生物英文文献.doc

生物英语短篇作文高中作文Biological Diversity。

Biological diversity, or biodiversity, refers to the variety of life on earth, including the variety of species, ecosystems, and genetic diversity within species. It is essential for the functioning of ecosystems and the services they provide, such as food, clean water, and air, and for human well-being.Biodiversity is threatened by human activities such as habitat destruction, pollution, overexploitation of resources, and climate change. These activities have led to the extinction of many species and the degradation of ecosystems, which in turn threatens human well-being.To protect biodiversity, we need to take actions at different levels. At the individual level, we can reduce our ecological footprint by consuming less, using renewable energy, and supporting conservation efforts. At thecommunity level, we can promote sustainable practices, such as organic farming and eco-tourism, and protect natural habitats. At the national and international level, we need policies and regulations that promote sustainable development, protect endangered species and habitats, and address the root causes of biodiversity loss.Conserving biodiversity is not only a moral and ethical duty, but also a practical necessity for our survival and well-being. We depend on biodiversity for our food, medicines, and other resources, and for the services that ecosystems provide. By protecting biodiversity, we are also protecting our own future.In conclusion, biodiversity is a crucial aspect of our planet's health and well-being. It is threatened by human activities, but we can take action to protect it. By conserving biodiversity, we are not only protecting the natural world, but also our own future.。

生物给人类的科技贡献英文作文English:Biology has made significant contributions to technology in numerous ways. One of the most important ways is through the field of medicine, where biological discoveries have led to the development of vaccines, antibiotics, and various medical technologies that have saved countless lives. Biotechnology, a branch of biology, has also revolutionized many industries with advancements in genetic engineering, pharmaceuticals, agriculture, and environmental protection. The study of genetics has paved the way for personalized medicine and gene therapy, providing solutions to genetic disorders and improving the overall quality of healthcare. Furthermore, biomimicry, which involves learning from nature to design innovative technologies, has led to the creation of new materials, structures, and systems that are more efficient, sustainable, and resilient. Overall, biology continues to play a crucial role in driving technological advancements that benefit society as a whole.中文翻译:生物学在许多方面为技术的发展做出了重要贡献。

生物医学工程医疗仪器论文中英文资料外文翻译文献Present status and problems of domestic medical instrument engineering. Biomedical EngineehngIn recent years considerable progress has been achieved in domestic medical instrument engineering. Many plants and scientific-research organizations of machine-making and the defense industry have changed their profile toward production of medical equipment.However, medical equipment often meets a reluctant market because of funding cuts in health services. Medical organizations often cannot satisfy even their basic requirements for diagnostic and therapeutic devices.Also, health service organizations tend to buy foreign rather than domestic medical equipment because the former are easily available on the domestic market and prices for both are comparable because of inflation.The transition to a market economy in Russia has had substantial impact on the relations between domestic manufactur- ers and consumers of medical devices. The spectrum and quality of available items has been significantly extended in recentyears. It should be also noted that available models of medical devices are continuously updated, which makes them sufficiently competitive.Representative information on the updating dynamics of domestic medical equipment is summarized in Table 1. The data were provided by the VNIIMP-VITA Joint-Stock Company, which compiles a data bank of such information.Generally, new items account for 37% of total production of medical devices. Routinely produced devices (duration of production, 2-5 years) account for 28%. Medical devices of long-term production (5-10 years) account for 17% and obsolete nomenclature (devices produced for more than 10 years) accounts for 18%.It is seen from Table 1 that in recent years there has been considerable progress in the updating of production of medical equipment. For example, according to the VNIIMP-VITA Joint-Stock Company, the share of the items that have been produced for no longer than 5 years in 1988 did not exceed 35%, while now it is 65%. For the items that have been produced for more than 10 years such shares are 40 and 18%, respectively.Updating of produced medical devices was encouraged by the increase in the number of designers and manufacturers, particularly those of former defense industry facilities. In collaboration with foreign partners they set up joint ventures for producing medical equipment.Analysis of the updating of the various groups of medical equipment is of substantialinterest.It is seen from Table 1 that detoxication devices contribute dominantly to the group of items that have been updated within the standard period of up to 5 years (100% of production, including modern devices for hemodialysis and hemosorption).Comparatively high updating indices are observed for devices for functional diagnosis: 72% of these devices have been produced for no longer than 5 years, and obsolete devices account for only 9% of total production. However, it should be noted that although production of some obsolete devices has been terminated, equipment of similar functional capacity is still urgently needed.Relatively low updating indices are observed among the devices for intensive care and resuscitation: 16% of new items and comparatively many obsolete devices (26%). Among new models apparatuses for artificial lung ventilation are worth mention. However, some apparatuses, which have been developed long ago are still on the market because they have good performance, are quite reliable, and still are in demand. This reduces the updating index of the group as a whole.All-Russian Scientific-Research Institute for Medical Instrument Engineering, Rusaian Academy of Medical Sciences (VNIIMP-VITA Joint-Stock Company), Moscow. Translated from Meditsinskaya Tekhnika, No. 1, pp. 4-9, January-February, 1996. Original article submitted August 23, 1995.0006-3398/96/3001-0001515.00 y Plenum Publishing CorporationTABLE 1. Updating of Basic Groups of Medical Devices and Apparatuses (% of total nomenclature)这里有个表The lowest updating indices are observed for devices for examining a patient's body structures. These are: ophthalmological, otolaryngological, and anthropometric devices, endoscopes, etc. The share of obsolete devices is high (44%), while the devices which have been produced for no more than 5 years account for only 20% of total production.It should be noted that these results on medical equipment updating are important general estimates, although they do not take into consideration specific achievements and shortcomings in the production of individual items. Therefore, some corresponding amendments are required.Our survey of available information, including the VNIIMP-VITA Joint-Stock Company data bank, materials presented at various exhibitions, and recent literature, shows that domestic medical industry has developed a number of original medical devices and apparatuses which were designed to replace similar obsolete models. However, many types of important and necessary medical devices still do not meet contemporary requirements, and some types of devices are not produced at all.For example, in recent years production of some sophisticated medical devices (apparatuses for intensive care, resuscitation, and anesthesiology; devices for artificial lung ventilation, respiratory narcosis devices, extracorporeal circulation) significantly rose, particularly at the former defense industry facilities, and their quality has been significantly improved. The functional performance of the devices is generally on par with foreign analogs.Perfusion units have also been improved and their production has expanded. This allowed the demand of the health service organizations for such equipment to be satisfied completely. Modern domestic hemodialysis devices (Renart-10, Renan- 10RT, etc.) have beendeveloped and brought into wide clinical practice.The development and production of diagnostic magnetic resonance imaging systems (Obraz-3, TOROS) are considerable breakthroughs in domestic medical industry. This substantially extends diagnostic capacities of many health service organizations and provides them with topical diagnosis previously unavailable domestically, although it is quite common in developed foreign countries.Domestic medical industry has begun production of pulse oximeters; these are of particular use in surgery and resuscita-tion. This bridged a substantial gap in the spectrum of available domestic medical devices.The Bilitest bilirubin meter, which has been recently developed and produced in Russia, fully meets the requirements of maternity and children's hospitals in devices for diagnosing jaundice.A high-standard radioimmunochemical laboratory was opened at the VNIIMP-VITA Joint-Stock Company to supply customers with necessary radioimmunochemical assay kits.A number of high-quality medical devices and instruments have been developed at the electronic industry plants andinstitutes. The following devices are particularly worthy of mention:- artificial cardiac valves of the Emitron Plant, which are on par with the best foreign analogs;- pH meters (Istok State Scientific-Manufacturing Association);- Ikar long-term (up to 24 h) cardiomonitors with electronic memory (Kometa Central Scientific-Manufacturing Association);- radiothermographs and racliothermoscopes for detecting deeply located thermal fields in the human body (Oktyabr' Manufacturing Association and Design Bureau for Ecological and Medical Equipment);- original thermal imaging system (Institute of Radioelectronics and Automatics, Russian Academy of Sciences; OPTROS, Ltd.);- original computer-assisted system Cardiac Rhythms for monitoring oatient condition and pulsimetry (Institute of Chemical Physics, Russian Academy of Sciences; Ekos, Ltd.);- video system for endoscopic imaging (Zenit Scientific-Manufacturing A~sociation; Elektron Scientific-Research and Manufacturing Association);- streamlined technology for producing disposable and reusable syringes, injection needles, and surgical threads.A number of other problems of domestic medical instrument-making industry have been successfully solved in recent years.For example, the number and quality of therapeutic devices, particularly for laser therapy, is quite sufficient. Research studies are carried out by many organizations including former defense industry facilities. Technologies which have been developed for other purposes give fruitful results in medical industry.According to our data, more than 150 models of such medical devices have been developed over the last 5 years. Some 100 of them are commercially available. Although domestic medical devices are often superior ot foreign analogs in terms of working performance and they are definitely less expensive, many of them are not in short demand and are virtually not used.However, this activity in many other areas of medical instrument engineering cannot beconsidered as sufficiently successful and rational.It should be noted that many newly developed models of domestic medical devices compare unfavorably with foreign analogs. This is particularly the case for X-ray and ultrasonic devices, electrocardiographic monitors, laboratory equipment, etc. Nevertheless, according to the VNIIMP-VITA Joint-Stock Company databank, certain positive trends have been observed in recent years even in these areas. However, most problems still remain unsettled and the conditions required to solve them have not yet been established.It is important to note that the serially produced X-ray apparatus RUM-20 (Mosrentgen Joint-Stock Company) has been significantly updated. The updated model RUM-20M-SG312 is commercially available in combination with the Sapfir domestic image intensifier or an image intensifier of a French manufacturer. The Kruiz fiat image intensifier has been developed at theAll-Russian Scientific-Research Institute for Medical Instrument Engineering in collaboration with MELZ Manufacturing Association and Mosrentgen Joint-Stock Company. This device is designed to replace existing fluorescent screens in the X-ray diagnostic apparatuses RUM-10, RUM-20, RUM-20M, and others. The use of the Kruiz image intensifier significantly increases image information content and allows threefold decrease in the radiation load on patients and medical personnel.The G 202-5 system for lit-par-lit raster imaging of patients in lying position has been developed at the Mosrentgen Joint-Stock Company. This device is commercially available with the PURS power source. It allows both manual and automatic X-ray photography and organ-oriented X-ray examination.The RTS-61 mobile X-ray video diagnostic apparatus has been developed at the Elektron Scientific-Research and Manufacturing Association. This device is designed to be used in surgery, orthopedics, and traumatology.Among the defense industry facilities which have reoriented their production to medical market the Scientific-Research Institute for Electromechanics (Istra) is worth mention. In collaboration with Phillips (Germany) and borrowing their technology and circuitry, the Institute for Electromechanics developed the Mammodiagnost mammographic scanner, which meets international standards of operating performance.The Rentgen-48 X-ray tomographic diagnostic systems with a rotary support table and the Rentgen-60 X-ray diagnostic systems with a remote control support table have been developed at the Sevkavrentgen Plant and received positive recognition by practicing physicians.The models of X-ray diagnostic devices listed above are examples of achievements of domestic medical industry.However, many important and significant problems of the development of domestic medical X-ray equipment remain unsettled, and it is unreasonable to expect that they will be solved in the foreseeable future unless special measures are taken.For example, the most common RUM-20 X-ray apparatuses with the Sapfir image intensifier are equipped with the obsolete X-ray image converter REP-1. To replace the REP-1 image converter, the Moscow Plant for Electronic Tubes has developed the Buer image converter of improved design. This device offers better image contrast, reduced clark background noise, and has an output fiberoptic window of improved design. However, the Buer image converter is not yet commercially available.Digital X-ray diagnostic devices are not yet commercially available from domesticmanufacturers either.The Design Bureau for Medical Engineering in collaboration with Medtekh, Ltd. (Novosibirsk) have developed the Diaskan X-ray digital scanner. Serial production of this device is in progress at the Design Bureau for Medical Engineering.However, devices of sufficient quality are not yet commercially available.Domestic medical industry does not produce X-ray tomographs. Their production in Chelyabinsk has been suspended.Electrocardiographic monitors are very important devices for functional diagnosis. However, domestic medical industry fails substantially behind leading foreign manufacturers and there is a disproportion in the development and production of necessary devices and apparatuses. Many automatic systems for ECG processing, including syndromal diagnosis, have been developed, but they trove not been tested and are of little demand. However, simple three-channel electrocardiographs of mass- scale application are not produced by domestic manufacturers.Foreign manufacturers offer various ultrasonic scanners and sophisticated imaging systems. Domestic manufacturers produce only simple devices with manual sector-by-sector scanning and a few simplified models with linear electronic scanning.Some positive results have been achieved in the development of endoscopic devices. These achievements are mainly due to the collaboration between LOMO and some companies from Japan. However, even these devices require further improvement of quality and reliability.Although the level of production of domestic laboratory equipment has noticeably risen in recent years, it is still too little to meet the demand. The number of organizations involved in the development of such equipment has risen. However, the available devices are simple and have limited functional capacity. Many important devices (e.g., automatic analyzers and simple routine devices) are not produced at all.Devices for blood transfusion and preparing blood substitute solutions are still in short supply (40 million items have been produced, while the demand is 200 million). The demand in dialyzers and polymer infusion systems reaches 100 and 150 million items, respectively, although such systems are not produced at all.The correspondence between production and demand, quality and technical performance, and adequate testing of medical production are put in the forefront under conditions of a market economy. The problem of competition with foreign manufacturers is also quite important because of increasing import of medical equipment and reduced sale of the production of domestic manufacturers. In this connection, the following circumstances should be taken into consideration.There is a considerable disproportion between production and demand of some groups of medical devices. For example, there is :~ huge surplus of laser therapeutic devices and their excessive development. Systems for syndromal electrocardiographic diagnosis, magnetotherapy, and electrostimulation are also in excessive supply. However, simple electrocardiographs, routine laboratory equipment, and some other ordinary but necessary devices of mass-scale application are not produced by domestic manufacturers. These disadvantages cause significant economic losses and present difficulties in the development of health service. Domestic and foreign experience show that these problems can be solved by adequate marketing, but this is in its infancy in the domestic medical industry.It should be noted that foreign companies place special emphasis on marketing and market research. They evaluate actual and pending demand as well as consumer requirements. Thefeedback between consumer and manufacturer gives valuable information on the improvement of the product quality and working performance. The marketing service in most leading companies is of paramount importance. The development of a new product often starts from marketing survey rather than from engineering or design research. Many domestic organizations of medical instrument engineering require cardinal measures for increasing the level of marketing.Testing of medical devices also requires substantial improvement. Considerable experience of foreign manufacturers of medical equipment should be taken into account. It should be noted, however, that this experience is often neglected by domestic manufacturers. Technical testing of medical equipment in foreign companies is usually carried out by independent laboratories which assess performance and quality. The specialists of the laboratories may also give recommendations for further improvement of the tested equipment. The basic goal of the testing is to check if the performance of the device matches its specifications and to conclude if the device can be used in medical organizations. However, the specialists of the laboratories usually go beyond this goal and issue comparative reviews of products of different companies. Such reviews contain the following information:- description of tested device, its specifications, and price;- results of technical testing, correspondence between specifications and actual performance, advantages and disadvan- tages, recommendations for improvement (if necessary);- comparative analysis of similar devices and apparatuses produced by different manufacturers. Such analysis is usually concluded by a most preferable model, which is recommended to medical organizations on the basis of functional capacity, reliability, and economic reasons.In the USA, activity of testing laboratories is controlled by governmental, nongovernmental, and independent nonprofit organizations.In Russia, the problem of balance between the demand in medical devices, their production by domestic manufacturers, and import is of considerable importance.The opinion of the Head of the Department of Medical Industry, Russian Ministry of Health and Medical Industry, Yu. F. Doshchitsin, which was published in the weekly "Meditsinskii Biznes" (No. 9, 1995), is that the requirements of Russian medical market must be met by domestic devices, including products of high technology. Russian medicine should not rely on imported devices alone. We certainly agree with this opinion.The total volume of medical equipment purchased from abroad is presently several times greater than purchases from domestic manufacturers. This situation is definitely unacceptable. Cardinal measures are required to boost and stimulate economically domestic manufacturers of medical equipment. This is particularly important for manufacturers of life support systems and devices for military medicine.However, positive aspects of contacts with foreign manufacturers of medical equipment should not be disregarded. International cooperation is very common in foreign practice, but it is clearly insufficient in Russia.International cooperation in medical industry is particularly vital in such areas as computer technology, microprocessors, and electronic engineering. Lack of sufficiently high-quality domestic computers and microprocessors presents considerable problems in the development of sophisticated medical devices and apparatuses.In recent years a number of domestic organizations established joint ventures withleading foreign manufacturers of medical devices. These joint ventures produce high-technology devices on the basis of imported circuitry, modules, and individual finished units. For example, VNIIMP-VITA produces ultrasonic doppler scanners, Kursk Manufacturing Association Pribor in collaboration with Frezenius (Germany) produces mobile apparatuses for hemodialysis and hemosorption, LOMO and some companies from Japan established a joint venture for manufacturing flexible endoscopes of improved design, Moscow Manufac- turing Association EMA produces ultrasonic diagnostic devices, etc.It seems reasonable to continue and extend mutually profitable contacts between domestic and foreign manufacturers of medical equipment.Active participation and patronage of the Russian Ministry of Health and Medical Industry as well as the Russian Government and local authorities are needed to solve the problems of medical industry listed above and to implement programs of development and production of high-quality domestic medical devices.References[1] V. A. Viktorov，V. P. gundarov，A. P. yurkevich. Present status and problems of domestic medical instrument engineering. Biomedical Engineehng~ V oL 30, No. 1, 1996.[2]All-Russian Scientific-Research Institute for Medical Instrument Engineering, Rusaian Academy of Medical Sciences (VNIIMP-VITA Joint-Stock Company), Moscow. Translated from Meditsinskaya Tekhnika, No. 1, pp. 4-9, January-February, 1996. Original article submitted August 23, 1995.国内医学仪器工程的现状和存在的问题近年来，国内在工程医疗器械实现取得了很大进展。

食品微生物检验英文参考文献1．贾英民主编，食品微生物学.高等职业教育教材，北京:中国轻工业出版社，2004.91. jiayingmin, food microbiology Textbooks for higher vocational education, Beijing: China Light Industry Press, September 20042．万萍主编，食品微生物基础与实验技术.高职高专食品类教材系列，北京:科学出版社，2. edited by Wan Ping, fundamentals and experimental techniques of food microbiology Food textbook series for higher vocational colleges, Beijing: Science Press,2004two thousand and four3.翁连海主编﹐食品微生物基础高职教材，北京:高等教育出版社，2005.43. Weng Lianhai, editor in chief, higher vocational textbook of food microbiology, Beijing: Higher Education Press, April 20054．苏世彦．食品微生物检验手册．北京:中国轻工业出版社，1998.104. sushiyan. Manual for microbiological examination of food. Beijing: China Light Industry Press, 1998.105．牛天贵．食品微生物学实验技术.北京:中国农业大学出版社，2002.85. Niu Tiangui. Experimental technology of food microbiology Beijing: China Agricultural University Press, August 20026．高鼎主编，食品微生物学，北京:中国商业出版社，1996.56. Gao Ding, chief editor, food microbiology, Beijing: China business press, 1996.5 7．谢梅英主编，食品微生物学，北京:中国轻工业出版社，2004.57. Xie Meiying, food microbiology, Beijing: China Light Industry Press, May 20048.中华人民共和国国家标准.食品卫生检验方法(微生物部分).北京:中国标准出版社，8. national standards of the people's Republic of China Methods for hygienic inspection of food (microbiological part) Beijing: China Standards Press,2003two thousand and three9.杨洁彬．食品微生物学.北京:北京农业大学出版社，19959. yangjiebin. Food microbiology Beijing: Beijing Agricultural University Press, 199510.沈萍主编﹒微生物学北京:高等教育出版社，200011.高东主编﹒微生物遗传学济南:山东大学出版社，199610. microbiology, edited by Shenping, Beijing: Higher Education Press, 200011 Gao Dong. Microbial genetics. Jinan: Shandong University Press, 1996。

