quran of Qud writer has noticed some lexical and structural similarities between the quran of Quds and Dehei dialect (Boreh and Basheh) in central plateau of Iran or Arismani-Badroodi dialect. Among the words which are used in daily dialogues of the people in this geographical area, some words are found from Avestan, Pahlavi, Middle Persian, and Dari Persian languages which have allocated special status to themselves in this dialect. Considering these similarities, it is concluded that there is some compatibility between this dialect of central area and the dialect of the Quran of Quds and the researcher has tried to somewhat show this compatibility in the Table 1. In this regard, there are some other similarities between the words of Quran of Quds and those of DEhei or Raji dialect in central plateau of Iran (Arismani – Badroodi); some of these similarities are hereby referred to. vei rah tou (birahe) Quds, p. 98, (verā) : bi rah, Varoh (birahe) :wrong way mehravan (mehraban) Quds, p.224, (mehravun) : Kind baŝid: beravid, Quds, p.199, bashide, bashi, and bashe (boro) : go (This word is still very common among people in past, present, and imperatives for first, second and third persons. nishanand, Quds, p. 98,(naŝnavend) : (they) don’t hear roodkade(vadi), Quds, p. 198, (ruxuna) : river Today instead of the word khane(home), kadeh is used. az, Quds, p. 377, (zāv) : again mahakon, Quds, p. 130, (menhā) kon : destroy, separate, vanish dar shahand, Quds, p. 407, (barŝend) : they go out dar sho, Quds, p. 301, (darŝen, darŝe): come in zoorfaraz, Quds, p.130, (zurhen) :powerful, strong Hand, Quds, p. 49 (they are): today in Raji dialect it is used as (nen)they are), and (hein, heim) : we are “They ask you when the resurrection day is” Kai: when, bohad: bod: bu; is and kainiz means when. Bi (but), Quds, p. 98: abi, bi Sord: ladder, Quds, p. 68, the word sord is obsolete. Abi and bi are very common in this dialect. Ima bid (we will bw) : amabin: amabim and imaim (we are), Quds, p. 298 :ama, ama im Nanad(they are not), Quds, p. 229 ; (na?end), (nahen), it meant ladder before, but now the word Sorod dar is still used among the Arsmani-Badroodi speakers. Doorzan(lier), Quds, p. 68 : (dorvejin) In Raji, Badroodi-arismani and Natanzi dialect, z is changed to j in many cases, such as :

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Zan (jen): woman, tondo tiz (Teij) : fast, Abriz Quds, and some of their common words and (catchment) which is used as (?Owrij). (Dehghanian, expressions were referred to. Considering such 2007:22). common words it could be inferred that during the p.51) lifetime of the translator of the Quran of Quds, at) َﻻﻣﺴﺘُ ّﻤﺎﻟﻨﺴﺎء In the Quran of Quds the expression is translated as: bemajidid women. Majididan means least 70 to 80 percent of the language used in central to touch. Today in Arismani –Badroodi dialect, plateau of Iran has been probably similar to the infinitives (vamojidan) and (bamojidan) mean to language of geographical domain of the Quran of suck. Quds in the structure of applied words. Linguistic and gadbakht(unlucky), Quds, p.195 : (vadbaxt) structural features of translation of the Quran of Quds sharestan(town), Quds, p. 191 : (ŝārestun) with the ancient dialect of Dehei (Arismani- shooyan(husbands), Quds, p. 3 : (ŝuy and šu) Badroodi) which is itself a combination of several VI niaz(rich), Quds, p. 412: (veniyāz) languages support this opinion and approves of it. Prefixes vi- and va- are still used in Raji-Arismani dialect: va che (poor), va kho(for nothing) va Andaze Acknowledgements: (infinite) Authors is grateful to Natanz branch, Noon (then), Quds, p. 412 : Noon vaght : then Islamic Azad University,Natanz, Iran for financial var godar (at the passage), Quds, p. 410 : godār support to carry out this work. :neck var(next), Quds, p. 410 : (var) Corresponding Author: vazgard(return), Quds, p. 411 : vāgel Ali Akbar Nargesian Prefix va- is used a lot such as: vāŝte : must, vāke: Natanz branch, Islamic Azad University,Natanz, Iran open, vāxord(stopped), vāgaŝt:returned [email protected] Bazhdar(flame), Quds, p. 400: (baraxŝ) Another common feature of Dehei dialect (Arismani- References Badroodi) and the language of Quds Quran is that 1. Aryanpur, Manoochehr.1973. A History of none of them has used utterances (mar), (mar … ra), Persian Literature. Tehran: Kayhan Press. (hami) and (andar). They have both used beginning 2. Chejne, A. (1969) The Arabic Language: Its spellings (abi) and (bi). Role in History, University of Minnesota Press, Minneapolis. 4. Discussions 3. Clawson, Patrick. 2005.Eternal Iran. Macmillan. Since the translation style of the Quran of 4. Lazard, Gilbert, 1971"Pahlavi, Pârsi, dari: Les Quds is expressive, word-by-word, and under the langues d'Iran d'apès Ibn al-Muqaffa" in R.N. lines, therefore the translator has used some new Frye, Iran and Islam. In Memory of the late words and some words made by him and the words Vladimir Minorsky, Edinburgh University Press. derived from Arabic language and from the region’s 5. Mahootian Shahrzad, (1997). Persian. London: geographical domain. Careful and cautious use of Routledge. ISBN 0-415-02311-4. Persian equivalences has made the translation 6. Qur'an Quds Razavi Library reserves the particularly valid but the main point is the structural manuscript and the number 54. Quran, translated compatibility and lexical similarity of the Quran of by Naser Makarem Shirazi in Qom. Quds and the ancient dialect of Dehei(Arismani - 7. Rypka, Jan. 1968.History of Iranian Literature. Badroodi). By studying and doing research on Reidel Publishing Company. different dialects in several geographical regions the 8. Windfuhr, Gernot. 2009.The Iranian Languages. researcher included that in central plateau of Iran and Routledge.. p. 418. in the deserts there were people who had the same dialect as that of geographical region of the Quran of

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Heat Shock Protein-70 and -27 Expressions as Parameters of Early Diagnosis and Disease Progression in Hepatocellular Carcinoma

Amal Fawzy1, Hatem Attia2, Fatma A Khalaf3, Eman Abd El Sameea4, Mahmoud A El Tahawy4, Mohamed Farag4 and Fatma Younis5

Departments of Clinical Pathology1, National Cancer Institute-Cairo University Clinical Pathology2, Biochemistry3 & Hepatology4, National Liver Institute-Menoufyia University & Tropical Medicine5, Faculty of Medicine- Al Azhar University [email protected]

Abstract: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Despite remarkable advances in diagnostic and therapeutic techniques, the incidence of HCC is still on the increase. The role of liver biopsy in diagnosis of HCC has declined. However, with recent advances in genomics and proteomics a great number of potential serum and tissue markers have been identified and are being developed as new candidate markers for both diagnosis and prognosis of HCC, and may increase the need for liver biopsy. The aim of this study is to investigate the role of HSP70 and HSP27 expressions in early detection of HCC and to find their relation to parameters of disease progression. The study was conducted on 76 patients, 42 have proved HCC and 34 patients with liver cirrhosis (LC) without HCC. Routine laboratory investigations were done including: liver function tests, complete blood counts, serum alpha fetoprotein (AFP), hepatitis B and HCV hepatitis markers, and HCV-RNA levels. HSP70 and 27 expressions were determined in liver sampling by Real-Time PCR. Overexpression of HSP70 was detected in 92.86% of HCC which is statistically significantly higher compared to LC (2.94%) cases, and overexpression of HSP27 was significantly increased in HCC cases (57.14%) as compared to LC patients (8.82%). The overexpression of HSP70 was associated with early HCC diagnostic parameters (tumour size) and prognostic criteria (vascular invasion and tumour grade, Overexpression of HSP27 was associated with tumor size and tumor number, but not associated with each of AFP, vascular invasion and tumor grade. Conclusion: From the above results, we conclude that, found expressions of HSP70 and HSP27 may play an important role in hepatocarcinogenesis, and especially HSP70 can contribute tumor progression. We thus suggest that HSP70 may represent a good molecular target for treatment of HCV-related HCC. [Amal Fawzy, Hatem Attia, Fatma A Khalaf, Eman Abd El Sameea, Mahmoud A El Tahawy, Mohamed Farag and Fatma Younis. Heat Shock Protein-70 and -27 Expressions as Parameters of Early Diagnosis and Disease Progression in Hepatocellular Carcinoma. Life Sci J 2013;10(1):262-268] (ISSN:1097-8135). http://www.lifesciencesite.com. 40

Key Words: Hepatocellular carcinoma, HSP70, HSP27, Expression, RT-PCR.

1. Introduction of HSP70 also leads to significant protection against Stress or heat shock proteins (HSPs) are a set of cell apoptosis [8]. HSP70 levels block the apoptotic highly conserved proteins with an essential defense pathway at different levels. HSP70 reduces or blocks mechanism for protecting cells from various caspase activation and suppresses mitochondrial environmental damages [1]. HSPs have been damage and nuclear fragmentation [9]. HSP70 can classified into six major families and designated also prevent caspase-independent apoptosis pathways. nomenclature according to their approximate HSP70 prevents cell death under conditions in which molecular weight: HSP100, HSP90, HSP70, HSP60, caspase activation does not occur, because of the HSP40, and small HSPs including HSP27 [2]. addition of exogenous caspase inhibitors [10]. The HSPs are inducible in response to various There were several reports about HSP70 physiologically or pathologically stressful conditions, expression in malignant tumors, such as breast cancer including carcinogenesis [3]. The functions of the [11], lung cancer [12], prostate cancer [13], and HSPs in the tumorigenesis have been implicated in carcinoma of the uterine cervix[14]. The majority of the regulation of cell cycle progression and apoptosis the published results demonstrated HSP70 [4], in multidrug resistance [5], and as a modulator of overexpression correlated with poor prognosis and p53 function [6]. resistance to therapy [15-18]. HSP27 belongs to the HSP70 is a housekeeping gene that assists with a small heat shock protein family. Its structure and variety of vital intracellular Chaperoning functions. function are thought to be modulated by Expression of HSP70 increases under conditions of phosphorylation mediated by MAPK2. Intracellular environmental cellular stress [7] and overexpression HSP27 plays an anti-apoptotic role through

262 Life Science Journal 2013;10(1) http://www.lifesciencesite.com interaction with Bid or cytochrome c [19] and also ultrasonography and triphasic C.T. abdomen. The has a main role as a chaperone, preventing the group of LC were 21 males and 13 females with age aggregation of misfolded proteins. As such, HSP27 range 46-66 years, they were selected according to may contribute to the pathogenesis of human diseases clinical examination, abdominal ultrasonography, such as cancer, autoimmune diseases, neurological laboratory investigations and liver biopsy findings. disorders (e.g., Alzheimer’s disease), and Exclusion criteria for patients: Any patients with cardiovascular diseases, where HSP27 has been cancer other than HCC, septicaemia, chronic investigated as a biomarker for myocardial ischemia inflammatory disorders, chronic heart disease were [20]. Increased levels of HSP27 were detected in a excluded. None of the selected patients had received number of cancers, especially hormone-sensitive local or systemic therapy for HCC before. neoplasm [21]. Previous studies about the clinical Local ethics committee approval and informed implications of HSP27 showed different results consent from all participants in this study were according to tumor types [21, 22]. obtained prior to testing. Hepatocellular carcinoma (HCC) is one of the world’s most common malignancies, especially in Laboratory investigations: Asia and southern Africa. Most HCCs are associated Serum levels of AST, ALT, albumin, total and with chronic liver diseases resulted from hepatitis B direct bilirubin, alkaline phosphatase and gamma or C viral infection, and the processes of chronic glutamyle transpeptidase (GGT) were done using inflammation and fibrosis act as a stressful condition. Integra-400 (Roche-Germany). Prothrombin HSPs induced in response to this stress condition may concentration was done by Fibrintimer (Roche- contribute to hepatocarcinogenesis [22, 23]. Germany). Complete blood counts were measured by Over- expression of 4 members of the HSP Sysmix K-21 automatic cell counter (Japan). AFP family in hepatitis C virus (HCV)-related serum level was measured by an automated Eleceyes hepatocellular carcinoma (HCC) samples by (Roche-Germany). HBV markers and HCV proteomic analysis was reported [24]. HSP70 is one antibodies were assayed by EIA (COBAS-Amplicore, of these 4 members and displayed the most increased Germany). HCV-RNA levels were analyzed by real levels in cancerous tissues compared to levels in time polymerase chain reaction using a commercial corresponding noncancerous liver tissues by kit (Roche Diagnostic, Branchburg, NJ) according to proteomic analysis. There have been a few the manufacturer's instructions. comprehensive studies of the expression of HSP70 or HSP27 in HCC [22, 25]; however its prognostic Sampling and Real-Time Quantitative RT-PCR relevance remains controversial. In addition, there Analysis of HSPs have been few studies on HSP expressions in Real-time PCR was performed using association with tumor cell proliferation or apoptosis noncancerous and cancerous liver biopsies taken from in HCCs. patients who underwent surgical liver resection. Part of liver sample was stored in liquid nitrogen Aim of the study: immediately after the operation and kept at -80ºC This aimed to investigate the expressions of until RNA extraction and the other part was fixed in HSP70 and HSP27 in early detection HCC and to 10% neutral buffered formalin and embedded in investigate their relation to clinicopathologic paraffin for histological studies. parameters of disease progression. Total ribonucleic acid (RNA) was isolated from liver tissue using Trizol reagent (Invitrogen, 2. Patients and methods: Carlsbad, CA, USA) following the protocol The current study was conducted on 76 recommended by the manufacturer. Reverse patients (51 males and 25 females, age range 46- 71 transcription reactions were performed using a Rever years), 42 of them have HCC and 34 patients with Tra Ace alpha-First Strand cDNA Synthesis Kit liver cirrhosis (LC) without HCC. They attend (Toyobo, Osaka, Japan). Briefly, 1 µg of total RNA, 1 outpatient and inpatient clinics of the Hepatology µL of oligo dT-primer, and 2 µL of dNTPs were Department-National Liver Institute- Menoufiya incubated at 65°C for 5 min, then 10 µL of a cDNA University, and Tropical Medicine Department of Al synthesis mixture was added and this mixture was Zahraa and National Cancer Institute from July 2010 incubated at 50°C for 50 min. The reaction was till April 2012. terminated by adding 1 µL of RNaseH and incubating The HCC group includes: 30 males and 12 the mixture at 37°C for 20 min., all RNA samples females with age range 51-71 years, diagnosed were treated with DNase I to remove genomic DNA. according to clinical examination, laboratory and Real-time quantification of HSPs mRNA radiological investigations including abdominal transcripts was performed as reported previously [26].

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The PCR reaction was carried out in a tube containing of 34 cases of LC. HSP27 was overexpressed in 3 a mixture of 2 µL cDNA, 12.5 µL 2 X SYBR Green cases (8.82%) of LC and in 24 (57.14%) out of 42 (Applied Biosystems), 0.5 µL of 25 nM sense and HCC, which is significantly higher (<0.01) than LC antisense primers, and H2O up to final volume of 25 cases (Table 3). µL. For standardization of the amount of RNA, In HCC cases, overexpression of HSP70 was expression of glyceraldehyde-3-phosphate associated with early HCC diagnostic parameters dehydrogenase (GAPDH) in each sample was (tumor size, p<0.05) and prognostic criteria: vascular quantified by using GAPDH primers. invasion (p<0.05), tumor grade (p<0.01), while not The primer sets used for HSPs and GAPDH are the related to AFP levels or tumor number (Table 4). following: Overexpression of HSP27 was associated HSP70: 5′-AGGCCGACAAGAAGAAGGTGCT-3′ significantly with (tumor size, p<0.05) and tumor (forward) number, and not associated with each of AFP, 5′-TGGTACAGTCCGCTGATGATGG-3′ vascular invasion and tumor grade (p>0.05) (Table 5). (reverse) HSP27: 5′-CCCACCCTCTATCACGGCTAC-3′ Table (1) Patients characteristics (forward) Parameters HCC patients LC patients 5′-GGGCTCAACTCTGGCTATCTC-3′ (N=42) (N=34) (reverse) Age (years) : GAPDH: 5′-GAAGGTGAAGGTCGGAGTC- 3′ (M±SD) 59.2±10.8 54.6±11.3 (forward) Gender: 5′-CCCGAATCACATTCTCCAAGAA-3′ Male 30 (71.4% ) 21 (61.8% ) (reverse) Female 12 (28.6%) 13 (38.2%) Mode of infection: Surgical procedures 24 (57.2%) 21 (%) The PCR conditions were 1 cycle at 50°C for 2 Blood transfusion 5 (11.9%) 2 (%) minutes, 1 cycle at 95°C for 10 minutes, then 40 Drug abuse 3 (7.1%) 4 (%) cycles at 95°C for 15 seconds and 60°C for 1 minute. Unknown causes 10 (23.8%) 7 (%) Real-time detection of the emission intensity of Ultrasonographic Data: Hepatomegaly 31 (73.8%) 25 (73.5%) SYBR Green was performed with an ABI prism 7700 Splenomegaly 27 (64.3%) 21 (61.8%) Sequence Detector (Perkin-Elmer Applied Ascites 4 (9.5%) 2 (5.9%) Biosystems), as reported previously [27]. Multiple Hepatitis markers: negative water blanks (no template control) were HCV antibodies 36 (85.7%) 30 (88.2%) included in every analysis. Samples were also tested HBS Ag 6 (14.3%) 4 (11.8%) Child-Pugh classification: to ensure that they were negative for DNA using a Class A 13 (31.0%) 14 (41.2%) complete master mix without reverse transcriptase (no Class B 21 (50.0%) 15 (44.1%) amplification control). No amplification was observed Class C 8 (19.0%) 5 (14.7%) for these controls, indicating the specificity of the assays for the respective mRNAs. Table (2) Comparison between laboratory data of Statistical analysis: patient groups Parameters HCC LC Data were coded and summarized using SPSS patients patients Test of p- (statistical package for Social Sciences) version 13.0 (N=42) (N=34) Sign. value for Windows. Quantitative variables were described M±SD M±SD using mean ± standard deviation and categorical data ALT (U/L) 76.5± 21.4 68.7±18.2 1.97 >0.05 by using frequency and percentage. Comparison AST (U/L) 97.2±32.2 72.2±20.4 2.75 >0.05 2 GGT (U/L) 57.1 ±11.9 35.2±10.3 3.86 <0.05 between groups was done using Chi square (Χ ) test ALP (U/L) 108.2±33.7 43.2±8.1 5.72 <0.01 for qualitative variables, student t test for normally T.bilirubin 3.63 ±1.95 5.66 ±3.4 2.07 >0.05 distributed variables and Mann Whitney (U) test for (mg/dl) none normally distributed quantitative variables. P S.albumin 2.98±0.35 3.01±0.68 1.13 >0.05 value <0.05 was considered statistically significant. (g/dl) Proth.Con 62.7± 10.2 58.6±9.7 1.84 >0.05 3. Results: (%) The clinical, radiological and laboratory HB (g/dl) 11.4 ± 2.3 10.9 ± 1.6 1.93 >0.05 characteristics of the patients are demonstrated in WBCs 8.6 ± 3.5 7.5± 2.8 2.01 >0.05 9 table (I), the comparison between patient groups are (X10 /L) Platelets 132.6 ± 24.5 114.2 ± 2.65 >0.05 shown in table (2). (X109/L) 15.2 Overexpression of HSP70 was detected in 39 AFP (ng/ml) 408.5±367.5 18.43±7.41 6.73 <0.001 (92.86%) out of 42 cases of HCC which is P<0.05 & <0.001 are statistically significant, p>0.05 is statistically significant compared to LC (p<0.001), in statistically non significant. which one case only (2.94%) has overexpression out

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Table (3) Comparison between patient groups as regard HSP mRNA expressions. Parameters HCC patients LC patients (N=42) (N=34) X2 p-value No % No % HSP70 mRNA: Overexpression 39 92.86% 1 2.94% 12.73 <0.001 Minimal expression 3 7.14% 33 97.06% HSP27 mRNA: Overexpression 24 57.14% 3 8.82% 8.51 <0.01 Minimal expression 18 42.86% 31 91.18% P<0.01 & <0.001 are statistically significant.

