The Science of Biology Chapter12

10: The nature of viruses has been apparent only within the last half century, and the first step on this path of discovery was taken by the Russian botanist Dmitri Lvanovsky in 1892 when he studying the tobacco mosaic disease.Viruses are very small entities, ranging in size from 0.02 to 0.3 microns. Unlike the organisms making up the five taxonomic kingdoms of the living world, viruses are acellular, they don’t consist of cells and conduct energy metabolism-they don’t produce ATP and incapable of fermentation , cellular respiration or photosynthesis. As for these, the questions of the viruses origin arise. Do they represent a primitive ‘nearly living’ stage in the evolution of life? Or are they organisms which have lost all cellular components except the nucleus? Could viruses simply be fragments of genetic material derived from cellular organisms? No one really knows the answers to these questions, but we do know that viruses have been around for a long time, and that almost every form of life is susceptible to viral attack.The basic units of a virus consist of nucleic acid surrounded by a capsid or coat, composed of one or at most a few kinds of proteins. These proteins are so assembled as to give the virion a characteristic shape. As they bud through host cell membranes, many animal viruses also acquire a membrane consisting of lipids and proteins, and many bacterial viruses have specialized ‘tails’made of protein. The viral nucleic acid is usually a single molecule and may be composed of either DNA or RNA, but not both. DNA or RNA can be double-stranded or single-stranded.Viruses are obligate intracellular parasite, that is why they must depend upon specific hosts for their reproduction and development. The cells of animals, plants and bacteria can all serve as hosts to viruses. Animal viruses attach to special sites on the plasma membrane of the host cell and are then taken up by endocytosis. A given virus can infect only those cells that have a receptor site for that virus. After the membrane breaks down, the viral protein capsid is broken down by cellular enzymes before the viral nucleic acid, in addition, the viral nucleic acid serves to direct the synthesis of new capsid protein by the protein-synthesizing system of the host, and the capsid combine with new viral nucleic acid spontaneously; and in due course, the new virions are released by the host cell.Plant viruses and bacteriophages must get through a cell wall as well as the host plasma membrane. Infection of a plant usually results from attack by a virion-laden insect vector. The insect uses its proboscis to penetrate the cell wall, and the virions the escape from the insect into the plant. Bacterial viruses are often equipped with tail assemblies that inject the nucleic acid into the host bacterium while the protein coat remains outside. Once inside the host cell, the virus genes takes over the metabolic machinery of the cell and generate their own.Sometimes, viral DNA does not immediately take control of the host metabolism, but insert itself into the host chromosome and present as ‘silent’ provirus until the host cell is exposed to some environmental insult, such as ultraviolet light or radiation.If the viral nucleic acid is RNA, replication needs special enzymes to make the process of RNA-to-RNA synthesis occurs. Some RNA viruses called retroviruses do not carry outRNA-to-RNA transcription. Instead, their RNA is transcribed into DNA is immediately, this reaction is catalyzed by reverse transcriptase, then newly formed DNA is inserted into host DNA and then transcribed into RNA and at last new viruses are produced.After replication and combination, most viruses are released by lysis of the host cell. But in other cases, like that of the retroviruses, viruses are released by extrusion, a process similar tobudding thereby the virus becomes enveloped in a small piece of cell membrane as it moves out of the cell. Lysis result in the destruction of the cell, but extrusion allows the cell to remain alive and continue to produce new viruses for a long period of time.A common way to classify viruses is to separates them first on the nature of the nucleic acid component (DNA or RNA) and then on whether the nucleic acid in the virion is single-ordouble-stranded. Further levels of classification depend on such factors as the overall shape of the virus and the symmetry of the capsid. Most capsid may be categorized as helical, icosahedral and so on. Another level of categorization is based on the presence or absence of membranous envelope around the virion; still further subdivision relies on capsid size and other criteria.18: Within a cell, energy is needed at every stage to drive the reactions that keep life in normal states. On the earth, almost all the energy that fuels life today comes from the sun and is captured in the process of photosynthesis by plants. Most nonphotosynthetic organisms obtain energy by ingesting