Fabrication and Mechanical Properties of Dense/Porous β-TricalciumPhosphate BioceramicsFaming Zhang1, a , Jiang Chang 1, b*, Jianxi Lu 1, 2, c , Kaili Lin 1, d 1 Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, ChineseAcademy of Sciences, Shanghai 200050, China 2 Shanghai Bio-lu Biomaterials Company, Shanghai 200335, China a star.zhang@, b* jchang@,c ir2bberck@,d lklsic@Keywords: Bioceramics, calcium phosphate, bone regeneration, weight bearing sitesAbstract: Attempt t o increase the mechanical properties of porous bioceramics, a dense/porous structured β-TCP bioceramics that mimic the characteristics of nature bone were fabricated. Experimental results show that the dense/porous structured β-TCP bioceramics demonstrated excellent mechanical properties with compressive strength up to 74 MPa and elastic modulus up to 960 MPa, which could be tailored by the dense/porous cross-sectional area ratio obeying the rule of exponential growth. The interface between the dense and porous bioceramics is connected compactly and tightly with some micropores distributed in the matrix of both porous and dense counterparts. The dense/porous structure of β-TCP bioceramics may provide an effective way to increase the mechanical properties of porous bioceramics for bone regeneration at weight bearing sites.IntroductionVarious methods for bone defect treatments have been developed using biological or synthetic grafts. The synthetic alternatives are promising grafts for their unlimited availability and without risk of disease transmission [1]. Calcium phosphate bioceramics, especially hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), have been extensively explored as bone grafts due to their compositions are similar to the inorganic components of nature bone [2]. The β-TCP bioceramics is well known as a biodegradable material demonstrated clinical efficacy. The porous β-TCP bioceramics is a structurally biomimetic of the cancellous bone, whose porous network could allow tissue to ingrowth exhibiting nicer osteoconductive properties. However, the porous β-TCP shows weak mechanical properties, which limit its application as bone grafts. The macrostructure feature of nature bone is porous cancellous bone inside with dense compact bone surrounding outside, which provides excellent biomechanical properties. Carotenuto et al [3] have prepared dense/porous layered HA bioceramic for orthopedic device coating by tape casting technique, whereas the bulk dense/porous bioceramics were rarely reported. Therefore in present study, a dense/porous structured β-TCP bioceramics that mimics the characteristics of nature bone were fabricated, and the microstructure and mechanical properties of such bioceramics were studied.ExperimentalThe β-TCP powders were synthesized by chemical precipitation reaction. The dense/porous structured β-TCP bioceramics were prepared by injected molding and subsequently pressureless sintering. The shrinkage rate of both porous and dense parts during sintering process was measured at different temperatures. X-ray diffraction (XRD) with Cu K α radiation was used to characterize the phase composition of the ceramics. The microstructures observation of the bioceramic samples was performed on a scanning electron microscopy (SEM).The compressive strength was conducted with a mechanical tester at 0.5 mm/min crosshead speed. The elastic modules were reanalyzed from the slope of the compressive strength-strain curve.All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of the publisher: Trans Tech Publications Ltd, Switzerland, . (ID: 159.226.129.129-19/09/06,02:35:46)Results and DiscussionThe major problem in preparation of the dense/porous bioceramics is the interface adhesion between the dense and porous parts because of their different shrinkage rate during sintering process. The shrinkage rate of dense and porous bioceramics at different temperatures was measured and the results are shown in Fig.1. It can be noticed that the porous β-TCP bioceramics exhibit much higher shrinkage rate than the dense counterpart. The porous bioceramics shows about 23% shrinkage in radial direction; in contrast, the dense bioceramics presents about 17% shrinkage. It can be calculated that from 850 o C to 1100 o C, the porous β-TCP bioceramics shows about 17% shrinkage rate and almost the same with that of the dense counterpart from 600 o C to 1100 o C. So as to avoiding the shrinkage differences, the porous β-TCP bioceramics were pre-sintered at 850 o C, then the dense bioceramics were injected surrounding the porous ceramics, finally the composites were pressureless sintered at 1100 o C for 5 hours and the dense/porous structured β-TCP bioceramics were obtained.Fig.1 The radial shrinkage rate of the porous and dense β-TCP bioceramicsThe phase composition of the as prepared bioceramics was analyzed by X-ray diffraction. The XRD results show that the high temperature sintered β-TCP preserved their original β phase without transform into their α-TCP phase, as shown in Fig.2. Because the α-TCP though bioactive, have proven less useful as bone regeneration materials due to their excessively high resorption rate than the β-TCP phase. And none of the other impurity phases can be detected in the XRD patterns; resultantly, high purity β-TCP bioceramics were prepared.Fig.2 X-ray diffraction pattern of the prepared bioceramics.Fig.3 shows the optical and SEM micrographs of the prepared dense/porous β-TCP bioceramics samples. It is clear to see that the inner porous structure mimics the cancellous bone to some extent, and outer side dense structure mimics the compact bone, as shown in Fig.3(a) and indicated by theS h i n k a g e (%)Temperature (o C)1020304050607080100200300400500600 2theta (deg.)I n t e n s i t y (c p s )arrows. Fig.3 (b) shows the interface of the dense/porous β-TCP bioceramic, it can be found that the interface between the dense and porous bioceramics is connected compactly and tightly. In the porous part, the macropore size is about 500 μm in diameter; the diameter of the interconnected pores is about 100 μm. Additionally, the porosity of the porous parts is about 72%, and the interconnectivity is more than 95%. The microstructure of the macroporous wall was shown in Fig.3(c); it is obvious that there are some micropores with diameter of 1 μm distributed uniformly in the porous wall. As the results, the microstructure of porous part of the bioceramics is a combination of macroporous and microporous. Contrastively, the microstructure of the dense bioceramics shows refined particle size and few micropores, as exhibited in Fig.3(d). The dense compact part is much denser than the porous cancellous part.Fig.3 The dense/porous β-TCP bioceramic sample (a), the microstructure of dense/porous interface(b), the macroporous wall (c) and dense compact bone (d).The variation of the compressive strength and Elastic modulus of the bioceramics with different dense/porous cross-sectional area ratio (S dense /S porous ) was illustrated in Fig 4. It is exhibited that the compressive strength increases from 10 MPa to 74 MPa with the dense/porous ratio from 0.1 to 4.7 obeying rule of exponential growth. And the elastic modulus has been increased form 180 MPa to 960 MPa with the dense/porous ratio increment, also following exponential growth. Evidently, the value of the porous bioceramics is only about 2.0 MPa and the elastic modulus is about 20 MPa, indicated by the square in Fig.4. It has been achieved about 5 to 37 times increment in the mechanical properties by the dense/porous structure design. The mechanical properties of the dense/porous bioceramics could be tailored by the dense/porous cross-sectional area ratio.Porous materials always have poor mechanical properties. Applications of calcium phosphates in the body have been limited by their low strength and numerous techniques have been investigated in attempts to retain their useful bioactive properties whilst providing more suitable mechanical properties for particular applications. These include the reinforcement of β-TCP using HA fiber orbioglass additives [4, 5]; however these techniques are limited for the porous calcium phosphate Compact bone Cancellousbone (b)(c) (d)using in the load bearing sites’ bone regeneration. In this study, excellent mechanical properties of the porous β-TCP bioceramics have been achieved by the dense/porous structured design. The compressive strength of human femoral cancellous bone, weight bearing sites, is in the range of 25~90 MPa, so the dense/porous structured β-TCP is comparable to the strength of human femoral cancellous bone. The high interconnective porous structure of the dense/porous β-TCP bioceramics could allow the tissue ingrowths, and the dense structure could bear the load to some extent. The dense/porous structure of β-TCP bioceramics may provide a simple but effective way to increase the mechanical properties of porous bioceramics for the bone regeneration applications at weight bearing sites.Fig.4 The variation of the compressive strength and elastic modulus of the bioceramics withdifferent dense/porous cross-sectional area ratio. ConclusionsThe dense/porous structured β-TCP bioceramics were prepared and revealed excellent mechanical properties with compressive strength from 10 to 74 MPa and elastic modulus from 180 to 960 MPa, which is 5 to 37 times higher than that of the pure porous β-TCP and comparable to the strength of human femoral cancellous bone. The interface between the dense and porous bioceramics is connected compactly and tightly. The dense/porous structure of β-TCP bioceramics may provide a simple but effective way to increase the mechanical properties of porous bioceramics for weight bearing site’s bone regeneration.AcknowledgementFinancial supports from the Shanghai Postdoctoral Scientific Key Program and the Science & Technology Commission of Shanghai Municipality of China (No.04DZ52043) are greatly acknowledged.References:[1] Niedhart C, Maus U, Redmann E, Schmidt-Rohlfing B, Niethard FU, Siebert CH: J BiomedMater Res Vol. 65A (2003), p.17[2] Hench Larry L: Journal of the American Ceramic Society Vol. 81(1998), p.1705[3] Carotenuto G: Advanced Performance Materials Vol. 5(1998), p.171[4] Hassna R. R. Ramay, Zhang M.: Biomaterials Vol. 25(2004), p.5171[5] Ashizuka M, Nakatsu M, Ishida E: Journal of the Ceramic Society of Japan, v 98(1990), p.204. 01020304050607080012345020040060080010001200E l a st i c M o d u l u s (M P a ) C o m p r e s s i v e S t r e n g h (M P a )S dense /S porous。

Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryfindings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantification of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsflanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-specific primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-specific primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Purification 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-specific primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into afilter cartridgewithin a collection bel thefilter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the samefilter.17.Centrifuge at10,000rpm for15s at RT.18.Collect thefiltrate(theflow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theflow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newfilterbel thefilter as small RNA.When you have >700μL of thefiltrate mixture,apply it in successive appli-cation to the samefilter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theflow-through and repeat until all of thefiltratemixture is passed through thefilter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to thefilter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theflow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to thefilter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theflow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer thefilter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of thefilter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave thefilter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube briefly.3.Incubate for1h at37◦C.3.3.Purification 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a purificationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard thefiltrate(theflow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of thefilter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let thefilter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube briefly.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-specific primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube briefly.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-specific primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which thefluorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method amplifies over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-specific primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefficiency.Good efficiencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially amplifies maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). 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2.4. Chemical and microbial analyses Analysis of DM and CP concentration in the experimental diets, excreta and probiotic products was done according to AOAC (1990 methods (930.05 and 976.05, respectively. The GE was measured by using the bomb calorimeter (model 1261, Parr Instrument Co., Moline, IL, and chromium concentration was determined with an automated spectrophotometer (Jasco V-650, Jasco Corp., Tokyo, Japan according to the procedure of Fenton and Fenton (1979. The microbiological assay of faecal samples (d 14 and 28 and intestinal digesta (d 28 was conducted by culturing in different media for the determination of total anaerobic bacteria (Tryptic soy agar, Bifidobacterium spp. (MRS agar, Lactobacillus spp. (MRS agar+0.02% NaN3+0.05% L-cystine hydrochloride monohydrate, Clostridium spp. (TSC agar and coliforms (violet red bile agar. The microbiological assay of probiotic products was also carried out by culturing technique. The L. acidophilus was enumerated using MRS agar+0.02%NaN3+0.05% L-cystine hydrochloride monohydrate, B. Subtilis by using plate count agar, S. cerevisiae and A. oryzae by potato dextrose agar. The anaerobic conditions during the assay of anaerobic were created by using gas pack anaerobic system (BBL, No. 260678; Difco, Detroit, MI. The tryptic soy agar (No. 236950, MRS agar (No. 288130, violet red bile agar (No. 216695, plate count agar (No. 247940, and potato dextrose agar (No. 213400 used were purchased from Difco Laboratories (Detroit,MI, and TSC agar(CM0589 was purchased from Oxoid (Hampshire, UK. The pH of probiotic products was determined by pH meter (Basic pH Meter PB-11, Sartorius, Germany.2.5. Small intestine morphology Three cross-sections for each intestinal sample were prepared after staining with azure A and eosin using standard paraffin embedding procedures. A total of 10 intact, welloriented crypt-villus units were selected in triplicate for each intestinal cross-section as described previously (Jin et al., 2008. Villus height was measured from the tip of the villi to the villus crypt junction, and crypt depth was defined as the depth of the invagination between adjacent villi. All morphological measurements (villus height and crypt depth were made in 10-μm increments by using animage proce ssing and analysis system (Optimus software version 6.5, Media Cybergenetics, North Reading, MA.2.6. Statistical analysesAll the data obtained in the current study were analyzed in accordance with a rand omized complete block design using the GLM procedure of SAS (SAS Inst. Inc., C ary, NC. In Exp. 1, one-way analysis of variance test was used and when signific ant differences (Pb0.05 were determined among treatment means, they were separ ated by using Duncan's multiple range tests. In Exp. 2, the data were analyzed as a 2×2 factorial arrangement of treatments in randomized complete block design. T he main effects of probiotic products (LF or SF, antibiotic (colistin or lincomycin, a nd their interaction were determined by the Mixed procedures of SAS. However, as the interaction (probiotic x antibiotic was not statistically significant (Pb0.05, it wa s removed from the final model. The pen was the experimental unit for all analysis in both experiments. The bacterial concentrations were transformed (log before st atistical analysis.3.1. Experiment 13.1.1. Growth performance and apparent total tract digestibilityDietary treatments had no effect on the performance of pigs during phase I (Table 3. However, during phase II and the overall experimental period, improved (Pb0.05 ADG, ADFI and G:F were observed in pigs fed PC, LF and SF dietswhen compared with pigs fed NC diet. Moreover, pigs fed PC and SF diets had hi gher (Pb0.05 ADG and better G:F than pigs fed LF diet during phase II and the o verall experimentalperiod. The dietary treatments had no influence on the ATTDof DM and GE; however, pigs fed PC and SF diets had greater ATTD of CP whe n compared with pigs fed NC and LF diets (Table 4.3.1.2. Bacterial population in faecesDietary treatments had no effect on the faecal total anaerobes and Bifidobacterium spp. population at d 14 and 28, and Lactobacillus spp. at d 14 (Table 5. However, pigs fed PC (d 14 and 28 and SF (d 28 diets had less (Pb0.05 faecal Clostridium spp. and coliforms than pigs fed NC diet.Moreover, pigs fed SF diet had greater (Pb0.05 faecal Lactobacillus spp. populatio n (d 28 than pigs fed NC, PC and LF diets.3.2. Experiment 23.2.1. Growth performance and apparent total tract digestibilityDuring phase I, pigs fed SF diet consumed more feed than pigs fed LF diet, wher eas the ADG and ADFI were similar between pigs fed LF and SF diets (Table 6. During phase II and the overall experimental period, pigs fed SF diet showed better ADG(Pb0.01, ADFI (Pb0.01 and G:F (Pb0.05 thanpigs fed LF diet. Howev er, different antibiotics had no effect on the performance of pigs. Pigs fed SF diet had greater ATTD of DM and CP during phases I and II (Pb0.01 and 0.001, respe ctively when compared with pigs fed LF diet (Table 7.However, different antibiotics had no effect on the ATTD of DM, CP and GE.3.2.2. Bacterial population in intestinePigs fed SF diet had greater (Pb0.05 Lactobacillus spp. And less Clostridium spp. (Pb0.01 and coliform (Pb0.05 population in the ileum than pigs fed LF diet (Table 8. Additionally,higher (Pb0.05 caecal Bifidobacterium spp. Population was observed in pigs fed SF diet. Antibiotics had no effect on the ileal microbial population; however, pigs fed colistin diet had less number of Bifidobacterium spp. (Pb0.05 and coliforms (Pb0.01 inthe cecum, whereas, feeding of lincomycin diet resulted in reduced (Pb0.05 caecal Clostridium spp.population.3.2.3. Small intestinal morphologyThe different probiotic products and antibiotics had no influence on the morphology of different segments of the small intestine, except for the greater (Pb0.05 villus height:crypt depth at the jejunum and ileum noticed in pigs fed lincomycin diet (Table 9.4.DiscussionPrevious studies on probiotics lack information on the method of production used, however, the preparation of probiotics by LF method is fairly common (Patel et al., 2004. The probiotic products used in the present study differedfrom the previous reports in that harvested probiotic microbes were added directly to the diets. In this study, the microbial biomass grown on the CB was directly sprayed onthe carrier (corn and soybean meal to obtain LF probiotic product. In case of the SF probiotic product, corn and soybean meal was used as a substrate during fermentation and as a carrier of probiotic microbes. We have reported previously that multi-microbe probiotic product prepared by SF method was better than the probiotic product prepared by submerged liquid fermentation in improving performance, nutrient retention and reducing harmful intestinal bacteria in broilers (Shim et al., 2010. In the current study, LF and SF method was used and corn–soybean meal was used as a substrate forthe growth of potential probiotic microbes under optimum conditions.2.4 化学和微生物分析在试验日粮干物质和粗蛋白含量的分析中,排泄物和益生菌产品是根据AOAC(1990方法(分别为930.05和976.05 分析。