Table (4) Comparison between HSP70 mRNA expression and prognostic data of HCC patients Parameters HSP70 Overexpression (N=39) No % X2 P-value AFP levels: <100 ng/ml 18 46.15% 2.82 >0.05 ≥100 ng/ml 21 53.85%) Tumor size: <5 cm 7 17.95% 4.16 <0.05 >5cm 32 82.05% Tumor number: Single 11 28.21% 2.01 >0.05 Multiple 28 71.79% Vascular invasion: Absent 6 15.38% 4.09 <0.05 Present 33 84.62% Tumor grade: G 1 2 5.13% 7.21 <0.01 G 2 9 23.08% G 3 28 71.79% P<0.05 & <0.01 are statistically significant, p>0.05 is statistically non significant.

Table (5) Comparison between HSP27 mRNA expression and prognostic data of HCC patients Parameters HSP27 Overxpression (N=24) No % X2 P-value AFP levels: <100 ng/ml 11 45.83% 2.11 >0.05 ≥100 ng/ml 13 54.17% Tumor size: <5 cm 5 20.83% 5.21 <0.05 >5cm 19 79.17% Tumor number: Single 8 33.33% 4.89 <0.05 Multiple 16 66.67% Vascular invasion: Absent 10 41.67% 1.59 >0.05 Present 14 58.33% Tumor grade: G 1 11 45.83% 2.75 >0.05 G 2 7 29.17% G 3 6 25.00% P<0.05 is statistically significant, p>0.05 is statistically non significant.

4. Discussion: been suggested to contribute to tumorigenesis [22, Heat-shock proteins have dual roles as a 29]. modifier to protein activities and as a central regulator Previous studies have shown up-regulated in both cell proliferation and apoptosis [27, 28]. expression of HSP70 and HSP27 in tumor cells, Among them, HSP70 and HSP27 are often including those of HCC. HSP70 and HSP27 among overexpressed in cells of various cancers and have HSPs are of special relevance in human cancer

265 Life Science Journal 2013;10(1) http://www.lifesciencesite.com inhibiting apoptosis [22, 30] .They were frequently studies, shown that HSP70 antisense oligomers can stained in the cytoplasm and nuclei of tumor cells, but specifically inhibit tumor cell proliferation by not in non-neoplastic hepatocytes [30]. inducing apoptosis [40,41]; these results suggest that Although early HCC is defined on the basis of anti-apoptotic functions of HSP70 may play an histopathologic [31] and clinical studies [32], the important role in tumor cell proliferation and tumor molecular changes that occur in early HCC are not progression in HSP70 over-expressed tumors like well understood. Early HCC is characterized by an HCC. Another plausible role for HSPs in increase of cell density and growth [33], but no tumorigenesis is as a modifier of protein activities positive molecular marker has yet been identified. [42, 43]. The tumor suppressor p53 protein, involved Gene expression profiling is a promising method for in the control of cell proliferation, represses finding molecular markers useful for the diagnosis of transcription by direct protein-protein interaction with early cancer [26]. the promoter region of the human HSP70 gene [28]. Our results revealed that, overexpression of Youshida et al. [25] study reported that HSP70 m RNA were detected in 92.86% of early immunohistochemically confirmed that HSP70 HCC cases, while the expression was noticed in protein levels are significantly higher in progressed 2.94% of LC cases without HCC. Overexpression of HCC than in early HCC and a significant association HSP27 m RNA was detected in 57.14% of HCC cases was found between immuno-reactivity for HSP70 and and in 8.82% of LC cases. Several studies have high histological grade of HCV-related HCC. HSP70 evaluated expression of HSP70 in HCC [22, 24-26 plays an important role as a chaperone of intracellular and 30]. Consistent with our findings, all studies have peptide antigens in cancer immunotherapy. reported overexpression of HSP70 in HCC tissues. Chuma et al. [26] further showed that HSP70 Chuma et al. [26] revealed significant overexpression could be a sensitive marker for the differential of HSP70 in early HCC compared with precancerous diagnosis of early HCC from precancerous lesions or lesions. noncancerous liver tissue, a diagnosis that is difficult Overexpression of HSP70 also has been even for pathologists because of the very well observed in cultured cells transformed with c-myc or differentiated histology with minimum atypia seen in H-ras, or cotrans formed with H-ras/p53, suggesting early HCC. Indeed, HSP70-p53 complexes can be involvement of HSP70 in transformation or detected in extracts from human cancer tissues [22]. progression [34, 35]., Yokoyama et al. [36] listed Cui et al. [44] has reported that coexpression of anti- examples of HSP70 upregulation in human HCC HSP70 and anti-p53 was shown in 3 out of 12 HCC tissues, and Yin [34], and Chuma et al.,[26] tissues (25%). Therefore studying the coordinated described overexpression of HSP70 in HCC. It is expression of HSPs with p53 protein may be helpful possible that HSP70 expression increases as a result to understand the roles of HSPs in the regulation of of tumorigenesis. For example, a stressful p53 function. environment in early HCC (nutrient depletion and As an apoptosis inhibitor, HSP is overexpressed hypoxia resulting from insufficient blood supply [32], in human HCC tissues and correlated with may stimulate HSP synthesis. carcinogenesis, progression and prognosis of HCC. Effendi & Sakamoto [37] detected Enhancement of intracellular HSP is closely related to immunoreactivity of HSP70 ranging up to 80% in the formation and development of HCC and a vital most cases of early HCC, while no or only focal and marker indicating the progression and aggravation of faint nuclear staining was observed in the HCC [45]. noncancerous background liver tissue. HSP70 is a In the current study, HSP70 overexpression was housekeeping gene that assists with a variety of vital specifically detected in HCC cases and showed a intracellular chaperoning functions. Expression of close relationship to the pathologic parameters related HSP70 increases under conditions of environmental to tumor progression, such as large tumor size, portal cellular stress [7] and overexpression of HSP70 also vein invasion, and high tumor grade, Joo et al. [22] leads to significant protection against cell apoptosis reported the same results as regard HSP70. Also, [38]. This is consistent with pathological processes Youshida et al. [25] found that HSP70 expression such as oncogenesis. Although it is not yet clear, the may be related to tumor differentiation and tumor stressful environment of early HCC (nutrition growth in HCV-related HCC. Yao et al. [30] added depletion, and hypoxia resulting from insufficient that, HSP70 was correlated with high Ki-67 labeling blood supply) may stimulate the expression of indices, large tumor size, presence of portal vein HSP70. invasion, and high tumor stage. HSP27 was The roles of HSP70 in promoting tumor cell significantly related to the subgroup of HBV- proliferation were previously observed in the various associated HCCs, but not to others. types of carcinoma [33, 39], but not in HCC. Some

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Our results reported that, the overexpression of 7- Mayer MP and Bukau B: Hsp70 chaperones: cellular HSP27 was associated significantly with tumor size functions and molecular mechanism. Cell Mol Life Sci. and tumor number, and not associated with each of 2005; 62(6):670-684. Review AFP, vascular invasion and tumor grade. Seung et al. 8-Padmini E and Lavanya S: HSP70-mediated control of endothelial cell apoptosis during pre-eclampsia. Eur J [46] reported that HSP27 expression was related to Obstet Gynecol Reprod Biol. 2011; 156(2):158-164. histologic grade and the survival of patients with 9- Stankiewicz AR, Lachapelle G, Foo CP, Radicioni SM HCC. While, Harimoto et al. [47] found that, the and Mosser DD: Hsp70 inhibits heat-induced apoptosis HSP27 expression did not correlated with upstream of mitochondria by preventing Bax clinicopathologic factor in the overall group of HCCs. translocation. J Biol Chem. 2005,18; 280 (46):38729- Also, Joo et al. [22] were not able to find any 38739. correlation between HSP27 immuno-reactivity and 10-Schmitt E, Gehrmann M, Brunet M, Multhoff G and the other parameters examined. Garrido C: Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy, J Leukoc Biol. 2007 ;81(1):15-27. Conclusion: 11- Sarkar R, Mukherjee S, Biswas J and Roy M: In conclusion, expressions of HSP70 and HSP27 Sulphoraphane, a naturally occurring isothiocyanate may play an important role in hepatocarcinogenesis, induces apoptosis in breast cancer cells by targeting and especially HSP70 showed a close relationship to heat shock proteins Biochem Biophys Res Commun. the pathological parameters associated with tumor 2012,10: S0006-291X(12)01733-0. progression. Our results could be additional evidence 12- Suzuki K, Ito Y, Wakai K, Kawado M, Hashimoto S, that HSP70 expressions can contribute to not only Seki N, Ando M, Nishino Y, Kondo T, Watanabe Y, hepatocarcinogenesis but also tumor progression, but Ozasa K, Inoue T,Tamakoshi A: Serum heat also is expected to be an ideal target for the therapy of shock protein 70 levels and lung cancer risk: a case- hepatocellular carcinoma. Nevertheless, further control study nested in a large cohort study. Cancer Epidemiol Biomarkers Prev. 2006; 15(9):1733-1737. analysis is still necessary to carefully evaluate the 13-Lu S, Tan Z, Wortman M, Lu S and Dong Z: Regulation roles of these molecular pathology candidates in early of heat shock protein 70-1 expression by androgen hepatocarcinogenesis. receptor and its signaling in human prostate cancer cells. Int J Oncol. 2010; 36(2):459-467. Corresponding author 14- Zhao EF, Bao L, Liang L and Li D: Expressions of heat Amal Fawzy shock protein in cervical cancer and precancerosis, Departments of Clinical Pathology1, National Cancer Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2006; Institute-Cairo University 22(2):250-253. [email protected] 15. Boroughs LK, Antonyak MA, Johnson JL and Cerione RA: A unique role for heat shock protein 70 and its binding partner tissue transglutaminase in cancer cell References: migration. J Biol Chem 2011, 286(43):37094–37107. 1-Walsh D, Li Z, Wu Y and Nagata K.: Heat shock and the 16- Malusecka E, Krzyzowska-Gruca S, Gawrychowski J, role of the HSPs during neural plate induction in early Fiszer-Kierzkowska A, Kolosza Z and Krawczyk Z: mammalian CNS and brain development, Cell Mol Life Stress proteins HSP27 and HSP70i predict survival in Sci. 1997 ;53(2):198-211. non-small cell lung carcinoma. Anticancer Res 2008, 2-Morimoto RI and Santoro MG.: Stress-inducible 28(1B):501–506. responses and heat shock proteins: new pharmacologic 17- Thubashini M, Malathi N and Kannan L: Expression of targets for cytoprotection. -Nat Biotechnol. 1998; heat shock protein70 in oral submucous fibrosis and 16(9):833-838. oral squamous cell carcinoma: an immunohistochemical 3-Mosser DD and Morimoto RI.: Molecular chaperones and study. Indian J Dent Res 2011, 22(2):256–259. the stress oncogenesis. Oncogene. 2004;23(16):2907- 18-.Lu S, Tan Z, Wortman M and Dong Z: Regulation of 2918. heat shock protein 70–1 expression by androgen 4-Akerfelt M, Trouillet D and Mezger VSistonen L.:Heat receptor and its signaling in human prostate cancer cells. shock factors at a crossroad between stress and Int J Oncol 2010, 36(2):459–467. development. Ann N Y Acad Sci. 2007; 1113:15-27. 19- Bruey JM, Ducasse C, Bonniaud P, Ravagnan L, Susin 5-Paduch R, Jakubowicz-Gil J and Niedziela P: Hepatocyte SA, Diaz-Latoud C, Gurbuxani S, Arrigo AP, Kroemer growth factor (HGF), heat shock proteins (HSPs) G, Solary E and Garrido C: Hsp27 negatively regulates and multidrug resistance protein (MRP) expression in cell death by interacting with cytochrome c. Nat Cell co-culture of colon tumor spheroids with normal cells Biol. 2000; 2(9):645-652. after incubation with interleukin-1beta (IL-1beta) and/or 20- Heidari-Bakavoli AR, Sahebkar A, Mobara N, camptothecin (CPT-11). Indian J Exp Biol. 2010; Moohebati M, Tavallaie S, Rahsepar AA, Kazemi A, 48(4):354-364. Alavi MS, Ghayour-Mobarhan M and Ferns GA: 6-Habib GM: p53 regulates Hsp90beta during arsenite- Changes in plasma level of heat shock protein 27 after induced cytotoxicity in glutathione-deficient cells. Arch acute coronary syndrome.Angiology. 2012;63(1):12-6. Biochem Biophys. 2009; 481(1):101- 109.

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21- Elpek GO, Karaveli S, Simşek T, Keles N and Aksoy N: 35- Gorgoulis VG and Halazonetis TD: Oncogene-induced Expression of heat-shock proteins hsp27, hsp70 and senescence: the bright and dark side of the response hsp90 in malignant epithelial tumour of the ovaries. Curr Opin Cell Biol. 2010; (6):816-827. APMIS. 2003; 111(4):523-530. 36- Yokoyama Y, Kuramitsu Y, Takashima M, Fujimoto M, 22- Joo M, Chi JG and Lee H : Expressions of HSP70 and Iizuka N, Terai S, Okita K, Sakaida I, Oka M, Raynes HSP27 in Hepatocellular Carcinoma J Korean Med Sci. DA, Guerriero V and Nakamura K: Increased 2005; 20(5): 829–834. expression of heat shock protein-binding protein 1 and 23- Ciocca DR and Calderwood SK: Heat shock proteins in heat shock protein 70 in human hepatocellular cancer: diagnostic, prognostic, predictive, and treatment carcinoma tissues. Mol Med Report. 2008; 1(2):197- implications.Cell Stress Chaperones. 2005 summer; 201. 10(2):86-103. Review 37- Effendi K and Sakamoto: MMolecular Pathology in 24- Takashima M, Kuramitsu Y, Yokoyama Y, Iizuka N, Early Hepatocarcino-genesis. Oncology 2010; 78:157– Toda T, Sakaida I, Okita K, Oka M and Nakamura K: 160. Proteomic profilingof heat-shock protein 70 family 38-Simard JP, Reynolds DN, Kraguljac AP, Smith GS and members as biomarkers for hepatitis C virus-related Mosser DD: Overexpression of HSP70 inhibits cofilin hepatocellular carcinoma. Proteomics 3:2487-2493, phosphorylation and promotes lymphocyte migration in 2003. heat-stressed cells. J Cell Sci. 2011; 124(Pt 14):2367- 25- Yoshida S, Hazama S, Tokuno K, Sakamoto K, 2374. Takashima M, Tamesa T, Torigoe T, Sato N and Oka 39- Malusecka E, Zborek A, Krzyzowska-Gruca S and M: Concomitant Overexpression of Heat-shock Protein Krawczyk Z: Expression of heat shock proteins HSP70 70 and HLA Class-I in Hepatitis C Virus-related and HSP27 in primary non-small cell lung carcinomas. Hepatocellular Carcinoma, Anticancer Res. An immunohistochemical study. Anticancer Res 2001; 2009;29(2):539-544. 21: 1015-1021. 26- Chuma M, Sakamoto M, Yamazaki K, Ohta T, Ohki M, 40- Kaur J and Ralhan R: Differential expression of 70-kDa Asaka M and Hirohashi S: Expression profiling in heat shock protein in human oral tumorigenesis. Int J multistage hepatocarcinogenesis: identification of Cancer 1995; 63: 774-779. HSP70 as a molecular marker of early hepatocellular 41-Zhao ZG, Ma QZ and Xu CX: Abrogation of heat-shock carci-noma. Hepatology 2003; 37: 198-207. protein (HSP)70 expression induced cell growth 27- Kim LS and Kim JH: Heat shock protein as molecular inhibition and apoptosis in human androgen- targets for breast cancer therapeutics J Breast Cancer. independent prostate cancer cell line PC-3m. Asian J 2011; 14(3):167-174. Androl. 2004; 6(4):319-324. 28- Zylicz M, King FW and Wawrzynow A: Hsp70 42- Mosser DD and Morimoto RI: Molecular chaperones interactions with the p53 tumour suppressor protein. and the stress of oncogenesis. Oncogene 2004; 23: EMBO J 2001; 20: 4634-4638. 2907-2918. 29- Jolly C and Morimoto RI: Role of the heat shock 43- Manal Ismail Abd-Elghany, Khaled Mahran and Nabiel response and molecular chaperones in oncogenesis and Mohie: Expression of heat shock protein 70 and cell death. J Natl Cancer Inst., 2000; 92: 1564-1572. transforming growth factor beta in hepatocellular 30- Yao DF, Dong ZZ and Yao M: Specific molecular carcinoma. El Menia Med., 2009, Bul., 20, No(1). markers in hepatocellular carcinoma, Hepatobiliary 44- Cui CW, Yang SJ, Liu YP, Liu YF. Interaction between Pancreat Dis Int. 2007;6(3):241-247. p53 and HSP70 in human hepatocellular carcinoma 31- Hirohashi S, Ishak KG, Kojiro M, Wanless IR, Theise tissues. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2003; ND, Tsukuma H, Blum HE, et al.: Hepatocellular 19: 195-196. carcinoma. In: Hamilton SR and LA Aaltonen, Eds. 45-Seon-Hee Yim and Yeun-Jun Chung: Overview of Pathology and Genetics of Tumors of the Digestive Biomarkers and Molecular Signatures in HCC Cancers System. Lyon: IARC Press, 2000:159-172. 2010, 2: 809-823. 32- Kudo M: Morphological diagnosis of hepatocellular 46- Seung Oe Lim, Sung Gyoo Park, Jun-Hi Yoo, Young carcinoma: special emphasis on intranodular Min Park, et al.: expression of heat shock proteins hemodynamic imaging. Hepatogastroenterology.1998; (HSP27, HSP60, HSP70, HSP90, GRP78, GRP94) in 45:1226-1231. hepatitis B virus-related hepatocellular carcinomas and 33- Park YN: Update on precursor and early lesions of dysplastic nodules. World J Gastroenterol 2005; hepatocellular carcinomas. Arch Pathol Lab Med. 2011; 11(14):2072-2079. 135(6):704-715. 47- Harimoto N, Shimada M, Aishima S, Kitagawa D, Itoh 34- Yin Y, Qin Q, Zhang W, Zhao J, Zhang C and Yu J: S, Tsujita E, Maehara S, Taketomi A, Tanaka S, Overexpression of heat shock protein 70 and Shirabe K and Maehara Y: The role of heat shock spontaneous cancer cell apoptosis in hepatocellular protein 27 expression in hepatocellular carcinoma in carcinoma. Zhonghua Gan Zang Bing Za Zhi Japan: special reference to the difference between 2001;9:84-85. hepatitis B and C. Liver Int 2004; 24: 316-321.