photosynthetic organisms or others that have themselves ingested photosynthetic organisms, and the energy stored by photosynthesis is usually released through a process known as respiration. In this chapter, we shall discuss these two processes.Photosynthesis is a logical starting point for our discussion of the basic energy transformation of life. In simple terms photosynthesis consists of the reduction of atmospheric CO2 to carbonhydrate by use of light energy, with an associated release of oxygen from water. This reaction can be summarized by the following generalized equation.Like many other physiological processes, photosynthesis consists of a number of sequential steps:①trapping of light energy by chloroplasts; pigments other than chlorophyll(e.g carotenoids)play an accessory role in photosynthesis by transferring energy to chlorophyll a. ②splitting of water and release of high-energy electrons and o2.③electron transfer leading to generation of chemical energy in the form of ATP and the reducing power as NADPH2.④terminal steps involving expenditure of energy of ATP and the reducing power of NADPH2 to fix CO2 molecules, and finally convert this compounds into more complex carbohydrates, such as sucrose, starch, cellulose and so on.Carbon dioxide is an exceedingly energy-poor compound, whereas carbohydrates isenergy-rich. Photosynthesis, then, converts light energy into chemical energy. In chemical terms, the energy is said to be stored by the addition of one more electron-stores energy in the substance being reduced.Although, photosynthesis can occur in any chlorophyll-containing parts of the plant, leaves that expose the greatest area of green tissue to the light are the principal organs of photosynthesis. Through a microscope it can be seen that the outer surfaces of the leaf have a layer of epidermis, which is covered by waxy layer of cuticle. The region between the upper and lower epidermis constitutes the mesophyll portion of the leaf. The cells of mesophyll contain many chloroplasts, which is the organelles that photosynthesis takes place. The CO2 required for photosynthesis can enter through some holes called stomata between the spaces of mesophyll cell. Chloroplast is bounded by two concentric membrane and a third set of internal membranes that form a series of flattened, interconnected sacs known as thylakoids, where chlorophyll moleculesand most of the electron-transport-chain molecules are located. The light reaction, in which light energy is trapped and converted into chemical energy, takes place on the thylakoid membrane. The dark reaction, in which CO2 is reduced to carbohydrates, occurs in the more fluid stroma that surrounds the thylakoid sacs.19: It is evident that the phenomenon of inheritance and variation is of universal important in the living world. To understand it, we must explore how hereditary material expresses itself in new combinations, and whether principles can be formulated about so complex event. This exploration is the study of the branch of biology known as genetics.Some of the basic concepts of heredity grew out of experiments performed by Gregor Mendel in the mid-1800s. Mendel spent most of his lifetime as a monk in an Austrian monastery and during this time he cultivated garden peas, and did a series of experiments to study inheritance in plants. We now know that owing to his elaborate design, unique technique, Mendel formulated two excellent Laws of Inheritance from his breeding experiments on the garden peas: one is the Law of Segregation, the other is the Law of Independent Assortment.The Law of Segregation emphasize on single traits and it can be stated as follows: the inheritance of each individual trait is determined by hereditary factors (genes), each organism possesses two inheritance factors for each character, when gametes are formed, the two factors separate into separated gametes. An offspring formed by the fusion of two gametes therefore receives one factor for each character from each parent. The Law of Independent Assortment involving two or more traits and states that during gametes formation, when two or more genes are involved in a cross, the alleles of one gene are inherited independently of the alleles of another gene. His great achievement laid a solid foundation for the genetics. In order to further understanding of these two laws, some details can be known either:1.Hereditary traits are controlled by discrete units that pass unchanged from generationto generation. For example, the trait white flower seems to disappear in the F1generation, but reappears on the F2 progeny, and that there are no intermediate colors,only red or white.2.Each trait is produced by two hereditary factors. This is a necessary assumption toaccount for the way in which a trait such as flower color appears in successivegenerations in a predictable ratio.3.When two contrasting hereditary factors are present in an organism, such asred-flower color and white-flower color, only