REVIEWThe genus Codonopsis (Campanulaceae):a review of phytochemistry,bioactivity and quality controlJing-Yu He •Na Ma •Shu Zhu •Katsuko Komatsu •Zhi-Yuan Li •Wei-Ming FuReceived:7May 2014/Accepted:18July 2014/Published online:7August 2014ÓThe Japanese Society of Pharmacognosy and Springer Japan 2014Abstract Codonopsis ,in the family Campanulaceae,is a genus containing 42species of dicotyledonous herbaceous perennial plants,predominantly found in Central,East and South Asia.Several Codonopsis species are widely used in traditional medicine and are considered to have multiple medicinal properties.Among the Codonopsis species,Codonopsis pilosula (Franch.)Nannf.and nceolata (Sieb.et Zucc.)Benth.&Hook.f.ex Trautv.are more popular than others according to the findings,especially phytochemical and bioactive studies.Phytochemical research shows that Codonopsis species contain mainly polyacetylenes,phenylpropanoids,alkaloids,triterpenoids and polysaccharides,which contribute to multiple bioac-tivities.However,the mechanisms of their bioactivities need to be further elucidated.The less popular Codonopsis species remain to be studied and exploited.In addition,although a series of methods for the quality evaluation of Codonopsis species have been developed,a feasible and reliable approach to the efficacious and safe use of various Codonopsis species is still needed,with considering botanical origin,chemical constituents and bioactive effects.This review aims to provide up-to-date and com-prehensive information on the phytochemistry,bioactivity and quality control of medicinal plants in the genus Codonopsis and to highlight current gaps in knowledge,which is useful for the wider development of the Codon-opsis genus.Keywords Codonopsis ÁPhytochemistry ÁBioactivity ÁQuality controlIntroductionCodonopsis ,which is a dicotyledonous genus of herba-ceous perennial plants in the family Campanulaceae,has 42species predominantly distributed in Central,East and South Asia;40Codonopsis species can be found in China [1].However,Codonopsis pilosula (Franch.)Nannf.,C.pilosula Nannf.var.modesta (Nannf.)L. D.Shen, C.tangshen Oliv.and nceolata (Sieb.et Zucc.)Benth.&Hook.f.ex Trautv.are the only species widely used,of which the fresh or dried roots are collectively regarded as famous herbal medicines and have been used in folk medicine for hundreds of years.Codonopsis Radix is pre-scribed as the dried roots of C.pilosula ,C.pilosula var.modesta and C.tangshen in the Chinese Pharmacopoeia (2010edition)[2].It is called ‘‘Dangshen’’in Chinese and ‘‘Tojin’’in Japanese,and has been used in traditional Chinese medicine for replenishing qi (vital energy)defi-ciency,strengthening the immune system,improving poor gastrointestinal function,gastric ulcer and appetite,decreasing blood pressure,etc.,and is sometimes used as a substitute for Ginseng (Panax ginseng C.A.Mey.)[3,4].The roots of other Codonopsis species,including C.tu-bulosa ,C.subglobosa ,C.clematidea and nceolata ,are reported to be used as substitutes for Codonopsis Radix in some regions [3]. nceolata ,commonly called bonnet bellflower,is a herb with high value in traditional Chinese medicine and its root is also becoming popular as aJ.-Y.He ÁN.Ma ÁZ.-Y.Li ÁW.-M.Fu (&)Guangzhou Institute of Advanced Technology,Chinese Academy of Sciences,1121Haibin Rd.,Nansha Dist.,511-458Guangzhou,People’s Republic of China e-mail:wm.fu@S.Zhu ÁK.KomatsuDivision of Pharmacognosy,Department of MedicinalResources,Institute of Natural Medicine,University of Toyama,2630Sugitani,Toyama 930-0194,JapanJ Nat Med (2015)69:1–21DOI10.1007/s11418-014-0861-9special vegetable in some Asian countries[5]nceo-lata has been used for the treatment of bronchitis,asthma, cough,tuberculosis,dyspepsia and psychoneurosis[6–8]. Phytochemical researches have revealed that the roots of Codonopsis species contained alkaloids,phenylpropanoids, triterpenoids,polyacetylenes,flavones,organic acid,poly-saccharides,etc.[9–54].Among them,polyacetylenes, phenylpropanoids,alkaloids,triterpenoids and polysac-charides are considered to be the major constituents and responsible for most of the activities found in the plants of this genus.The chemical profile varied greatly between species and sample collections may cause these Codon-opsis species to possess diverse bioactivities.Some com-pounds belonging to thesefive chemotypes have been evaluated for potential biological activity and pharmaco-logical mechanisms.However,the pharmacological mechanisms of these Codonopsis species related to bio-logical activity and clinical application remain largely unexplained.Additionally,the toxicity of Codonopsis has not been reported in the scientific literature.As many investigations indicated that a variety of chemical constituents contributed to the effects of Codon-opsis species,the quantitation of bioactive components becomes urgent for ensuring the efficacy of Codonopsis species.In the Chinese Pharmacopoeia(2010edition),only lobetyolin is used as the chemical marker for identification of Codonopsis Radix,which seems useless for many Codonopsis species involved[2].Hence,a number of studies have attempted to develop accurate,sensitive and selective analytical methods for qualitative and quantitative evaluation of Codonopsis materials.To provide information benefiting traditional uses and scientific studies,this review summarizes and evaluates the available phytochemical and bioactive properties of Codonopsis genus reported by the literature.In addition, for the efficacious and safe uses of Codonopsis,the pro-gress of research on quality evaluation of various Codon-opsis species is also presented.Chemical constituents of genus Codonopsis Phytochemical studies have been performed on Codonopsis species plants over the last30years all around the world. Only some of the different Codonopsis species plants have been explored for obtaining information on chemo-taxo-nomical identification,isolation and identification of vari-ous important chemicals from this genus and comparison of the chemicals in different plants or species.C.pilosula,C.tangshen,nceolata and C.clemati-dea have been widely investigated in their phytochemistry; more than100compounds have thereby been isolated and identified.On the other hand,few compounds in C.cordifolioidea,C.nervosa,C.thalictrifolia,C.xundian-ensis and C.tubulosa are reported because they are found only in selected regions.The components in other Codonopsis species have not yet been reported since these Codonopsis species may be difficult to collect and/or be scarce.To date,polyacetylenes,phenylpropanoids,alka-loids,triterpenoids,etc.have been isolated and character-ized from the different parts of these Codonopsis species plants.The names of these constituents,the plant and the parts from which they are derived are summarized in Table1.The structures of the compounds are shown in Figs.1,2,3,4,5,6and7.AlkaloidsThe pyrrolidine alkaloids codonopsine(1),codonopsinine (2),codonopsinol(3)and radicamine A(4)were isolated from the aerial parts of C.clematidea[9,10].Two pyr-rolidine alkaloids,codonopyrrolidiums A(5)and B(6), were isolated from the roots of C.tangshen[13],and were also found in the roots of C.pilosula and C.pilosula var. modesta[12,14].In addition,the pyrrolidine alkaloids codonopsinols A(7),B(8),C(9)and the glycoside,cod-onopiloside A(10)were obtained from the roots of C. pilosula[11].Codotubulosine B(11)was found in the roots of C.tubulosa[15].Other alkaloids,n-9-formyl harman(12),norharman (13),1-carbomethyl carboline(14),1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid(15)and tryptophan(16),were isolated from the roots of nceolata[16,17,19]. Tryptophan(16),perlolyrine(17)and nicotinic acid(18) were obtained from the roots of C.pilosula[18,20,21]. The common compounds uracil(19)and adenosine(20) were found in the roots of C.pilosula and the roots of C. pilosula and C.tangshen,respectively[13,18,22]. PhenylpropanoidsThe phenylpropanoids tangshenosides I(21),II(22),III (23)and IV(24)werefirst isolated from C.tangshen[23, 25].Tangshenoside V(25),tangshenoside VI(26)and codonosides A(27)and B(28),considered to be the characteristic components,were isolated from C.tangshen [13,26].Tangshenoside VI(26)was also isolated from the aerial parts of C.nervosa[27].Recently,tangshenoside VIII(29)has been obtained from the roots of nceolata [24].In addition,12phenylpropanoids,cordifoliketones A (30)and B(31),sinapinaldehyde(32),coniferaldehyde (33),coniferoside(34),isoconiferin(35),nervolans B(36) and C(37),dillapiole(38),1-allyl-2,6-dimethoxy-3,4-methylenedioxybenzene(39),4-allyl-2-(3-methylbut-2-en-1-yl)phenol(40)and sachaliside(41),were isolated from the roots of C.cordifolioidea[28,29].Syringin(42)has been commonly found in5Codonopsis species[21,23,30–32].TriterpenesThree new triterpenyl esters,codonopilates A(43),B(44) and C(45),together with seven known triterpenoids, 24-methylenecycloartanyl linolate(46),24-methylene-cycloartan-3-ol(47),friedelin(48),1-friedelen-3-one(49), stigmast-7-en-3-one(50),taraxerol(51)and stigmast-7-en-3-ol(52),were isolated from the CHCl3-soluble fraction of the methanol extract of C.pilosula[14].Additionally,a-spinasterol(53)was obtained from C.pilosula,C.tang-shen,nceolata and C.thalictrifolia[32–35],and tar-axeryl acetate(54)was obtained from C.pilosula, C. tangshen and C.clematidea[10,34,35].The oleanan-type bisdesmoside with sugars at C-3and C-28,codonolaside (55),codonolasides I(56),II(57)and III(58),and their prosapogenins,eclalbasaponin XIII(59)and echinocystic acid3-O-b-D-glucuronopyranoside(60),were isolated from the roots of nceolata[36].The triterpene saponins, lancemasides A(61),B(62),C(63),D(64),E(65),F(66) and G(67),have also been isolated from the roots of C. lanceolata cultivated in Korea[19].Codonolaside IV(68), codonolaside V(69),foetidissimoside A(70),aster saponin Hb(71),oleanolic acid(72),echinocystic acid(73)and stigmasterol(74)were found in the roots of nceolata [19,30,34,37–39].Foetidissimoside A(70)and rubiprasin B(75)were isolated from the aerial parts of C.clematidea [10].For the aerial parts of C.thalictrifolia,isolation of a-spinasterol(53)and b-amyrin acetate(76)was reported [32].Zeorin(77)and lupeol(78)were isolated from the whole plants of C.nervosa[40].PolyacetylenesIsolation and identification of lobetyolin(79),lobetyolinin (80)and lobetyol(81)from the roots and aerial parts of plants belonging to the genus Codonopsis have also been reported[10,12,18,25,27,29,32,34,41,42].Three new polyacetylene glucosides,cordifolioidynes A(82),B(83) and C(84),were isolated from a95%ethanol extract of the roots of C.cordifolioidea[29].Recently,cordifoli-oidynes B(83)has also been found in C.pilosula,C. pilosula var.modesta and C.tangshen,which are the botanical sources of Codonopsis Radix[12].FlavonesChrysoeriol(85),tricin(86),wogonin(87)and luteolin (88)were isolated from the roots of C.xundianensis Wang ZT and Xu GJ,which grows in Yunnan Province,China [43].Luteolin(88),kaempferol(89),luteolin-5-O-b-D-glucopyranoside(90),luteolin-7-O-b-D-gentiobioside(91), apigenin-7-O-b-D-glucopyranoside(92)and luteolin-7-O-Table1Compounds in Codonopsis speciespound names Species Part of the plant ReferencesAlkaloids1Codonopsine C.clematidea Aerial parts[9]2Codonopsinine C.clematidea Aerial parts[9]3Codonopsinol C.clematidea Aerial parts[10]4Radicamine A C.pilosula Roots[11]C.clematidea Aerial parts[10]5Codonopyrrolidium A C.pilosula Roots[12]C.pilosula var.Roots[12]modestaC.tangshen Roots[13]6Codonopyrrolidium B C.pilosula Roots[14]C.pilosula var.Roots[12]modestaC.tangshen Roots[13]7Codonopsinol A C.pilosula Roots[11]8Codonopsinol B C.pilosula Roots[11]9Codonopsinol C C.pilosula Roots[11]10Codonopiloside A C.pilosula Roots[11]11Codotubulosine B C.tubulosa Roots[15]12n-9-Formyl harman nceolata Roots[16]13Norharman nceolata Roots[16]141-Carbomethyl carboline nceolata Roots[16] 151,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid nceolata Roots[17]16Tryptophan C.pilosula Roots[18]nceolata Roots[19]17Perlolyrine C.pilosula Roots[20]18Nicotinic acid C.pilosula Roots[21]19Uracil C.pilosula Roots[18]20Adenosine C.pilosula Roots[22]C.tangshen Roots[13]Phenylpropanoids21Tangshenoside I C.pilosula Roots[12]Roots[12]C.pilosula var.modestaC.tangshen Roots[23]nceolata Roots[24]22Tangshenoside II C.tangshen Roots[23]nceolata Roots[24]23Tangshenoside III C.tangshen Roots[25]nceolata Roots[24]24Tangshenoside IV C.tangshen Roots[25]nceolata Roots[24]25Tangshenoside V C.tangshen Roots[26]26Tangshenoside VI C.tangshen Roots[26]C.nervosa Aerial parts[27]27Codonoside A C.tangshen Roots[13]28Codonoside B C.tangshen Roots[13]29Tangshenoside VIII nceolata Roots[24]30Cordifoliketone A C.cordifolioidea Roots[28]31Cordifoliketone B C.cordifolioidea Roots[28] 32Coniferaldehyde C.cordifolioidea Roots[29] 33Sinapinaldehyde C.cordifolioidea Roots[29] 34Coniferoside C.cordifolioidea Roots[29] 35Isoconiferin C.cordifolioidea Roots[29] 36Nervolan B C.cordifolioidea Roots[28] 37Nervolan C C.cordifolioidea Roots[28] 38Dillapiole C.cordifolioidea Roots[28] 391-Allyl-2,6-dimethoxy-3,4-methylenedioxybenzene C.cordifolioidea Roots[28] 404-Allyl-2-(3-methylbut-2-en-1-yl)phenol C.cordifolioidea Roots[28] 41Sachaliside C.cordifolioidea Roots[29] 42Syringin C.pilosula Roots[21]C.tangshen Roots[23]nceolata Roots[30]C.nervosa Aerial parts[31]C.thalictrifolia Aerial parts[32]Triterpenes43Codonopilate A C.pilosula Roots[14] 44Codonopilate B C.pilosula Roots[14] 45Codonopilate C C.pilosula Roots[14] 4624-Methylenecycloartanyl linolate C.pilosula Roots[14] 4724-Methylenecycloartan-3-ol C.pilosula Roots[14] 48Friedelin C.pilosula Roots[14]C.tangshen Roots[12]nceolata Roots[33] 491-Friedelen-3-one C.pilosula Roots[14] 50Stigmast-7-en-3-one C.pilosula Roots[14] 51Taraxerol C.pilosula Roots[14]C.tangshen Roots[34] 52Stigmast-7-en-3-ol C.pilosula Roots[14] 53a-Spinasterol C.pilosula Roots[35]C.tangshen Roots[34]nceolata Roots[33]C.thalictrifolia Aerial parts[32] 54Taraxeryl acetate C.pilosula Roots[35]C.tangshen Roots[34]C.clematidea Aerial parts[10] 55Codonolaside nceolata Roots[36] 56Codonolaside I nceolata Roots[36] 57Codonolaside II nceolata Roots[36] 58Codonolaside III nceolata Roots[36] 59Eclalbasaponin XIII nceolata Roots[36] 60Echinocystic acid-3-O-(60-O-methyl)-b-D-glucuronopyranoside nceolata Roots[36] 61Lancemaside A nceolata Roots[19] 62Lancemaside B nceolata Roots[19] 63Lancemaside C nceolata Roots[19] 64Lancemaside D nceolata Roots[19] 65Lancemaside E nceolata Roots[19]66Lancemaside F nceolata Roots[19] 67Lancemaside G nceolata Roots[19] 68Codonolaside IV nceolata Roots[37] 69Codonolaside V nceolata Roots[38] 70Foetidissimoside A nceolata Roots[19]C.clematidea Aerial parts[10] 71Aster saponin Hb nceolata Roots[39] 72Oleanolic acid nceolata Roots[30] 73Echinocystic acid nceolata Roots[30] 74Stigmasterol C.tangshen Roots[34]nceolata Roots[33] 75Rubiprasin B C.clematidea Aerial parts[10] 76b-Amyrin acetate C.thalictrifolia Aerial parts[32] 77Zeorin C.nervosa Whole plants[40] 78Lupeol C.nervosa Whole plants[40] Polyacetylenes79Lobetyolin Codonopsis pilosula Roots[18]Roots[12]C.pilosula var.modestaC.tangshen Roots[25]nceolata Roots[41]C.clematidea Aerial parts[10]C.cordifolioidea Roots[29]C.nervosa Whole plants[27]C.thalictrifolia Aerial parts[32] 80Lobetyolinin C.pilosula Roots[18]Roots[12]C.pilosula var.modestaC.tangshen Roots[12]C.clematidea Aerial parts[10] 81Lobetyol C.pilosula Roots[42]Roots[12]C.pilosula var.modestaC.tangshen Roots[34]C.cordifolioidea Roots[29] 82Cordifolioidyne A C.cordifolioidea Roots[29] 83Cordifolioidyne B C.pilosula Roots[12]Roots[12]C.pilosula var.modestaC.tangshen Roots[12]C.cordifolioidea Roots[29] 84Cordifolioidyne C C.cordifolioidea Roots[29] Flavones85Chrysoeriol C.xundianensis Roots[43] 86Tricin C.xundianensis Roots[43] 87Wogonin C.xundianensis Roots[43]88Luteolin C.nervosa Whole plants[40]C.thalictrifolia Aerial parts[32]C.clematidea Aerial parts[10]C.xundianensis Roots[43] 89Kaempferol C.nervosa Whole plants[40] 90Luteolin-5-O-b-D-glucopyranoside C.nervosa Aerial parts[27]C.thalictrifolia Aerial parts[32] 91Luteolin-7-O-b-D-gentiobioside C.nervosa Aerial parts[27]C.thalictrifolia Aerial parts[32] 92Apigenin-7-O-b-D-glucopyranoside C.nervosa Aerial parts[31]93Luteolin-7-O-b-D-glucopyranosyl(1?6)-[(6000-O-caffeoyl)]-b-D-glucopyranoside C.nervosa Whole plants[40] C.clematidea Aerial parts[10]94Hesperidin C.pilosula Roots[35] Organic acids95Succinic acid C.pilosula Roots[44]C.nervosa Aerial parts[31] 963-O-caffeoylquinic acid C.nervosa Aerial parts[31]C.thalictrifolia Aerial parts[32] 975-O-caffeoylquinic acid C.nervosa Aerial parts[31] 984-(b-D-Glucopyranosyl)-benzoic acid C.nervosa Aerial parts[31] 99Caffeic acid C.thalictrifolia Aerial parts[32] 100Linoleic acid C.thalictrifolia Aerial parts[32] 1019,10,13-Trihydroxy-(E)-octadec-11-enoic acid C.pilosula Roots[35] 102Shikimic acid nceolata Roots[33] 103Vanillic acid C.tangshen Roots[34] Other compounds104Atractylenolide III C.pilosula Roots[21] 1055-Hydroxymethyl-2-furaldehyde C.pilosula Roots[14]C.tangshen Roots[34] 106Angelicin C.pilosula Roots[44] 107Psoralen C.pilosula Roots[44] 108Emodin C.pilosula Roots[18] 109Geniposide C.pilosula Roots[17] 110Hexyl-b-D-glucopyranoside C.pilosula Roots[18] 111Butyl-b-D-fructournanoside C.pilosula Roots[18] 112b-Sitosterol C.pilosula Roots[35]C.nervosa Whole plants[40] 113b-Daucosterol C.pilosula Roots[35] 114Hexyl-b-gentiobioside C.tangshen Roots[25] 115Hexyl-b-sophoroside C.tangshen Roots[25] 116(E)-2-hexenyl-b-sophoroside C.tangshen Roots[25] 117(E)-2-hexenyl-a-L-arabinopyranosyl(1?6)-b-D-glucopyranoside C.tangshen Roots[25]C.clematidea Aerial parts[10] 118Cordifoliflavane A C.cordifolioidea Roots[45] 119Cordifoliflavane B C.cordifolioidea Roots[45] 120Lanceolune A nceolata Roots[46] 121Lanceolune B nceolata Roots[46] 122Lanceolune C nceolata Roots[46]b-D-glucopyranosyl(1?6)-[(6000-O-caffeoyl)]-b-D-gluco-pyranoside(93)were obtained from C.nervosa[27,31,40],and luteolin(88),luteolin-5-O-b-D-glucopyranoside(90)and luteolin-7-O-b-D-gentiobioside(91)were alsofound in the aerial parts of C.thalictrifolia[32].In addi-tion,luteolin(88)and luteolin-7-O-b-D-glucopyrano-syl(1?6)-[(6000-O-caffeoyl)]-b-D-glucopyranoside(93)were isolated from the aerial parts of C.clematidea[10].Hesperidin(94)was only isolated from the roots of C.pilosula[35].Organic acidsTo date,succinic acid(95),3-O-caffeoylquinic acid(96),5-O-caffeoylquinic acid(97)and4-(b-D-glucopyranosyl)-benzoic acid(98)have been found in C.nervosa[31].Caffeic acid(99),linoleic acid(100)and3-O-caffeoyl-quinic acid(96)were isolated from C.thalictrifolia[32].Succinic acid(95)and9,10,13-trihydroxy-(E)-octadec-11-enoic acid(101)were isolated from C.pilosula[35,44].Shikimic acid(102)and vanillic acid(103)were onlyobtained from the roots of nceolata and C.tangshen,respectively[33,34].Other compoundsAtractylenolide III(104),5-hydroxymethyl-2-furaldehyde(105),angelicin(106),psoralen(107),emodin(108),ge-niposide(109),hexyl-b-D-glucopyranoside(110),butyl-b-D-fructournanoside(111),b-sitosterol(112)and b-dau-costerol(113)were isolated from the roots of C.pilosula[14,17,18,21,35,44].Hexyl-b-gentiobioside(114),hexyl-b-sophoroside(115),(E)-2-hexenyl-b-sophoroside(116),(E)-2-hexenyl-a-L-arabinopyranosyl(1?6)-b-D-glucopyranoside(117)and5-hydroxymethyl-2-fur-aldehyde(105)were isolated from the roots of C.tangshen[25,34].Cordifoliflavanes A(118)and B(119)were iso-lated from the roots of C.cordifolioidea[45].Three newbenzofuranylpropanoids,lanceolunes A(120),B(121)andC(122),as well as a new cerebroside,codonocerebrosideA(123),have been isolated from the roots of nceolata[41,46].(E)-2-Hexenyl-a-L-arabinopyranosyl(1?6)-b-D-glucopyranoside(117),3-oxo-a-ionol-b-D-glucopyranoside (124)and1,6-hexanediol,3,4-bis(4-hydroxy-3-methox-yphenyl)(125)were isolated from the aerial parts of C. clematidea[10].In addition,sweroside(126)and b-sitos-terol(112)were obtained from the whole plants of C. nervosa[40].Nutritive constituents including amino acids and trace elements in C.pilosula have been reported[47]. Essential oilsAs one of the important compositions,essential oils of several Codonopsis species have been reported.In the essential components from C.pilosula,50of66separated components were identified by GC–MS,mainly containing 1,2-benzonedicarboxylic acid dibatyl-ester(12.45%), heptedecanoic acid(8.10%)and2,4,5-triisopropyl styrene (7.62%)[48].Using the GC–MS method,54peaks were separated and37of them were identified in the essential components extracted from C.clematidea,in which the most abundant component was methyl hexadecanoate(30.40%)[49].The essential oils from the whole plants ofC.thalictrifolia,as a traditional Tibetan medicine,were analyzed by GC–MS,and45of60separated components were identified by comparing their mass spectra,in which the main principles were palmitic acid(43.5%)and linolic acid(18.3%)[50].In the essential oils extracted from the fresh and dried roots of C.cordifolioidea,63compounds were identified by GC–MS analysis,indicating that linolic acid(21.9%),retene(11.4%),pentadecane(7.4%), methyl9,12,15-octadecatrienoate(6.8%)and heneicosyl-cyclopentane(3.8%)were the main components[51]. PolysaccharidesLarge-molecule components in Codonopsis species were also studied.A water-soluble polysaccharide with a molecular mass of1.19104Da was obtained from the roots of C.pilosula and its structure investigation revealed that this polysaccharide had a backbone consisting of (1?3)-linked-b-D-galactopyranosyl,(1?2,3)-linked-a-D-galactopyranosyl and(1?3)-linked-b-D-rhamnopyr-anosyl residues and were branched with two glycosyl123Codonocerebroside A nceolata Roots[41] 1243-Oxo-a-ionol-b-D-glucopyranoside C.clematidea Aerial parts[10] 1251,6-Hexanediol,3,4-bis(4-hydroxy-3-methoxyphenyl) C.clematidea Aerial parts[10] 126Sweroside C.nervosa Whole plants[40]residues composed of a-L-arabinose-(1?5)-a-L-arabi-nose,whose C-1linked residues at the O-2position of galactosyl along the main chain in the ratio of1:1:2:1:1[52].Another polysaccharide with a molecular mass of 7.49104Da was isolated from C.pilosula and its com-ponents were galactose,arabinose and rhamnose in themolar ratio of1.13:1.12:1.Its main chain was shown to be (1?3)-linked-b-GalpNAc,(1?3)-linked-a-Rhap and (1?2,3)-b-Galp[53].Furthermore,a pectic polysac-charide with a molecular mass of1.459105Da was at first isolated from C.pilosula,and its structural analysis revealed that this polysaccharide is composed of rhamnose, arabinose,galactose and galacturonic acid in the molar ratio of0.25:0.12:0.13:bined with chemical and spectroscopic analyses,its structure was proposed to be 1,4-linked-a-D-GalpA and1,4-linked-a-D-GalpA6Me interspersed with rare1,2-linked-b-L-Rhap,1,2,6-linked-a-D-Galp and terminal a-L-Arap[54].BioactivitiesAlthough there is information on the uses of many Codonopsis species in traditional medicine,only bioactiv-ity studies on C.pilosula and nceolata have been reported frequently,which proved their importance as medicinal plants.Bioactivity studies on other Codonopsis species such as C.clematidea and C.cordifolioidea were scarce.The studies generally referred to the bioactive effects of aqueous,methanol and ethanol extracts,as well as their further purified fractions,flavones,saponins and polysaccharides.Codonopsis pilosulaAnti-tumor activityThe polysaccharide from C.pilosula(10l g/mL)was able to inhibit the activities of human gastric adenocarcinoma cells and hepatoma carcinoma cells[55].A pectic poly-saccharide(50,100,200and400l g/mL)exhibited marked cytotoxicity to human lung adenocarcinoma A549cells,in a dose-dependent manner[54].Anti-diabetic activityAfter mice were orally administered the polysaccharide from C.pilosula for a week,Fu et al.[56]found that three different doses of polysaccharide(100,200and300mg/ kg/day)could effectively decrease fasting blood glucose and insulin in serum,enhance superoxide dismutase(SOD)activity and reduce the content of malondialdehyde(MDA) in serum.It was therefore considered to possess a signifi-cant hypoglycemic effect in diabetic mice by improving insulin resistance.He et al.[57]showed that the aqueous extract of the roots of C.pilosula(equal to4.5g raw material/kg/day)might retard the progression of diabetesby reducing the blood glucose level and preventing the increase of aldose reductase activity in streptozotocin-induced diabetic mice after3days of oral administration.Anti-aging activityXu et al.[58]found that after mice were orally administrated the polysaccharide from C.pilosula for8weeks,the poly-saccharide(50and150mg/kg/day)was able to increase the thymus index and spleen index as well as the activities of SOD in serum and liver,glutathione peroxidase and nitric oxide synthase particularly in kidney,while decreasing MDA in serum and liver and lipofuscin in brain.Its post-ponement of senility might be related to raising immunity, eliminating free radicals and anti-lipoperoxidation. Effects on gastric mucosaLiu et al.[59,60]found that the water-soluble fraction from the roots of C.pilosula(equal to10g raw material/ kg)had a significant protective effect on gastric mucosal damage caused by alcohol,0.6N HCl and0.2N NaOH, and suggested that the pharmacological mechanism was related to the synthesis and/or release of prostaglandins in gastric mucosa.To date,Song et al.[61]found that lob-etyolin at the oral dose of1.5mg/kg had an effect on decreasing the ulcer index and the level of gastrin and increasing the level of6-keto-prostaglandin F1a in rats with gastric ulcer induced by ethanol,and suggested that lobetyolin played a protective role in gastric mucosa injury. Effects on blood systemAqueous extracts of C.pilosula(500l g/mL)potently inhibited erythrocyte hemolysis[62].In addition,after ischemia–reperfusion injury rats received8mg/100g body weight of a solution of saponins via intraperitoneal injec-tion,the results showed that the increase in SOD levels was accompanied by decreases in MDA,serum creatinine and blood urea nitrogen levels;bcl-2mRNA and protein levels were raised in transplanted kidneys from treated animals, while bax mRNA and protein levels were reduced.The apoptosis index was significantly decreased in transplanted kidneys from treated animals relative to untreated controls. These results clearly demonstrated protective effects on ischemia–reperfusion injury after kidney transplantation, which might be explained by decreasing lipid peroxidation and inhibition of apoptosis[63].Effects on immunityZhang and Wang[64,65]found that6days of oral administration of the polysaccharide from C.pilosula (800mg/kg/day)had effects on immunosuppressed mice induced by cyclophosphamide,including increasing the thymus and spleen index and the phagocytic activity of peritoneal macrophages and recovering the activity of a-naphthyl-acetate esterase in peripheral lymphocytes.In an immunological study in vitro,a water-soluble polysaccha-ride(50,100and200l g/mL)could stimulate concanavalin A-or lipopolysaccharide(LPS)-induced lymphocyte pro-liferation in a dose-dependent manner[52].In addition,the methanol extract of C.pilosula(1mg/mL)inhibited inducible nitric oxide synthase and protein oxidation in LPS-stimulated murine RAW264.7macrophage cells[66].Effects on nervous systemTotal alkaloids(30l g/mL)caused a significant enhancement of nerve growth factor-induced neurite outgrowth in PC12 cells as well as an increase in the phosphorylation of mitogen-activated protein kinase[67].Moreover,Pan et al.[68]orally administered alkaloids from C.pilosula(1mg/kg/day)to mice for4days after they suffered from amnesia by scopol-amine,and found that the alkaloids were effective against the decrease in acetylcholine.Other chemicals,saponins from Codonopsis Radix,were reported to have protective effects on the damage to astrocytes induced by hypoxia/hypoglycaemia and reoxygenation,and were able to inhibit the necrosis of astrocytes at three different concentrations(5.2,52and 520l g/mL)[69].Additionally,the polysaccharide from C. pilosula(1.1mmol/mL)also had marked protective effect on neural stem cell injury induced by sodium thiosulphate[70]. Other bioactivitiesThe extract of C.pilosula(20,40and60l g/mL)signifi-cantly attenuated angiotensin II(AngII)-induced insulin-like growth factor II receptor(IGFIIR)promoter activity.C.pilosula also reversed Ca2?influx,mitochondrial outer-membrane permeability and apoptosis increased by AngII plus Leu27-IGFII which was applied to enhance the AngII effect.Molecular markers in the IGFIIR apoptotic pathway and IGFIIR-Gaq association were down-regulated by C. pilosula.However,p-BadSer136and Bcl-2were increased. The results suggested that C.pilosula could suppress the AngII plus Leu27-IGFII-induced IGFII/IGFIIR pathway in myocardial cells[71].Codonopsis lanceolataAntioxidant activityThe water-soluble fraction and the n-butanol-soluble frac-tion of ethanol extract of nceolata showed。