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Use of Fourier Transform Infrared Spectroscopy to Study Cadmium-Induced Changes in Strongylocentrotus nudus gonad

ZhenlinWei

Biology Science Department, Dezhou University, Dezhou, Shandong, 253023, CHINA; [email protected]

Abstract: In this paper, the effects of 0.1 and 0.01 mM cadmium on Strongylocentrotus nudus gonad were studied by means of fourier transform infrared spectroscopy. The second derivative spectra and curve-fitting analysis revealed adverse effects of cadmium stress on the metabolism of lipids and proteins in gonad. For lipids, cadmium treatment shifted bands position to higher wavenumer and decreased bands area, especially those bands contributed from CH2 stretching vibration group, suggesting that the structure of lipids constituents were disordered and the contents decreased. Taken account to proteins, cadmium resulted in increasing fraction of β sheet structure and decreasing fraction of α helix, as well as declining protein contents. Compared with 0.01 mM treatment, the higher concentration showed more significant effects, such as bands position shifting to a larger extent and formation of 1692 cm-1 band within Amide I regions. The results demonstrated that FTIR spectroscopy is a promising tool for detection of cadmium induced changes in the context of molecular structure observation in urchin gonad. [Weil ZL. Use of Fourier Transform Infrared Spectroscopy to Study Cadmium-Induced Changes in Strongylocentrotus nudus gonad. Life Sci J 2013;10(1):269-276] (ISSN:1097-8135). http://www.lifesciencesite.com. 41

Keywords: Cadmium stress; Strongylocentrotus nudus; Gonad; Fourier Transform Infrared Spectroscopy

1. Introduction Although numerous studies were performed Following the development of metal- on sea urchin under Cd stress by means of working industries and the extensive use of heavy biochemical and genetic methods, the majority of metal- containing chemical fertilizer, the problem of research focused on sperm, embryo and larvae rather heavy metal pollution on environment has emerged than the gonad tissue, which serves as edible and and became increasingly attractive (Soualili et al., reproductive tissue where the sperm and egg cells are 2008). In fact, there are growing studies about the generated. In fact, this organ, due to its large volume heavy metal pollutants in the marine environment and and its function of gametogenesis, is a prime target on the accumulation in tissues of various organisms for heavy metal stress. Therefore, the Cd induced (D’Souza et al., 2008; Das, 2000; Dumas and Miller, damage will adversely affect the physiological 2003; Esslemont et al., 2003; Fernandez and Beiras, activity. It has known that Cd could interfere on 2001). Among the three most polluting heavy metals, gametogenesis, inhibit embryogenesis, induce cell cadmium (Cd) is thought to be the most toxic to apoptosis, and interrupt development at different organisms and poses the greatest threat to the stages (Agnello et al., 2007, Au et al., 2001). environment (D’Souza et al., 2008; Henczová et al., However, these phenomena are most likely of results 2006). of cadmium stress instead of the initial events to As being sensitive to heavy metal stress, trigger the following results on exposure to Cd. different sea urchin species have been used to sensor Unlike conventional methods, Fourier transform seawater pollutant and to elucidate the mechanism of infrared spectrometry (FTIR) is a convenient and heavy metal toxicity (Agnello et al., 2007; Garman et easier handing method to detect the conformational al., 1997; Kobayashi and Okamura, 2004; Nacci et changing of biological molecular components and al., 2000; Quiniou et al., 1999; Roccheri et al., 2002; these conformational disordering as well as Soualili et al., 2008; Soualili and Guillou, 2009). subsequent disturbed intramolecular or Experimental evidence suggests that exposure to Cd intermolecular interaction is the key for inducing may cause a cascade of events including generation malfunction. of reactive oxygen species (Howlett and Avery, The vibration of chemical bond absorbs 1997; Waisberg et al., 2003), depletion of glutathione radiation in the IR region between 4,000 and 400 cm- (Shimizu et al., 1997), inhibition of enzymes 1. Each functional group in a molecule has involving in DNA synthesis and repair (Giaginis et characteristic absorption frequencies in the IR al, 2006), and DNA single-strand breaks (Schröder et spectrum. Particularly, FTIR can be used to probe the al., 1999), eventually resulting in cellular damages structure of biological composition or its chemical and genetic mutation (Henczová et al., 2004; group, and the composition and structure of Waisberg et al., 2003) . molecular functional group can be determined by

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analyzing the position, width and intensity of subtracted from the samples automatically. The acquired spectra in a complex biological system acquisition parameters were resolution of 4 cm-1 when rely on certain algorithms (Corte et al., 2010; with 32 scans co-added. All spectra were normalized Yee et al., 2004). and baseline-corrected with Omnic7 software with The advantages of FTIR method enable it to default settings, and smoothed with smoothing factor examine the initial response to stress with high of nine. The spectra for control and Cd treated sensitivity through acquire rapidly spectra from very samples were calculated from 3 repeats and each small amounts of samples (Corte et al., 2010), and used a different individual. These replicates were offers a fast method to fingerprint the global cellular averaged and these averaged spectra for each sample features under specific conditions (Alvarez-Ordónez were then used for further data and statistical et al., 2010). Compared with traditional physiology analysis. methods, the FTIR method has its superiority when Since the original spectra yield rather broad analyze certain biological samples, because the FTIR bands in specific regions, mathematical data could detect the initial response of cells to biotic and treatments such as second-derivative analysis has to abiotic stimuli even before obviously physiological be applied to resolve the fine details of interested events were detected, as well as provides structural bands (Wolkers et al., 1998). Second-derivative information on biological molecular thus reflecting spectra to determine the position of the band as the mechanism of damages and adaptive of cells starting parameters for the curve-fitting procedure concerning to heavy metal. Consequently, FTIR were calculated with Omnic 7 software using 11 spectroscopy, together with infrared microscopy, is point Savitsky-Golay algorithm. With curve fitting an important technique to study the cellular changes analysis, one not only resolves the individual at molecular level in various biological samples component bands, but may also calculate the area (Alvarez-Ordónez et al., 2010; Cakmak et al., 2006). from each of the individual bands. Therefore, curve- In the current study, the effects of Cd fitting analysis has become an important tool for treatment on urchin gonad were investigated by qualitative and quantitative analysis of infrared monitoring the conformational changing of functional spectra. Curve-fitting of the spectra was performed groups of protein and lipids with FTIR spectroscopy. with the professional PeakFit V4.12 software Second derivative spectra and curve-fitting analysis (http://www.seasolve.com) under its AutoFit Peaks II of IR spectrum could acquire accurate data, thus Second Derivative mode with default settings, thus allowing quantitatively analyze these functional the precise band position and the area of band were groups. The presented results provided novel and obtained as the results. The functional groups of essential information on the Cd induced damage on bands were assigned with Knowitall 7.8 software and urchin gonad. referenced other literatures. Statistical analysis was 2. Material and Methods performed using SPSS 11.5 software. Differences Sea urchin Strongylocentrotus nudus with were analyzed by the student's t-test. A probability approximately 5 cm in diameters were collected in level (p-value) of less than 0.05 is regarded as the coast of Dalian (38°54'45" N, 121°36'09" E) of significant. liaoning province, P.R.CHINA, during 15th -17th 3. Results June, in 2011. The animals were acclimated in Under Cd stress, urchin gonad appeared laboratory with filtered natural seawater for a week, obviously changing in their color, control gonad is and then treated with 0.01 or 0.1 mM cadmium light yellow, whereas Cd treated gonad showed dark chloride solutions for 24h. For control and two yellow, and 0.1 mM Cd has more darker color treatments, 10 individuals were cultured in three compared with 0.01 mM Cd treatment. In this tanks at 22±1 °C, supplied with 50 L O2-saturated research, FTIR spectra were acquired to detect the filtered natural sea water. After treatment, the gonads conformational changes and content variations of the of female urchins were separated from whole body, functional groups contributed from protein and lipids wash two times with filtered natural seawater, and of sea urchin gonad due to 0.01 and 0.1 mM Cd were freeze dried to remove moisture. stresses, and the average spectra of control and Tablets for FTIR spectroscopy were treated samples were showed in Figure 1. Generally, prepared in Agate mortars, dried samples were the spectra were complex and consist of several ground to fine powders and mixed with KBr (1:100 bands arising from the vibration of different groups p/p). Spectra were collected by a FTIR-NEXUSTM belonging to proteins, lipids, carbohydrates and (Nicolet, America) instrument. The absorbance nucleic acids, indicating that the gonad enrich in spectra were measured between 400 and 4000 cm-1 biochemical compositions. at room temperature (25±1°C). The background spectra collected under identical conditions were

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Figure 1. Comparative spectra of control, 0.01 mM Cd and 0.1 mM Cd Upper: native spectra in the regions of 4000-400 cm-1; Under: the second derivative spectra in the regions of 1800- 1200 cm-1. Each curve represents the average spectrum of three individuals.

Accordingly, the majority of dominant bands absorption bands within this region to appeared several were fall into two distinct wavenumber regions, namely prominent bands. The bands between 1500 and 1000 3000-2800 cm-1 and 1700-800 cm-1, which mainly cm-1 were of the “fingerprint” region (Palaniappan represented the contribution of groups of lipids, and Renju, 2009b), amide Ⅲ and the function group of proteins and carbohydrates (D’Souza et al., 2008). In nucleic acid and carbohydrates contributed to these -1 detail, the band located around 3295 cm represents N- absorption bands in samples. Overall, the spectrum of H stretching vibrations that are mainly caused by control and Cd treated samples differ in the shape of proteins (Palaniappan and Vijayasundaram, 2009a; absorbance curve, indicating to obvious changes in Wolkers et al., 1998). The bands between 3000 and structure and contents of biological components due to -1 2800 cm mainly represent C-H stretching vibrations stress. that are caused by lipids (D’Souza et al., 2008). The The original spectra faced complex multi- protein absorption bands mainly located between 1700 -1 component bands which overlapped into a broad and 1500 cm contained amide I and amide II bands unresolved absorption, and the individual component (Warnau et al., 1996), but overlapped with other absorptions from condensed phase spectra cannot be

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resolved by increasing spectral resolution, causing of control and two treatments, thus PeakFit v4.12 difficulty in band differentiation and their assignment. software were used to refine these spectra through Therefore, the second-derivative spectra and curve- curve-fitting analysis. Selecting of this option enable us fitting processes were performed to distinguish to find and fit hidden peaks by looking for local overlapped peaks and achieve more precise calculation minima in smoothed second derivative spectra, and on band position and their area. The advantage of this provide more precise fitting results. process lies in that the data is de-noised to a great After curve-fitting, six, six and eight bands extent and overlapping is largely minimized (D’Souza were visible in control, 0.01 mM Cd and 0.1 mM Cd, et al., 2008). respectively, suggesting tremendous changes in these The examination of the second derivative regions under Cd stress (Fig.2). Overall, the bands spectra for control and cadmium treated samples profile of 0.01 mM Cd was more similar to control than revealed several differences (Fig.1), including peak 0.1 mM Cd, 6 bands were visible both in control and location shifting and peak height variation, especially 0.01 mM Cd with a generally slight up-wavenumber in the regions from 1500 to 3200 cm-1, indicated that shifting. The dominant bands of control and 0.01 mM -1 obvious conformational changes were occurred for Cd at 2958 and 2875 cm were assigned to CH3 -1 lipids and proteins. The spectrum of 0.01 mM Cd was stretching vibration, 2925 and 2854 cm to CH2 more similar to control than 0.1 mM Cd, demonstrated stretching vibration (Palaniappan and Renju, 2009b), that the higher concentration of cadmium stress while two weak intensive bands at 2979/2981 cm-1 was induced more obviously spectra changing and due to C-H asymmetric stretching vibration. For 0.1 significantly affected urchin gonad physiological mM Cd, compared with control, two newly emerged process. The region 3300 to 3000 cm-1 is characteristic weak bands were around 2815 and 2839 cm-1 for C-H stretching vibrations of C≡C, C=C and Ar-H, accompanied by 3 bands position shifting, band around while the region from 3000 to 2700 is dominated by the 2875 to 2870 cm-1, band of 2895 to 2898 cm-1, and -1 C-H stretching vibrations of -CH3, CH2, CH and CHO band of 2979 to 2981 cm , respectively. functional groups respectively (Dumas and Miller Curve-fitting result showed Cd stress 2003; Howlett and Avery, 1997). In this research, the decreased the total area of four dominant bands, from bands within these two regions were considered for 336.774±3.3336 for control to 36.279±0.7451 (p>0.05) analyzing of lipids. for 0.01 mM Cd, and 29.817±2.3641 (p<0.05) for 0.1 A medium intense band was found around mM Cd. Hence, the qualitatively analyzing of the -1 3012 cm-1 in all spectra, which assigned to the bands of 3012, 2958, 2925, 2875 and 2854 cm vibrating stretching of C=H group belongs to un- confirmed that Cd adversely affected gonad lipid saturated lipids (Guillén and Cabo, 1999), and the metabolism and the higher concentration resulted in existing of this peak was well consistent with the decreasing of lipid content to a larger extent. In conclusion that urchin gonad is rich in several types of addition, the curve-fitting results revealed that un-saturated lipids (Zhu et al., 2010). cadmium treated with 0.1 mM (0.1 mM Cd) increased The comparative spectra of control with that the CH3/CH2 ratio from 28.81% to 30.28%, which of 0.01 mM Cd and 0.1 mM Cd showed similar band reflected the increased disorder status of lipids. The width and peak positions, likely implying that the increased CH3/CH2 ratio is also a sign of higher fatty composition of gonad un-saturated lipids was kept acyl chain unsaturation, which has known to occur with stable to somewhat. However, the band area of 3012 fatty acyl chain peroxidation (Petibois and Deleris, cm-1 of control differ with that of 0.01 mM Cd and 0.1 2004). In the last place, cadmium induced band mM Cd based on curve-fitting analyzing, achieving position to higher value were visible, which suggested 0.6992±0.019 for control, 0.6121±0.023 (p<0.001) for that treatment of Cd decreases the fluidity of the lipid 0.01 mM Cd and 0.5031±0.008 (p<0.001)for 0.1 mM acyl chains. Cd. As this band being a useful indicator of the A prominent band located at 2896 cm-1 that belongs different degrees of un-saturation in acyl chains of to -OCH3 group stretching vibration phospholipids, the results implied that Cd stress (http://www.chem.uni-potsdam.de) was revealed in all decreased unsaturated lipids contents or reduced the spectra. Curve-fitting results demonstrated a weak degrees of un-saturation, and the higher concentration increasing of band area following the Cd stress was of Cd resulted in more obvious effects. occurring, from 111.469±0.2901 for control to Within 3000-2800 cm-1 region, second 11.529±0.8506 (p>0.05) for 0.01 mM Cd, and derivative spectra revealed three prominent bands 11.878±0.8429 (p>0.05) for 0.1 mM Cd, also located at 2961, 2925 and 2852 cm-1 in control, these indicating a aggravating of lipid redox status (Wei et bands were evident in 0.01 mM Cd and 0.1 mM Cd al., 2009). with wavenumber shifting to a certain extent. However, a shoulder band around 2902 cm-1 was found in spectra

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Figure 2. Curve fitting results of regions from 2800 to 3000 cm-1. The marker “original” represents the raw spectrum after baseline correction; “Generated” represents the overall curve as the results of curve fitting; the last figure represents the comparative second derivative spectra for control, 0.01 mM Cd and 0.1 mM Cd. A: curve fitting result of Control; B: curve fitting result of 0.01 mM Cd; C: curve fitting result of 0.1 mM Cd; D: second derivative spectra of Control, 0.01 mM Cd and T0.1 mM Cd in the region of 2800 to 3400 cm-1.

Table1. Results of curve fitting of the regions from 1600-1700 cm-1 Control 0.01 mM Cd 0.1 mM Cd S. A. B.P. Area % Area B.P. Area % Area B.P. Area % Area 1613 2.02±0.060 8.13 1614 2.06±0.022d 8.64 1615 1.75±0.066a 9.08 β sheet 1626 4.11±0.258 16.59 1626 4.13±0.278d 17.30 1631 4.88±0.480c 25.30 β sheet 1638 4.48±0.199 18.06 1638 4.29±0.317d 17.98 - - - β sheet 1650 4.54±0.375 18.33 1651 4.40±0.359d 18.45 1651 5.56±0.292b 28.82 α-helix 1662 4.43±0.241 17.87 1662 4.12±0.191c 17.27 1668 3.75±0.106a 19.41 Turn 1675 3.26±0.090 13.15 1675 3.04±0.087b 12.73 1681 2.36±0.160a 12.22 Turn 1687 1.95±0.093 7.88 1687 1.82±0.131d 7.64 - - - Turn ------1692 0.95±0.041 4.93 β sheet B.P.: Band position; S.A.: Structure assignment; ap<0.001; bp<0.01; cp<0.05; dp>=0.05;

Absorption bands around 1746 cm-1 constant decreasing profile with increasing of cadmium correspond to isolated carbonyl group (COOR), concentration, from 4.2069±0.1584 for control to indicating ester-containing compounds commonly 3.9376±0.2022 (p>0.05) for 0.01 mM Cd treatment, found in membrane lipid. Lipids make up ~60-65% of and 3.4860±0.2238 (p<0.001) for 0.1 mM Cd a plasma membrane and have a major influence on its treatment. properties, thus cadmium stress that disturbs lipids The observations suggested that Cd induced structure or their metabolism would lead to functional changes in the conformational order of lipid acyl disorder of plasma membrane and induced a cascade of chains. It is well known that the CH2 anti-symmetric downstream events to fulfill its toxic effects on cells. and symmetric stretching vibrations give information The area changing trend of 1746 cm-1 also appeared a about the state-of-order of the hydrocarbon tails in

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lipids (Akkasa et al., 2007). The sign if it can increase position shifting of amide I bands to higher value are in the frequencies of these bands with Cd treatment useful indicator of the overall protein disordering under indicates that cadmium functions by disordering the experimental conditions (Yang et al., 2002). Thirdly, a lipid system by increasing the number of CH3 group newly emerging band located at 1692 cm-1 only in the (Alvarez-Ordónez et al., 2010). Additionally, the spectra of 0.1 mM Cd treatment, together with the characteristic band of CH2 shift to CH3 demonstrates increasing area of typical β-sheet (1614 and 1626 cm-1 the oxidative damage of lipid as common phenomena for control and 0.01 mM Cd treatment, 1615 and 1631 of cells under stress, and this shift is a key event to cm-1 for 0.1 mM Cd treatment), likely demonstrated induce several down-stream events as the reasons to that cadmium stress would induce the protein structure generate cellular damages. Another instance also to to irreversible protein aggregate (Wolkers et al., 1998). proved that Cd stress led to lipid peroxide, the band The result showed the area increased from around 2896 cm-1 appeared an increasing band area 37.35 to 40.48 after 0.01 mM Cd treatment with regard with the ascending Cd concentration, indicating that Cd to 0.01 mM Cd treatment, but decreased significantly speed up the lipid oxidation. Moreover, Cd stress to 31.81 when stressed with 0.1 mM Cd treatment, decreased lipid contents based on 3012, 2961, 2925, suggesting a waved variation pattern of the protein 2852 and 1746 cm-1 curve-fitting analysis. Taken content. Similar result reported previously also stated together, Cd stress resulted in changing of lipids that low dosage of Cd increased protein contents in a structure of urchin gonad. Since these lipid components short stress period (Ivanina et al., 2008). could be divided into plasma membrane related and Second derivative spectra analyzing in the energy metabolism related, thus Cd induced effects on regions between 3200 and 3400 cm-1 uncovered a weak lipids would involve in a wide range of biological band around 3299 cm-1, which correspond to amide A processes, which need to be experimental investigated stretching mode that can generally be associated with further. N-H and intermolecular O-H molecules (Akkasa et al., The amide I band of proteins is located in the 2007). Overall, this band position shifted slightly, region of 1700-1600 cm-1. The amide I absorption band being at 3299, 3299 and 3300 cm-1 for control, 0.01 is primarily due to the C=O stretching vibration of the mM Cd treatment and 0.1 mM Cd treatment, amide groups weakly coupled with the in-plane N-H respectively. Instead, the band area was decreased bending and C-N stretching (He et al., 1991). The significantly, from 9.85±0.2654 to 9.68±0.9106 amide I band of proteins is a complex composite which (p>0.05) (0.01 mM Cd) and to 6.80±0.3213 (p<0.001) consists of a number of component bands in terms of α- (0.1 mM Cd), suggesting a decline of protein contents helices, β-sheets (parallel pleated sheet and anti- induced by Cd stress. This observation was well in parallel pleated sheet), turns and random-coil structures agreement to the analyzing results for the bands within (D’Souza et al., 2008). The frequency of the absorption Amide I and II regions. It was not unexpected results maximum of these bands is conformational sensitive, since one of the most popular phenomena of Cd stress thus cadmium stress induced shifts of the spectra is of decreased protein contents. provided information about structural changes in the Considering the changes in Amide I and II proteins of samples. regions of control and Cd treated 1 and 2 samples, it As described by Palaniappan (2009a; 2009b), could be concluded that the protein synthesis is the whole Amide I and II area was analyzed by means sensitive to Cd stress in urchin gonad, and the stability of curve-fitting in this research. Curve-fitting results of protein secondary structures was able to change after uncovered some interesting observations. Firstly, seven Cd treatment. bands were found between 1700 and 1600 cm-1 for Similar to changing profile of lipids, proteins control and 0.01 mM Cd treatment, while only 6 bands components also appeared decreased bands area and for 0.1 mM Cd treatment (Table.1), the absent band in disordered structure. In this paper, Cd induced protein 0.1 mM Cd around 1638 and 1675 cm-1 infer to the β structural change was emphasized analyzed. Several sheet and Turn structure of protein, respectively. researches with FTIR spectra elucidated that heavy Secondly, an obvious band position shifting was metal could alter protein structure exhibiting up-shifted observed when compared the control with treatment 1- wavemunbers of bands within Amide I region and and 2. Giving an example, the band at 1662 cm-1 in increased proportion of β sheet (Henczová et al., 2008). control was shifted to 1662 cm-1 in 0.01 mM Cd Well consensus with that, obvious bands position treatment and to 1668 cm-1 in 0.1 mM Cd treatment. In shifting to higher value was evidenced, all 6 bands of proteins, the most important hydrogen bonds are those 0.01 mM Cd treatment were moved generally and the between peptide bonds. Generally, the positions of proportion of β sheet was increased accompanied with amide I bands reflect the degree of hydrogen bonding: decreasing of α helix. Unlike 0.01 mM Cd treatment, the higher the wavenumber, the weaker the H bonding component bands of 0.1 mM Cd treatment showed (i.e. the less-ordered protein structure). Therefore, these more diversity. Except for position shifting to a larger