one will be expressed. one will bedominant and the other recessive. In the case of the peas, red-flower color isdominant to white-flower color since only the hereditary character of red shows onthe F1 generation.4.Each parent contributes only one of the hereditary factors to each gamete. Moreover,as a consequence of segregation, equal numbers of gametes each kind are formed.5.when gametes unite at the fertilization, the two hereditary factors are broughttogether. Fertilization is a random union in the sense that equal numbers of thedifferent kinds of gametes are produced and it is a matter of chance how they willpair. This being true it should be possible, on a probability basis, to predict the ratioof various characteristics in the offspring. In the flower color experiment, forexample, the ratio of approximately three red-flowered plants to each white-floweredplant conform the expected or predicted ratio of 3 to 1.Today, Mendel’s theories seem rather obvious in the light of the discovery of DNA, genes, chromosomes and meiosis. But it should be remembered that Mendel did his work before all of these theories. He arrived at his great conclusions purely by reasoning from the patterns of inheritance he detected in his experiments. Of course, we know that a variety of influence, including environment, interaction of one gene with another, can cause the appearancechanges, and a single gene may have multiple effects on explaining the phenomenon of the testimony to the strength of the Mendel’s conclusions.20: we know that when a cell divides it must first duplicate the genetic information contained in its chromosomes, so that each daughter cell receive its own copy from the parental cell, but how the genetic information copied on a molecular level? Since 1928, Frederick Griffith published his experiments on pneumococci, a series of experiments led to the realization that DNA is the genetic material. Soon after then, scientists proposed that the linear order of nucleotides in DNA determines the order of amino acids in polypeptides, and therefore determines the structure and function of proteins. This work is now so well established that it is called the central dogma of molecular biology. The dogma states that the information contained in DNA molecules is transferred to RNA molecules, and then from the RNA molecules the information is expressed in the structure of proteins. A summary of this process is: DNA molecule is known to be composed of two nucleotide chains, each nucleotide are joined by covalent bonds between the sugar of one nucleotide and the phosphate group of the next nucleotide in the sequence. The nitrogenous bases are side groups of the chains, the two chains are held together by hydrogen bonds between nitrogenous bases (adenine=thymine and guanine= cytosine) in opposite directions, then, the ladder like double-chained molecule is coiled into a double helix. During replication, the two chains of the DNA separated, and each chain acts as a template for the synthesis of its new partner according to base pairing principle. This process is much like one might unzip a zipper and then produce two complete double-chained molecules, each identical in base sequence to the mother DNA molecule.The transcription of DNA into RNA begins when the enzyme of RNA polymerase binds to a sequence of nucleotides on the DNA called the promoter. Next, the two strands of DNA are separated, and one strand serves as the template for the formation of a complementary strand of RNA. RNA polymerase moves along the DNA and joins the complementary ribonucleotides to the growing RNA strand, one by one. The enzyme works only in the 3’ to 5’direction on its DNA template, assembling RNA in the 5’ to 3’ direction. This process is much like the replication of DNA, with one important difference. When DNA replication, once begin, usually copies all DNA in the cell, whereas RNA synthesis transcribes only selected proteins of the DNA. When the polymerase reaches a termination signal on the DNA, it leaves the DNA chain, and the RNA also detached.Sine DNA molecules are double-stranded, any section of DNA could, in principle, betranscribed into two different RNA molecules, one complementary to each strand. In practice, only one of the strands is transcribed in any one segment of DNA. The orientation of the promoter indicates which strand is to be transcribed because the promoter reads ‘correctly’ only on that strand. The codes for some genes lie on one DNA strand, and codes for other genes lie on its partner.There are four major stages in protein synthesis:1.activation: each amino acid is first activated by reacting with a molecule of ATP. Theactivated amino acid is then attached to its own particular tRNA molecule with theaid of an enzyme(a synthetase) that is specific for that particular amino acid and thatparticular tRNA molecule.2.initiation: this stage consists of three steps:①mRNA, which carries the informationnecessary to synthesize one protein