常用生物医学英文期刊刊名缩写与全称对照表序号期刊缩写名称期刊名称中译刊名原版刊号1AMA Agricultural Mechanizationin Asia亚洲农业机械化655D582J. AOAC. Int.AOAC. International Journal 国家分析化学家协会志652B043Acta Cytol.Acta Cytologica细胞学报591C06 4Acta Hort.Acta Horticultural园艺学报670LB535Agric. Equipment Int.Agricultural & EquipmentInternational世界农业与设备650C596Agric. Engin. Abs.Agricultural EngineeringAbstracts农业工程文摘654C037Agric. Met.Agricultural Meteorlolgy农业气象学652LB02 8Agric. Res.Agricultural Research农业研究650C76 9Agric. Syst.Agricultural Systems农业系统研究650C7610Agric. Water Mgt.Agricultural WaterManagement农业用水管理654LB5211Agriculture Ecosystems andEnvironment农业生态系统与环境715LB5212Agron. J.Agronomy Journal农学杂志650B02 13Amer. J. Bot.American Journal of Botany美国植物学杂志588B01 14Amer.J. Phys.American Journal of Physics美国物理学杂志530B06 15Amer. J. physiol.American Journal ofPhysiology美国生理学杂志595B0116Amer. J. Vet.Res.American Journal ofVeterinary Research美国兽医研究杂志693B0117Amer. Speech American Speech美国语410B03 18Am.Potato J.American Potato Journal美国马铃薯杂志660B01 19Analyt. Biochem.Analytical Biochemistry分析生物化学582B09 20Analyt. Chem.Analytical Chemistry分析化学546BO1 21Anim. Breed. Abs.Animal Breeding Abstracts动物育种文摘690C01 22Anim. Feed.Sci.T ech.Animal Feed Sicence &Technology家畜饲料科学与技术831LB0123Anim. Genet.Animal Geneties动物遗传学591C79 24Anim. Sci.Animal Science动物科学690C51 25Ann. Appl.Biol.Annals of Applied Biology应用植物学纪事581C01 26Ann. Bot.Annals of Botany植物学纪事588C0127Ann. Enomol. Soc. Am.Annals of the EntomologicalSociety of America美国昆虫学会纪事592B0128Ann. Rev. Biochem.Annual Review ofBiochemistry生物化学年鉴582B0529Ann. Rev. Entomol.Annual Review of Entomology昆虫学年鉴592B02 30Ann. Rev. Genet.Annual Review of Genetics遗传学年鉴581B1331Ann. Rev. Microbiol.Annual Review ofMicrobiology微生物学年鉴586B0232Ann. Rev. Pharm. Tox.Annual Review ofPharmacology and Toxiology药理学与毒物学年鉴633B0333Ann.Rev.Physiol.Annual Review of Physiology生理学年鉴595B0334Ann.Rev.Phyto.Annual Review ofPhytopathology植物病理学年鉴588B0935Ann.Rev.Plant.Physiol.Mol.Bio.Annual Review of PlantPhysiology and PlantMolecularBiology植物生理学与植物分子生物学年鉴588B0236Antomo. Engin.Antomotive Engineering机动车工程870B0237Appl.Environ.microbiol.Applied & EnvironmentalMicrobiology应用与环境微生物学586B0138App. Anim. Behav. Sci.Applied Animal BehaviourScience应用动物行为科学591LB6239Appl.Radiat.Isotop.Applied Radiation andIsotopes应用放射和同位素538C0140Arch. Environ. Health Archives of EnvironmentalHealth环境卫生纪要612B0541Arch.Virol.Archives of Virology病毒学文献586LE0142Aust.J.Agri.Res.Australian Journal ofAgricultural Research澳大利亚农业研究杂志650UA0143Aust.J.Bot.Australian Journal of Botany 澳大利亚植物学杂志588UA0144Aust.J.Soil.Res.Australian Journal of SoilResearch澳大利亚土壤研究652UA5345Aust.J. Plant.Physiol.Australian Journl of PlantPhysiology澳大利亚植物生理学杂志588UA0246Auto.Eng.Automotive Engineer汽车工程师873C76 47Avian.Dis Avian Diseases禽类疾病693B56 48Avian.Pathol.Avian Pathology禽类病理学693C63 49Biochem.Genet.BiochemicalGenetics生化遗传学581B60 50Biochem.Biochemistry生物化学582B0151Biochim.Biophys.Acta.(BBA)Biochimeca et BiophysicaActa (C)(BBA) :Molecuar CellResearch生物化学与生物物理学报:分子细胞研究582LB0652Biochim.Biophys.Acta.(BBA)Biochimica et BiophysicaActa (L)(BBA):Lipids & lipidMetabolismd生物化学与生物物理学报:类脂及其代谢582LB0653Biochim.Biophys.Acta.(BBA)Biochimica et BiophysicaActa (CR)(BBA):Reviews onCancer生物化学与生物物理学报:癌症评论582LB0654Biochim.Biophys.Acta.(BBA)Biochimica et BiophysicaActa:Gene Structure and生物化学与生物物理学报：基因结构582LB06Expression(N)(BBA)及其表述55 B.A.Biological Abstracts生物学文摘580B0256Biol.Fertil.Soil.Biology & Fertility of Soils 土壤生物学与土壤肥力652E0557Biol.Reprod.Biology of Reproduction生殖生物学580B15 58Biometrics生物统计学581B11 59Biom.Biometrika生物统计学581C07 60Biosci.Bioscience生物科学580B5161Biosci.Biotech.Biochem.Bioscience Biotechnology andBiochemistry生物科学、生物技术和生物化学652D5162Biosysems.Eng.Biosystems Engineering（Journal of AgriculturalEngineering Research）生物系统工程（原名：农业工程研究杂志）654C0163Biotech.Histochem.Biotechnic andHistochemistry生物工艺学与组织化学580B0964Biotech.Genet.Eng.Rev.Biotechnology and GeneticEngineering Reviews生物技术与遗传工程评论582C10565Biotech.Bioeng.Biotechology &Bioengineering生物技术与生物工程学580B1266Brit.J.Nutr.British Journal of Nutrition英国营养学杂志612C03 67Brit.Poult.Sci.British Poultry Science英国家禽科学690C7168Can.J.Anim.Sci.Canadian Journal of AnimalScience加拿大动物科学杂志690NA0169Can.J.Plant.Sci.Canadian Journal of PlantScience加拿大植物科学杂志650NA0270Can.J.Soil.Sci.Canadian Journal of SoilScience加拿大土壤科学杂志652NA0171Can.Vet.J.Canadian Veterinary Journal加拿大兽医杂志693NA52 72Cell Cell细胞581B17 73Cell Bio.Int.Cell Biology International国际细胞生物学581C68 74Cell.Tech.Cell T echnology 细胞工学581D72 75Cell.Immun.Cellular Immunlogy细胞免疫学581B18 76Cereal.Chem.Cereal Chemistry谷类化学582B11 77 C.A.Chemical Abstracts化学文摘540B01-A 78Chromosoma染色体581E0179Clin.Immun.Clinical Immunology临床免疫学631B0980College English大学英语410B04/doc/d210679737.html,munications in Soil Science & Plant Analysis土壤科学与植物分析通讯652B68/doc/d210679737.html,parative Biochemistry &Physiology,PartA:Comparative Physiolog比较生物化学与生理学,A辑:比较生理学582C0283Compute.Meth.Program.Biomed Compute Methods and Programs 生物医学的电~611LB51in Biomedicine子计算机方法与程序84Control.Instrum.Control and Instrumentation控制与仪表使用737C02 85Crop.Physiol.Abs.Crop Physiology Abstracts作物生理文摘652C04 86Crop.Sci.Crop Science农作物科学660B06 87Cryobiol.Cryobiology低温生物学581B59 88Curr.Microbiol.Current Microbiology当代微生物学586E05 89Current Problems inObstertrics,Geynecdogy &Fertility当前产科学，妇科学和生育问题646B0690Cytogenetics and GenomeResearch细胞遗传学与基因组研究581LD5391Cytologia细胞学581D0592Derwen.Biotech.Abs.Derwent BiotechnologyAbstracts生物技术文摘582C1193Develop.Development发育581C10 94Dev.Bio.Developmental Biology发育生物学580B05 95Diesel.Progr.Diesel Progress柴油机进展784B0296ELT J.ELT Journal(English LanguageTeaching Journal)英语教学杂志410C0297East.Europ.Econ.Eastern European Economics东欧经济学270B100 98Ecol.Entomol.Ecological Entomology生态昆虫学592C03 99Ecol.Monogr Ecological Monographs生态学专论581B01 100Ecol.Ecology生态学581B02,序号期刊缩写名称期刊名称中译刊名原版刊号101E lec.World.Electrical World电世界721B03102E ndorinol.Endocrinology内分泌学638B08103E ndocr.Rev.Endocrine Reviews内分泌评论638B37104E ng.Today.English Today今日英语410C104105E ntom.Entomophaga 食虫类昆虫杂志658F01106E nviron.Exp.Bot.Environmental &Experimental Botany环境与实验植物学588C06107E nviron.Entomol.EnvironmentalEntomology环境昆虫学592B68108Euphytical国际育种学660LB01109E ur.J.Plan.Pathol.European Journal ofPlant Pathology欧洲植物病理学杂志588LB03110E ur.J.Soil.Sci.European Journal of Soil 欧洲土壤科学652C68Science杂志111E xp.Bio.Med.Experimental Biology andMedicine实验生物学与实验医学会会报580B07112E xp.Cell.Res.Experimental CellResearch实验细胞研究581B03113E xp.Parasitol.ExperimentalParasitology实验寄生虫学585B01114E xp.Mol.Pathol.Experimental andMolecular Pathology实验与分子病理学631B06115F eed.Inter.Feed International国际饲料831B70 116F eedstuff Feedstuff饲料831B02 117F ert.Steril.Fertility & Sterility 生育与不孕613B02 118F ield.Crop.Abs.Field Crop Abstracts农作物文摘660C02 119F ield.Crop.Res.Field Crops Research大田作物研究660LB57 120F ood.Chem.Food Chemistry食品化学830C69 121F ood.Sci.Tech.Abs.Food Science andTechnology Abstracts食品科学与技术文摘830C06122F ood.Tech.Food Technology食品工艺学830B03 123G ene Gene基因581LB56 124G ene.Genet.Systics.Genes & Genetic Systics遗传学杂志581D01 125G enet.Genetics遗传学581B05 126Genome基因组581NA51 127G ent.Res.Gentical Research遗传学研究581C02 128Gleanings in Bee Culture养峰集刊690B02 129G rass.Forage.Sci.Grass & Forage Science 牧场与饲料科学690C60130H elminthol.Abs.HelminthologicalAbstracts:"Animal andHuman."蠕虫学文摘:"动物与人类"585C01131Heredity遗传581C05132H istochem.Cell.Bio.Histochemistry and CellBiology组织化学与细胞生物学581E04133H ort.Sci.Hort Science园艺科学670B100 134H ortic.Abs.Horticultural Abstracts园艺学文摘670C01 135Hrereditas遗传581KB01 136H uman.Rep.Human Reproduction人类生殖613C59 137Hybridoma杂交水稻631B89 138I mmunol.Immunology免疫学631C06 139I mmunol.T oday Immunology Today今日免疫学631LB56 140I mpl.Trac.Implement & Tractor农具与拖拉机655B02 141In Vitro离体培养594B53 142I ndex Vet.Index Veterinarius兽医文献索引693C02 143I ndian.J.Genet.Plant.Breed.I ndian Journal of 印度遗传学与660HA01Genetics & PlantBreeding作物育种杂志144I nfec.Immun.Infection and Immunity遗传与免疫631B63 145I nt.J.Plant.Sci.International Journal ofPlant Science国际植物科学杂志588B03146I nt.J.Paras.International Journalfor Parasitlogy国际寄生虫学杂志585C05147I nt.J.Food.Sci.T ech.International Journal ofFood Science andTechnology国际食品科学与技术杂志830C05148I nt.J.Syst.Sci.International Journal of Systems Science国际系统科学杂志737C52149I nt.Pest.Control International PestControl国际病虫害防治658C01150I ntervirol.Intervirology国际病毒学586LD01 151I rr.Drinage Abs.Irrigation and Drinage Abstracts灌溉与排水文摘654C01152J.Agr.Food Chem.Journal of Agricultural& Food Chemistry农业化学与食品化学杂志652B03153J.Anat.Journal of Anatomy解剖学杂志594C01 154J.Anim.Sci.Journal of AnimalScience家畜科学杂志690B03155J.Appl.Ecol.Journal of AppliedEcology应用生态学杂志581C08156J.Appl.Microbiol.Journal of AppliedMicrobiology应用微生物学杂志586C02157J.Appl.Appl.Meteor.Journal of Applied andApplied Meteorology气候学与应用气象学杂志564B06158J.Bact.Journal of Bacteriology细菌学杂志586B04 159J.Cell Sci.Journal of Cell Science细胞科学杂志581C60160J.Cereal Sci.Journal of CerealScience谷物科学杂志660C68/doc/d210679737.html,c.Journal of ChemicalEducation化学教育杂志540B53162J.Chromatogr.Sci.Journal ofChromatographic Science色谱科学杂志546B05163J.Dairy Res.Journal of DairyResearch乳品研究杂志834C01164J.Dairy Sci.Journal of Dairy Science乳品科学杂志834B01 165J.Econ.Ent.Journal of EconomicEntomology经济昆虫学杂志592B06166J.Environ.Qual.Journal of EnvironmentalQuality环境质量杂志650B06167J.Exper.Bot.Journal of ExperimentalBotany实验植物学杂志588C02168J.Food Proc.Pres.Journal of FoodProcessing &Preserration食品加工与保鲜杂志830B68169J.Food Sci.Journal of Food Science食品科学杂志830B02 170J.Immunol.Meth.Journal of ImmunologicalMethods免疫法杂志631LB05171J.Insect Ph.Journal of InsectPhysiology昆虫生理学杂志592C05172J.Inver.Pat.Journal of InvertebratePathology无脊椎动物病理学杂志592B05173J.Irrig.Drain.Eng.Journal of Irrigationand Drainage Engineering灌溉与排水工程杂志860B02-07174Journal of Linguistics语言学杂志410C06175J.Microb.Meth.Journal ofMicrobiological Methods微生物学方法杂志586LB55176J.Mol.Bio.Journal of MolecualrBiology分子生物学杂志582C09177Journal of Money. Creditand Banking货币,信贷和银行业务杂志297B77178J.Morphol.Journal of Morphology形态学杂志581B09 179J.Nutr.Journal of Nutrition营养学杂志612B07180J.Nutr.Bichem.Journal of NutritionalBiochemistry营养生物化学杂志612B102181J.Pharmac.Sci.Journal of Pharmaceutical Sciences药物科学杂志633B06182J.Plant Res.Journal of PlantResearch植物研究杂志588D01183J.Reprod.Dev.Journal of Reproductionand Development繁殖与发育杂志690D64184J.Soil Water Cons.Journal of Soil & Water Conservation水土保持杂志652B05185J.Virol.Journal of Virology病毒学杂志586B55 186J.Am.Oil Chem.Soci.Journal of the American Oil Chemists'Society美国油脂化学家会志821B03187J.Am.Soci.Hort.Sci.Journal of the American Society forHorticultural Science美国园艺科学会志670B03188J.Am.Vet.Medi.Associ.Journal of the AmericanVeterinary MedicalAssociation美国兽医学会志693B03189J.Sci.Food Agri.Journal of the Science ofFood & Agriculture食品与农业科学杂志830C01/doc/d210679737.html,.Journal of VeterinaryMedical Education兽医学教育杂志693B82191Landtechnik农业技术655E01192L /doc/d210679737.html,sers in Surgery &Medicine外科与内科激光应用611B100193L ife Sci.Life Sciences生命科学580B128194L iv.Produc.sci.Livestock ProductionScience畜产科学690LB51195M ana.Sci.Management Science管理科学714B63-A 196M eat Sci.Meat Science肉类科学834C59197M etab.Clin.Exp.Metabolism; Clinical &Experimental新陈代谢;临床与实验638B04198MeteorologicalApplications应用气象杂志564C52199M icrobiology Microbiology微生物学586C03200M icrobilogy Immunol.Microbiology&Immunology 微生物学与免疫学586D52。

A fern that hyperaccumulates arsenic(这是题目，百度一下就能找到原文好，原文还有表格，我没有翻译)A hardy, versatile, fast-growing plant helps to remove arsenic from contaminated soilsContamination of soils with arsenic,which is both toxic and carcinogenic, is widespread1. We have discovered that the fern Pteris vittata (brake fern) is extremely efficient in extracting arsenic from soils and translocating it into its above-ground biomass. This plant —which, to our knowledge, is the first known arsenic hyperaccumulator as well as the first fern found to function as a hyperaccumulator— has many attributes that recommend it for use in the remediation of arsenic-contaminated soils.We found brake fern growing on a site in Central Florida contaminated with chromated copper arsenate (Fig. 1a). We analysed the fronds of plants growing at the site for total arsenic by graphite furnace atomic absorption spectroscopy. Of 14 plant species studied, only brake fern contained large amounts of arsenic (As;3,280–4,980 p.p.m.). We collected additional samples of the plant and soil from the contaminated site (18.8–1,603 p.p.m. As) and from an uncontaminated site (0.47–7.56 p.p.m. As). Brake fern extracted arsenic efficiently from these soils into its fronds: plants growing in the contaminated site contained 1,442–7,526p.p.m. Arsenic and those from the uncontaminated site contained 11.8–64.0 p.p.m. These values are much higher than those typical for plants growing in normal soil, which contain less than 3.6 p.p.m. of arsenic3.As well as being tolerant of soils containing as much as 1,500 p.p.m. arsenic, brake fern can take up large amounts of arsenic into its fronds in a short time (Table 1). Arsenic concentration in fern fronds growing in soil spiked with 1,500 p.p.m. Arsenic increased from 29.4 to 15,861 p.p.m. in two weeks. Furthermore, in the same period, ferns growing in soil containing just 6 p.p.m. arsenic accumulated 755 p.p.m. Of arsenic in their fronds, a 126-fold enrichment. Arsenic concentrations in brake fern roots were less than 303 p.p.m., whereas those in the fronds reached 7,234 p.p.m.Addition of 100 p.p.m. Arsenic significantly stimulated fern growth, resulting in a 40% increase in biomass compared with the control (data not shown).After 20 weeks of growth, the plant was extracted using a solution of 1:1 methanol:water to speciate arsenic with high-performance liquid chromatography–inductively coupled plasma mass spectrometry. Almostall arsenic was present as relatively toxic inorganic forms, with little detectable organoarsenic species4. The concentration of As(III) was greater in the fronds (47–80%) than in the roots (8.3%), indicating that As(V) was converted to As(III) during translocation from roots to fronds.As well as removing arsenic from soils containing different concentrations of arsenic (Table 1), brake fern also removed arsenic from soils containing different arsenic species (Fig. 1c). Again, up to 93% of the arsenic was concentrated in the fronds. Although both FeAsO4 and AlAsO4 are relatively insoluble in soils1, brake fern hyperaccumulated arsenic derived from these compounds into its fronds (136–315 p.p.m.)at levels 3–6 times greater than soil arsenic.Brake fern is mesophytic and is widely cultivated and naturalized in many areas with a mild climate. In the United States, it grows in the southeast and in southern California5. The fern is versatile and hardy, and prefers sunny (unusual for a fern) and alkaline environments (where arsenic is more available). It has considerable biomass, and is fast growing, easy to propagate,and perennial.We believe this is the first report of significant arsenic hyperaccumulationby an unmanipulated plant. Brake fern has great potential to remediate arsenic-contaminated soils cheaply and could also aid studies of arsenic uptake, translocation, speciation, distribution and detoxification in plants. *Soil and Water Science Department, University ofFlorida, Gainesville, Florida 32611-0290, USAe-mail: lqma@†Cooperative Extension Service, University ofGeorgia, Terrell County, PO Box 271, Dawson,Georgia 31742, USA‡Department of Chemistry & SoutheastEnvironmental Research Center, FloridaInternational University, Miami, Florida 33199,1. Nriagu, J. O. (ed.) Arsenic in the Environment Part 1: Cyclingand Characterization (Wiley, New York, 1994).2. Brooks, R. R. (ed.) Plants that Hyperaccumulate Heavy Metals (Cambridge Univ. Press, 1998).3. Kabata-Pendias, A. & Pendias, H. in Trace Elements in Soils and Plants 203–209 (CRC, Boca Raton, 1991).4. Koch, I., Wang, L., Ollson, C. A., Cullen, W. R. & Reimer, K. J. Envir. Sci. Technol. 34, 22–26 (2000).5. Jones, D. L. Encyclopaedia of Ferns (Lothian, Melbourne, 1987).积累砷的蕨类植物耐寒，多功能，生长快速的植物，有助于从污染土壤去除砷有毒和致癌的土壤砷污染是非常广泛的。