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extent, one band was disappeared and one emerged, References which the latter was a good indicator for protein 1. Agnello M, Filosto S, Scudiero R, Rinaldi AM, aggregating (Kilimann et al., 2006). Based on the Roccheri MC. Cadmium induces an apoptotic response results, it was suggested that under experimental in sea urchin embryos. Cell Stress Chaperones conditions, the gonad proteins were more sensitive than 2007;12:44-50. 2. Akkasa SB, Severcan M, Yilmaz O, Severcan F. Effects lipids to Cd stress, because the conformational of lipoic acid supplementation on rat brain tissue: An diversity of proteins was more obvious than lipids. It FTIR spectroscopic and neural network study. Food was a reliable hypothesis that Cd could directly bind to Chem. 2007;105:1281-1288. proteins with interaction of -SH group of proteins and 3. Alvarez-Ordónez A, Halisch J, Prieto M. Changes in replace Zn2+ which critical for protein maintain the Fourier transform infrared spectra of Salmonella enzymes activity and structure stabling, both results in enterica serovars Typhimurium and Enteritidis after target protein disorder (Yepiskoposyan et al., 2006). adaptation to stressful growth conditions. Int. J Food Moreover, Cd induced transcriptomic alternation also Microbl. 2010;142:97-105 generated novel proteins that showed different 4. Au DW, Reunov AA, Wu RS. Reproductive impairment structures (Georg and Gomes, 2007). of sea urchin upon chronic exposure to cadmium. Part II: Effects on sperm development. Environ. Pollut. 4. Discussions 2001;111:11-20. The analysis of the FTIR spectra collected 5. Cakmak G, Togan I, Severcan F. 17-Estradiol induced from urchin gonad treated with Cd revealed that lipids compositional, structural and functional changes in and proteins were sensitive to stress. The results of the rainbow trout liver, revealed by FT-IR spectroscopy: A current study have provided insight on the Cd induced comparative study with nonylphenol. Aqua. Toxicol. conformational changes of biomolecular including 2006; 77:53-63. lipids and proteins, as well as on the content variation 6. Corte L, Rellini P, Roscini L, Fatichenti F, Cardinali G. of these components. From a physiological aspect, the Development of a novel, FTIR (Fourier transform FTIR analysis construct a direct link between the infrared spectroscopy) based, yeast bioassay for toxicity testing and stress response study. Anal. Chimica Acta. functional biomolecular and the physiological status 2010; 659:258-265. under heavy metal stress, because the macromolecular 7. D’Souza L, Devi P, Shridhar DM, Naik CG. Use of characteristics and their contents are fundamental fourier transform infrared (FTIR) spectroscopy to study factors related with several physiological process cadmium-induced changes in Padina Tetrastromatica whereby the cell maintain its normal development and (Hauck). Anal. Chem. Insights 2008; 3:135-143. growth. Therefore, in this first study on Cd induced 8. Das AK. Trace metal status in marine biological abnormal of lipids and proteins, partial mechanism of samples: a review. Int. J Environ.Pollut.2000; 13:208- Cd stress on urchin gonad were elucidated, and some 225. observations of this paper need biochemical and 9. Dumas P, Miller L. The use of synchrotron infrared microspectroscopy in biological and biomedical molecular biological research for further analyzing. investigations. Vib. Spectrosc.2003; 32:3-21. FTIR spectroscopy offers a fast and efficient 10. Esslemont G, Harriott VJ, McConchie DM. Variability tool for detection of qualitative and quantitative Cd of trace-metal concentrations within and between induced changes in the context of molecular structure colonies of Pocillopora damicornis. Mar.Pollu.Bull. level in urchin gonad. In addition, it has the potential as 2000; 40:637-642. an accurate and sensitive technique for research on 11. Fernandez N, Beiras R. Combined toxicity of dissolved heavy metal stress by means of detecting the mercury with copper, lead and cadmium on conformational changing of biological molecular embryogenesis and early larval growth of the components and these conformational disordering as Paracentrotus lividus sea urchin. Ecotox. 2001; 10:263- 271. well as subsequent disturbed intramolecular or 12. Garman GD, Anderson SL, Cherr GN. Developmental intermolecular interaction is the key for inducing abnormalities and DNA-protein crosslinks in sea urchin malfunction. embryos exposed to three metals. Aquat.Toxicol. 1997; Acknowledgements: 39:247-265. The author thanks Dr. Xu Junxiao for his 13. Georg RC, Gomes SL. Transcriptome analysis in technical support. response to heat shock and cadmium in the aquatic fungus Blastocladiella emersonii. Eukaryotic Cell 2007; Corresponding Author: 6:1053-1062. 14. Giaginis C, Gatzidou E, Theocharis S. DNA repair Dr. Zhenlin Wei systems as targets of cadmium toxicity. Toxicol. Appl. Department of Biology science Pharmacol.2006; 213, 282-290. Dezhou University 15. Guillén, MD, Cabo N. Usefulness of the frequency data Dezhou, Shandong 253023, China of the fourier transform infrared spectra to evaluate the E-mail: [email protected] degree of oxidation of edible oils. Food Chem.1999;47:709-719.

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16. He WZ, Newell WR, Haris PI, Chapman D, Barber J. sponge Suberites domuncula. Arch. Environ. Contam. Protein secondary structure of the isolated photosystem Toxicol.1999; 36:47-55. II reaction center and conformational changes studied by 30. Shimizu M, Hochadel JF, Waalkes MP. Effects of Fourier transformation infrared spectroscopy. Biochem. glutathione depletion on cadmium-induced 1991; 30:4552-4559. metallothionein synthesis, cytotoxicity, and proto- 17. Henczová M, Deér AK, Komlósi V, Mink J. Detection oncogene expression in cultured rat myoblasts. J. of toxic effects of Cd on different fish species via liver Toxicol. Environ. Health 1997; 51:609-621. cytochrome P450-dependent monooxygenase activities 31. Soualili D, Dubois P, Gosselin P, Pernet P, Guillou M. and FTIR spectroscopy. Anal. Bioanal. Chem.2006; Effects of glutathione depletion on cadmium-induced 385: 652-659. metallothionein synthesis, cytotoxicity, and proto- 18. Henczová, M, Deér AK, Filla A, Komlósi V, Mink J. oncogene expression in cultured rat, Paracentrotus Effects of Cu and Pb on different fish species: Liver lividus, as a bioindicator. ICES J Mari. Sci. 2008; 1-8. cytochrome P450-dependent monooxygenase activities 32. Soualili D, Guillou M. Variation in the reproductive and FTIR spectra. Comp.Biochem. Physiol. C 2008; cycle of the sea urchin Paracentrotus lividus in three 148:53-60. differently polluted locations near Algiers (Algeria). 19. Howlett NG, Avery SV. Induction of lipid peroxidation Mar.Biodiversity Records 2009; 1-6. during heavy metal stress in Saccharomyces cerevisiae 33. Stohs SJ, Bagchi D, Hassoun E, Bagchi M. Oxidative and influence of plasma membrane fatty acid mechanisms in the toxicity of chromium and cadmium unsaturation. Appl.Environ.Microb. 1997; 63:2971- ions. J. Environ. Pathol. Toxicol. Oncol. 2001;19:77-88. 2976. 34. Waisberg M, Joseph P, Hale B, Beyersmann D. 20. Ivanina AV, Cherkasov AS, Sokolova IM (2008) Effects Molecular and cellular mechanisms of cadmium of cadmium on cellular protein and glutathione synthesis carcinogenesis. Toxicol. 2003;192:95-117. and expression of stress proteins in eastern oysters, 35. Warnau M, Temera Ali, Jangoux M, Dubois P, Iaccarino Crassostrea virginica Gmelin. J Exp.Biol. 211:577-586. M, De Biase A, Temara, A, Pagano G. Spermiotoxicity 21. Kilimann KV, Doster W, Vogel RF, Hartmann, C, and embryotoxicity of heavy metals in the echinoid Gänzle MG. Protection by sucrose against heat-induced Paracentrotus lividus. Environ. Toxicol. Chem. 1996; lethal and sublethal injury of Lactococcus lactis: An FT- 15:1931-1936. IR study. Biochimica et Biophysica Acta 2006; 36. Wei ZL, Dong L, Tian ZH. Fourier transform infrared 1764:1188-1197. spectrometry study on early stage of cadmium stress in 22. Kobayashi N, Okamura H. Effects of heavy metals on clover leaves. Pak. J. Bot. 2009; 41:1743-1750. sea urchin embryo development. 1. Tracing the cause by 37. Wolkers WF, Bochicchio A, Selvaggi G, Hoekstra FA. the effects. Chemosphere 2004; 55:1403-1412. Fourier Transform infrared microspectroscopy detects 23. Nacci D, Serbst J, Gleason TR, Cayula S, Thursby G, changes in protein secondary structure associated with Munns Jr WR, Johnston RK. Biological responses of the desiccation tolerance in developing maize embryos. sea urchin, Arbacia punctulata, to lead contamination Plant Physiol. 1998; 116:1169-1177. for an estuarine ecological risk assessment. J Aquatic 38. Yang J, Yen HE. Early Salt Stress Effects on the Ecosystem Stress and Recovery 2000; 7:187-199 changes in chemical composition in leaves of ice plant 24. Palaniappan PLRM, Vijayasundaram V. The FT-IR and Arabidopsis. A fourier transform infrared study of the brain tissue of Labeo rohita due to arsenic spectroscopy study. Plant Physiol. 2002; 130:1032- intoxication. Microchemi. J 2009; 91:118-124. 1042. 25. Palaniappan PLRM, Renju VB. FT-IR study of the 39. Yee N, Benning LG, Phoenix VR, Ferris FG. effect of zinc exposure on the biochemical contents of Characterization of metal-Cyanobacteria sorption the muscle of Labeo rohita. Infrared Phys.Techn. 2009; reactions: A combined macroscopic and infrared 52:37-41. spectroscopic investigation. Environ. Sci. Technol. 26. Petibois C, Deleris G. Oxidative stress effects on 2004; 38:775-82. erythrocytes determined by FT-IR spectrometry. Analyst 40. Yepiskoposyan H, Egli D, Fergestad T, Selvaraj A, 2004; 129:912-916. Treiber C, Multhaup G, Georgiev O, Schaffner W. 27. Quiniou F, Guillou M, Judas A. Arrest and delay in Transcriptome response to heavy metal stress in embryonic development in sea urchin populations of the Drosophila reveals a new zinc transporter that confers bay of brest (Brittany, France): link with environmental resistance to zinc. Nucleic Acids Res. 2006; 34:4866- factors. Mar.Pollu.Bull. 1999; 38: 401-406. 4877. 28. Roccheri MC, Agnello M, Bonaventura R, Matranga V. 41. Young, LG, Nelson L. The effects of heavy metal ions Cadmium induces the expression of specific stress on the motility of sea urchin spermatozoa. Biol. proteins in sea urchin embryos. Bull.1974;147:236-246. Biochem.Biophys.Res.Co. 2004; 321:80-87. 42. Zhu BW, Qin L, Zhou DY, Wu HT, Wu J, Yang JF, Li 29. Schröder HC, Hassanein HM, Lauenroth S, Koziol C, DM, Dong XP, Murata Y. Extraction of lipid from sea Mohamed TA, Lacorn M, Steinhart H, Batel R, Müller urchin (Strongylocentrotus nudus) gonad by enzyme- WE. Induction of DNA strand breaks and expression of assisted aqueous and supercritical carbon dioxide HSP70 and GRP78 homolog by cadmium in the marine methods. European Food Res. Techn. 2010; 230:737- 743. 11/6/2012

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An Epidemiological Study of Nosocomial Infections in Tabriz Children’s Hospital Based on National Nosocomial Infection Surveillance System (NNIS)

Shahram Abdoli Oskouie 1, Mohammad Ahangarzadeh Rezaee 1, Kamyar Ghabili 2, Farahnaz Firoozi 3

1. Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 2. Physical Medicine and Rehabilitation Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3. Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran [email protected]

Abstract: Nosocomial infections are major public health problems particularly in developing countries. These are one of the most important causes of mortality and morbidity in hospitals. Therefore, the purpose of this study was to estimate the epidemiology of NIs in a university-affiliated pediatric hospital in Northwest of Iran. The epidemiology of nosocomial infections were evaluated in Tabriz children hospital during a 12 month period from May 2008 to May 2009 using national nosocomial infections surveillance system definitions. One hundred and three patients (56.3% males and 43.7% females) were diagnosed with nosocomial infections. The incidence of nosocomial infection was 1.33 per 100 hospital discharges and 0.34 days per 100 hospital days. Frequencies of nosocomial infections by type were as follows bacteremia (68.9%), urinary tract infection (13.6), lower respiratory tract infection (9.7%), and wound infection (7.8%). Neonates ward was the highest portion of nosocomial infections followed by NICU and hematology wards. The most common pathogenic organisms were Coagulase-negative Staphylococcus (35%), Klebsiella (20.4%), Serattia (9.7%), E. coli (6.8%), and Pseudomonas (5.8%). The mean hospital stay was 25.54±17.64 days. The difference in the mean stay days for patients with nosocomial infection and non-infected patients (3.9±1.5 days) was statistically significant (P<0.0001). More than one-fourth of these patients expired. Our study emphasizes the need for implementing further health associated infections surveillance systems with proper methods to prevent and manage infections in hospitalized patients. [Oskouie SA, Ahangarzadeh Rezaee M, Ghabili K, Firoozi F. An Epidemiological Study of Nosocomial Infections in Tabriz Children’s Hospital Based on National Nosocomial Infection Surveillance System (NNIS). Life Sci J 2013;10(1):277-279] (ISSN:1097-8135). http://www.lifesciencesite.com. 42

Keywords: Nosocomial infection; children; national nosocomial infection surveillance system

1. Introduction result, preventive measures have been subjected to Nosocomial infection (NI) is a major new and additional study. In Iran, as in most complication of hospital care in both adults and developing countries, the true burden of NIs is not children. Health care associated infections are known (Askarian and Gooran, 2003). Therefore, the important causes of mortality, substantial morbidity, purpose of this study was to estimate the prolonged hospital stay, and increased costs (Fanos epidemiology of NIs in a university-affiliated and Cataldi, 2002). Accelerated improvements in pediatric hospital in Northwest of Iran. diagnostic and therapeutic procedures have helped 2. Material and Methods significant progress in medical era but plentiful using In a prospective and analytic-descriptive of invasive methods cause many health care problems study, epidemiological characteristics of NIs were every day. In the United States between 5% and 10% evaluated in Tabriz Children’s Hospital, a university- of hospitalized patients in acute care units suffer NI. affiliated hospital, during a 12 month period between The frequency of NI in children seems to be lower May 2008 and May 2009 using national nosocomial and is negatively correlated with age ranging from 7- infections surveillance (NNIS) system definitions. 9 % for infants younger than 1 year of age to 1.5 % to NIs were reviewed and data were collected from 4 % for hospitalized 10 year old children (Fanos and patients hospitalized in various wards of the hospital Cataldi, 2002). including pediatric intensive care unit (PICU), NI monitoring is very difficult especially neonatal intensive care unit (NICU), neonate unit, with limited resources, but it is vital if infection and surgery, infectious diseases, hematology and control measures are to be appropriately implemented internal medicine wards. and assessed. Nowadays substantial progress has The CDC-NNIS system defines an NI as a been made in measuring the burden of nosocomial localized or systemic condition resulting from an infections in pediatric patients, particularly in certain adverse reaction to the presence of an infectious populations such as critical care units and after agent or its toxin, and not present or incubating at the certain procedures (Khan and Celik, 2001). As a time of admission to the hospital (Garner et al., 1988;

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Horan et al., 2008). Patients who were not infected or Pseudomonas (5.8%), and Candida (1.9%). The were not in incubation period at the admission time frequency of antimicrobial resistant isolated and had positive culture of other criterion after third organisms was high. Half of isolated S. aureus was day of admission were defined as cases of NI in our meticillin resistant. Klebsiella was resistant to third study. Infection control nurse reviewed reports of generation cephalosporins (87%), aminoglicosides fever, changes in surgical wounds, prescription of (80%), and imipenem (52%). All isolated new antibiotics or any change in antibiotic regimen Pseudomonas were resistant to third generation and probable case of NI based on physician’s clinical cephalosporins and imipenem. More than one-fourth suspicions daily and all positive cultures weekly. The of the patients expired with the diagnosis of NI nurse presented all suspicious cases to infection (Figure 1). control physician (a subspecialist of pediatric Considering total number of hospitalized infectious diseases) and after confirming the patients (7730 cases) and total number of hospitalized diagnoses, NIs were registered. The data included the day (30147 days), the incidence of NIs was 1.33 name, age, sex, ward, location of the infection, (infection per 100 hospital discharges), 0.34 organism and its susceptibility pattern to current (infection per 100 hospital days). antibiotics, and infection onset dates. Admission and 30 29.6 28.6 discharge times and the patient’s final condition at 26.2 the discharge time were collected as well. Four types 25 of NIs were selected including bacteremia, lower 20 respiratory tract infections (LRTIs), urinary tract 15 12.5 infections (UTIs), and post-surgical wound 10 infections. 10 Data were presented as mean ± standard 5 deviation (SD), or percentage. Statistical analysis was 0 Septisemia Urinary tract Lower respiratory Wound infection Overall performed with SPSS for windows version 13.0 using infection tract inf independent-samples t-test. A P<0.05 was considered statistically significant. Figure 1. Mortality rates among the studied patients 3. Results In this study 103 patients (58 boys and 45 4. Discussion girls) were reviewed as NIs cases. Overall we The overall incidence of 1.33 of NIs in this recorded 71 (68.9%) cases of bacteremia, 14 (13.6%) study is consistent with the results of Canadian and cases of UTIs, 10 (9.7%) cases of LRTIs, and 8 European studies. Ford-Jones et al (1989) reported (7.8%) cases of wound infections. The mean age of average incidence of 0.17% to 14% according to age the patients with NIs was 21.60±39.32 months. The and pediatric specialty. Salamati et al (2006) mean hospital stay was 25.54±17.64 days. The computed the incidence of NIs in NICU, neonates difference in the mean hospital stay days of the and neonatal surgery units as 2.9%, 0.3%, and 1.7%, patients with NI and non-infected patients (3.9±1.5 respectively. Technological advances in surfactant days) was statistically significant (P<0.0001). replacement therapy have improved the survival rate We recorded that neonate unit was the most of premature infants with very low birth weights and common ward of the hospital regarding the NI (32%) thereby increasing the risk of NIs. Welliver and followed by NICU (22.3%), hematology unit McLaughlin (1984) reported the attack rate of the (16.5%), and PICU (12.6%). Mechanical ventilation entire hospital population surveyed was 4.1 NIs per was used in 42.3% of patients with bacteremia, 100 discharged patients. Raymond and Aujard (2000) 42.9% with UTI, 90% with LRTIs, and 12.5% with reported overall incidence of 2.5% ranging from 1% wound infection. Furthermore, urinary catheter was in general pediatric units to 23.6 in PICU. The NIs used in 23.9%, 85.7%, 30% and 12.5% of these incidence of 1.33 in our study is lower than the report patients, respectively. In addition, total parenteral of 7.7% for 1623 Australian children (Burgner et al., nutrition was administered for 29.6% of patients with 1996). Burgner et al (1996) revealed that in contrast bacteremia, 42.9% with UTI, 70% with LRTIs, and to other studies, younger children were not at 37.5% with wound infections. Laparatomy (23.2%), increased risk of NI, admission into neonate unit umbilical catheter (11.6%), central venous lines rather than age per se was associated with increased (7.2%), and venous cutdown (7.2%) were the most risk. The frequency of NI in adult units is higher 5% common invasive procedures performed in these of 8.4% in Germany and 10.4% in France. patients. The most common pathogenic organisms We recorded that bacteremia was the most were coagulase-negative Staphylococci (35%), common type of NI (69.8%), which was consistent Klebsiella (20.4%), Serattia (9.7%), E. coli (6.8%), with that of the reports of Urrea et al. (2003) (51.7%),