molecule, attached to the 40s ribosome. ②theanticodon of the first tRNA binds to the codon of the mRNA that represents theinitiation signal. ③the 60s ribosome now combines with 40s body. The 60s bodyhas two binding site: one is P site where the growing peptide chain will bind, theother is A site where the incoming tRNA will bring the next amino acid in.3.elongation: at this point the A site is vacant, and each of the 20 tRNA can come inand try to fit itself in. but only one can be the right anticodon that corresponds to thenext codon on the mRNA. In the next elongation, the whole ribosome moves onecodon along the mRNA. Simultaneously with this move, the dipeptide is translocatedfrom the A site to the P site, while the empties tRNA dissociates and goes back to thetRNA pool to pick up another amino acid. After the translocation, the A site isassociated with the next codon on the mRNA. These elongation steps are repeateduntil the last amino acid is attached.4.termination: after the last translocation, the next codon reads ‘stop’ (UAA,UAG orUGA). No more amino acid can be added. Releasing factors then cleave thepolypeptide chain from the last tRNA in a mechanism not yet fully understood. ThetRNA itself is released from the P site, and the whole mRNA is released from theribosome.23：within multiple-cellular organisms, cells of different tissues divide at very differentrates, this process is strictly controlled so that cells divide only when necessary. Occasionally, however, the genetic control fail, a cell begins to grow and divide without restrain, until its offspring begin to crowed surrounding cells and interfere with tissue functions. The alteration has spawned a tumor. Tumor cells divide not only at a horrendous rate, but not respond to the controls telling them when to stop. They will not stop as long as conditions for growthremain favorable. If the tumor cells remain localized, it is said to be benign. Sometimes,however, a tumor cell can metastasize and then grow and divide in other organs of the body, such tumor cells are said to be malignant, or cancerous by definition. A cancer cell has the following characteristics:1.Almost all cancer cells have an abnormal number of chromosomes.2.Cancer cells tend to have a rather spherical shape, making them more mobile thannormal cells.3.Profound abnormalities in the plasma membrane. Membrane transport andpermeability are amplified. Some proteins at the surface are lost or altered, and newone appears.4.Profound changes in the cytoplasm. The cytoskeleton shrinks, becomesdisorganized, or both. Enzyme activity shifts.5.Abnormal growth and division.6.Diminished capacity for adhesion to substrates. Secretions needed for adhesiondwindle; cells cannot become properly anchored in the parent tissue.Our current understanding of cancer suggests that most cancer arise from a single abnormal cell, but how this cell produced? According to multistep hypothesis, we know that the development of a cancer is a long-term process, and it is a result of several independent genetic mutations occurring in the same cell that convert a normal cell into a malignant cell. These mutations can be caused by mutagenic agents known as carcinogens. Carcinogens include numberous natural and synthetic compounds, X-rays, gamma rays and ultraviolet radiation, which may cause single base changes, deletions, insertions and result in cancerous.Genes that cause cancer are called oncogenes. Research results show that more than fifty oncogenes have now been isolated from a variety of animals. In each case, an identical gene sequence, called a proto-oncogene, has been discovered in the normal DNA of the host animal—and the cells carrying them rarely become cancerous. Proto-oncogenes, which is highly conserved in diverse species, is an inherent parts of vertebrate DNA, and it codes for proteins necessary in normal cell functioning. It may become cancer-causing genes only on those occasions when specific mutations altered its structure or its expression.How oncogene-encoded proteins induce cancer? In normal cells, extracellular signals trigger cell division. At the very least, signaling mechanisms of the pathways controlling division must include the following:①growth factors (molecules that carry growth signals from one cell to another);②signal receptors at the membrane surface of the receiving cell;③molecules that transmit signals from the cell surface to specific targets inside. As to cancerous cells, these mechanism is destroyed, this may be generally foreshadows cell division and then cause it cancerous.24: within the past three decades the ability to alter the genetic characteristics of organisms has been enhanced by the advent of modern recombinant DNA technology. The aim of this technology is to impart some new characteristics or functions to an organism by transferring into its cells the DNA of a different organism. This technology can make an organism obtain desired characteristics or functions in short