Animal(2012),6:10,pp1620–1626&The Animal Consortium2012doi:10.1017/S1751731112000481The effect of chitooligosaccharide supplementation on intestinal morphology,selected microbial populations,volatile fatty acid concentrations and immune gene expression in the weaned pig A.M.Walsh,T.Sweeney,B.Bahar,B.Flynn and J.V.O’Doherty-School of Agriculture,Food Science and Veterinary Medicine,University College Dublin,Lyons Research Farm,Newcastle,Co.Dublin,Ireland(Received24March2011;Accepted29January2012;First published online2March2012)An experiment(complete randomised design)was conducted to investigate the effects of supplementing different molecular weights (MW)of chitooligosaccharide(COS)on intestinal morphology,selected microbial populations,volatile fatty acid(VFA)concentrations and the immune status of the weaned pig.A total of28piglets(24days of age,9.1kg(6s.d.0.80)live weight)were assignedto one of four dietary treatments for8days and then sacrificed.The treatments were(1)control diet(0ppm COS),(2)control diet plus5to10kDa COS,(3)control diet plus10to50kDa COS and(4)control diet plus50to100kDa COS.The COS was included in dietary treatments at a rate of250mg/kg.Tissue samples were taken from the duodenum,jejunum and ileum for morphological measurements.Digesta samples were taken from the proximal colon to measure lactobacilli and Escherichia coli populations and digesta samples were taken from the caecum and proximal colon for VFA analysis.Gene expression levels for specific cytokines were investigated in colonic tissue of the pig.Supplementation of different MW of COS had no significant effect on pig performance during the post-weaning period(days0to8;P.0.05).The inclusion of COS at all MW in the diet significantly reduced faecal scores compared with the control treatment(P,0.01).Pigs fed the10to50kDa COS had a higher villous height(P,0.05)and villous height:crypt depth ratio(P,0.05)in the duodenum and the jejunum compared with the control treatment.Pigs fed the5to10kDa COS had a lower lactobacilli population(P,0.05)and E.coli population(P,0.05)in the colon compared with the control group.Pigs offered the5to10kDa COS had significantly lower levels of acetic acid and valeric acid compared with the control group(P,0.05). The inclusion of different MW of COS had no significant effect on the expression of the cytokines tumour necrosis factor-a,Interleukin (IL)-6,IL-8and IL-10in the gastro-intestinal tract of the weaned pig.The current results indicate that a lower MW of5to10kDa COS possessed an antibacterial activity,while the higher MW of10to50kDa was optimum for enhancing the intestinal structure. Keywords:chitooligosaccharide,pig,microbiology,intestinal morphologyImplicationOur results indicate that the inclusion of chitooligosaccharides (COSs)in piglet diets may moderate several gut health para-meters that contribute to some of the common problems that occur after weaning in the absence of in-feed antibiotics.It was observed that COSs with a molecular weight(MW)of5to 10kDa were more effective in reducing Escherichia coli populations while a MW of10to50kDa enhanced the intestinal structure.IntroductionThe weaning period imposes profound social and environ-mental stresses on the piglet such as removal from the sow,change in diet and mixing of piglets from different litters. Numerous studies have reported that there is a reduction in villous height(villous atrophy)and an increase in crypt depth (crypt hyperplasia)after weaning,which leads to increased susceptibility to intestinal gut dysfunction(Spreeuwenberg et al.,2001;Pierce et al.,2006).The post-weaning period is characterised by a reduction in feed intake,poor growth rates,diarrhoea and an increased risk of disease(Lalles et al., 2007).These negative effects on piglet growth during the weaning period were managed by growth-promoting anti-biotics.However,the European Union placed a total ban on the use of in-feed antibiotic growth promoters on the1st January2006due to public concerns regarding bacterial resistant and human health issues. Chitooligosaccharides(COS)may be a potential viable alternative to traditional antimicrobials in animal production.-E-mail:john.vodoherty@ucd.ie 1620Chitosan is a natural biopolymer derived by alkaline deacety-lation of chitin,which is the principal component of protective cuticles of crustaceans such as crabs,shrimps,prawns,lobsters and cell walls of some fungi such as aspergillus(Qin et al., 2006).Both chitin and chitosan are biopolymers composed of glucosamine and N-acetylated glucosamine(2-acetylamino-2-deoxy-D-glucopyranose)units linked by b(1to4)glycosidic bonds(Koide,1998).Low molecular weight(MW)COS is a water-soluble derivative of chitosan due to shorter chain lengths(Kim and Rajapakse,2005).Recently,both chitosan and its derivatives have generated considerable interest due to their biological activities,including antimicrobial,antitumour, immunoenhancing effects and the acceleration of wound healing(No et al.,2002;Liu et al.,2006)There is considerable variation in the literature on the biological properties of COS (Jeon et al.,2001;Liu et al.,2006).Most of this variation is partly due to the widely different MW used across studies.It is hypothesised that the biological properties of COS may be influenced by its MW and COS will enhance selected indices of health in weaned piglets.Material and methodsAll procedures described in this experiment were conducted under an experimental licence from the Irish Department of Health in accordance with the cruelty to Animals Act1876 and the European Communities(Amendments of the Cruelty to Animals Act1976)Regulations.Experimental dietsThe experiment was designed as a complete randomised block design and comprised four dietary treatments.Thedietary treatments were as follows:(1)control diet(0ppm COS),(2)control diet plus5to10kDa COS,(3)control diet plus10to50kDa COS and(4)control diet plus50to100kDa COS.The COS was sourced from Kitto Life Co.Ltd(Kyungki-do,Seoul,Korea)and was supplemented in the experimental diets at a concentration of250ppm.The diets were fed for 8days ad libitium,after which time the pigs were humanely sacrificed.The diets were formulated to have similar diges-tible energy(16MJ/kg)and standardised ileal digestible (SID)lysine(14g/kg)contents.All amino acids requirements were met relative to SID lysine(National Research Council, 1998).The ingredient composition and chemical analysis of the dietary treatments are presented in Table1.Animals and managementA total of28piglets(progeny of large white3(large white3landrace sows))were selected from a commercial pig unit at24days of age.The piglets had a weaning weight of9.1kg(s.d.50.80)and were blocked on the basis of litter of origin and live weight(n57).The piglets were individu-ally housed in fully slated pens(1.7m31.2m).They were individually fed and had ad libitum access to feed and water. The house temperature was thermostatically controlled at 308C throughout the experiment.This study was not a growth performance study but some performance data were recorded.The piglets were weighed at the beginning of the experiment(day0)and at the end of the experiment(day8). Food was available up to thefinal weighing and all remaining food was weighed back for the purpose of cal-culating feed efficiency.Pigs were observed for clinical signs of diarrhoea and a scoring system was applied to indicate the presence and severity of this as described by Pierce et al. (2006).Faeces scores were assigned daily for individual pigs from day0and continued until day8.The following faeces scoring system was used:15hard faeces,25slightly soft faeces in the pen,35soft,partially formed faeces,45loose, semi-liquid faeces and55watery,mucous-like faeces.Gut morphological analysisThe piglets were humanely sacrificed on day8by a lethal injection of Euthatal(pentobarbitone sodium BP–Merial Animal Ltd,Sandringham House,Essex,UK)at a rate of1ml/ 1.4kg BW.On removal of the digestive tract,sections of the duodenum(10cm from the stomach),the jejunum(60cm from stomach)and the ileum(15cm from caecum)were excised andfixed in10%phosphate-buffered formalin.The preserved segments were prepared using standard paraffin-embedding techniques.The samples were sectioned at5m m Table1Composition and chemical analysis of experimental diets (as-fed basis)Items Starter diet* Ingredient(g/kg)Whey permeate125.0 Wheat444.2 Soya bean meal142.5 Whey protein isolate130.0 Full-fat soybean80.0 Soya oil65.0 Vitamins and minerals 5.0 Lysine HCL 4.5 DL-methionine 1.6L-threonine 2.2 Analysis(g/kg,unless otherwise stated)DM892.5 CP(N36.25)224.2 GE(MJ/kg)18.2 Ash43.7 NDF110.3 Lysine-16.5 Methionine and cysteine-9.9 Threonine-10.7 Tryptophan- 2.5 Calcium-8.0 Phosphorous- 6.0 DM5dry matter;GE5gross energy.Starter diet provided(mg/kg completed diet):Cu,175;Fe,140;Mn,47;Zn, 120;I,0.6;Se,0.3;retinol,1.8;cholecalciferol,0.025;alpha-tocopherol,67; phytylmenaquinone,4;cyanocobalamin,0.01;riboflavin,2;nicotinic acid,12; pantothenic acid,10;choline chloride,250;thiamine,2;pyridoxine,0.015.*COS was included in dietary treatments T2–T4at a rate of250mg/kg.-Calculated for tabulated nutritional composition(Sauvant et al.,2004).Chitooligosaccharide in piglet diets1621thickness and stained with haemotoxylin and eosin(Pierce et al.,2006).Villous height and crypt depth were measured on the stained sections(43objective)using a light micro-scopefitted with an image analyser(Image Pro Plus,Media Cybernetics,Buckinghamshire,UK).Measurements of15well oriented and intact villi and crypts were taken for each seg-ment.Villous height was measured from the crypt–villous junction to the tip.Crypt depth was measured from the crypt–villous junction to the base.Results were expressed as the mean villous height or crypt depth in micrometres. Intestinal microfloraFor microbial analysis,digesta samples(,1061g)were aseptically recovered from the proximal colon of each pig immediately post slaughter.Digesta samples were stored in sterile containers(Sarstedt,Wexford,Ireland),placed on ice and transported to the laboratory within2h.A1.0g sample was removed from the digesta sample,serially diluted (1:10)in9.0ml aliquots of maximum recovery diluents (Oxoid,Basingstoke,UK)and spread plated(0.1ml aliquots) onto selective agars,as follows:Lactobacillus spp.were isolated on de Man,Rogosa and Sharp(MRS)agar(Oxoid) with an overnight(18to24h)incubation at378C in an atmosphere enriched with5%CO2,as recommended by the manufacturers(Oxoid).The Escherichia coli species were isolated on MacConkey agar(Oxoid)following aerobic incubation at378C for18to24h(O’Doherty et al.,2010). Target colonies of Lactobacilli and E.coli were identified by Gram stains and colony morphology(Salanitro et al.,1977). The API50CHL(BioMerieux,Biomerieux,Craponne,France) kit was used to confirm suspect Lactobacilli spp.Suspect E. coli colonies were confirmed with API20E(BioMerieux, France).This API system identifies the suspect colonies by measuring their ability to produce cytochrome oxidase. Typical colonies of each bacteria on each agar were counted, log transformed and the numbers of bacteria were expressed per gram of digesta after being serially diluted.Volatile fatty acid(VFA)analysisSamples of digesta from individual pigs were taken from the caecum and the proximal colon to measure the VFA concentration and molar proportions of VFAs.The VFA con-centrations in the digesta were determined using gas liquid chromatography according to the method described by Pierce et al.(2007).A1-g sample was diluted with distilled water (2.53weight of sample)and centrifuged at14003g for4min(Sorvall GLC–2B laboratory centrifuge,Dupont, Wilmington,DE,USA).Then,1ml of the subsequent super-natant and1m l of internal standard(0.5g3-methyl-n-valeric acid in1l of0.15mol/l oxalic acid)were mixed with3ml of distilled water.Following centrifugation to remove the precipitate,the sample wasfiltered through Whatman 0.45m m polyethersulphone membranefilters into a chromato-graphic sample vial.A1-m l sample was injected into a model 3800Varian gas chromatograph with a25m30.53mm i.d. megabore column(coating CP-Wax58(FFAP)–CB(no. CP7614))(Varian,Middelburg,the Netherlands).RNA extraction and complementary DNA(cDNA)synthesis Tissue samples were collected from the mesenteric side of the colon,rinsed with ice-cold sterile phosphate-buffered saline(Oxoid)and stripped of overlying smooth muscle cells. Approximately1to2g of the porcine colon tissue was cut into small pieces and placed in tubes containing15ml of RNAlater(Applied Biosystems,Foster City,CA,USA)and immediately stored at2208C pending RNA extraction.Total RNA was extracted from colon tissue samples(25mg)using a GenElute Mammalian Total RNA Miniprep Kit(RTN70, Sigma-Aldrich,St Louis,MO,USA)according to the manu-facturer’s instructions.To eliminate possible genomic DNA contamination,total RNA samples were subjected to DNAse I(AMPD1,Sigma-Aldrich)treatment according to the man-ufacturer’s protocol.Then RNA purification was performed using a phenol–chloroform extraction method(Chomczynski and Sacchi,2006).The total RNA was quantified using a NanoDrop-ND1000Spectrophotometer(Thermo Fisher Scien-tific,Wilmington,DE,USA)and the purity was assessed by determining the ratio of the absorbance at260and280nm. All total RNA samples had260/280nm ratios above1.8.In addition,RNA integrity was verified by visualisation of the18 and28S ribosomal RNA bands stained with ethidium bromide after gel electrophoresis on1.2%agarose gels(Egel,Invitro-gen Inc.,Carlsbad,CA,USA).Total RNA(1m g)was reverse transcribed(RT)using the RevertAid H minusfirst strand cDNA synthesis kit(Fermentas GmbH,St Leon-Rot,Germany)with oligo dT primers.Thefinal RT product was adjusted to a volume of120m l using nuclease-free water.Real-time quantitative PCRAll primers for the selected cytokines,genes such as Inter-leukin-1a(IL-1a),IL-6,IL-10,tumour necrosis factor(TNF-a) and the reference genes b-actin(ACTB),b2-microglobin (B2M),glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and peptidylprolyl isomerise A(PPIA)are presented in Table2. Amplification was carried out in a reaction volume of20m l containing10m l SYBR Green Fast PCR Mastermix(Applied Biosystem),forward and reverse primer mix(1m l),8m l DEPC treated water and1m l of template cDNA.Quantitative real-time PCR was carried out using an ABI PRISM7500Fast sequence detection system for96-well plates(Applied Biosys-tem).The thermal cycling conditions were as follows:an initial denaturation step at958C for10min,40cycles of958C for15s, followed by608C for1min.Dissociation analyses of the PCR product were performed to confirm the specificity of the resulting PCR products.All samples were run in triplicate.The cycle threshold value(C t)is defined as the fractional cycle number at whichfluorescence passes thefixed threshold.The mean C t values of triplicates of each sample were used for calculations.Normalisation of quantitative PCR dataNormalisation of the C t values obtained from real-time PCR was performed by(i)transforming the raw C t values into relative quantities using the formula,relative quantities5 (PCR efficiency)D C t,where D C t is the change in the C t valuesWalsh,Sweeney,Bahar,Flynn and O’Doherty 1622of the sample relative to the highest expression (minimum C t value),(ii)using geNorm,a normalisation factor was obtained from the relative quantities of four most stable housekeeping genes (GAPDH,B2M,ACTB and PPIA)and (iii)the normalised fold change or the relative abundance of each of the target genes was calculated by dividing their relative quantities by the normalisation factor.Statistical analysisThe experimental data were analysed as a randomised block design using the GLM procedure of SAS (2004).The individualpig served as the experimental unit.Food intake was inclu-ded as a covariate in the model for villous height,crypt depth and villous height to crypt depth ratio in the digestive tract.The microbial counts were log transformed.The data were checked for normality using the Proc Univariate function of SAS.The means were separated using the Tukey–Kramer Test.Probability values of ,0.05were used as the criterion of statistical significance.All results are presented in the tables as least square means 6standard error of the means (s.e.).ResultsPerformance and faecal scoringThe average faecal scores of the pigs are presented in Table 3.The supplementation of different MW of COS had no significant effect on the growth performance of the pig during the 8-day experimental period (P .0.05).However,the inclusion of COS at all MW in the diet significantly reduced faecal scores com-pared with the control treatment (P ,0.01).MicrobiologyThe effect of COS supplementation at different MW on selected microbial populations in the colon of the pig is shown in Table 3.Pigs offered diets containing 5to 10kDa COS had a lower E.coli number compared with the control (P ,0.05)and the 50to 100kDa COS (P ,0.05)treatments.The 10to 50kDa treatment had a neumerically lower E.coli number compared with the control group (P 50.09).Pigs offered diets containing 5to 10kDa COS had a significantly lower population of lacto-bacilli in the colon compared with the control group (P ,0.05)and the 50to 100kDa COS diet (P ,0.01).Pigs offered 50to 100kDa COS had a higher lactobacilli number than pigs offered 10to 50kDa COS (P ,0.05).The supplementation of different MW of COS had no significant dietary effect on the lacto-bacilli :E.coli ratio in the colon of the pig.Table 2Porcine-specific primers used for real-time PCR 1.Forward primer sequence (50-30)Gene 2.Reverse primer sequence (50-30)T m (8C)IL-6 1.AGACAAAGCCACCACCCCTAA59.82.CTCGTTCTGTGACTGCAGCAGCTTATC 62.7IL-8 1.TGCACTTACTCTTGCCAGAGAACTG 61.92.CAAACTGGCTGTTGCCTTCTT 61.7IL-10 1.GCCTTCGGCCCAGTGAA 57.62.AGAGACCCGGTCAGCAACAA 59.4TNF-a 1.TGGCCCCTTGAGCATCA55.22.CGGGCTTATCTGAGGTTTGAGA 60.3GAPDH 1.CAGCAATGCCTCCTGTACCA 62.22.ACGATGCCGAAGTTGTCATG 62.1B2M 1.CGGAAAGCCAAATTACCTGAAC 59.02.TCTCCCCGTTTTTCAGCAAAT 60.0ACTB 1.CAAATGCTTCTAGGCGGACTGT 59.02.TCTCATTTTCTGCGCAAGTTAGG 60.0PPIA1.CGGGTCCTGGCATCTTGT58.02.TGGCAGTGCAAATGAAAAACTG56.5IL 5interleukin;TNF 5tumour necrosis factor;GAPDH 5glyceraldehyde-3-phosphate dehydrogenase;B2M 5b 2-microglobin;ACTB 5genes b -actin;PPIA 5peptidylprolyl isomerise A.Primers were designed using Primer Express TM software and were synthesisedby MWG Biotech (Milton Keynes,UK).Table 3Effect of COS supplementation at different MW on faecal scoring,selected microbial populations in the proximal colon and the total VFA concentration and the proportions of VFAs in the caecum of the weaned pig (least square means and s.e.;n 57)Dietary treatmentsControl 5to 10kDa 10to 50kDa50to 100kDas.e.SignificanceFaeces scoring Days 0to 84.06b 3.31a 3.44a 3.38a 0.124**Proximal colonic bacterial population (log cfu/g of digesta)Escherichia coli5.94b 4.34a 4.71a 5.81b 0.477*Lactobacilli spp.7.39bc 6.24a 6.56ab 7.56c 0.347*VFA concentrations in the caecum Total VFA (mmol/g of digesta)95.8770.25103.00116.7913.006ns Acetic acid 67.36b 45.77a 70.30b 79.20b 8.543*Propionic acid 19.8416.5123.3927.05 3.616ns Isobutyric acid 0.770.490.810.790.157ns Butyric acid 6.45 6.14 6.817.51 1.575ns Isovaleric acid 0.67b 0.45a 0.68b 0.83b 0.092*Valeric acid0.790.881.011.410.282nsCOS 5chitooligosaccharide;MW 5molecular weight;VFA 5volatile fatty acid.Probability of significance:*P ,0.05;**P ,0.01;ns,P ,0.05.Means with the same superscript alphabets within rows are not significantly different (P .0.05).Chitooligosaccharide in piglet diets1623Volatile fatty acidsThe effects of COS supplementation at different MW on the VFA concentrations in the caecum are shown in Table3.The supplementation of different MW of COS had a significant effect on the concentrations of acetic acid(P,0.05)and isovaleric acid(P,0.05)in the caecum.Pigs fed5to10kDa COS had lower levels of acetic acid and isovaleric acid compared with the control(P,0.05),10to50kDa COS (P,0.05)and50to100kDa COS(P,005).There was no significant effect of MW on VFA concentrations(P.0.05)in the proximal colon(data not shown).Gut morphologyThe effects of varying COS MW on villous height,crypt depth and the villous height:crypt depth ratios in the gastro-intestinal tract are shown in Table4.Pigs fed the10to 50kDa COS had a higher villous height in the duodenum and the jejunum compared with the control group(P,0.05), 5to10kDa COS(P,0.01)and50to100kDa COS diets (P,0.05).There was no effect of dietary treatment on crypt depth in the duodenum(P.0.05).Pigs offered the10to 50kDa COS had a higher villous height:crypt ratio in the duodenum and the jejunum compared with the control group(P,0.05)and the5to10kDa COS diet(P,0.01).Cytokine gene expression analysisThe effects of COS supplementation on the immune response in colon tissues of the pig are shown in Table5.The supplementation of different MW of COS had no significant effect on the expression of the cytokines TNF-a,IL-6,IL-8 and IL-10(P.0.05)in the gastro-intestinal tract of the pig. DiscussionThe hypothesis of the current experiment is that the biolo-gical properties of COS may be influenced by its MW and COS will enhance selected indices of health in weaned pig-lets.It was demonstrated in the current study that the lower MW of5to10kDa possessed antibacterial activity while the higher MW of10to50kDa was optimum for enhancing intestinal structure.Dietary supplementation of COS at the low MW of5to 10kDa decreased both lactobacilli and E.coli counts,while the10to50kDa COS numerically decreased E.coli popula-tions in the colon of the pig.In a study by Liu et al.(2008), COS supplementation at different concentrations reduced E. coli concentrations in the caecum of the weanling pig.E.coli is considered to be one of the most important causes of post-weaning diarrhoea in weaned pigs;therefore,a reduction inTable4Effect of COS supplementation at different MW on villous height,crypt depth and the villous height:crypt depth ratio in the gastro-intestinal tract of the weaned pig(least square means and s.e.)Dietary treatments Control5to10kDa10to50kDa50to100kDa s.e.Significance Covariate(intake) Villous height(m m)Duodenum284.0a256.0a326.3b266.2a17.38*ns Jejunum271.6a270.7a316.5b260.8a16.15*ns Ileum239.8268.3251.5242.915.07ns ns Crypt depth(m m)Duodenum305.7330.2280.1311.718.98ns ns Jejunum294.1298.4281.6268.420.93ns ns Ileum207.5242.8228.2239.811.29ns ns Villous:crypt depth ratioDuodenum 1.0a0.8a 1.2b0.9a0.08*ns Jejunum0.9a0.9a 1.2b 1.0ab0.06*ns Ileum 1.2 1.1 1.1 1.00.06ns nsCOS5chitooligosaccharide;MW5molecular weight.Probability of significance:*P,0.05;**P,0.01;ns,P,0.05.Means with the same superscript alphabets within rows are not significantly different(P.0.05).Table5Effect of COS supplementation at different MW on the immune response in unchallenged proximal colon tissues(leastsquare means of fold change in normalised relative gene expression with their s.e.;n57animals)Dietary treatments Control5to10kDa10to50kDa50to100kDa s.e.SignificanceColonTNF-a0.3660.3530.3760.3620.0568nsIL-60.2480.3590.3180.3220.0645nsIL-80.3850.5440.3700.3580.0797nsIL-100.3640.3420.3110.3070.0614ns COS5chitooligosaccharide;MW5molecular weight;TNF5tumour necrosis factor;IL5interleukin.Probability of significance:*P,0.05;**P,0.01;ns,P,0.05.Walsh,Sweeney,Bahar,Flynn and O’Doherty1624E.coli populations may reduce the incidence of diarrhoea in post-weaned pigs(Fairbrother et al.,2005).Although many species of E.coli are commensal,high levels of specific E.coli (like ETEC)will increase the risk of disease.Unfortunately, ETEC numbers were not measured in the current study.In the current study,the faecal score was decreased in pigs fed the COS diets compared with the control.These results suggest that the supplementation of the5to10kDa and10 to50kDa COS reduces E.coli populations in the colon, resulting in a lower faecal score in the post-weaning period. The50to100kDa COS led to a reduced diarrhoea score but no reduction in E.coli populations;therefore,this MW of COS may be working as a bulking agent to affect the faecal score.The50to100kDa COS may retard the rate of passage through the intestine and may have the ability to absorb water.In the current study,it was demonstrated that supple-mentation of5to10kDa COS had the strongest antimicrobial effect against both lactobacilli and E.coli.This is in agreement with other studies in which low MW COS(5to10kDa)were shown to possess strong antibacterial properties compared with higher MW COS and the antibacterial properties of COS increased at a low MW of,5kDa against Gram-negative such as E.coli(Zheng and Zhu,2003;Kittur et al.,2005).In a study by Liu et al.(2010),COS supplementation decreased E.coli populations compared with the control in the caecum of weaned pigs,while Jeon et al.(2001)observed a anti-microbial effect of COS against Gram-positive bacteria such as Lactobacilli under in-vitro conditions.To explain COS antibacterial activity,two mechanisms have been proposed.Thefirst mechanism is that the posi-tively charged COS reacts with negatively charged molecules at the microbial cell surface,thereby altering cell perme-ability(Chung and Chen,2008).Therefore,COS may interact with the membrane of the cell to alter cell permeability. However,as evident from the current study,this activity may differ with varying MW as the50to100kDa group had no inhibitive effect on the selected microbial populations,while the MWs of5to10kDa and10to50kDa COS had the strongest inhibitive effect.The other antibacterial mechan-ism is the binding of COS with DNA to inhibit RNA synthesis (Liu et al.,2004).It has been proposed that COS penetrates the nuclei of the bacteria and interferes with RNA and pro-tein synthesis.It is noteworthy that all the COS samples used in the current study were soluble in aqueous solutions.Kim and Rajapakse(2005)found that COS with a MW of .30kDa were not effective as antibacterial agents due to their poor solubility in aqueous solutions at a neutral pH. Volatile fatty acids are the major end products of bacterial metabolism in the large intestine(Macfarlane and Macfarlane, 2003).Both protein and carbohydrate fermentation contribute to the production of acetic acid;however,branched-chain fatty acids such as isovaleric acid are produced from protein fermentation(Mackie et al.,1998).In the current study,the5 to10kDa group had the lowest selected microbial populations while also reducing isovaleric acid and acetic acid concen-trations in the caecum.The shift in the production of the fermentation end products is reflected in the reduction of the selected microbial populations.The quantity of VFA produced depends on the amount and composition of the substrate and on the type of microbes present in the large intestine (van Beers-Schreurs et al.,1998).Reduced VFA concentrations indicate that lower amounts of substrate were fermented as a result of a lower microbial activity in the caecum(Htoo et al.,2007).Villous height is generally reduced and crypt depth is increased,which may explain the increased occurrence of diarrhoea and reduced growth after weaning(Pluske et al., 1996).The inclusion of10to50kDa COS in the present study was found to increase the villous height and villous:crypt depth ratio in the duodenum and also in the jejunum com-pared with the control group.Very little data have been published on the effects of COS MW on gut morphology in weaned piglets;thus,the exact mechanism for the increase in villous height and villous:crypt depth ratio is unclear.It may be hypothesised that low MW COS has the potential to promote intestinal morphology through cell proliferation. The COS has been shown to influence colonic cell prolifera-tion,crypt depth and crypt circumference in mice(Torzsas et al.,1996).A study carried out by Liu et al.(2008),on different con-centrations of COS,demonstrated that200mg/kg of COS increased villous height and villous:crypt ratio in the jeju-num and ileum(Liu et al.,2008).The possible explanation for this improved intestinal structure was that COS is com-posed of N-acetyl glucosamine(Kim and Rajapakse,2005), which may bind to certain types of bacteria and possibly interfere with their adhesion to the gut tissue of host animals (Ofek et al.,2003;Liu et al.,2008).This result is in agree-ment with Moura˜o et al.(2006),who reported that an increase in villi length in the ileum of weaned rabbits was correlated to a lower intestinal microflora.A decrease in bacteria load has been shown to increase the proliferation of epithelial cells,which leads to an improved intestinal mor-phology and increased villous height(Moura˜o et al.,2006). In the present study,in pigs fed the lower MW of5to10kDa COS,a strong antimicrobial effect on both Lactobacilli and E.coli populations was observed,with no effect on villous structure,while the higher MW of10to50kDa resulted in a reduction in E.coli numbers in comparison with the control and was optimum for improving villous integrity.There were no effects of COS supplementation in colon tissue on any of the cytokines analysed.This overall lack of an effect on these inflammatory cytokines implies that COS inclusion in the diet had no effects on immune gene expression of the pigs.Mori et al.(1997)also demonstrated that chitin and its derivatives do not stimulate the production of IL-6,IL-1and TNF-a byfibroblasts.In our study,no dif-ferences were observed on growth performance between days0and8post-weaning.In conclusion,MW is an important factor to consider when investigating the biological properties of COS.On the basis of the current study,the lower MW of5to10kDa possessed antibacterial activity while the higher MW of10to50kDaChitooligosaccharide in piglet diets1625。