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de Gentile et al. (2001) (46.1%), and Ben Jaballah et Corresponding Author: al. (2006) (68.2%). However, it was higher than the Dr. Kamyar Ghabili reports of Raymond and Aujard (2000) (36%), Physical Medicine and Rehabilitation Research Mühlemann et al. (2004) (37%), and Center, Tabriz University of Medical Sciences, Tantracheewathorn et al. (2007) (28.6%). Tabriz, Iran; Nonetheless, in these studies bacteremia was known E-mail: [email protected] as the most common type of infection. The rate of 13.61 for UTI in our study was References consistent with the report of Raymond and Aujard 1. Fanos V, Cataldi L. Nosocomial infections in pediatric and neonatal intensive care: an epidemiological update. Pediatr Med (2000) as 11%. The UTI frequency (13.61) was low Chir 2002;24:13-20. similar to that of the previous study and was lower 2. Khan MM, Celik Y. Cost of nosocomial infection in Turkey: an than for adults (30-40%) probably due to less estimate based on the university hospital data. Health Serv Manage Res 2001;14:49-54. frequent use of urinary catheters in children. The 3. Askarian M, Gooran NR. National nosocomial infection lower rate of LRTIs in our study (9.7%) is surveillance system-based study in Iran: additional hospital stay comparable with the results of de Gentile et al (2001) attributable to nosocomial infections. Am J Infect Control 2003;31:465-468. but is lower than the results of other studies. The 4. Garner JS, Jarvis WR, Emori TG, Horan TC, Hughes JM. CDC bacterial species responsible for NI differ in adult and definitions for nosocomial infections, 1988. Am J Infect Control pediatric studies. Coagulase-negative Staphylococci 1988;16:128-140. 5. Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance infect children more often than adults, and were the definition of health care-associated infection and criteria for pathogens isolated most frequently in our hospital specific types of infections in the acute care setting. Am J Infect (35%) especially in neonate and hematology units. Control 2008;36:309-332. 6. Ford-Jones EL, Mindorff CM, Langley JM, Allen U, Nàvàs L, Richards et al (1999) reported that Coagulase- Patrick ML, Milner R, Gold R. Epidemiologic study of 4684 negative Staphylococci (36%) were the most hospital-acquired infections in pediatric patients. Pediatr Infect common blood stream isolated in NIs, while in the Dis J 1989;8:668-675. 7. Salamati P, Rahbarimanesh AA, Yunesian M, Naseri M. study by Welliver and McLaughlin (1984) S. aureus Neonatal nosocomial infections in Bahrami Children Hospital. was the most commonly isolated pathogen. This Indian J Pediatr 2006;73:197-200. study also revealed that documented viral infections 8. Welliver RC, McLaughlin S. Unique epidemiology of nosocomial infection in a children's hospital. Am J Dis Child were more common than infections caused by gram 1984;138:131-135. negative bacilli. In our hospital, S. aureus was 9. Raymond J, Aujard Y. Nosocomial infections in pediatric isolated in 8.2% of the patients. Similar to the study patients: a European, multicenter prospective study. European Study Group. Infect Control Hosp Epidemiol 2000;21:260-263. by Raymond and Aujard (2000), the antimicrobial 10. Burgner D, Dalton D, Hanlon M, Wong M, Kakakios A, Isaacs resistance frequency of Klebsiella and other gram- D. Repeated prevalence surveys of paediatric hospital-acquired negative bacilli was high. Candida was a pathogen of infection. J Hosp Infect 1996;34:163-170. 11. Urrea M, Pons M, Serra M, Latorre C, Palomeque A. Prospective increasing importance. Infections due to antibiotic incidence study of nosocomial infections in a pediatric intensive resistant bacteria were associated with increased care unit. Pediatr Infect Dis J 2003;22:490-494. length of stay in PICU after onset of infection and 12. de Gentile A, Rivas N, Sinkowitz-Cochran RL, Momesso T, Iriart EM, Lopez E, Beck-Sague CM, Jarvis WR. Nosocomial increased mortality. Similar to the previous reports, infections in a children's hospital in Argentina: impact of a the difference between hospital days in patients with unique infection control intervention program. Infect Control NI and non-infected patients was statistically Hosp Epidemiol 2001;22:762-766. 13. Ben Jaballah N, Bouziri A, Kchaou W, Hamdi A, Mnif K, significant. In our study, incidence of NI was Belhadj S, Khaldi A, Kazdaghli K. Epidemiology of nosocomial underestimated because viral infections were not bacterial infections in a neonatal and pediatric Tunisian intensive included due to limitations in medical equipments care unit. Med Mal Infect 2006;36:379-385. 14. Mühlemann K, Franzini C, Aebi C, Berger C, Nadal D, Stähelin and also due to the absence of post-discharge J, Gnehm H, Posfay-Barbe K, Gervaix A, Sax H, Heininger U, surveillance. Increasing frequency of antimicrobial Bonhoeffer J, Eich G, Kind C, Petignat C, Scalfaro P. Prevalence resistant isolated emphasizes the necessity for of nosocomial infections in Swiss children's hospitals. Infect Control Hosp Epidemiol 2004;25:765-771. bacteriological monitoring of hospitalized children. 15. Tantracheewathorn T, Vititpatarapak N, Phumisantiphong U. In conclusion, repeated prevalence surveys Epidemiologic study of nosocomial bacterial infection of might enable simple and cost effective assessment of pediatric patients at BMA Medical College and Vajira Hospital. J NIs, facilitating appropriate infection control Med Assoc Thai 2007;90:258-265. 16. Richards MJ, Edwards JR, Culver DH, Gaynes RP. Nosocomial interventions. infections in pediatric intensive care units in the United States. Acknowledgements: National Nosocomial Infections Surveillance System. Pediatrics Authors are grateful to Dr. Farid Panahi and 1999;103:e39. Ms. Farzaneh Alikhah for their assistance in preparing this manuscript.

12/12/2012

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Promoting the recovery of neurologic function and antagonizing cell apoptosis in ICH model rats by Induced Pluripotent Stem Cell Transplantation*

Jing Qi1#, Jie Qin1#, Guangming Gong2,Bo Song1 , Huili Zhang1, Yanlin Wang1, Haiyun Qi1, Bo Yang3, Yi Zhang4, Yuming Xu1*, Shilei Sun1*

1. The Third Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China 2. Department of Microbiology and Immunology, College of Basic Medicine, Zhengzhou University, Zhengzhou, Henan 450001, China 3.Neurosurgery Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou Henan 450052, China 4.Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. [email protected]

Abstract: Intracerebral hemorrhage (ICH) is a major disease that seriously threatens human health; it features high fatality rate and disability rate and often leaves severe neurologic dysfunction even after recovery. The effect of induced pluripotentstem cells (iPSCs) transplantation on the recovery of the neurologic dysfunction of ICH is still uncertain. We prepared ICH rat models by injecting collagenase to the striatum of rats through stereotaxic apparatus, injected derived from ICH patients around the hematoma to evaluate the dynamic changes of neurologic function on day 1, 3, 7, 14 and 28 after ICH treatment by iPSCs transplantation with modified neurological severity score (mNSS) table, and studied the cell apoptosis changes around the hematoma. We found out that the neurological scores of rats were obviously improved and the apoptotic cells were significantly reduced compared with the model group. Our research results showed that iPSCs transplantation can effectively promote the recovery of neurologic function of ICH rats and antagonize cell apoptosis induced by encephalorrhagia. [Qi J, Qin J, Song B, Zhang HL, Wang YL, Yang YK, Qi HY, Gong GM, Xu YM, Sun SL. Promoting the recovery of neurologic function and antagonizing cell apoptosis in ICH model rats by Induced Pluripotent Stem Cell Transplantation. Life Sci J 2013;10(1):280-284] (ISSN:1097-8135). http://www.lifesciencesite.com. 43

Key words：induced pluripotent stem cells (iPSCs), intracerebral hemorrhage (ICH), apoptosis

1.Introduction also avoid the ethical issues brought by embryonic After intracerebral hemorrhage(ICH), the stem cells, reduce immunological rejection, and secondary lesion is also the main cause of hemorrhagic provide possibilities of clinical application for brain injury in addition to the physical injury resulted autologous stem cell gene treatment(Lindvall and from the hematoma’s oppressing the surrounding brain Kokaia 2011). tissues(Steiner and Bosel 2010). Histiocyte death Cell apoptosis largely depends on the activation resulted from secondary lesion after ICH has important of cysteinyl aspartate-specific protease (caspase) clinical significance, and cell apoptosis as a gene- family, in which caspase-3 is a key enzyme in regulated manner of cell death may be one of the mammalian cell apoptosis (Uccelli, Benvenuto et al. important parts(Felberg, Grotta et al. 2002). ICH is 2011). Recently, its effects in the neuron apoptosis short of effective treatment means, and satisfactory have been observed by people, and it has also been curative effects are unable to be achieved even if the confirmed by experiments that caspase-3 participates in hematoma is timely eliminated. various pathologic changes of brain injury, including Stem cell treatment is one of the possible transient cerebral ischemia, cerebral trauma and treatment methods for ICH. Induced pluripotent stem epilepsy and is also an important neuronal apoptosis cells (iPSCs) are a kind of pluripotent stem cell that can effector. However, in this experiment, Oct4, Sox2, Klf4 reprogram somatic cells to embryonic stem cell-like and c-Myc genes were transformed into ICH patients’ cells through its ectopic expression of some skin fibroblasts by lentiviral vector and further transcription factors(Lunn, Sakowski et al. 2011). As a reprogrammed to iPSCs, TUNEL was used to conduct totipotent cell, it can improve the neurologic functions apoptotic cell counting and to observe the changes of of rats by means of neuron replacement, anti- caspase-3, and the possible mechanisms of ICH patient inflammation, anti-apoptosis, angiogenesis promotion, derived iPSCs transplantation improving neurologic myelin sheath and axon regeneration, and neuro- function of ICH rats were discussed from the protection, but the specific mechanism is still not perspective of anti-apoptosis, which has not been clear(Marchetto, Winner et al. 2010). In addition, it can reported in any literature yet.

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2. Materials and Methods 2001; Lu, Chen et al. 2003) was performed 24h after 2.1 cells the operation, and rats scored 8-12 in mNSS. IPSCs comed from our laboratory, and the cells were authorized by Ethics Committee of Zhengzhou 2.3 Behavioral Tests University. Two days before recovery, we prepared a mNSS scoring (including movement, sensation, layer of mouse embryonic fibroblasts cells(MEF) balance and reflection) was adopted to evaluate the feeders at 2.5×104 cells per well of 6-well plates using neurologic function deficit and recovery status of the fibroblasts medium(Dulbecco's modified Eagle's rats, and each group was scored on day 1, 3, 7, 14 and medium(DMEM)(Invitrogen) supplemented with 10% 28 after operation fetal bovine serum (FBS) (Invitrogen),1% non-essential amino acids (NEAAs) (Invitrogen),and 100IU/ml 2.4 Immunohistochemical Staining penicillin/streptomycin (Invitrogen)). 4-5 days,iPSCs Samples of rat brain tissues were extracted in clones were seen on MEF in hESC medium each group after 14 days of modeling; brain tissues of (DMEM/F12 supplemented with 20% KnockOut 3-5 rats were fixed with paraformaldehyde fixation, Serum Replacement (Invitrogen), 1mM glutamine then dehydrated with gradient sucrose, frozen and cut (Invitrogen), 1% NEAA, 0.1 mM β- into coronal slices (12μm thick). Each group of slices mercaptoethanol(Sigma-Aldrich), and 4 ng/ml bFGF were conducted with immunofluorescence staining of (Invitrogen))and sub-cultured every 6-7 days or so. brdu (rabbit anti-human as primary antibody and goat alkaline phosphatase test (AP Staining) was performed. anti-rabbit,FITC conjugated conjugated as second Cells were fixed in 4% paraformaldehyde for 1-2 antibody, Santa), and the immunohistochemical minutes. Aspirated and rinsed with 1 ×Rinse Buffer. staining of caspase-3(rabbit anti-rat as primary Added stain solution (BufferA: BufferB: antibody and goat anti-rabbit as second antibody). BufferC=100:38:28) enough to cover the well. The TUNEL assay immunofluorescence detection of plate was incubated in dark at room temperature for 15 apoptosis(Roche). minutes. Then rinsed with 1 × Rinse Buffer and The slices were re-warmed, fixed with 4% observed under the microscope Cell. paraformaldehyde at RT for 5min, washed with PBS for 3 times and 5min/time, heated (92~98℃) in 2.2 Experimental animal grouping and modeling 0.01mol/ L pH6.0 citrate buffer solution for 30min for Animal feeding and all relevant follow-up antigen retrieval and cooled naturally at RT for 30min; experimental procedures were authorized by Ethics then the slices were washed with PBS for 5min for 3 Committee of Zhengzhou University and the Sprague- times and sealed with 5% BSA at RT for 60min; Dawley (SD) rats were provided by Henan Provincial subsequently, first antibody (1:100-1:500) was Animal Center. 20 male rats(200-250g) were randomly dropwise added on the slices and the slices were left at divided into two groups: ten as a intracerebral 4℃ over night and then washed with PBS for 5min for hemorrhage PBS group (PBS group, after the models 3 times; then second antibody (1:500) was added at RT were identified as successful 24h after the operation， for 1h; afterwards, the slices were washed with PBS for (10uL)PBS were injected by stereotaxic puncture, ten 5min for 3times; for immunohistochemistry, color was as a iPSCs intervention group(iPSCs group, after the developed with DBA for 5min, and the slices were re- models were identified as successful 24h after the stained with haematoxylin and sealed. operation ，(1-2×105/uL) iPSCs were injected by stereotaxic puncture). iPSCs were incubated with 20 2.5 Statistical Analysis umol/L BrdU (Sigma-Aldrich) for 48 hours before SPSS 13.0 statistical software was adopted to transplantation. the rats were fixed on rat brain analyze the data and all measurement data were stereotaxic apparatus (Narishige SN-3, Tokyo, Japan) indicated in mean ± standard deviation ( X ± S ), where, at a prone position, and by referring to the repeated measurement and analysis was conducted on references(Jeong, Chu et al. 2003) (Fujiwara, mNSS data lines, Student t-test was conducted on the Mandeville et al. 2011) and the Rat Brain Stereotaxic rest of the data. Data was considered statistically Graph of George Paxinos, needles were inserted significant if P <0.05. perpendicularly for 6mm at 0.2mm back bregma and 3mm left, with the target point at the left corpus 3. Results striatum. (0.5U/2u L) collagenaseⅦ (Sigma- 3.1 iPSCs preparations Aldrich)was injected slowly with a 10 μL microsyringe, IPSCs clones were seen on day 5 after recovery and the needle was retained for ten minutes and then (Figure.1A). Then AP staining was performed and the withdrawn slowly.Then the wound surface was cleaned stained cells presented modena, hence positive and the scalp was suture. mNSS scoring(Chen, Li et al. (Figure.1B).

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3.5 Expression of caspase-3 caspase-3 was also expressed in each group and the expression level around the hematoma was higher. Cells were considered positive if the cytoplasm was stained brownish yellow granules; for negative cells, only the karyon was stained blue. The expression of caspase-3 in iPSCs group was relatively descreased compared with PBS group. The results showed that the Figure 1. IPSCs clone (A). (100 ×) Alkaline difference of positive cell number between iPSCs phosphatase staining of iPSCs(B) (100 ×) . group and PBS group had statistical significance (P＜ 0.05). (Figure.3E-F,G) 3.2 Behavioral Tests After the rats were induced to ICH by stereotaxic collagenase injection, the rats showed contralateral hemiplegy among other neurologic function deficits soon. Symptoms and physical signs reached the peak 1~3d after the operation and then the rats recovered gradually. Compared with model group, the mNSS scoring of iPSCs group on day 7 after operation were different. The difference of mNSS scoring on day 14,28was notable and significant (P＜0.05), (Figure.2).

PBS group iPSCs group 12

4 mNSS scorces TUNEL Caspase-3 2 70 0 60 1 3 7 14 28 50 * Time Points(day) 40 30

Figure 2. MNSS scoring at each time point for each 20 * group. Since day 14, scoring of iPSCs group is clearly 10 0

superior to that of PBS group, (p<0.05). The numbers of postive cell

PBS group 3.3 Brdu immunofluorescence staining G iPSCs group Under fluorescence microscope, it can be seen that groups Brdu immunofluorescence stained positive cells Figure 3. Brdu immunofluorescence stained positive obviously gathered and spread around the cells obviously gathered and spread around the hematoma(Figure.3A), and only few scattered on the hematoma(A) (400 ×), and only few scattered on the opposite side of hematoma(Figure.3B). opposite side of hematoma(B)(400 ×) .TUNEL stained positive cells presented green globular shape. The 3.4 TUNEL immunofluorescence staining apoptotic cells were mainly located around the nidus in TUNEL staining showed that a great number of PBS group(C)(200 ×).There were only few apoptotic apoptotic cells existed around the nidus 14d after ICH, cells seen in the iPSCs group(D)(200 ×).Numbers of and the apoptotic cells were mainly located around the apoptotic cells of iPSCs group(*) is higher than that of nidus in PBS group, while only few can be found on PBS group (p<0.05) (G). Immunohistochemical the opposite side. Besides, there were only few staining of caspase-3 (E) is PBS group, and (F) is apoptotic cells seen in iPSCs group; the difference had iPSCs group (400 ×). Numbers of positive cells of statistical significance (P<0.05). (Figure.3C-D,G). iPSCs group(*) is higher than that of PBS group (p<0.05) (G).

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research on ICH rat brain slices. The difference had 4. Discussion statistical significance. ICH has high incidence rate, high fatality rate and In recent studies, people have realized that high disability rate. For most survivors, various degrees Caspase-3 is a kind of cysteinyl aspartate-specific of neurological dysfunction may remain, yet at present, protease (caspase), and the proteolysis cascade it there is still no really effective treatment method for results selectively splits the substrate aspartate residue. neurological impairment after ICH(Steiner and Bosel Caspase-3 can activate the specific signal system and 2010). Therefore, in addition to further reinforcing the generate nuclear shrinking, DNA fragment formation prevention research on ICH and the emergency and other apoptosis, eventually control the occurrence measures in acute phase, seeking the effective and development of apoptosis by splitting the protein treatment method to promote the recovery of damaged kinase, nuclease and cytoskeleton(Huang, Xi et al. neurologic function is also the common objective of the 2002). This experiment showed that Caspase-3 in numerous medical workers. Stem cells, especially with iPSCs group is significantly reduced compared with the appearance of iPSCs, have become the potential, that of PBS group. Besides, it is reported in some possible effective method for ICH treatment(Lindvall literature that Caspase-3 also plays a role in cell and Kokaia 2011). apoptosis by destroying the anti-apoptosis factor of cell In this experiment, ICH models were successfully (such as bcl-2) and the cell structure (such as structural prepared by injecting collagenase to the striatum of rats protein actin, fodrin, lamin). Some scholars have through stereotaxis. After modeling, the symptoms and proved with immunohistochemical double mark signs of neurologic deficit of rats were typical and method that Caspase-3 and TUNEL staining coexist could sustain for about 3 days, after which, there were around the hemorrhagic nidus, and also confirmed that various degrees of recovery.We transplanted iPSCs 95% are neurons and less than 5% are neuroglial cells. into the experimental ICH rat brains and observed the We adopt contiguous slices to separately conduct treatment effects. It was observed that transplanted Caspase-3 immunohistochemistry and TUNEL staining, iPSCs could survive in brain tissue and were mainly and both the results are basically consistent in terms of scattered around the hematoma through Brdu range and change of positive cells(Wu, Hua et al. 2002). biomarker tracing, promoting that iPSCs could migrate Apoptosis mechanism may participate in some towards the damaged parts of tissue. For iPSCs group, neuronal damages after ICH and the activation of the scoring of neurologic function was superior to that caspase-3 has important regulation and control effect in of model group since day 7, and notably higher than the process of neuronal apoptosis after ICH. the latter on day 14,28. The difference had statistical In this experiment, we took the ICH model rats significance. This showed from the point of as the object of study and adopted iPSCs for behavioristics that iPSCs transplantation does have transplantation. The results showed that iPSCs certain treatment effects in the ICH models. transplantation can effectively improve the neurologic After ICH, the hematoma not only triggers mass dysfunction of ICH model rats. The mechanism may effect in the surrounding area, but also causes three relate to apoptosis regulation and control. This study pathologic changes: neuron and spongiocyte death, provides reliable preclinical theoretical basis for the vasogenic cerebral edema and damage of blood brain further discussion of iPSCs transplantation, and barrier(Lu, Chen et al. 2003). The exact mechanism eventually promotes its clinical application. relevant to pathologic changes of ICH is still not determined, and identifying the characteristics of the Acknowledgements: cell damage and death after ICH is helpful for This work was supported by General Project of researching the treatment measures of neurologic Natural Science Foundation of China (81070920) (To damage. During cell apoptosis, nuclear chromatin DNA Y.M.X.). Authors are very grateful to National Natual double strands are broken into plenty of DNA Science Foundction of China for financial support. fragments containing 3’-OH ends. Under the effect of terminal deoxynucleotidyl transferase (TDT)(Gong, Corresponding authors: Boulis et al. 2001), these fragments integrate with Shilei Sun, M.D. and Yuming Xu,Ph.D., M.D. fluorescein-marked nucleotide and then the peroxidase- Third Department of Neurology, First Affiliated conjugated anti-fluorescein antibody, thus promoting Hospital of Zhengzhou University, Zhengzhou 450052 the apoptotic cell staining, which is called terminal Henan Province, China deoxynucleotidyl transferase-mediated dUTP nick end Email: [email protected] labeling, i.e. TUNEL staining(Kitaoka, Hua et al. 2002). and [email protected] We observed that the number of apoptotic cells in #these authors are thought to have equal contributions. iPSCs group was obviously reduced compared with that of the PBS group by conducting TUNEL staining