spans, so it has far-reaching potential and has been widely used in agriculture, medicine and so on. Four basic steps are involved in recombinant DNA technology.First, DNA from the donor organisms is cut into small, manageable pieces. To do this, special enzymes are required, the enzymes that cut DNA are called restriction enzymes, each of which hasthe capability to recognize a specific nucleotide base sequence and to cut each DNA strand whenever the sequence occurs. After cutting, there would be a mixture of DNA segments with‘sticky ends’. Of all these fragments, only one may be of interest.The second step in this technology involves joining the pieces of DNA to a vector that acts as a carrier to transport the gene into a new host cell. There are two types of vectors are commonly used: plasmids and bacteriophages. Plasmids are small, circular DNA molecules in the cytoplasm of bacteria and can replicate independently. The initial procedure is similar by using these two vectors: the donor DNA and ‘vector’ DNA are cut with the same restriction enzyme to create the same sticky ends, then the two types of DNA are mixed, complementary base pairing occurs between the sticky ends, and the ends are joined by ligase. But when bacteriophages act as vectors, some protein are to be added next, the protein then combine with the DNA to form bacteriophages, the bacteriophages then infect other bacterial cells by attaching to the cells and injecting their DNA, and this process is called transduction.Once inside the bacteria, the plasmid or bacteriophage replicates autonomously to produce many copies of itself and the foreign DNA it carries. These copies are sometimes referred to as a gene library. Because thousands of identical copies can be produced, the entire library must be searched to find the one clone that has the desired gene. Generally, there are several techniques, including radioactive probe and specific antibody, are used to solve these problems. When the copies have been identified, the bacteria with the desired gene can be cultured, creating limitless numbers of bacteria.Plasmids and bacteriophages are useful vectors to transport DNA into bacterial cells, but such vectors cannot normally enter eukaryotic cell. How can we do about this next? At present, using microinjection, shotgun procedure or bacterium agrobacterium can insert genes into host cells. Of all these methods, microinjection and shotgun techniques have been successfully used in introducing DNA into plant cell or animal cell. But unfortunately, both two ways have a very low success rate. The bacterium agrobacterium can infects a plant cell efficiently and inserts its plasmids into the host’s chromosomes, but not all plants are susceptible to the crown gall bacterium. So we can select considerable ways to cope with special questions.Of course, it must be remembered that getting the DNA into the cell is only the beginning, so at last, we must use proper control elements to stimulate transcription and allow the host cells to multiple large numbers of descendants, and the descendants are screened to commercially utilize.Although, the recombinant DNA technology seems difficult, genetically engineered plants with herbicide-insect and disease-resistance are come into fruit, and gene transfers to domesticated animals have given us many advantages. Looking towards future, some researchers have suggested using this technology to transfer genes for the fixation of atmospheric nitrogen into crop plants, thus eliminating the need for the application of nitrogenous fertilizers.翻译10：病毒病毒的性质在过去的半个世纪已明显，以及在这一发现道路上的第一步是由俄罗斯植物学家DmitriLvanovsky在1892年时研究的烟草花叶疾病。

第1课知识的悖论The Paradox of KnowledgeThe greatest achievement of humankind in its long evolution from ancient hominoid ancestors to its present status is the acquisition and accumulation of a vast body of knowledge about itself, the world, and the universe. The products of this knowledge are all those things that, in the aggregate, we call "civilization," including language, science, literature, art, all the physical mechanisms, instruments, and structures we use, and the physical infrastructures on which society relies. Most of us assume that in modern society knowledge of all kinds is continually increasing and the aggregation of new information into the corpus of our social or collective knowledge is steadily reducing the area of ignorance about ourselves, the world, and the universe. But continuing reminders of the numerous areas of our present ignorance invite a critical analysis of this assumption.In the popular view, intellectual evolution is similar to, although much more rapid than, somatic evolution. Biological evolution is often described by the statement that "ontogeny recapitulates phylogeny"--meaning that the individual embryo, in its development from a fertilized ovum into a human baby, passes through successive stages in which it resembles ancestral forms of the human species. The popular view is that