Journal of Molecular Catalysis B:Enzymatic 57(2009)262–269Contents lists available at ScienceDirectJournal of Molecular Catalysis B:Enzymaticj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /m o l c a tbRole of the support surface on the loading and the activity of Pseudomonas fluorescens lipase used for biodiesel synthesisAndrea Salis ∗,Mani Shankar Bhattacharyya,Maura Monduzzi,Vincenzo SolinasDipartimento di Scienze Chimiche,Universitàdi Cagliari -CSGI,Cittadella Universitaria,S.S.554bivio Sestu,09042Monserrato (CA),Italya r t i c l e i n f o Article history:Received 25July 2008Received in revised form 23September 2008Accepted 25September 2008Available online 10October 2008Keywords:Support surface LipaseAdsorption Loading Biodiesela b s t r a c tThe present work investigates the influence of the support surface on the loading and the enzymatic activity of the immobilized Pseudomonas fluorescens lipase.Different porous materials,polypropylene (Accurel),polymethacrylate (Sepabeads EC-EP),silica (SBA-15and surface modified SBA-15),and an organosilicate (MSE),were used as supports.The immobilized biocatalysts were compared towards sun-flower oil ethanolysis for the sustainable production of biodiesel.Since the supports have very different structural (ordered hexagonal and disordered)and textural features (surface area,pore size,and total pore volume),in order to consider only the effect of the support surface,experiments were performed at low surface coverage.The different functional groups occurring on the support surface allowed either physical (Accurel,MSE,and SBA-15)or chemical adsorption (Sepabeads EC-EP and SBA-15–R-CHO).The surface-modified SBA-15(SBA-15–R-CHO)allowed the highest loading.The lipase immobilized on the MSE was the most active biocatalyst.However,in terms of catalytic efficiency (activity/loading)the lipase immobilized on the SBA-15,the support that allowed the lowest loading,was the most efficient.©2008Elsevier B.V.All rights reserved.1.IntroductionAlmost 25years ago Zaks and Klibanov firstly found that enzymes,in particular lipases,could work in non-aqueous media [1],even though with a catalytic activity usually lower by compar-ison with that in conventional water media.Considerable efforts have been made in order to decrease the activity gap between aqueous and non-aqueous media.Biocatalyst engineering aims to develop different enzyme formulations –soluble or insoluble –able to improve enzyme performance towards specific applications [2].Among different approaches,enzyme immobilization on porous materials seems to be very effective,because they are able to spread enzyme molecules on their high surface areas.This is of funda-mental importance in non-aqueous media since enzyme powders tend to aggregate;thus only a small fraction of enzyme molecules,those present on the aggregate surface,can work [2].The mate-rials used as supports generally affect enzyme performance.Both enzyme loading and activity in non-aqueous media depend on the morphological features (surface area and pore size)and the surface nature of the support [3].The surface area can be fully used only if pore size is larger than enzyme size.Surface coverage is an impor-∗Corresponding author.Tel.:+390706754362;fax:+390706754388.E-mail address:asalis@unica.it (A.Salis).tant parameter that indicates how much of the available support surface is used by the immobilized enzyme.High surface coverage may result in a high activity per support mass unit,nevertheless,since surface area increases with pore size decreasing,limitations due to substrate diffusion inside the pores may occur.The surface nature of the support can affect the enzyme activity through both direct and indirect effects.The former are due to the type,strength,and orientation of enzyme–support interactions.The latter are due to the substrate/product interaction with the support,thus affect-ing substrate/product partitioning between the medium and the enzyme substrate-binding site.More importantly,the partitioning of water between the enzyme and the medium due to the support hydrophilic/hydrophobic character should be considered [3].Lipases (EC 3.1.1.3)are surface-active enzymes whose natural function is triglyceride hydrolysis.They belong to the large struc-tural family (␣/␤-hydrolases)that comprises a wide variety of enzymes (proteases,esterases,etc.)whose activities rely mainly on a catalytic triad usually formed by Ser,His,and Asp residues.Bacte-rial lipases have been classified into eight families on the basis of the amino acid sequences and some fundamental biological properties [4].Lipases display a common feature that is a high hydrophobic surface in proximity of the active site that,in water solution,is buried by an amphiphilic amino acid chain (the lid)[5].The first step of the catalytic path involves a substantial conformational change of the lipase architecture (lid opening)that leads to the adsorption of the lipase at the oil/water interface through the hydrophobic1381-1177/$–see front matter ©2008Elsevier B.V.All rights reserved.doi:10.1016/j.molcatb.2008.09.015A.Salis et al./Journal of Molecular Catalysis B:Enzymatic57(2009)262–269263surface.Thus,lipases prefer water-insoluble substrates and display high affinities towards hydrophobic surfaces.Lipases are among the most used enzymes in biotechnology because of their high versa-tility mainly in non-aqueous media[6].Among these applications biodiesel synthesis is receiving great attention as demonstrated by the high number of recent papers in thefield[7–31].Biodiesel is a mixture of fatty acid methyl(or ethyl)esters that is cur-rently used as a substitute of diesel fuel coming from petrol. Current production processes are energy consuming and produce unwanted by-products(i.e.soaps)that complicate the purification steps[32,33].Instead,the production of biodiesel through bio-catalysis is a sustainable method that does not produce any waste. Moreover the process is carried out at low temperature(i.e.30◦C) and atmospheric pressure[34].The present work is aimed to investigate the role of the nature of the support surface on the loading and the activity of Pseudomonas fluorescens lipase.The immobilized biocatalysts were compared towards the ethanolysis of sunflower oil for biodiesel production. P.fluorescens lipase was chosen since it seems to be one of the best enzymes for this application[31,35].The materials–namely:SBA-15(pure silica),SBA-15–R-CHO(surface-modified SBA-15),MSE (organosilicate),Accurel MP1004(polypropylene)and Sepabeads EC-EP(polymethacrylate carrying surface epoxy groups)–were used as supports for enzyme immobilization.All these materials carry different surface functional groups that can affect the type of enzyme–support interactions and thus the catalytic activity.As it will be shown below,the morphological features of the used mate-rials are very different,therefore,a low enzyme surface coverage was used to focus on the effect of the surface functional groups only.Consequently a small fraction of the available surface area, likely mainly the external part,was involved so that the effects due to pore size and extent of surface area would be negligible.2.Experimental2.1.ChemicalsLipase(triacylglycerol acyl hydrolase,EC 3.1.1.3)from P.fluorescens(lipase AK)was purchased from Amano enzymes (Japan).Tetraethylorthosilicate(98%),Pluronic copolymer123, 3-aminopropyltrimethoxysilane(97%)and glutaraldehyde(50%) were purchased from Aldrich.Bradford reagent and bovine serum albumin(98%)were from Sigma.Buffer salts,Na2HPO4(99%)and NaH2PO4(99%),acetonitrile and methylene chloride(HPLC grade) were from Merck.Ethanol(99.9%)was purchased from J.T.Baker. Karl-Fischer solution was purchased from Riedle de Haen.Accurel MP1004polypropylene powder and Sepabeads EC-EP powder were kind gifts of Membrana GmbH Accurel Systems(Obernburg,Ger-many),and Resindion SRL,Mitsubishi Chemical Co.(Milan,Italy) respectively.2.2.Characterization of supportsSBA-15and MSE mesoporous materials were synthesised according to what previously reported[36,37].The surface mod-ification of SBA-15mesoporous silica was carried out in two steps[38]:first the amino function–reaction with aminopropy-ltrimethoxysilane–then the aldehyde function–reaction with glutaraldheyde–were introduced.Upon modification with glu-taraldehyde,the colour of the support changed from white to red.Textural analysis of supports was carried out on a Thermoquest-Sorptomatic1990,by determining the N2adsorption/desorption isotherms at77K.Before analysis,samples were out-gassed overnight at40◦C prior to measurement.The specific surface area,the total pore volume and the pore size distribution were assessed by the Brunauer-Emmett-Teller(BET)[39]and the Barret-Joyner-Halenda(BJH)[40]methods,respectively.2.3.Pseudomonasfluorescens lipase immobilizationEnzyme immobilization was carried out by suspending125mg of the preliminary sieved support powder(120mesh)in10mL of an enzyme solution(5mg/mL)in phosphate buffer(0.1M,pH8).The suspension was kept under gentle stirring at constant temperature (298K),until the equilibrium of the process was reached.The sus-pension was centrifuged and washed twice with5mL of phosphate buffer(0.1M,pH8).The amounts of lipase adsorbed by the different supports were quantified in terms of loading,that can be expressed both as mg protein/g support,L P,and LU/g(1LU=1␮mol/min)sup-port,L A,by determining the protein content or the lipase activity of the enzyme solution at the beginning and at the end of the adsorp-tion process,respectively.These two different ways to express the loading were used since different proteins generally occur in the commercial preparation.Thus,L P measures the behaviour of all the proteins in the immobilising solution,while L A focuses only on the target enzyme,that is the lipase.Determination of protein content was carried out according to the Bradford method[41].A volume of50␮L of the lipase solution was mixed with950␮L of phosphate buffer solution0.1M at pH8 and1mL of the Bradford reagent.After exactly6min,absorbance was read by mean of a Cary50spectrophotometer at =595nm. The blank was obtained by mixing1mL of the buffer solution with 1mL of the Bradford reagent.Protein content was estimated by mean of a calibration curve obtained using BSA(98%)as protein standard.Lipase activity was measured by the pH-stat method,using a 718Stat Titrino equipment from Metrohm(Herisau,Switzerland).A sample of100␮L of lipase solution was added to a gum arabic-stabilised emulsion of tributyrin in distilled water at25◦C.The pH was maintained at7.0by titration with10mM sodium hydroxide solution.The substrate emulsion was prepared by homogenising a mixture of tributyrin(3mL),distilled water(47mL)and an emulsification reagent(10mL)at18,000rpm for1min by an Ultra-Turrax homogeniser.The emulsification reagent was prepared by dissolving gum arabic(6.0g),glycerol(54mL),NaCl(1.79g)and KH2PO4(0.041g)in distilled water(40mL)[42].The enzymatic activity expressed as LU(lipase units)is defined as the amount of enzyme that produces1␮mol of butyric acid per min.2.4.Pre-equilibration of substrates and enzymes at the desired water activityPre-equilibration was carried out by putting for several days the vials containing sunflower oil,ethanol,and the enzyme prepa-rations inside closed vessels containing saturated salt solutions at25◦C.The chosen water activities(a w)were0.113(LiCl),0.328 (MgCl2),0.529(Mg(NO3)2),0.708(SrCl2),and0.973(K2SO4).Water content of the pre-equilibrated reagents was measured through a Karl–Fischer737KF Coulometer from Metrohm(Herisau, Switzerland).The knowledge of the water content of the pre-equilibrated ethanol was necessary to calculate the amount of wet ethanol to be weighed to keep constant the molar ratio between the reagents.2.5.Biocatalytic ethanolysis of sunflower oilA typical substrate mixture was obtained by mixing2g(2.28mmol)of sunflower oil,a suitable amount of ethanol in the molar ratio(alcohol:oil)8:1,both pre-equilibrated at the desired264 A.Salis et al./Journal of Molecular Catalysis B:Enzymatic 57(2009)262–269Fig.1.Functional groups occurring on the surface of the supports used for lipase immobilization.water activity,in 4mL screw-capped vials with Teflon-lined septa.The reactions were carried out at 30◦C and started by adding 125mg of immobilized lipase to the substrates mixture.Reac-tion vials were shaken through a horizontal shaking water bath at 80oscillations min −1.Samples (5␮L)were withdrawn at different times and 40␮L of the internal standard solution (trilaurin in hex-ane 3×10−2M)was added.The resulting mixtures were diluted to the final volume of 1.2mL with hexane and analyzed by HPLC.All reactions were performed at least in duplicate.2.6.HPLC analysisHPLC analysis was performed using a Lichrospher 100RP-8end capped,5␮m column (Merck,Darmstadt,Germany)and moni-tored by an evaporative light scattering detector Sedex 75(Sedere,France).Analyses were carried out at 35◦C,at constant flow of 1.6mL/min with the following solvent gradient.After 2min of running pure acetonitrile,a linear gradient to 20%methylene chlo-ride was achieved over 3min.This mixture was run for 5min when the gradient to pure acetonitrile was achieved over 4min.This was run for one additional minute.A good separation was obtained for triglycerides coming from sunflower oil (five peaks:8.7;9.4;10.1;11.1and 12.0min),trilaurin (7.0min);mono-and di-glycerides (three peaks:4.3;5.0and 5.8min)and the fatty acid ethyl esters (two peaks:2.3and 2.4min).Sunflower oil conversion and ethyl ester yields were calculated according to calibration curves obtained with the internal standard (trilaurin)method.All analyses were performed in triplicate with reproducibility within 3%.3.Results and discussionIn consideration of the different nature of the materials used to immobilize the enzyme,some characterizations had to be pre-liminarily performed.This was done in order to choose the best operational conditions which should emphasize the role of the surface on the immobilization and on the catalytic performance.Particles of the same size,obtained through preliminary sieving (120mesh)were used,then the supports were characterized in terms of surface area and pore size in order to choose a low value of loading.Thus,only the external surface of the mesoporous mate-rials is assumed to be involved in the adsorption of the enzyme.The indirect effect due to water partitioning was minimised comparing the biocatalysts performance at the optimal water activity.3.1.Supports characterizationAll the materials used in the present work (SBA-15,surface mod-ified SBA-15,MSE,Accurel MP1004,and Sepabeads EC-EP)were previously used as supports for enzyme immobilization [38,43–45].By the chemical point of view,SBA-15is an inorganic material since is constituted by pure silica,two are organic polymers (Accurel polypropylene and Sepabeads EC-EP polymethacrylate),and MSE has a hybrid nature being an organosilicate.However it should be remarked that,rather than the chemical composition of the bulk,the functional groups occurring on the surface are important for the present work.As shown in Fig.1,these are silanols for the SBA-15surface,methyl and methylene groups at the Accurel surface,whereas those at the surface of MSE are both silanols and methy-lene groups.Aldehydic groups occur at the surface of functionalized SBA-15and epoxy groups at that of Sepabeads EC-EP.Table 1reports the specific surface area (A BET ),the maximum of the pore size dis-tribution and the total pore volume of the materials obtained by the N 2adsorption/desorption isotherms.3.1.1.Mesoporous materialsSBA-15was first synthesized by Stucky and coworkers [36].It is a silica-based mesoporous structure constituted by cylindrical channels organized with a hexagonal pattern.The surface area,determined by the BET method [39]was 766m 2/g;the total pore volume was 2.14cm 3/g (Table 1).The pore size distribution was mono-modal with a maximum centered at the value of 6.9nm.Table 1Morphological properties of the mesoporous materials determined through N 2adsorption/desorption isotherms.SupportA BET (m 2/g)aMaximum of pore size distribution (nm)bTotal pore volume (cm 3/g)Maximal surface coverage (%)Actual surface coverage (%)SBA-15766 6.9 2.10.530.13Modified SBA-15213 3.60.4 2.1 1.1MSE1400 3.91.50.320.12Accurel polypropylene28Macroporous 0.5168.0Sepabeads EC-EP polymethacrylate862–800.45.22.6a Calculated by the BET method [39].bCalculated by the desorption branch by mean of BJH method [40].A.Salis et al./Journal of Molecular Catalysis B:Enzymatic57(2009)262–269265parison among thefirst twenty amino acid sequence of Pseudomonasfluorescens lipase and other bacterial lipases from subfamilies I1,I2and I3.The SBA-15surface was modified following a previously reported procedure[38].This consists of two steps,where at first the–NH2functional group is introduced(reaction of the silanols with aminopropyltrimethoxysilane),then the reaction with glutaraldehyde introduces the–CHO functional group.The functionalizing procedure alters the SBA-15morphology signifi-cantly.Both pore size,surface area and total pore volume decreased being3.6nm,213m2/g and0.4cm3/g,respectively.The synthesis of MSE organosilicate was reported for thefirst time by Bao et al.[37].The texture of this material is similar to that of SBA-15since it has the same type of uniform cylindri-cal pores arranged through a hexagonal pattern.Differently from SBA-15,MSE silicon atoms are alternatively connected by means of–Si–O–Si–and–Si–CH2–CH2–Si–groups(Fig.1).The presence of the methylene groups confers a partial hydrophobic character to the material.The pore size distribution,displayed a very narrow peak at3.9nm.The surface area was1400m2/g,and the total pore volume was1.45cm3/g(Table1).3.1.2.Macroporous materialsAccurel polypropylene is a commercial support whose charac-terization was previously reported[44].Here it is recalled that both macropores and mesopores occur.The maximum of the pore size distribution is around6␮m for macropores and9.0nm for meso-pores.The specific surface area(A BET)was around28m2/g and the total pore volume was around0.5cm3/g.The Sepabeads EC-EP is a commercial polymeric support(poly-methacrylate)carrying surface epoxy groups.Hence it is able to bind enzymes covalently through the amino groups of lysine residues occurring at the enzyme surface.The polymer had a very wide distribution of pore size(3–50nm).The surface area of this material was86m2/g,and the total pore volume was about 0.4cm3/g(Table1).rmation available in the literature about lipase AK (Pseudomonasfluorescens lipase):an attempt of classification Microbial lipases are generally inactivated by short-chain alco-hols,i.e.methanol or ethanol used for biodiesel synthesis.The transesterification activity of Candida antarctica lipase B decreased when the methanol:oil molar ratio exceeded3:2[46].Previous studies showed that Pseudomonas lipases have high tolerance towards methanol and ethanol used in triglyceride alcoholysis [8,20,35,47].We have recently shown that lipase AK–a com-mercial preparation of P.fluorescens lipase(Pfl)–immobilized on polypropylene was able to work with a8:1(methanol:oil)molar ratio[35].Moreira et al.found that the same lipase,immobilized on silica–PVA composite was still active although a very high(18:1) ethanol:oil molar ratio was used[31].The different behaviour of lipases coming from different microbial sources with respect to the inhibition caused by short-chain alcohols should be searched in the different enzyme structures.Unfortunately the structure of Pflhas not yet been resolved,thus no speculation about struc-ture/activity can be done at the present time.Nevertheless,starting from the information available in the literature a classification of this enzyme may be attempted.This enzyme was characterized by the Amano researchers that isolated the strain No.924—identified as P.fluorescens AK102.A molecular weight of about33kDa was estimated[48].The isoelec-tric point p I=4,the pH stability range4<pH<10,and the optimum pH of activity in the range8<pH<10were determined[48].As additional information the N-terminal sequence of thefirst20 amino acids was reported(ADDYATTRYPIILVHGLTGT)[48].Fig.2reports the comparison among the sequences of thefirst twenty amino acids of Pfland other four bacterial lipases from three different subfamilies.Eighteen of them follow the same sequence homology as those of lipase from Burkolderia cepacia(for-merly Pseudomonas cepacia),15of those from Burkholderia glumae (formerly Chromobacterium viscosum)lipase,9of those from Pseu-domonas aerouginosa lipase,and only1of those from P.fluorescens SIK W1lipase.Thefirst two lipases belong to subfamily I.2,the third to subfam-ily I.1the fourth to subfamily I.3(Table2).The sequence homology of Pfl,although limited to thefirst20amino acids,is very high if compared to those of lipases from Burkholderia cepacia and Burkholderia glumae.Moreover,as reported in Table2,the lipases of the subfamily I.2are characterized by having almost the same number of amino acids(∼320)and a molecular weight of33kDa [4].Hence,on the basis of the available data,that is the molecular weight(33kDa)and thefirst20amino acid sequence homology,it may be hypothesised that Pflbelong to subfamily I.2of microbial lipases.Table2Classification of some bacterial lipases.Sub-family Structure Pdbfile Number of amino acids MW(kDa)Ref.Pseudomonas aeruginosa I.1Yes1EX928530[56] Pseudomonasfluorescens(AK102)–No––33[57] Burkholderia cepacia I.2Yes3LIP32033[5] Burkholderia glumae I.2Yes2ES431933[58] Pseudomonasfluorescens SIK W1I.3No–44950[59]266 A.Salis et al./Journal of Molecular Catalysis B:Enzymatic57(2009)262–269 3.3.Estimation of support surface coverageMorphological features can affect both the immobilization andthe catalytic processes.This is particularly important for meso-porous supports since the pore and the enzyme sizes are similar.On the contrary,Accurel and Sepabeads EC-EP are not affected bythis problem since they have very large pores(Table1).It is known from structural data that Burkholderia cepacialipase(Bcl)is a globular enzyme with approximate dimensions of3nm×4nm×5nm[5].Since Pfland Bcl have the same molecularweight the dimensions of Pflare likely to be similar to those of Bcl.Thus,assuming that Pflis spherical with an enzyme diameter(d e)equal to5nm,A enzyme–the specific surface area of the support,occupied by the immobilized enzyme(m2/g)–can be calculatedaccording to Eq.(1):A enzyme=d e22×n enzyme(1)where (d e/2)2is the surface occupied by a single enzyme molecule and n enzyme is the number of enzyme molecules per gram of support obtained according to Eq.(2):n enzyme=L p1000×MW enzymeN A(2)here L P is the protein loading is(12.4mg/g);MW enzyme is the molec-ular weight of Pfl(33,000g×mol−1),N A is the Avogadro number. This allows to estimate the surface coverage–S cov(%)–of the immo-bilized lipase:S cov(%)=A enzymeA BET×100(3)here A BET is the specific surface area(m2/g)of the supports.The results of the calculations are reported in thefifth column of Table1. The mesoporous supports:MSE,SBA-15,and SBA-15–R-CHO have very large surface areas and thus very low surface coverages,being 0.32%,0.53%,and2.1%,respectively.Accurel and Sepabeads EC-EP have low surface areas(28and86m2/g),thus surface coverages were equal to15.9%and5.2%,respectively.On the basis of these surface coverage values,Pflis expected to be bound mainly on the external surface of the mesoporous sup-ports.Indeed,it is reasonable that during the adsorption process enzyme macromoleculesfirst interact with free external adsorp-tion sites.It can reasonably be assumed that the structure does not affect the loading and the activity of the immobilized lipase.As a consequence,the chemical nature of the support surface could be the main parameter involved in the immobilization and catalysis processes.3.4.Immobilization of Pseudomonasfluorescens lipaseThe Pflwas immobilized on thefive characterized supports. Fig.3reports the comparison of the loadings–L A and L P–obtained from the quantification of the immobilization exper-iments on the different supports.The higher the loading,in particular L A,the higher the enzyme–support affinity.The affin-ity of Pflin terms of both L A(Fig.3a)and L P(Fig.3b)for thefive supports follows the series:SBA-15–R-CHO>Accurel>Sepabeads EC-EP>MSE>SBA-15.Depending on the functional groups located on the support surface,different kind of interactions between the enzyme and the support surface should take place.On the basis of the nature of their surface,the supports SBA-15,Accurel polypropylene,and MSE are expected to interact through physical(electrostatic,hydro-gen bonds,dipole–dipole and hydrophobic)interactions.Accurel polypropylene is the most hydrophobic material whereas thesilica parison among loadings of Pflimmobilized on different supports.(a)L A and(b)L P.Enzyme immobilization was carried out by suspending125mg in10mL of an enzyme solution(5mg/mL)in phosphate buffer(0.1M,pH8).The suspension was kept under gentle stirring at constant temperature(298K),until the equilibrium of the process was reached.based SBA-15is the most hydrophilic(Fig.1).The organosilicate MSE,due to the simultaneous presence of both polar–OH and apo-lar–CH2–groups,has an intermediate hydrophobic/hydrophilic character.As shown in Fig.3a,L A decreases as hydrophilicity increases,confirming the affinity of lipases toward hydrophobic surfaces[44,49–52].The low loading obtained for SBA-15can be explained by its hydrophilic nature and by the surface electrical charges carried by both the support and the enzyme.These charges depend on both the isoelectric points(p I)of the enzyme and of the sup-port and on the pH of the immobilizing solution[38,43].Since Pflhas a p I=4and SBA-15has a p I=3.7[43],both the support and the lipase are expected to be negatively charged at pH8. Hence,unfavourable electrostatic interactions are likely to occur. The immobilization pH was chosen on the basis of the optimal pH for catalytic activity.Indeed,in non-aqueous media the enzyme is expected to maintain the‘memory’of the pH of the immobilising solution.In other words,the enzyme amino acid residues retain the same charges they had in the immobilizing solution[53].The chosen pH should allow for a high activity even though,not nec-essarily,for a high loading.In any case,although the net charge of the enzyme at pH8is negative,there might be some regions of it where positive charges are still present,thus allowing the formation of favourable electrostatic interactions with the SBA-15 surface.In addition,other kinds of forces,such as hydrogen bonds and dipole–dipole interactions should be responsible of the reached loading.The intermediate loading measured for Pflon MSE mesoporous organosilicate agrees with the simultaneous presence of both hydrophilic and hydrophobic groups on MSE surface[54].A.Salis et al./Journal of Molecular Catalysis B:Enzymatic 57(2009)262–269267The groups occurring on the surface of the SBA-15–R-CHO (alde-hydic)and Sepabeads EC-EP (epoxy)can establish covalent bonds with the –NH 2groups of the lysine residues occurring at the enzyme surface.A slightly higher value of L A was reached by the SBA-15–R-CHO compared to Sepabeads EC-EP.A less net distinc-tion was observed in terms of L P ,being the differences within the error bars.On the basis of these results,it can be concluded that,at the investigated loadings,the textural properties (surface area and pore size)do not affect the immobilization process significantly.Indeed a high loading is observed for Accurel,that has the lowest surface area (A BET =28m 2/g).Moreover,comparing L A of physisorbed lipases on mesoporous materials,MSE loading is higher than that measured for SBA-15,although the pore size of the latter (6.9nm)is larger than that of the former (3.9nm).This might confirm that enzymes do not deeply penetrate the pores,and the external surface is mainly involved in the adsorption.Similar considerations can be done if L P instead of L A is considered.3.5.Catalytic measurements3.5.1.Effect of water content on transesterification enzymatic activityThe ethanolysis of sunflower oil was the reaction chosen to evaluate the role of the support.The reactions were carried out in solvent-free conditions,with a molar ratio ethanol:oil =8:1,T =30◦C,atmospheric pressure,and the same mass of immobilized biocatalyst (125mg).In this environment the hydration level of the enzyme is one of the fundamental parameters to be considered,since it strongly affects the enzyme activity [53].Water allows for the internal enzyme flexibility due to polypeptide chain motion,which is needed by the enzyme to be catalytically active.In the case of immobilized enzymes,the partitioning of water among the com-ponents of the system (enzyme,support and reagent mixture)strongly depends on the support hydrophilic/hydrophobic bal-ance.Therefore,to avoid problems related to water partitioning,before starting the reactions,the different components of the sys-tem were pre-equilibrated to fixed values of water activity (a w )[53].Table 3reports the water content of the pre-equilibrated ethanol as determined by Karl-Fischer coulometric measurements.The water content ranged between 5.9%(a w =0.113)and 32%(a w =0.973).After pre-equilibration,the enzyme activity as a func-tion of water activity was determined by adding to the reagents mixture a weighed amount of the pre-equilibrated Pflimmobi-lized on Accurel polypropylene.Fig.4shows the results of this study.At low a w values,Pfldisplayed a low activity;as water activ-ity increased,also enzyme activity increased until a maximum (104␮mol min −1)at a w =0.529was reached.A further increase of a w produced an enzyme activity decrease.Therefore a w =0.529was used in the experiments aimed to compare the effect of the support surface on the biocatalyst performance.Table 3Water content of pre-equilibrated ethanol at different water activities (a w )deter-mined through Karl–Fischer coulometric measurements.Salta w Water content ±S.D.(%)LiCl0.113 5.9±0.4MgCl 2·6H 2O 0.32813±1Mg(NO 3)·6H 2O 0.52919±1SrCl 2·6H 2O 0.70827±2K 2SO 40.97332±2Fig.4.Enzymatic activity of Pflimmobilized on polypropylene vs.water activ-ity (a w ).Sunflower oil (2g)and methanol were mixed in the stoichiometric ratio 1:8.Immobilized Pfl(125mg)was added to the substrates mixture pre-equilibrated at different a w values,and incubated at 30◦C with a constant shaking (80oscillations min −1).parison among immobilized biocatalysts towards biodiesel synthesisThe comparison among the different biocatalytic systems was made by following ethyl esters yield (Fig.5)as a function of time.All the immobilized biocatalysts were active towards sunflower oil ethanolysis.However the different biocatalysts showed differ-ent performance.For instance,considering ethyl esters yields at 8h of reaction time,the mol%values are 91for Pfl-MSE,88for Pfl-Sepabeads EC-EP and 84for the others biocatalysts.The dif-ferent yield curves presented different slopes,and thus different biocatalyst activities.Activity values were calculated considering the initial rate of formation of the ethyl esters.As shown in Fig.6,the series of immobilized biocatalysts activity followed the order:Pfl-MSE >Pfl-SBA-15>Pfl-Sepabeads EC-EP >Pfl-Accurel >Pfl-SBA-15–R-CHO.Remarkable differences are observed in the catalytic activity of Pflimmobilized on the different supports.For instance the Pfl-MSE is almost twice more active than the Pfl-SBA-15–R-CHO.It is evident that the enzymatic activity can be modulated by the different surface–enzyme intermolecular interactions.3.6.Role of support surface oncatalytic efficiencyThe catalytic activities reported in Fig.6were obtained using the same mass of immobilized preparation.But,this does not take into account the different loadings.Thus,in order to compare the direct effects of functional groups of the different supports on enzyme performance the catalytic efficiency (activity/mg of loaded pro-Fig. 5.Time course of enzymatic biodiesel synthesis.Sunflower oil (2g)and methanol were mixed in the stoichiometric ratio 1:8.Immobilized Pfl(125mg)was added to the substrates mixture pre-equilibrated at a w =0.529,and incubated at 30◦C with a constant shaking (80oscillations min −1).。