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References 13. Uccelli, A., F. Benvenuto, A. Laroni and D. Giunti. 1. Chen, J., Y. Li, L. Wang, Z. Zhang, D. Lu, M. Lu Neuroprotective features of mesenchymal stem and M. Chopp . Therapeutic benefit of intravenous cells.Best Pract Res Clin Haematol.2011; 24(1): administration of bone marrow stromal cells after 59-64. cerebral ischemia in rats. Stroke.2010;32(4): 14. Wu, J., Y. Hua, R. F. Keep, T. Schallert, J. T. Hoff 1005-1011. and G. Xi. Oxidative brain injury from 2. Felberg, R. A., J. C. Grotta, A. L. Shirzadi, R. extravasated erythrocytes after intracerebral Strong, P. Narayana, S. J. Hill-Felberg and J. hemorrhage. Brain Res.2002; 953(1-2): 45-52. Aronowski . Cell death in experimental intracerebral hemorrhage: the "black hole" model of hemorrhagic damage. Ann Neurol. 2002; 51(4): 517-524. 12/22/2012 3. Fujiwara, N., E. T. Mandeville, X. Geng, Y. Luo, K. Arai, X. Wang, X. Ji, A. B. Singhal and E. H. Lo. Effect of normobaric oxygen therapy in a rat model of intracerebral hemorrhage. Stroke. 2011; 42(5): 1469-1472. 4. Gong, C., N. Boulis, J. Qian, D. E. Turner, J. T. Hoff and R. F. Keep. Intracerebral hemorrhage- induced neuronal death. Neurosurgery.2001; 48(4): 875-882; discussion 882-873. 5. Huang, F. P., G. Xi, R. F. Keep, Y. Hua, A. Nemoianu and J. T. Hoff. Brain edema after experimental intracerebral hemorrhage: role of hemoglobin degradation products. J Neurosurg.2002; 96(2): 287-293. 6. Jeong, S. W., K. Chu, K. H. Jung, S. U. Kim, M. Kim and J. K. Roh. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke.2003; 34(9): 2258-2263. 7. Kitaoka, T., Y. Hua, G. Xi, J. T. Hoff and R. F. Keep. Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage. Stroke.2002; 33(12): 3012-3018. 8. Lindvall, O. and Z. Kokaia . Stem cell research in stroke: how far from the clinic? Stroke.2011; 42(8): 2369-2375. 9. Lu, M., J. Chen, D. Lu, L. Yi, A. Mahmood and M. Chopp. Global test statistics for treatment effect of stroke and traumatic brain injury in rats with administration of bone marrow stromal cells. J Neurosci Methods. 2003;128(1-2): 183-190. 10. Lunn, J. S., S. A. Sakowski, J. Hur and E. L. Feldman. Stem cell technology for neurodegenerative diseases. Ann Neurol. 2011; 70(3): 353-361. 11. Marchetto, M. C., B. Winner and F. H. Gage. Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases. Hum Mol Genet.2010; 19(R1): R71-76. 12. Steiner, T. and J. Bosel. Options to restrict hematoma expansion after spontaneous intracerebral hemorrhage. Stroke .2010;41(2): 402-409.

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Contact lens care and disease characteristics in keratoconus

John Ching-Jen Hsiao1,2,*, Shyan-Tarng Chen1,2

1 School of Optometry, Chung Shan Medical University, Taichung City, Taiwan 2 Department of Ophthalmology, Chung Shan Medical University Hospital, Taichung City, Taiwan

[email protected]

Abstract: Keratoconus is a noninfalmmatory, progressive corneal disorder characterized by localized conical protrusion, apical thinning, irregular astigmatism, and central corneal scarring. These changes in cornea often lead to some unique clinical symptoms and signs which usuallycan’t be seen in normal eyes. Contact lens care of keratoconus is more challenging than care of normal eyes because the need for special diagnostic trial lenses, experienced clinician and more professional chair time.In this article, we will focus on how to diagnose Keratoconus based on its disease characteristics and the complexity of contact lens care for it. The model of 3 point touch lens fittingwill also be discussed in detail.[John Ching-Jen Hsiao, Shyan-Tarng Chen. Contact lens care and disease characteristics in keratoconus. Life Sci J. 2013, 10(1):285-287](ISSN:1097-8135).http://www.lifesciencesite.com. 44

Keywords: Keratoconus, Irregular astigmatism, Contact lens care

1. Introduction Visual acuity was assessed for each eye with Keratoconus is a progressive, often bilateral, Snellen vision test chart viewed at 3 meter with non-inflammatory ocular diseasecharacterized by mirror reflection set up. Corneal topography was steepening, apical thinning, and central scarring of performed using Dicon topographer (Paradigm the cornea. Patients’ vision with keratoconus, usually Medical, USA) and keatometry reading using Topcon deteriorate as the corneal protrusion worsen due to keartometer. Contact lenses used were Rose K rigid more irregular astigmatism and central corneal scar. gas permeable lenses specially designed for The onset of Keratoconus typically begin at puberty keratoconic eyes. and early adulthood and likely to progress until late in life and thus affects adults’ visual quality in their 3. Results and Discussion daily activities. It is widely accepted that the corneal At the early stage of keatoconus, patients will distortions induced by keratocconus increase the find a slowly but continuously decrease in both aided eye’s optical aberrations and that the resultant and unaided vision; this is because of the increase of degradation in retinal image quality reduced visual irregular astigmatism and frequent changes in the acuity and contrast sensitivity.1,2 The classic clinical symptoms and signs of keratoconus include refractive errors as the condition progresses.Often worsening of best corrected spectacle acuity, patients will complain seeing diplopia (double vision), fluctuating visual acuity, increased regular and mild photophobia, ghosting, flare from lights at night irregular corneal astigmatisms, an inferiorly and mostly important of all,and the reduced vision displaced, thinned protrusion of the cornea, Vogt’s with spectacles.The diagnosis of keratoconus is based striae in the posterior corneal stroma, scars in on many clinical findings, and they include: Bowman’s layer, and Fleischer’s ring. These signs The early signs: become more prevalent as the disease progresses.3,4 Distorted retinoscopy reflex, distorted and In this paper, we will discuss some of the symptoms steeper keratomety mires, distorted photo- and signs mentioned above and the lens fitting keratoscopy rings, inferior steeping in corneal philosophy of 3 point touch model for keratoconus. topography maps, apical corneal thinning and cone formation of the cornea. 2. Material and Methods The later signs: The following examinations were performed: Advanced Cone Formation: usually find on Visual acuity with and without correction, manifest patients when his or her average ketatometry reading refraction, keratometry, corneal topography, falls between 50.00 to 60.00 diopters. The profile of complete slitlamp biomicroscopy examination and the cone can take shape in Nipple cone, Oval cone, and Globus cone, with the last one being the most many trail rigid lens fittings with fluorescein dye. large in size and most difficult to fit contact lens.

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Munson’s Sign of Lower Lid: The lower lid is soft contact lens and spectacle have no effect on the pushed downward when a patient with severe corneal distorted surface of the keratoconic cornea and hence ectasia looks down. This can be easily seen by preserving the majority of aberration induced by comparing the both lids when patients with irregular astigmatism. In contrast, rigid contact lens keratoconus look in a downward gaze (Fig.1). improves vision in eyes with keratoconus is that tears Fleischer Iron Ring: Iron deposition in the fill the gap between the irregular corneal surface, cornea, resulting in brown-colored rings or lines, are allowing the smooth inner surface of the lens to act as thought to be caused by the absorption of the metal the new smooth surface of the cornea, thereby into the basal epithelium layer from local stagnant reduces the majority of the irregular astigmatism. tears.5This brown ring usually encircles the base of It is widely accepted that the three point touch the ectatic cone of the keratoconus. One can easily method is the most suitable philosophy for see with slit lamp under cobalt blue light. keratoconus rigid lens fitting. The three point of Apical scar formation: When the severity of the cone reaches to a certain level that may cause the touch refer to the positions on the lens found at the 3 integrity of cornea to disrupt and induce scar. This is o’clock position, the 9 o’clock position, and the may also associated with rigid lens fitting if patients central part of the lens. This can be easily analyzed had been wearing the lens as the treatment of by looking the corneal topography (Fig. 2) and the keratoconus. corresponding rigid lens fitted photography using 3 Increased visibility of corneal nerve fibres: This point touch method and Rose K rigid lens (Fig.3) of is also due to the stress on cornea associated with the our example patient with keratoconus. The red, progression of corneal ectasia. steeper regions at the middle of topography of both Vogt’s straie: Vertical lines that occur in the eyes match with the central light touch bearing area posterior stroma, also induced by the stress of the in the photos. The fitting philosophy emphasize on cone. The striae can disappear upon induced pressure, the lens must rest lightly against the cone apex and either via examiner‘s finger or Q-tip on patients’ the majority of lens weight is supported by the nasal eyelid (Fig.1). and temporal zone by the mid-periphery of the back All these clinical signs would be more obvious surface. The objectives of the lens fitting are to and easier to detect as the condition of keratoconus provide best possible visual acuity, minimal progresses. interference to corneal physiology and to optimize lens comfort. Some case studies have led to hypothesis that apical clearance fitting accelerate ectatic progression, whereas some other opposing studies suggest that via apical support, the cornea may acquire more central scar. To balance the 2 above hypotheses, one must be careful not to impose excessive mechanical pressure on the cone when the fitting technique of 3 point touch method of lens is used.

Fig. 1. Spherical orthokeratology lens fitted on a cornea with high astigmatism.

The treatment of keratoconus includes correction with spectacles at early stages, the use of contact lens at the moderate stage, and finally cross- linking or keatoplasty at severe cases. Although corneal transplantation has been successful in the surgical management of keratoconus, most of the patients with this condition are still managed with Fig. 2. Topography of Keratoconus OU. contact lenses for visual rehabilitation. 6,7In general,

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7. Lembach RG. Use of contact lenses for management of keratoconus.Ophthalmol Clin North Am 2003; 16:383–394.

1/14/2013

Fig. 3. “3 point touch” rigid lens fitted photography in corresponding to the corneal topography above, using and Rose K rigid lens.

4. Conclusion Due to the significant impact of keratoconus to patients’ quality of lives, it is important to diagnosis the disease and to deal with it with appropriate treatment whenever suitable. Rigid Gas Permeable contact lens remains the choice of treatment for keratoconus because of its ability to reduces irregular astigmatism, to improve visual acuity and its’ non- surgical in nature. Although contact lens care of keratoconus possess more challenges than that of the normal eyes, the benefit patients received from enhanced visual acuity is priceless.

Corresponding Author: John Ching-Jen Hsiao, OD, FIACLE. School of Optometry, College of Medical Sciences and Technology, Chung Shan Medical University, No. 110, Jianguo N. Rd., Taichung City 402, Taiwan. E-mail:[email protected]

References 1. Lim L, Wei R, Tan D & Chan W. Evaluation of higher order ocular aberrations in patients with keratoconus. J Refract Surg 2007; 23: 825–828. 2. Maeda N, Fujikado T, Kuroda T et al. Wavefront aberrations measured with Hartmann-Shack sensor in patients with keratoconus. Ophthalmology 2002; 109:1996–2003. 3. Krachmer JH, Feder RS, Belin MW. Keratoconus and related non-inflammatory corneal thinning disorders. Surv Ophthalmol 1984; 28:293–322. 4. Zadnik K, Gordon MO, Barr JT, Edrinton TB. The CLEK study group Biomicroscopic signs and disease severity in keratoconus. Cornea 1996; 15:139–46. 5. Gass JD. The iron lines of the superficial cornea; Hudson-Stahli line, Stocker’s line and Fleischer’s ring. Arch Ophthalmol 1964; 71: 348-358. 6. Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998; 42:297–319.

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The Influence of Fermentation by Different Lactobacillus on the Free Radical Scavenging Activity of Burdock and Variations of Its Active Components

Chi-Ting Horng1,2, Shih-Chiang Lee3, Rei-Chu Chang4, Wan-Ping Lee 5, Feng-Lang Lin2, Chin-Wen Hsu6, Fu-An Chen2,*

1 Department of Ophthalmology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, ROC 2 Department of Pharmacy & Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung, Taiwan, ROC. 3 Dong Yuan Biotech Pharmaceutical Co. Ltd., Kaohsiung, Taiwan. 4 Department of Food Science and Technology, Tajen University, Pingtung, Taiwan, ROC 5 Department of Nursing, Tajen University, Pingtung, Taiwan, ROC 6 Department of Surgery, Kaohsiung Armed Force General Hospital, Kaohsiung, Taiwan, ROC [email protected] Abstract: Burdock (Arctium lappa L.) is a nutritious plant which is commonly cultivated in Taiwan and Japan. The purpose of this study is to explore the effect of fermentation by different lactobacillus on the free radical scavenging activity of burdock and variations of its active components. Four lactobacillus as Lactobacillus casei subsp. casei (Orla-Jensen) Hansen and Lessel (BCRC No.10697), Lactobacillus delbrueckii subsp. bulgaricus (Orla-Jensen) Weiss et al (BCRC No.10696), Lactobacillus plantarum subsp. plantarum (Orla-Jensen) Bergey et al (BCRC No.10069) and Streptococcus thermophilus (Orla-Jensen) (BCRC No.14086) were used to ferment burdock for 48 hours. The amount of lactic acid bacteria (LBA), sweetness, pH, total polyphenols and the free radical scavenging activity, using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay were measured. The result showed Lactobacillus casei subsp. casei (Orla-Jensen) Hansen and Lessel (BCRC No.10697) had better effect on LBA populations, total polyphenols and free radical scavenging activities compared with other three lactobacillus. This result provides important information on developing fermented burdock antioxidant dietary supplements. [Chi- Ting Horng, Shih-Chiang Lee, Rei-Chu Chang, Wan-Ping Lee, Feng-Lang Lin, Chin-Wen Hsu , Fu-An Chen. The influence of fermentation by different lactobacillus on the free radical scavenging activity of burdock and variations of its active components. Life Sci J. 2013;10(1):288-292] (ISSN:1097-8135). http://www.lifesciencesite.com. 45

Keywords: Burdock、Lactobacillus、Fermentation、Free radical scavenging activity、Total polyphenols

1. Introduction blood cholesterols levels and immune competence, Burdock (Arctium lappa L.) has long been which could be resulted from the modification and/or cultivated as a vegetable in Taiwan for dietary use1. creation of nutrient, botanically-active components and Burdock is also used as a folk medicine as a diuretic microbial metabolites 18-20. The present study is thus to and antipyretic2. It has become a popular health drink examine the influence of fermentation by different in Taiwan in the last decade. Several studies have lactobacillus on the free radical scavenging activity of reported that the root of burdock possesses various burdock and variations of its active components. pharmaceutical activities including antibacterial activity3,4, desmutagenic activity5, antioxidant ability6- 2. Material and Methods 9, hepatoprotective effect10,11, gastroprotective Material activity12,13, hypoglycemic activity 14,15, hypolipidemic Burdock (Arctium lappa L.) was obtained from activity 16, sexual behavior enhancement15 and anti- the Gueilai Community Developmental Institute in inflammatory activity16, among which the Pingtung County, southern Taiwan. Lactobacillus： gastroprotective activity, hepatoprotective efficacy, Lactobacillus casei subsp. casei (Orla-Jensen) 7 anti-inflammatory activity , and antioxidant activity Hansen and Lessel (BCRC No.10697), Lactobacillus 8 are associated with the free radical scavenging activity . delbrueckii subsp. bulgaricus (Orla-Jensen) Weiss et Fermentation using yeast or lactic acid bacteria al (BCRC No.10696), Lactobacillus plantarum subsp. has long been applied in food industry due to its plantarum (Orla-Jensen) Bergey et al (BCRC beneficial effects in flavor development, in inhibition No.10069) and Streptococcus thermophilus (Orla- of spoilage bacteria and pathogens, in intestinal health Jensen) (BCRC No.14086) were purchased from the and other health benefits related to cancer prevention, Bioresource Collection and Research Center (BCRC),

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Hsinchu, Taiwan. De Man, Rogosa, Sharpe (MRS) solutions and expressed as mg gallic acid /g BFL. All broth was the product of Difco (Becton, Dickinson measurements were done in triplicate. and Company, USA). Methanol and acetone were the product of Tedia and Mallinckrodt (USA), DPPH free radical scavenging activity of BFL repectively. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) The free radical scavenging activity of BFL was and gallic acid were purchased from Sigma (USA). evaluated using DPPH free radical-scavenging assay Folin-ciocalteu reagent was purchased from Merck as described previously22. A stock solution (1 mg/mL) (Germany). All other chemicals were of analytical of each extract was prepared and diluted with reagent grade. methanol into various concentrations. An aliquot of 50 L of each dilution was transferred into a 96-well Burdock fermentation microplate (NUNC, Roskilde, Denmark). A working Six hundred grams of the root of burdock was solution of DPPH (250 M) in methanol was freshly extracted by 3 L of hot distilled water for 30 min at prepared and then an aliquot of 150 L was added to 100 °C. The aqueous burdock extract solution was each well. The DPPH scavenging percentage was filtered through filter paper and a filter funnel. After measured at 490 nm on an ELISA reader cooling, 100 mL of aqueous burdock extract solution (ThermoLabsystems, Cheshire, UK after incubation was placed into a bottles and further sterilization in for 0.5 h. Each dilution was performed at least in an autoclave. four lactobacillus strains as mentioned triplicate. previously were inoculated for fermentation at 37 °C. The physicochemical property, total polyphenols 3. Results and discussion content and free radical scavenging activity of In the present study, four lactobacillus strains as burdock ferment liquid (BFL) were determined after Lactobacillus casei subsp. casei (Orla-Jensen) fermentation for 48 hrs. Hansen and Lessel (BCRC No.10697), Lactobacillus delbrueckii subsp. bulgaricus (Orla-Jensen) Weiss et al (BCRC No.10696), Lactobacillus plantarum subsp. Physicochemical properties of BFL plantarum (Orla-Jensen) Bergey et al (BCRC Lactic acid bacteria (LBA) populations were No.10069) and Streptococcus thermophilus (Orla- counted using standard methods according to CNS Jensen) (BCRC No.14086) were used to ferment 10890. Ten fold dilutions beginning with 1 ml of a burdock and the effects of fermentation by different sample were added to 9 ml of normal saline solution LBA on the functional ingredients and free radical -1 (0.85% NaCl) to obtain a 10 dilution. Appropriate scavenging activity were measured in order to find dilutions were used for the pour plate counting of the appropriate fermentation conditions of LAB. The medium used was de Man Rogosa Shape lactobacillus. The general physicochemical properties (MRS) for incubation for 48 h at 37±1℃ and then after each of the total viable LBA count and the counting LAB in term of CUF/mL. The pH of BFL burdock fluid had been inoculated with the four was measured by means of a Mettler Toledo Delta lactobacillus. After fermentation, the amount of total 320 pH meter (Mettler-Toledo, Greifensee, viable bacterial in burdock ferment liquid (BFL) was Switzerland). Sugars content of BFL were measured and the result was 10697 ＞ 10069 ＞ determined using an Atago digital hand-held 10696≒14086 (Fig. 1). For a probiotic product to be refractometer (Tokyo, Japan) in terms of ºBrix. Three beneficial, it must contain at least 106 CFU/mL of replicates were used for BFL sample. viable LBA according to the criteria of probiotic products in Taiwan. Determination of total polyphenols in BFL Fig. 1 shows the LBA in the four lactobacillus Total polyphenols in BFL were measured strains after 48 hours fermentation were higher than spectrophotometrically using the Folin–Ciocalteu 106 CFU/mL, and the colony 10697 has the largest reagent based on a colorimetric oxidation/reduction 13,21 amount of lactic acid bacteria compared to other reaction . 1 mL of Folin–Ciocalteu reagent lactobacillus strains. These results can be applied to (diluted 10 times with water) was added to 0.2 mL of the development of probiotic products. The pH diluted aqueous acetone sample. After that, 0.8 ml of among lactobacillus strains were between 2-5 which 7.5 % Na2CO3 was added and mixed thoroughly. The is similar to juice and carbonated drinks and the absorbance was measured at 765 nm (Hitachi, Tokyo, result was 14086＞10696＞10697＞10069 (Fig. 2). Japan) after 0.5 h of standing. The amount of total On the sweet determination standard, higher than 15 polyphenols was calculated as a gallic acid equivalent degree is defined as highly sweetened; between 12-15 from the calibration curve of gallic acid standard degree is defined as very sweet; between 10-12degree