nitrogen /ˈnaɪtrədʒən/ n. [化学] 氮photosynthetic /fəʊtəʊsɪnˈθetɪk/ adj. [生化] 光合的；光合作用的photosynthesis /ˌfəʊtəʊˈsɪnθəsɪs/ n. 光合作用botany /ˈbɒtəni/ n. 植物学；（特定地区的）植物（生态）botanist /ˈbɒtənɪst/ n. 植物学家lichen /ˈlaɪkən/ n. 地衣；青苔 vt. 使长满地衣fungus /ˈfʌŋɡəs/ n. 真菌，真菌类植物；霉菌；（尤指鱼的）真菌感染algae /ˈældʒiː/ n. 水藻，海藻acquatic /əˈkwɑːtɪk/ adj. 水生的，水栖的；与水生动植物有关的；水的，水上的 n. （尤指适于在水塘或水族馆中生活的）水生植物（或动物）bacterium /bækˈtɪriəm/ n. 细菌organism /ˈɔːrɡənɪzəm/ n. 生物，有机体，（尤指）微生物；有机组织，有机体系rodent /ˈroʊdnt/ n. 啮齿目动物（如老鼠等） adj. （与）啮齿动物（有关）的primate /ˈpraɪmeɪt/ n. 灵长目动物（包括人、猴子等）mammal /ˈmæml/ n. 哺乳动物reptile /ˈreptaɪl; ˈreptl/ n. 爬行动物；（非正式）卑鄙的人predator /ˈpredətər/ n. 捕食性动物； 掠夺者，掠夺成性的人（或组织）prey /preɪ/n. 猎物，捕获物artery /ˈɑːrtəri/ n. 动脉； vein /veɪn/ n. 静脉；（植物的）叶脉fingernail /ˈfɪŋɡərneɪl/ n. 手指甲 scale /skeɪl/ n.鳞，鳞片；鱼鳞状物claw /klɔː/ n. （动物的）爪，爪子 horn /hɔːrn/ n. 角；角质nourish /ˈnɜːrɪʃ/ v. 养育，滋养；培养 clam /klæm/ n. 蛤，蛤蜊crab /kræb/ n. 螃蟹，蟹肉beaver 狸 pond 池塘 puddle 水坑 snail 蜗牛 shrimp 虾hormone 荷尔蒙 intestine 肠 corn 谷物 squash 南瓜bean 豆类植物 nectar花蜜 pollen花粉 hive 蜂巢moss 苔藓 hibernate 冬眠 penguin 企鹅 reef 礁 coral 珊瑚beak 鸟嘴 enzymes 酵母 larvae 幼虫 tadpole 蝌蚪 caterpillar 毛虫grasshopper 蚱蜢 toad 蟾蜍 herbicide 除草剂secretion（n.） secrete（v.）分泌pancreas 胰腺 odor 气味 roe 鱼卵 caviar 鱼子酱 raccoon 浣熊gland 腺体 cricket 蟋蟀。

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Mobile Genetic Elements 2:6, 267-271; November/December, 2012; © 2012 Landes BioscienceLETTER TO THE EDITORLETTER TO THE EDITORGAA repeats were shown to be the most unstable trinucleotide repeats in the pri-mates genome evolution by comparison of orthologous human and chimp loci.2 The instability of the GAA repeat in the first intron of the frataxin gene X25 is particu-larly well studied since it causes an inher-ited disorder, Friedreich ataxia (FRDA).3-6 I n Friedreich ataxia, once the length of the GAA repeat inside the frataxin gene (FXN GAA) reaches a certain threshold, the combined probability of its expan-sions and deletions in progeny of affected parents is about 85%.7 Deletions and con-tractions of the repeat in intergenerational transmissions can reach hundreds of base pairs.7 However, the FXN GAA repeat is much more stable in somatic cells.8 I t is relatively stable in blood, but shows some instability in dorsal root ganglia,9 which is responsible for some of the neurodegen-erative symptoms of Friedreich ataxia.5 GAA repeats were shown to be stable in FRDA fibroblasts cell lines and neuronal stem cells.10The question why the FXN GAA repeat is so much more stable in somatic cells than in intergenerational transmis-sions remains open. Recent studies in FRDA iPSCs that are closer to embryonic cells than somatic cells models, showed expansions of the GAA repeat with 100% probability.10,11 It is intriguing that all cells Complexes between two GAA repeats within DNA introduced into Cos-1 cellsMaria M. KrasilnikovaPennsylvania State University; University Park, PA USAKeywords: replication, GAA repeat, Friedreich ataxia, genome instability, chromatinCorrespondence to: Maria M. Krasilnikova; Email: muk19@ Submitted: 10/08/12; Revised: 12/09/12; Accepted: 12/10/12/10.4161/mge.23194in the iPSC cell lines that were analyzed were synchronously adding about two GAA repeats in each replication.The studies focused on the FXN GAA repeat provided many valuable insights; however, human genome contains many other GAA repeats: the human X chro-mosome, for instance, contains 44 GAA stretches with more than 100 repeats in each. About 30 GAA repeats were detected on the chromosome 4.12 GAA repeats mostly originated from the 3' end of the poly A associated with Alu elements.13I t is not known what makes repeats with the GAA motif most unstable com-pared with other trinucleotide repeats. It is possible that GAA repeats instability is caused by their ability to form non-B DNA structures. In vitro, GAA repeats can form triplexes,14,15 and sticky DNA structures.16 At the same time, hairpins 17 and paral-lel duplexes 18 have also been observed. When transcription is going through a GAA repeat, it can also form an R-loop, a DNA-RNA complex that leaves one of the complementary strands single-stranded.19 However, it is unclear whether these struc-tures indeed form in mammalian cells. If we assume that the instability of the GAA repeat is indeed associated with the struc-ture formation, it is still unclear why the structures would form in early embryo-genesis when the GAA expansion event in Friedriech ataxia is believed to occur,7 and do not form in somatic cells where the GAA repeat was shown to be more stable. I n our recent study, we hypothesize that the differences in chromatin structure are at least partially responsible for the differ-ences in the GAA repeats stability.1The propensity of GAA repeat to form a triplex structure may strongly depend on the structure of chromatin at the repeat and surrounding area.1 Consistent with other studies, we observed that formation of chromatin at an SV40-based plasmid introduced into mammalian cells occurs gradually: 8 h after transfection there are only occasional nucleosomes at the plas-mid, while by 72 h the nucleosome struc-ture is already regular.20 Our analysis of replication stalling at the repeat revealed that the repeat affects replication only in the first replication cycle, when chromatin is still at the formation stage. We believe that replication stalling at GAA is caused by a triplex structure that the GAA repeat adopts during transfection or inside the cell. In the subsequent replication cycles, replication was completely unaffected by the presence of the repeat, which is likely to be due to the inhibition of triplex for-mation by tight chromatin packaging.1Here we show the data that strengthen our previous observations and extend it to one more structure: a complex betweenWe have recently shown that GAA repeats severely impede replication elongation during the first replication cycle of transfected DNA wherein the chromatin is still at the formation stage.1 Here we extend this study by showing that two GAA repeats located within the same plasmid in the direct orientation can form complexes upon transient transfection of mammalian Cos-1 cells. However, these complexes do not form in DNA that went through several replication rounds in mammalian cells. We suggest that formation of such complexes in mammalian genomes can contribute to genomic instability.We studied replication of several SV40-based plasmids that contained two (GAA)57 repeats located at different posi-tions in Cos-1 cells (Figs. 1–3). I n each case, the cells were transiently transfected with 1 μg of each plasmid, and replication intermediates were isolated after about 30 h. This allowed us to observe replica-tion arcs that resulted from the plasmids that replicated for more than two rounds. However, some residual amount of the first replication cycle arcs can also be registered. Replication stalling at GAA repeats only occurs during the first repli-cation cycle of an SV40-based plasmid,1 hence we did not observe it in our system.For each of the plasmids, and for all patterns of restriction digests that we studied, we observed complexes between the two (GAA)57 repeats (indicated by red arrows in each of the figures). The migration of those complexes was differ-ent depending on the digest pattern, so the complexes were at different positions in 2D gel patterns, in agreement with our expectations based on their shapes.We did not observe replication stalling associated with these complexes. When this complex is formed, it migrates signifi-cantly slower on 2D gels; the replication of plasmids that contain such complexes should result in an extra replication arc originating from the complex position. However, the number of molecules that form this complex is significantly lower than the overall number of plasmids, and there may be not enough material to observe their replication.For the situation when the two GAA repeats were located in two different frag-ments upon PvuI digest (Fig. 1), we showed that the spot 3 (Fig. 1D ) (also indicated by an arrow in Fig. 1A ) con-tains both fragments: the spot hybridized to the probes corresponding to either of them (Fig. 1A and B ). However, the com-plex appears at the position that migrates slower than unreplicated plasmid in the second dimension upon AflI I I and ScaI digest when both repeats belong to the same fragment (Fig. 2). We suggest that this fragment contains a loop generated by the interaction between the two GAA repeats (Fig. 2C ) that slows it down in the second dimension, and has little effect on the mobility in the first dimension sinceThe method of two-dimensional elec-trophoresis 22,23 allowed us to analyze the replication progression through a DNA fragment containing two GAA repeats. In this method, the replication intermediates are isolated under non-denaturing condi-tions, digested by a restriction enzyme, and separated on two consecutive gel runs in perpendicular directions. The first direction runs in 0.4% agarose (that separates mostly by mass), and the second direction runs in 1% agarose (that sepa-rates by both mass and shape of a DNA molecule).two GAA stretches. Two GAA repeats has been shown to readily form complexes, such as “sticky DNA,” in vitro,16 but it is not obvious whether it can also form inside the mammalian cells. The sticky DNA requires more than one GAA stretch to form.21 We studied the interaction of two GAA repeats located within the same plas-mid, but since the human genome contains at least several hundreds of long GAA motif repeats,12,13 this structure can theoretically form in the genomic environment as well. However, more experiments are needed to detect its formation in genome.Figure 1. A complex between two (GAA)57 repeats within the same plasmid. Plasmid replication intermediates were isolated from Cos-1 cells 30 h after transient transfection. Intermediates were digested by restriction enzymes indicated in the plasmid maps, and separated by two-dimension-al neutral-neutral agarose gel electrophoresis as described previously.1 The gel was transferred to a nylon membrane and hybridized to one of the probes indicated in the plasmid maps as green lines. A position of the complex at the 2D gel pattern depends on the restriction digest of the replication intermediates (indicated by a red arrow). (A ) Two-dimensional electrophoresis of replication intermediates digested by PvuII that places each repeat within a separate fragment. The membrane was hybridized with probe 1 indicated in (C ). The names of the plasmids GAAGAA, GAACTT, etc., reflect the orientation of the GAA repeats within the plasmid (GAAGAA means that the two GAA stretches are in the direct orientation). (B ) The same membrane was stripped of probe 1, and re-hybridized with probe 2 (C ). (C ) The scheme of the plasmid that was used in the experiment in (A and B ). Two different fragments that resulted from the PvuII digest are shown in red and blue. The positions of GAA repeats are shown in black. They can be in the direct or the reverse orientations in this plasmid. (D ) The scheme of the 2D gel in (A ). Spot 1: unreplicated blue fragment, spot 2: unreplicated red fragment that appears because of the cross-contamination of probe 1 with probe 2 due to their preparation from the same plasmid with a restriction digest. Spot 3: a complex between the red and the blue fragments. Spikes 3-2 and 3-4 may result from double-stranded breaks in the plasmid during transfection that make one of the arms of the complex in spot 3 shorter.it has the same mass as the unreplicated fragment.The complexes formed only when the two GAA stretches were positioned on a plasmid as direct repeats (GAAGAA and CTTCTT in the plasmid names). The inverted repeats GAACTT and CTTGAA did not form complexes as shown in Figures 1 and 2. This is in agreement with the sticky DNA formation in supercoiled plasmids containing two GAA repeats that has been previously shown in vitro.16 In sticky DNA, the two GAA strands that are in the antiparallel orientation, and the CTT strand, form a stable complex stabilized by Mg2+-dependent reverse-Hoogsteen triads. However, the sticky DNA complex fell apart upon heating in the presence of EDTA, which removes the Mg2+ ions necessary for its stability,16 while we did not detect any changes in spot 3 upon heating the intermediates with EDTA (Fig. 3B). We suggest that in our case the complex may be different from the canonical sticky DNA. It may be based on Hoogsteen base pairing where the Mg2+ is not needed and a slightly acidic pH has a stabilizing effect.24 It has been shown that this type of structure forms within long GAA stretches in vitro even at a pH that is close to neutral.15 We also cannot exclude that the complexes are hemicatenated molecules connected at GAA repeats with Watson-Crick pairing.25The complexes between the two GAA repeats persisted only until the plasmids went through one replication round. DpnI restriction enzyme is a frequent-cutter that digests all DNA that contains strands syn-thesized in bacteria: it cleaves DNA that is methylated at GATC by dam methyl-ase, which is only present in bacteria, but not in mammalian cells. Extensive DpnI digest that we performed, cleaved the ini-tial DNA used in transfection, as well as the products of the first replication cycleA complex between the two repeats within the same fragment slow down its progres-sion in the second dimension of the 2D gel. The same plasmid as in the Figure 1 was used in this experiment, however, they were digested with different enzymes. (A) Replication intermediates were digested with AflIII and ScaI, placing both repeats within the same fragment. The complex of two GAA repeats results in a slowly migrating structure that is shown by a red arrow. (B) A map of the digest of the same plasmid as in Figure 1 with restriction enzymes ScaI and AflIII. Here both of the repeats are located within the same fragment shown in blue. (C) The scheme of the 2D gel in . Spots 1 and 2 are the same as in Figure 1D. Spot 3: a looped intermediate that resulted from the interaction of the two GAA repeats.A complex between the two GAA repeats does not form in plasmids that went through more than two replication rounds in mammaliancells. Two-dimensional gels of replication intermediates of a plasmid containing two (GAA)57 repeats were obtained as described in the Figure 1) Two-dimensional gel of replication intermediates digested by AflIII (placing the two repeats at two different fragments). A red arrow indi-cates the position of the complex between the two GAA-containing fragments. (B) The same replication intermediates were incubated at 80°C in the presence of 10 mM EDTA for 10 min; the pattern of the 2D gel did not change. (C) The same intermediates were additionally digested with10 units of DpnI for 2 h prior to loading. The spot at the position indicated by an arrow in Figure 1 A is not present in this picture. An additional spot that appeared in this pattern is likely not a part of the pattern, and is probably due to some contamination. (D) A map of the plasmid that was used inFigure 1A and B. Spot 1, unreplicated blue fragment; spot 2, unreplicated red fragment (which appeared due to contamination of probe 1 with other plasmid sequences). Spot 3, a complex between the blue and the red fragments. A very faint duplicate Y arc from replication of the second fragment originates from spot 2. The spikes originating from spot 3 can be interpreted the same way as11. Ku S, Soragni E, Campau E, Thomas EA, Altun G, Laurent LC, et al. Friedreich’s ataxia induced plu-ripotent stem cells model intergenerational GAA·TTC triplet repeat instability. Cell Stem Cell 2010; 7:631-7; PM I D:21040903; /10.1016/j.stem.2010.09.014.12. Siedlaczck I , Epplen C, Riess O, Epplen JT. Simple repetitive (GAA)n loci in the human genome. Electrophoresis 1993; 14:973-7; PM I D:7907288; /10.1002/elps.11501401155.13. Chauhan C, Dash D, Grover D, Rajamani J, MukerjiM. Origin and instability of GAA repeats: insights fromAlu elements. J Biomol Struct Dyn 2002; 20:253-63;PMID:12354077; /10.1080/07391102.2002.10506841.14. Gacy AM, Goellner GM, Spiro C, Chen X, Gupta G, Bradbury EM, et al. GAA instability in Friedreich’sAtaxia shares a common, DNA-directed and intraal-lelic mechanism with other trinucleotide diseases. MolCell 1998; 1:583-93; PMI D:9660942; http://dx.doi.org/10.1016/S1097-2765(00)80058-1.15. Potaman VN, Oussatcheva EA, Lyubchenko YL, Shlyakhtenko LS, Bidichandani SI, Ashizawa T , et al.Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH. NucleicAcids Res 2004; 32:1224-31; PMID:14978261; http:///10.1093/nar/gkh274.16. Sakamoto N, Chastain PD, Parniewski P , OhshimaK, Pandolfo M, Griffith JD, et al. Sticky DNA: self-association properties of long GAA.TTC repeats inR.R.Y triplex structures from Friedreich’s ataxia. MolCell 1999; 3:465-75; PMID:10230399; http://dx.doi.org/10.1016/S1097-2765(00)80474-8.17. Heidenfelder BL, Makhov AM, Topal MD. Hairpinformation in Friedreich’s ataxia triplet repeat expansion.J Biol Chem 2003; 278:2425-31; PMI D:12441336;/10.1074/jbc.M210643200.18. LeProust EM, Pearson CE, Sinden RR, Gao X. Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich’s ataxia. J MolBiol 2000; 302:1063-80; PM D:11183775; http:///10.1006/jmbi.2000.4073.19. McIvor EI, Polak U, Napierala M. New insights into repeat instability: role of RNA•DNA hybrids. RNABiol 2010; 7:551-8; PMI D:20729633; http://dx.doi.org/10.4161/rna.7.5.12745.20. Chandok GS, Kapoor KK, Brick RM, Sidorova JM, Krasilnikova MM. A distinct first replication cycleof DNA introduced in mammalian cells. NucleicAcids Res 2011; 39:2103-15; PMID:21062817; http:///10.1093/nar/gkq903.21. Sakamoto N, Ohshima K, Montermini L, Pandolfo M, Wells RD. Sticky DNA, a self-associated complexformed at long GAA*TTC repeats in intron 1 of thefrataxin gene, inhibits transcription. J Biol Chem2001; 276:27171-7; PMI D:11340071; http://dx.doi.org/10.1074/jbc.M101879200.22. Krasilnikova MM, Mirkin SM. Analysis of tripletrepeat replication by two-dimensional gel electro-phoresis. Methods Mol Biol 2004; 277:19-28;PMID:15201446.23. Friedman KL, Brewer BJ. Analysis of replicationintermediates by two-dimensional agarose gel elec-trophoresis. Methods Enzymol 1995; 262:613-27; PM I D:8594382; /10.1016/0076-6879(95)62048-6.24. Frank-Kamenetskii MD, Mirkin SM. T riplex DNA structures. Annu Rev Biochem 1995; 64:65-95; PM D:7574496; /10.1146/annurev.bi.64.070195.000433.25. Lucas I, Hyrien O. Hemicatenanes form upon inhibition of DNA replication. Nucleic Acids Res 2000; 28:2187-93; PM D:10773090; /10.1093/nar/28.10.2187.26. McLay DW, Clarke HJ. Remodelling the paternal chromatin at fertilization in mammals. Reproduction 2003; 125:625-33; PM D:12713425; /10.1530/rep.0.1250625.because they contain one strand synthe-sized in bacteria.The replication intermediates digested with DpnI did not contain the spot 3, cor-responding to the complex between two GAA stretches (Fig. 3C ). We suggest that the absence of the complex is due to the chromatin coverage of the plasmid that accompanies replication. This is similar to our observation that GAA repeats only block replication during the first replica-tion round, until the chromatin is formed. The replication blockage that we have pre-viously observed is consistent with forma-tion of a triplex that occurs in transfected DNA only prior to nucleosome cover-age.1 Here, the complex between the two (GAA)57 repeats also occurred only with-out the chromatin structure. The absence of the complex in replicated DNA also shows that the complexes that we observe are not an artifact of the isolation and sub-sequent treatment of our intermediates, since then they would exist in at least some fraction of the replicated DNA as well.The question remains whether the non-B DNA structures can form within GAA repeats in mammalian cells since their formation requires DNA stretches that are not folded in chromatin. A win-dow when these complexes can form during development is the spermatogen-esis when the maturing sperm chromatin changes from nucleosome- to protamine-bound assembly.26 Another opportunity to form complexes comes when the chroma-tin of a sperm and an egg restructure after the fusion of the gamets.26,27 This is asso-ciated with degradation of protamines and nucleosome deposition, as the zygote DNA may lack a compact chromatin structure.28 It should be noted that the expansions in Friedreich ataxia were traced to the early divisions of the zygote.7An opportunity for the complexes to form may also exist in cancer cells. It is known that some regions of their genome are overmethylated and convert in hetero-chromatin, while other regions are under-methylated, which may promote a loose chromatin packaging.29,30It is not clear whether the two-repeats complexes would compete with triplex structure formation within each individ-ual (GAA)57 repeat. It is possible that both of them exist and contribute to overallgenomic instability. However, a separate study is necessary to determine whether these structures indeed have a biological role.Disclosure of Potential Conflicts of InterestNo potential conflicts of interest weredisclosed.Acknowledgments This study was supported by NI H grant GM087472, and research grant from Friedreich’s Ataxia Research Alliance toMMK.References 1. Chandok GS, Patel MP , Mirkin SM, Krasilnikova MM.Effects of Friedreich’s ataxia GAA repeats on DNAreplication in mammalian cells. Nucleic Acids Res2012; 40:3964-74; PM D:22262734; http://dx.doi.org/10.1093/nar/gks021.2. Kelkar YD, Tyekucheva S, Chiaromonte F , MakovaKD. The genome-wide determinants of humanand chimpanzee microsatellite evolution. GenomeRes 2008; 18:30-8; PMI D:18032720; http://dx.doi.org/10.1101/gr.7113408.3. Sharma R, Bhatti S, Gomez M, Clark RM, MurrayC, Ashizawa T, et al. The GAA triplet-repeat sequencein Friedreich ataxia shows a high level of somaticinstability in vivo, with a significant predilection forlarge contractions. Hum Mol Genet 2002; 11:2175-87; PM I D:12189170; /10.1093/hmg/11.18.2175.4. Pandolfo M. The molecular basis of Friedreichataxia. Adv Exp Med Biol 2002; 516:99-118;PM D:12611437; /10.1007/978-1-4615-0117-6_5.5. Pandolfo M. Friedreich ataxia. Arch Neurol 2008;65:1296-303; PM I D:18852343; http://dx.doi.org/10.1001/archneur.65.10.1296.6. Campuzano V, Montermini L, Moltò MD, PianeseL, Cossée M, Cavalcanti F , et al. Friedreich’s ataxia:autosomal recessive disease caused by an intronic GAAtriplet repeat expansion. Science 1996; 271:1423-7; PM ID:8596916; /10.1126/sci-ence.271.5254.1423.7. De Michele G, Cavalcanti F , Criscuolo C, Pianese L,Monticelli A, Filla A, et al. Parental gender, age at birthand expansion length influence GAA repeat intergener-ational instability in the X25 gene: pedigree studies andanalysis of sperm from patients with Friedreich’s ataxia.Hum Mol Genet 1998; 7:1901-6; PMI D:9811933; /10.1093/hmg/7.12.1901.8. De Biase I , Rasmussen A, Monticelli A, Al-MahdawiS, Pook M, Cocozza S, et al. Somatic instability of theexpanded GAA triplet-repeat sequence in Friedreich ataxia progresses throughout life. Genomics 2007;90:1-5; PMID:17498922; /10.1016/j.ygeno.2007.04.001.9. De Biase I , Rasmussen A, Endres D, Al-Mahdawi S,Monticelli A, Cocozza S, et al. Progressive GAA expan-sions in dorsal root ganglia of Friedreich’s ataxia patients.Ann Neurol 2007; 61:55-60; PMID:17262846; http:///10.1002/ana.21052.10. Du J, Campau E, Soragni E, Ku S, Puckett JW,Dervan PB, et al. Role of mismatch repair enzymesin GAA·TTC triplet-repeat expansion in Friedreichataxia induced pluripotent stem cells. J Biol Chem2012; 287:29861-72; PMID:22798143; http://dx.doi.org/10.1074/jbc.M112.391961.29. Watanabe Y, Maekawa M. Methylation of DNAin cancer. Adv Clin Chem 2010; 52:145-67; PM D:21275343; /10.1016/S0065-2423(10)52006-7.30. Kulis M, Esteller M. DNA methylation and cancer.Adv Genet 2010; 70:27-56; PMID:20920744; /10.1016/B978-0-12-380866-0.60002-2.27. Spinaci M, Seren E, Mattioli M. Maternal chromatinremodeling during maturation and after fertilization in mouse oocytes. Mol Reprod Dev 2004; 69:215-21; PM ID:15293223; /10.1002/mrd.20117.28. I mschenetzky M, Puchi M, Gutierrez S, MontecinoM. Sea urchin zygote chromatin exhibit an unfolded nucleosomal array during the first S phase. J Cell Biochem 1995; 59:161-7; PM D:8904310; /10.1002/jcb.240590205.。

中英文对照资料外文翻译文献译文一：柑桔属类胡萝卜素生物合成途径中七个基因拷贝数目及遗传多样性的分析摘要：本文的首要目标是分析类胡萝卜素生物合成相关等位基因在发生变异柑橘属类胡萝卜素组分种间差异的潜在作用；第二个目标是确定这些基因的拷贝数。

本实验应用限制性片段长度多态性（RFLP）和简单序列重复（SSR）标记法对类胡萝卜素生物合成途径中的七个基因进行了分析。

用[R-32P]dCTP标记PSY，PDS，ZDS，LCY-b，LCY-e，HY-b和ZEP cDNA片段作为作探针，使用若干限制性内切酶对来自25种柑桔基因型基因组DNA的限制性片段长度差异进行了分析。

而对于SSR标记，设计两对引物分别扩增LCY-b和HY-b基因的表达序列标签（ESTs）。

在这7个基因中，LCY-b只有1个拷贝，而ZDS存在3个拷贝。

利用RFLP和SSR分析发现基因的遗传多样性与核心分子标记一致。

RFLP和SSR对PSY1，PDS1，LCY-b和LCY-e14个基因的分析结果足以解释这几个主要的商业栽培种的系统树起源。

此外，我们的分析结果表明，不同种类柑橘中类胡萝卜素积累的番茄红素环化酶LCY-b和LCY-e1等位基因存在种间差异。

前人报道PSY，HY-b和ZEP基因与种间类胡萝卜素含量差异密切相关，但本实验发现这些等位基因并不起关键作用。

关键词：柑桔；类胡萝卜素；生物合成基因；基因变异；系统发育前言类胡萝卜素是植物光合组织中普遍存在的一类色素。

在色素蛋白复合体中，它们作为光敏元件进行光合作用，并且防止过强光照强度引起的灼伤，并在园艺作物果实，根，或块茎色泽和营养品质上起着十分重要的作用。

事实上，其中一些微量营养素是维生素A的前体，是人类和动物的饮食必不可少的组成部分。

由于具有抗氧化性，类胡萝卜素在预防慢性疾病也发挥着重要的作用。

类胡萝卜素生物合成途径现在已经明确。

类胡萝卜素通过核酸编码的蛋白酶在质体中合成。

其直接前体是牻牛儿基牻牛儿基焦磷酸（GGPP，该前体同时也是赤霉素，质体醌，叶绿素，维生素K，维生素E的前体）。