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as defined as slightly sweet; between 8-10 degree as The present study is to further evaluate the functional defined as a bit sweet; and less than 8 degree is ingredient and the free radical scavenging activity of defined as non sweetness. There was no significantly burdock after fermentation with 14086, 10069, 10696 different in measuring of sweetness among the four and 1069 strains at 370C for 48 hours. The result of lactobacillus strains and the results of the four stains the total polyphenols showed the total polyphenols is were all under degree 8 (Fig. 3). 10697≒10696≒14086＞10069 (Fig. 4). On DPPH

70 free-radical scavenging ability, the result showed the DPPH free-radical scavenging ability of BFL is 60 10697≒10069＞14086＞10696 (Fig. 5). 50 ) 6 120 40 100 30

CFU/mL (x10 CFU/mL 20 80

10 60 0 10069 10696 10697 14086 40 Lactobacillus strains

Fig. 1. Comparison of the total viable lactic acid Total polyphenols (mg/g) 20 bacteria among different fermented burdock with 48 h fermentation. 0 10069 10696 10697 14086

6 Lactobacillus strains Fig. 4. Comparison of the total polyphenols 5 among different fermented burdock with 48 h

4 fermentation. Total polyphenols were expressed as mg gallic acid /g BFL. 3 pH

80 2 70 1 60 0 10069 10696 10697 14086 50 Lactobacillus strains 40 Fig. 2. Comparison of the pH among different fermented burdock with 48 h fermentation. 30 1.5 20 scavenging activityDPPH (%) 10 1.2 0 10696 10697 14086 10069 0.9 Lactobacillus strains

Brix o Fig. 5. Comparison of the free radical scavenging 0.6 activity among different fermented burdock with 48 h fermentation. 0.3

In conclusion, the present study used four 0.0 10069 10696 10697 14086 different lactobacillus to ferment burdock to explore Lactobacillus strains the effect of different lactobacillus on functional Fig. 3. Comparison of the sweetness among ingredient and free radical scavenging activity of different fermented burdock with 48 h fermentation. burdock. The overall results showed that lactobacillus Burdock contains polyphenols such as 10697 strain has better effect on LBA populations, chlorogenic acid, caffeic acid, isochlorogenic acid total polyphenols and free radical scavenging and dicaffeoylquinic acids which has been reported to 8 activities. The results can applied for developing have effect on anti-free radicals . There are many burdock probiotic products with free radical diseases are associated with free radicals and scavenging activity. consuming antioxidants food could reduce the incidence of the free radicals related diseases 23,24.

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Corresponding Author: potentiated by carbon tetrachloride. Journal of Fu-An Chen, Ph.D. Biomedical Science, 9, 401-409. Department of Pharmacy & Graduate Institute of 12. Dos Santos, A.C., Baggio, C.H., Freitas, C.S., Pharmaceutical Technology, Lepieszynski, J., Mayer, B., Twardowschy, A., Tajen University, 20 Wei-Shin Road, Yan-Pu, Missau, F.C., dos Santos, E.P., Pizzolatti, M.G., Pingtung, Taiwan, ROC. Marques, M.C.(2008). Gastroprotective activity of E-mail: [email protected] the choroform extract of the roots from Arctium lappa L. J Pharmacy and Pharmacology, 60, 795- References 801. 1. Han, C. M. (1995). Cultivation management of 13. Chen, F. A., Lee S.C., Chao H.R., Fu W.C., Hsu burdock in Kao-Ping Area. Agricultural World, M.C., Horng C.T., Wang C.C., Matsui, H., 145, 55-57. Agoramoorthy, G. (2009). Effects of burdock 2. Kan, W. S. (1981). Pharmaceutical Botany. extract preparation on gastric mucosal protection. National Research Institute of Chinese Medicine, Asian Journal of Chemistry, 21, 3015-3022. Taiwan, p549. 14. Chang, TH, Liu, IM, Horng, CT, Tsai, FC, Kuo, 3. Chow, L. W., Wang, S. J. & Duh, P. D. (1997). DH, Shieh, PC, Lee, SC, Shiang, JC, Chen, F A, Antibacterial activity of burdock. Food Science, (2012). Beneficial effects of the burdock ferment 24, 195-202. liquid on diabetic disorders in STZ-induced 4. Holetz, F.B., Pessini, G.L., Sanches, N.R., Cortez, diabetic rats. Life Science Journal-Acta Zhengzhou D.A., Nakamura, C.V., Filo, B.P. (2002). University Overseas Edition, 9(2): 823-831. Screening of some plants used in the Brazilian 15. Cao, JF, Zhang, PY, Xu, CW, Huang, TT, Bai, folk medicine for the treatment of infectious YG, Chen, KS, (2012). Effect of aqueous extract diseases. Memórias do Instituto Oswaldo Cruz, 97, of Arctium lappa L. (burdock) roots on the sexual 1027-1031. behavior of male rats. 12:8. BMC Complement. 5. Morita, K., Nishijima, Y. & Kada, T. (1984). A Altern. Med.. doi:10.1186/1472-6882-12-8. desmutagenic factor isolated from burdock 16. Lee, YJ, Cho, DH, Cho, GH, Kim, JS, Kang, DG, (Arctium lappa Linne). Mutation Research, 129, Lee, HS (2012). Arctium lappa ameliorates 25-31. endothelial dysfunction in rats fed with high 6. Duh, P. D. (1998). Antioxidant activity of burdock fat/cholesterol diets. 12:116 doi:10.1186/1472- (Arctium lappa Linne): its scavenging effect on 6882-12-116. free-radical and active oxygen. Journal of the 17. Lu, M., Toshima, Y., Wu, X., Zhang, X., Cai, Y. American Oil Chemists' Society, 75, 455-461. (2007). Inhibitory effects of vegetable and fruit 7. Lin, C. C., Lin, J. M., Yang, J. J., Chuang, S.C. & ferment liquid on tumor growth in Hepatoma-22 Ujiie, T. (1996). Anti-inflammatory and radical inoculation model. Asia Pacific Journal of scavenging effect of Arctium lappa. The American Clinical Nutrition, 16:443-446. Journal of Chinese Medicine, 24, 127-137. 18. Ojokoh AO (2007) Effect of fermentation on the 8. Chen, F. A., Wu, A. B. & Chen, C. Y. (2004) The chemical composition of mango (Mangifera indica influence of different treatments on free radical R) peels. African Journal of Biotechnology, scavenging activity of Burdock and the variation 6:1979-1981. of its active components. Food Chemistry, 86, 19. Sobowale AO, Olurin TO, Oyewole OB (2007). 479-484. Effect of lactic acid bacteria starter culture 9. Lou, Z., Wang, H., Li, J., Chen, S., Zhu, S., Ma, fermentation of cassava on chemical and sensory C., Wang, Z. (2010). Antioxidant activity and characteristics of fufu flour. African Journal of chemical composition of the fractions from Biotechnology, 6, 1954-1958. burdock leaves. Journal of Food Science, 75, 20. Bae, EA, Trinh, H.T., Rhee, Y.K., Lee, Y.C., Kim, C413-419. D.H. (2008) Antiallergic effect of ginseng 10. Lin, S. C., Chung, T. C., Lin, C. C., Ueng, T. H., fermented with Ganoderma lucidum. Journal of Lin, Y. H., Lin, S. Y., et al. (2000). Ginseng Research, 32:57-61. Hepatoprotective effects of Arctium lappa on 21. Negi, P.S., Jayaprakasha, G.K., Jena, B.S., (2003). carbon tetrachloride- and acetaminophen-induced Antioxidant and antimutagenic activities of liver damage. The American Journal of Chinese pomegranate peel extracts. Food Chem., 80, 393- Medicine, 28, 163-173. 397. 11. Lin, S. C., Lin, C. H., Lin, C. C., Lin, Y. H., Chen, 22. Chen, F. A., Wu, A. B., Shieh, P. C., Kuo, D. H. & C. F., Chen, I. C., et al. (2002). Hepatoprotective Hsieh, C. Y. (2006). Evaluation of antioxidant effects of Arctium lappa Linne on liver injuries activity of Ruellia Tuberosa. Food Chemistry, 94, induced by chronic ethanol consumption and 14-18.

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23. Ames, B. N. (1983) Dietary carcinogens and anticarcinogens: Oxygen radicals and degenerative disease. Sci. 221:1256-1263. 24. Leong, L. P. & Shui, G. (2002) An investigation of antioxidant capacity of fruits in Singapore markets. Food Chem. 76:69-75.

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Study the sources of mercury vapor in atmosphere as a threatening factor for human health and bio-filtering methods for removal of toxic pollution

Maryam K. Hafshejani1, Farzaneh Khandani2, Ramin Heidarpour3, Armin Arad*4, Sadegh Choopani5

1Shahrekord University of Medical Sciences, Shahrekord, Iran 2 Ahar Branch, Islamic Azad University, Ahar, Iran 3Department of applied chemistry & chemical engineering, Sofian branch, Islamic Azad University, Sofian, Iran 4North Khorasan University of Medical Sciences, Bojnurd, Iran 5Faraiand Sanat Sharif Co, Tehran, Iran Corresponding author email: [email protected]

Abstract: A global atmospheric transport model is used to calculate mercury concentrations in the atmosphere. In this study, a comprehensive analysis was made to describe the environmental behavior and pollution status of mercury (Hg). The most important aim in this study is to show mercury pollution sources and to introduce some new technologies for removing this dangerous material from air. In this paper, some bio technologies such as bio filtering method and bio adsorbent such as leaves of trees was introduced. The capacity and application of biotrickling filters for the removal of mercury vapor from simulated flue gases was expressed. It was resulted from paper that the most prominent source of mercury is the east of Asia and the southeast of Asia. The lowest source is concerned Australia. In this investigation it was found that activated carbon can be one of the best tools for removal of mercury vapor. The adsorbent uniquely combines a chelating ligand with an ionizing surface nano-layer on a mesoporous substrate was the other consequences of this study which can be as a suitable adsorbent for omitting this pollutant from atmosphere. [Hafshejani MK, Khandani, F, Heidarpour R, Arad A. Study the sources of mercury vapor in atmosphere as a threatening factor for human health and bio-filtering methods for removal of toxic pollution. Life Sci J 2013;10(1):293-296] (ISSN:1097-8135). http://www.lifesciencesite.com. 46

Key Words: Mercury, Atmosphere, Activated carbon, Biofiltering.

1. Introduction: Coal-fired plants for electricity generation are one of The industrial activities of human have the anthropogenic sources of emissions of arsenic and resulted in the pollution of soil, water, and selenium [4]. Lead (Pb) is the other prominent atmosphere with heavy metals. Heavy metals are the pollutant in marine environments. Before the most prominent dangerous groups of anthropogenic implementation of antipollution policies on environmental pollutants with high toxicity and automotive Pb >60% of its anthropogenic discharge persistence in the environment. Independent of the into the atmosphere originated from alkyl-leaded origin of the source, heavy metals can accumulate in additives in gasolines [5]. Although these groups of crops or plants and may lead to the damage and metals are present in soils, contamination comes from alteration of animal or human physiological functions local sources, mostly industry (mainly non-ferrous through the food chain [1]. Vegetation is an effective industries, but also power plants, iron and steel and indicator of the impact of a contamination source in chemical industries), agriculture (irrigation with its vicinity, because most plants have the capacity to polluted waters, use of fertilizers, especially accumulate these pollutions therefore their phosphates, contaminated manure, sewage sludge and concentrations are much higher than those in the air pesticides containing heavy metals), waste [2]. For example, Mercury and its compounds are incineration, combustion of fossil fuels and road included in the Title III listing of hazardous air traffic [6]. In the present study we report the results pollutants (HAP) by US EPA due to their potential of the measurements of air mercury pollution and our impact on human health. As a result, they are subject effort is to show the best ways for removal of this to standards and regulations such as clean air mercury contaminant. rule issued in 2005 [3]. For another example, Arsenic and selenium have been linked to harmful 2. Mercury Determination and Removal from toxicological impacts despite the fact that these Atmosphere: elements are essential trace elements for mans and Using H2S and an activated carbon as animals. These are among the toxic air pollutants adsorbent, the removal of Hg0 in simulated coal addressed in the 1990 Clean Air Act Amendments. combustion flue gases was examined in one study 293

Life Science Journal 2013;10(1) http://www.lifesciencesite.com which has been done in Japan at 2005. The following significantly greater amount of mercury vapor within consequences were obtained from this investigation: the CB systems. In the IOF technique, the addition of in the presence of both H2S and SO2, mercury air flow over the restoration demonstrated a removal was favored at lower temperatures (80–100 significant increase in mercury vapor released 0 0 C). At 150 C, presence of O2 was indispensable for compared to the sealed IOF technique [10]. In the 0 Hg removal from H2S–SO2 flue gas system. The IOF technique readings are initially higher but level partial oxidation of H2S with O2 to elemental sulfur off and remain relatively constant after about 24 h. and the clause reaction may contribute to the Hg0 The largest release of mercury vapor was reported in removal over activated carbon by the following the first reaction: Sad+Hg=HgS. The formation of elemental sulfur on the activated carbon was confirmed by a visual observation [6]. In the other investigation, The science men have shown that in the spring summer period the western Russian Arctic is under, the influence of regional and far continental anthropogenic sources of atmospheric mercury emissions. The concentrations of mercury in Murmansk, and over Kola and Moto sky Bays were associated with a primary direction of near surface winds from the nearest sources of mercury emission located in the Kola Peninsula region. The mercury concentrations are two-fold lower than the concentrations that are typical of the near surface air of the inner continental reserve territories (background areas), and are comparable to the concentrations measured in the air of the other Arctic regions [7]. Estimates of the global consumption of mercury by application and region in 2005 are shown in Figure 1. Two groups of anthropogenic sources of mercury might be distinguished: primary anthropogenic sources and secondary anthropogenic sources. Primary anthropogenic sources are those where mercury of geological origin is mobilized and released to the environment. The two main source categories in this group are mining (both for mercury and for other minerals) and extraction and burning of fossil fuels which contain mercury as a trace contaminant. Secondary anthropogenic sources are those where emissions occur from the intentional use of mercury, including mercury use in industrial processes, in products, in dental applications, or in artisanal and small-scale gold mining (ASGM) operations. Emissions to the environment from both primary and secondary sources can occur via direct discharge of exhaust gases and effluents, and through the generation of mercury-containing wastes [8]. For example, the regional mercury budget from anthropogenic and natural sources in Chongqing, an important industrial region in southwest China has been investigated [9]. A closed bottle technique (CB) and an intraoral flow (IOF) technique are two in vitro mercury vapor collection techniques. Both techniques Figure 1. The global consumption of mercury by indicated mercury vapor release was dependent on application and region in 2005 [8] volume. The largest bottle, 500 ml, yielded a 294

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Figure 2. Trends of mercury emissions to the air from coal combustion and cement manufacturing in Chongqing [9]

The annual trends in mercury emission to in USA at 2004. The adsorbent uniquely combines a atmosphere from this source are also presented in chelating ligand with an ionizing surface nano-layer Figure 2. The amount of mercury vapor released on a mesoporous substrate. This enables selective, drops by more than half after 5 h from trituration, multi-dentate adsorption of mercury directly from the which is in agreement with similar findings by other gas phase. A novel chelating adsorbent for the studies. [10]. removal of gaseous mercuric chloride directly from The feasibility of using biotrickling filters flue gases has been developed. for the removal of mercury vapor from simulated flue gases was evaluated by Indian Institute of Technology at 2007. In this study, early results on the control of mercury vapors in biotrickling filters were presented and discussed. The results highlight the highly complex nature of the mechanisms involved in mercury capture in biotrickling filters. At the same time, the potential of novel biosystems for the control of mercury vapor from flue gases is resulted. Further process optimization and detailed engineering of the best way to integrate the proposed mercury vapor control with existing or novel flue gas treatment are required. For the initial experiment, the gas stream consisted of mercury vapor (300–650 µg m_3) in air which was treated in the reactor containing sulfur oxidizing bacteria and operated at room temperature. Under these conditions, the biotrickling filter exhibited complete removal of mercury vapor at an initial empty bed residence times of 70 s. Subsequently, the gas residence time was gradually Figure 3. Inlet and outlet concentrations of mercury reduced to 35 s, 20 s and to 6 s which is seen in vapor in the SOB packed biotrickling filter and in the Figure 3 [11]. cell-free control [11] A novel adsorbent for the capture of mercuric chloride vapor from flue gases has been The adsorbent is composed of a structured developed in the investigation which has been done active nanolayer on a mesoporous silica substrate. Chelation is achieved through the use of a molten salt 295

Life Science Journal 2013;10(1) http://www.lifesciencesite.com coating on an immobilized chelating group. centre of Konya (Turkey). Atmospheric Elemental analyses indicate that the theoretical Environment 2006; 40:1122–1133. adsorption capacity for mercury is very high, 6. Pandey S K, Kim K H, Yim U H, Jung M C, approximately 33 mg/g. The adsorbent is suited for Kang C H. Airborne mercury pollution from a use in the lowtemperature end of the flue–gas large oil spill accident on the west coast of treatment train, with a maximum operational Korea. Journal of Hazardous Materials 2009; temperature of 135 ◦ C. Evaluation of the dynamic 164:380–384. adsorbent capacity for mercuric chloride showed very 7. Antón M A L, Somoano M D, Fierro J L G, efficient uptake and a minimum operating capacity of Tarazona M R M. Retention of arsenic and 12 mg Hg2+/g. Finally, the formation of chelating selenium compounds present in coal combustion complex between HgCl2 captured and cysteine and gasification flue gases using activated ligands was confirmed using Far-FTIR [12]. carbons. Fuel Processing Technology 2007; 88:799–805. 3. Conclusion 8. Migon C, Robin T, Dufour A L, Gentili B. In this paper the most important is to Decrease of lead concentrations in the Western introduce the various sources of mercury vapor which Mediterranean atmosphere during the last 20 can pollute the atmosphere. By investigating the other years. Atmospheric Environment 2008;42:815– studies in this field it is clear that Asia is the most 821. prominent of these sources. The two main source 9. Morimoto T, Wu S, Uddin M A, Sasaoka E. categories in this group are mining (both for mercury Characteristics of the mercury vapor removal and for other minerals) and extraction and burning of from coal combustion flue gas by activated fossil fuels which contain mercury as a trace carbon using H2S. Fuel 2005; 84:1968–1974. contaminant. Secondary anthropogenic sources are 10. Golubeva N, Burtseva L, Matishov G. those where emissions occur from the intentional use Measurements of mercury in the near-surface of mercury, including mercury use in industrial layer of the atmosphere of the Russian Arctic. processes, in products, in dental applications, or in The Science of the Total Environment 2003; artisanal and small-scale gold mining (ASGM) 306:3–9. operations. The best result that can be achieved from 11. Pacyna E G, Pacyna J M, Sundseth K, Munthe this investigation is that bio technologies can be low J, Kindbom K, Wilson S, Steenhuisen F, cost method for eliminating these pollutants. Maxson P. Global emission of mercury to the Biofiltring and activated carbons can be the best atmosphere from anthropogenic sources in 2005 adsorbent for removal of mercury vapor. These and projections to 2020. Atmospheric adsorbent can remove with high efficiency which was Environment 2010; 44:2487–2499. shown in Figure 3. 12. Wang D, He L, Wei S, Feng X. Estimation of mercury emission from different sources to Corresponding Author: atmosphere in Chongqing, China. Science of the Armin Arad Total Environment 2006; 366:722– 728. North Khorasan University of Medical Sciences 13. Nemea A L, McLaren J D, O’Brien W J. Bojnurd, Iran Investigation of two mercury vapor collection E-mail: [email protected] techniques. Dental Materials 1999;15:375–381. 14. Philip L, Deshusses M A. The control of References: mercury vapor using biotrickling filters. 4. Al-Alawi M M, Mandiwana K L. The use of Chemosphere 2008;70:411–417. Aleppo pine needles as a bio-monitor of heavy 15. Abu-Daabes M A, Pinto N G. Synthesis and metals in the atmosphere. Journal of Hazardous characterization of a nano-structured sorbent for Materials 2007;148:43–46. the direct removal of mercury vapor from flue 5. Ondera S, Dursunb S. Air borne heavy metal gases by chelation. Chemical Engineering pollution of Cedrus libani (A. Rich.) in the city Science 2005; 60:1901 – 1910.

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The Age Effects of Traffic Signs on Visual Performance

Chun-Wen Chao 1,*, Cheng Hung Huang 2, Tienwei Tsai 3

1Ph.D. Program of Design Science, Tatung University, Taipei 106, Taiwan; 2Department of Industrial Design, Tatung University, Taipei 106, Taiwan; 3Department of Multimedia Design, Chihlee Institute of Technology, New Taipei City 220, Taiwan Corresponding author email: [email protected]

Abstract: Since drivers aged 70-74 are twice as likely to die when involved in a crash compared to drivers aged 30-59 (for drivers aged 80 and older the risk is five times as high), their need when an accident occur differs from younger people, according to the study conducted by American Automobile Association (AAA, 2012). Other Researches also show senior drivers need significantly more light to see than young drivers. That’s because pupils get smaller and don’t widen as much in dark conditions and making senior harder to see. This diminished vision is a significant problem (Green, 2008). The goal of this paper is to identify the impact of legibility, luminance contrast of signs on driver’s ages. A series of experiments are conducted to investigate the effects of light and age on the threshold and the confident recognition of traffic signs. The experimental results have shown that subjects above the age of 65 have a decrease in visual search performance in the evening since the transmittance of short-wave region (blue lights) within visible range decreases with age because of the loss of luminance and color contrast. The nighttime, raining and evening tests revealed that the older group had significantly longer decision sight distance compared to the younger group (p <0.000, T-tests of 95% significance). The results indicate that observed age-related evening and raining legibility performance decreases visual acuity, which therefore significantly increase chances for an accident. This study helps highlight the fact that when people ignore visual performance in evening and raining conditions it may cause accidents. More importantly, our study has set up the foundation for future work with contributing a dedicated viewpoint for traffic signs and visual performance under evening and raining conditions. [Chun-Wen Chao, Cheng Hung Huang, Tienwei Tsai. The Age Effects of Traffic Signs on Visual Performance. Life Sci J 2013;10(1):297-302] (ISSN:1097-8135). http://www.lifesciencesite.com. 47

Keywords: Visual performance; luminance contrast; color temperature; age effects, traffic signs.

1. Introduction illustrating a range of comfort, with luminance on the A study on senior driving by AAA indicates that vertical axis and color temperature on the horizontal older driver involvements in police reported crashes (see Fig. 1). Researchers have also found out that the and fatal crashes by aged 65 and older will account for luminaries type, arrangement, and height can create 25% of the total driver fatalities in the US by year 2030. sufficient visual conditions in road lighting for traffic Due to the high accident casualty rate for elderly safety and comfort (Goodspeed and Rea,1999, drivers, many countries are constantly devoted to Schnell,2004, Smiley,2008). improving elderly driver safety. How to suppress the increase in traffic accidents has thus become a government challenge and needs to take a more dedicated study on this issue. To tackle this problem, the purpose of this paper was set to investigate the influence of light conditions on sign visibility for elderly drivers. Many traffic sign studies have researched on sign visibility (Kline, Ghali, and Kline, 1990), sign luminance (Graham and King, 1997), sign legibility (Schieber and Goodspeed, 1997) and sign comprehensibility (Madani, 2000). Common experiences express that luminance (light quantity) and color temperature (light quality) make various psychological impressions on human. The research of Figure 1. The curve illustrating a range of comfort with Kruithof (1941), Wade and Brožek (2001) on these luminance on the vertical axis and color temperature on psychological and interactive effects of color the horizontal. temperature and luminance produced a curve

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However, the influence of evening and raining illustrated in Fig. 2. light condition on sign legibility had not been fully addressed. In practice, it is found that there is often no additional light on the road and most drivers do not turn the head light on in the evening and raining. In that case, it might affect the ability of drivers to concentrate on traffic signs while driving and could substantially influence road user safety, driving effectiveness and overall traffic flow. Other characteristics that could affect legibility and recognition performance are discussed as follows. Previous researches have proposed some solutions to reduce reading time and improve the accuracy of traffic signs, such as enlarging the physical letter sizes on signs (Huang, Wang and Chen 2010; Mace), using highly -efficient retroreflective sheeting or increasing the sign luminance by adding assistance lighting (ASTM, 2009). Figure 2. Three combinations of signs were set up in Legibility luminance is an essential factor on vision the VR scenes by four color temperatures. modality. The minimum readable brightness and contrast depends on other factors, such as font type and Each subject was required to perform a visual size. search task under day time, evening, raining, and night Perception distance in driving can be defined as conditions, and then completed a workload assessment the minimum (visibility) distance of being able to based on five 7-point rating scales using the NASA - acquire the information conveyed from a sign. The TLX (Task Load Index) method, a multi-dimensional performance measured in this study was highly rating scale in which information about the magnitude correlated with the relative visual performance values. and sources of several workload-related factors are In a context directly related to highway signs, subjects combined to derive a sensitive and reliable estimate of were exposed to varying levels of surrounding workload (Hart and Staveland, 1988). complexity and luminance contrast. The source analysis is a relatively easy-to-read visual performance 2.1 Experiment Participants model (REA 1988 and Ouellette). This model provides A total of 30 subjects were selected into two a calculation of distance with an input parameter for groups: 15 younger subjects (aged 20~29 with an the speed and accuracy of visual information. To take a average of 27.4 years old, i.e., M = 27.4) and 15 older more detailed analysis, our experiment is based on the subjects (aged 65~78 with an average of 73.8 years old, following factors: the age of the subject, the size of the i.e., M = 73.8). Both groups participated in the same visual target, the brightness of the visual contrast tasks in the simulation experiment. between the target and its background, and the brightness around the visual target environment. 2.2 Pre-Experiment Preparation The VR scene was created in 3Ds Studio Max, 2. The Proposed System Framework and with all the elements for the creation of one perfect To investigate the effects of the traffic sign on image, including global illumination (Fritchman, 2005), visual search performance under different light anti-aliasing, and color temperature. A series of conditions, the experiment was designed to examine pictures were taken crossing through daylight, twilight, the decline in sensitivity to luminance contrast and raining, and night conditions from 2:00pm to 8:00pm color temperature by age in a VR simulation system. In on different days over the course of a month in order to our experiments, the same observation method with backup the rendering results. The camera was set to the subjects in the car is used in a correct and coherent way. wide-angle setting and the images were adjusted There were three kinds of signs used in the study: (1) A according to the response curve constructed in the white and circular one with a black number and a red laboratory. The results we carried out were almost border; (2) a white and triangular one with a black matched with those of real scenes by simulating symbol and a red border; (3) a red and octagonal one standard atmospheric conditions. It remains a challenge, with Chinese texts in white and a white border. Three however, that atmospheric model parameters can be combinations of number, text, shape, and color were automatically determined such that twilight renderings used. The symbols such as narrow road signs, Chinese perfectly match actual twilight recordings. Next, we set text stop signs, and speed limit signs were selected. up the color temperature with the Intensity/Color Different scenes generated in the VR system are Attenuation tool in the 3Ds Studio Max to reproduce

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the realistic scenes as (1) daylight: 6504 K, CIE D65, (2) evening: 4100K, CIE F11, (3) raining: 3400K, and 4. Statistical Analysis (4) night: 2860 K, CIE A, 120W incandescent light. The mean legibility distances of young and old The luminance contrast ratio was ranged as 8:1, 5:1, subjects were statistically significant (p <0.001) in 3.4:1, and 2.8:1, controlled by the contrast analyzer which young subjects had a better legibility skill than version tool (see Fig. 3). older subjects by 45%. Symbolic signs in any condition of light were better for legibility for all subjects in the simulation conditions. Analysis of experiment variance revealed the following:

(1) The difference between symbol and Chinese text background luminance contrast combination was statistically significant (p < 0.000). The effect of number and Chinese text background luminance contract combination was statistically significant (p <0.002). Cross-comparisons between different signs showed that Chinese text under any light conditions had considerably poorer legibility distances compared to other signs. On the other hand, symbols had best legibility at perception distance and 53% better than Chinese text as shown in Table 1. Figure 3: The contrast analyser tool and the color temperature tool in 3Ds Studio Max. Table 1. Multiple Comparisons of signs. P (I) signs (J) signs (I-J) SD Consequently, the selected background and value(a) legend luminance levels were intended to match the number text 3.370(*) .735 .002 credible luminance contrast levels in a real-world symbol 1.271 .679 .098 environment. The collected data was processed and -3.370(* text number .735 .002 analyzed with appropriate statistical techniques. The ) results of our study have presented more functional -2.099(* symbol .343 .000 information and could be used as a source of ) recommendations for better light quality and quantity, symbol number -1.271 .679 .098 and furthermore, to improve traffic sign perception. text 2.099(*) .343 .000

3. Experiment Procedures

The following experiment was performed under (2) Younger subjects showed faster response to the rendering light in the VR scene for the quality of the threshold and a higher percentage of incorrect illuminant to remain the real lights continuous. In this response by 25%. Younger subjects also had better literacy rates than older subjects by 54% under case, rheostats were not used to dim the light because the attendant would change the color or the quality of rainy condition, 42% at night, 40% in the evening, the light. We presented a real-time, fully-textured and and 12% in the afternoon. Moreover, the mean anti-aliased 3D graphical scene of a virtual world, confidence legibility distance of younger and older which was projected at a resolution of 1024×768 pixels subjects in the VR scenes were statistically by a projector on a single 120 inch screen in front of significant (p < 0.000), as summarized in Table 2. the subjects. The total horizontal field of view was 60 degrees and the vertical field of view was 46.8 degrees. The frame rate was constant at 60 Hz. A NI USB-6221 Table 2. Multiple Comparisons of ages. (I) P data acquisition device collected the dynamic data at (J) age M (I-J) SD 250 KS/s. The virtual road was a roadway about age value(a) Young 1,500m long with signs posting numbers (speed limit), old 3.205(* younge 32.89 .714 .000 Chinese text (stop), and symbols (lane ends). The older ) roadway was at a low-density traffic, mimicked with r two 3.5 m-wide sidewalks and their surrounding virtual environment. The driving speed was set at 5 km/h, which was controlled by the simulator.

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(3) Evening and raining confidence level interaction measure the performance of mental demand, was statistically insignificant. The effect of day and effort, and presence in our experiment. The effect night level interaction was statistically significant of the virtual space on subjects was a significant (p < 0.024). The effect of day and evening level main effect of age on the reaction time (p <0.023) interaction was statistically significant (p < 0.005). but the judgment in the VR world was not Multivariate test of the differences between light influenced by age (p > 0.19). More importantly, and age (p < 0.005) was also statistically on most subscales, older and younger adults did significant. not change in their judgment of subjective workload and senses of acting in the virtual space. Bigger estimates of subjective workload under low luminance conditions were found on all subscales (p <0 .001). 92% of younger subjects felt present in the virtual space and 89% of older subjects were captivated by the virtual world. The results of IPQ showed that the VR scenes produced realistic effects.

5. Experimental Results The results indicate that observed age-related

Threshold and confidence legibility distances under different light conditions. evening and raining legibility performance decreases Figure 4. The mean legibility distance of younger visual acuity results. Older subjects had a longer subjects. perception reaction time than younger ones (p <0.000). The nighttime, raining and evening tests revealed that the older group had significantly longer decision sight distance compared to the younger group (T-tests of 95% significance). However, younger subjects showed an earlier start of response execution relative to the threshold of response preparation and a higher percentage of incorrect response by 32% in the VR scene. Multivariate test of differences between light and age was statistically significant (p < 0.005), and the difference between signs and light was also statistically significant (p < 0.032). Signs with symbols possessed superior mean legibility in comparison to the Chinese text signs by 53% (p < 0.01) and numerical text of

Threshold and confidence legibility distances under different light conditions. speed limit signs by 49% (p < 0.01).

Figure 5. The mean legibility distance of older subjects. The poor legibility distances were provided by the letter/background combinations of Chinese character sign yielding contrast ratios of 3:1 and 5:1. From Fig. 4 and Fig. 5, it is concluded that Chinese text signs had poor legibility in the evening signs without any additional lighting in the evening had and night tests. Symbol signs can be an effective way poor legibility for older subjects (p < 0.003). Older to convey information if the subject is already familiar subjects need to come extra 60% closer to the signs with the meaning of the icon or the symbol. The higher under evening and raining condition to perceive them the contrast level, the better the recognition level for correctly. high contrast signs. At or above that level, the symbol recognition is simply driven by the visual acuity (4) Issues on subjective workload and Igroup performance of the reader, which can easily be Presence Questionnaires (IPQ) are discussed as predicted by reference to the resolution of the smallest follows. The NASA –TLX developed by Hart and relevant (critical) detail. Bigger and brighter signs are Staveland (1988) is a subjective workload more effective, and it took less time to provide a very assessment tool while the Igroup Presence high readability signs. It leads to a 45% enhancement Questionnaire (IPQ) is a scale for measuring the in reaction time by enlarging the size 25% of the sign sense of presence experienced in a virtual in the experiments. environment (VE). Subscales of the NASA-TLX by mean scores (on 7 point scale) were used to

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6. Discussion and Future Work raining day. Therefore, it is suggested that controlling It is obvious that visibility of traffic signs is lights by setting up environment light detector sensors critically important for driving safety; therefore, on highways, designing light detector sensors to age-related changes in the human visual system must automatically turn on the car headlights or use the night be taken into account in designing traffic signs to vision equipment when color temperature is under assure safe driving for older people. In this paper, to 4100K and luminance contrast ratio is under 5:1. Our address this issue, some age-related changes of visual study also helps highlight the fact that when people functions are investigated in relation to visibility of ignore visual performance in evening and raining traffic signs. It is concluded that traffic sign legibility conditions it may cause accidents. More importantly, under different light conditions may be affected by our study has set up the foundation for future work age-related vision. Practical suggestions are provided with contributing a dedicated viewpoint for traffic signs after discusses on the suitability of current traffic sign and visual performance under evening and raining standards. Moreover, according to the results of conditions. Potential applications of this study include questionnaires used in conducting this research, only observations of visual search behavior in the VR world 18% of younger drivers and 28% of older drivers for the design of better simulation experiments. would turn on their headlights in the evening; 22% of younger drivers and 34% of older drivers would turn Correspondence: on their headlights on a rainy day. It also indicates the [email protected]; importance of our study because all of these factors C.W. Chao is currently an assistant professor at Chihlee affect a driver’s vision and will therefore significantly Institute of Technology. increase chances for an accident. Evening driving, for instance, is in twilight conditions, in which eyes could find it particularly References difficult to differentiate relative brightness of red and 1. AAA Senior Driving 2012. http://seniordriving. blue objects, which phenomenon is called the “Purkinje aaa.com/understanding-mind-body-changes/visi effect” (Schnell, Aktan, Li, 2004). The “Purkinje on/light-requirements effect” is the tendency of the peak luminance intensity 2. ASTM D 4956-09e1:2009. Standard of the eyes to transfer to the blue end of the color specification for retroreflective sheeting for spectrum at low light beam levels (Purkinje,1825, traffic control. Wade and Brožek, 2001). This effect introduces 3. ERGO 2001. User's guide. Pasadena: Avery diversity in color contrast under different levels of Dennison Corporation. illumination. For example, in clear sunlight, flowers 4. Fritchman Thompson Barbara.: 2005. appear to be bright red against the green of their leaves, Astronomy hacks tips and tools for observing but in the same scene viewed in the evening, the color the night sky. contrast is reversed, with the red petals appearing a 5. Goodspeed, C. H., Rea, M. S.:1999. The dark red or black, and the leaves appearing relatively significance of surrounding conditions for bright. The phenomenon explains why signs in red will roadway signs, Journal of the Illuminating appear darker in the evening. The results also conveyed Engineering Society 28(1) :164-173. that evening driving results in poor perception accuracy 6. Graham, J. R., Fazal A. and King L. E.: 1997. distance for older drivers. Evening driving could be Minimum luminance of highway signs required afflicted due to the Purkinje effect because the human by older drivers, Transportation Research eye lens become yellow and the transmittance for the Record 1573:91-98. short-wave region in the visible range (blue lights) 7. Madani, H.AI.: 2000. Influence of drivers’ decreases with age (ERGO 2001). Therefore, it is comprehension of posted signs on their safety suggested that sufficient light should be provided for related characteristics, Accident Analysis and safe driving in the evening. Prevention 32:575-581. According to our study, we highly recommend 8. McLaughlin, P.M. and Murtha, S.J.: 2010 Jul. all drivers should turn on the headlights not only in the The effects of age and exogenous support on night but also in raining and evening conditions. Most visual search performance. Exp Aging Res visual conditions can be perceived with the road 36(3): 325-45. luminance levels which are even lower than the 9. Hart, S.G. and Staveland, T.E.: 1988. recommended today (Artal P, 2012). Such signs are Development of NASA-TLX (Task Load expected to engage driver’s attention and have the Index): results of empirical and theoretical potential to reduce driver workload and improve safety. research. In: Hancock PA, Meshkati N, Eds. Older people can process information from traffic signs Human Mental Workload. Amsterdam: efficiency by increasing light in the evening or on a North-Holland. pp.139- 178 .

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10. Huang, K-C., Wang, H-F., Chen ,C-C.: 2010. 16. Schieber, F. and Goodspeed, C.H.: 1997. Effects of shape, size, and chromaticity of Nighttime conspicuity of highway signs as a stimuli on estimated size in normally sighted, function of sign brightness, background severely myopic, and visually impaired students. complexity and age of observer, Proceedings of Perceptual and Motor Skills 110:931-940. the Human Factors and Ergonomics Society 11. Kline, T. J. B., Ghali, L.M. and Kline, D.W.: 41st Annual Meeting. Santa Monica, CA: 1990. Visibility distance of highway signs Human Factors and Ergonomics Society. pp. among young, middle-aged, and older observers: 1362-1366. icons are better than text, Human Factors 32: 17. Schnell, T., Aktan, F., Li, C.: 2004. Traffic sign 609-619. luminance requirements of nighttime drivers for 12. Kruithof, Arie and Tubular, Andries.: 1941. symbolic Signs. Transportation Research Luminescence Lamps for General Illumination. Record No. 1862: Journal of the Transportation Philips Technical Review 6 (3): 65–96. Research Board. pp.24-35. 13. Green, Marc Ph.D. Co-Authors: Merrill, J. 18. Schwarz, C., Cánovas C., Mira-Agudelo A.: Allen O.D., Ph.D., Abrams ,Bernard S. O.D., 2012. Night myopia studied with an adaptive Weintraub , Leslie O.D.: 2008.Contributor: optics visual analyzer, PLoS ONE 7(7): e40239. Odom, J. Vernon Ph.D. Forensic vVision with doi:10.1371 /journal. pone. 0040239 application to highway safety with CD-Rom 19. Smiley, A.: 2008. Driver behavior: A moving 3rd Edition. ISBN 10: 978-1-933264-54-7 target. TR News 254:19-24. US Department of Copyright Date Ed. Transportation. 14. Wade, Nicholas J and Brožek, Josef.: 2001 20. Mace, D.J., P.M. Garvey, and R.F Heckard.: Purkinje's Vision. Lawrence Erlbaum 1994. Relative visibility of increased legend Associates pgs 13. ISBN 9780805836424. size Vs. brighter materials for traffic signs. 15. Purkinje, JE. Neue Beiträge zur Kenntniss des FHWA-RD- 94-035, Federal Highway Sehens in Subjectiver Hinsicht.: 1825. Reimer Administration, Washington, D.C. Berlin. pp.109–110.

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