humankind has progressed from a state of innocent ignorance, comparable to that of an infant, and gradually has acquired more and more knowledge, much as a child learns in passing through the several grades of the educational system. Implicit in this view is an assumption that phylogeny resembles ontogeny, so that there will ultimately be a stage in which the accumulation of knowledge is essentially complete, at least in specific fields, as if society had graduated with all the advanced degrees that signify mastery of important subjects.Such views have, in fact, been expressed by some eminent scientists. In 1894 the great American physicist Albert Michelson said in a talk at the University of Chicago:While it is never safe to affirm that the future of Physical Science has no marvels in store even more astonishing than those of the past, it seems probable that most of the grand underlying principles have been firmly established and that further advances are to be sought chiefly in the rigorous application of these principles to all the phenomena which come under our notice .... The future truths of Physical Science ate to be looked for in the sixth place of decimals.In the century since Michelson's talk, scientists have discovered much more than the refinement of measurements in the sixth decimal place, and none is willing to make a similar statement today. However, many still cling to the notion that such astate of knowledge remains a possibility to be attained sooner or later. Stephen Hawking, the great English scientist, in his immensely popular book A Brief History of Time (1988), concludes with the speculation that we may "discover a complete theory" that "would be the ultimate triumph of human reason--for then we would know the mind of God." Paul Davies, an Australian physicist, echoes that view by suggesting that the human mind may be able to grasp some of the secrets encompassed by the title of his book The Mind of God (1992). Other contemporary scientists write of "theories of everything," meaning theories that explain all observable physical phenomena, and Nobel Laureate Steven Weinberg, one of the founders of the current standard model of physical theory, writes of his Dreams of a Final Theory (1992).Despite the eminence and obvious yearning of these and many other contemporary scientists, there is nothing in the history of science to suggest that any addition of data or theories to the body of scientific knowledge will ever provide answers to all questions in any field. On the contrary, the history of science indicates that increasing knowledge brings awareness of new areas of ignorance and of new questions to be answered.Astronomy is the most ancient of the sciences, and its development is a model of other fields of knowledge. People have been observing the stars and other celestial bodies since the dawn of recorded history. As early as 3000 B.C. the Babylonians recognized a number of the constellations. In the sixth century B.C., Pythagoras proposed the notion of a spherical Earth and of a universe with objects in it chat moved in accordance with natural laws. Later Greek philosophers taught that the sky was a hollow globe surrounding the Earth, that it was supported on an axis running through the Earth, and chat stars were inlaid on its inner surface, which rotated westward daily. In the second century A.D., Ptolemy propounded a theory of a geocentric (Earth-centered) universe in which the sun, planets, and stars moved in circular orbits of cycles and epicycles around the Earth, although the Earth was not at the precise center of these orbits. While somewhat awkward, the Ptolemaic system could produce reasonably reliable predictions of planetary positions, which were, however, good for only a few years and which developed substantial discrepancies from actual observations over a long period of time. Nevertheless, since there was no evidence then apparent to astronomers that the Earth itself moves, the Ptolemaic system remained unchallenged for more than 13 centuries.In the sixteenth century Nocolaus Copernicus, who is said to have mastered all the knowledge of his day in mathematics, astronomy, medicine, and theology, became dissatisfied with the Ptolemaic system. He found that a heliocentric system was bothmathematically possible and aesthetically more pleasing, and wrote a full exposition of his hypothesis, which was not published until 1543, shortly after his death. Early in the seventeenth century, Johannes Kepler became imperial mathematician of the Holy Roman Empire upon the death of Tycho Brahe, and he acquired a collection of meticulous naked-eye observations of the positions of celestial bodies chat had been made by Brahe. On the basis of these data, Kepler calculated that both Ptolemy and Copernicus were in error in assuming chat planets traveled in circular orbits, and in 1609 he published a book demonstrating mathematically chat the planets travel around the sun in elliptical orbits. Kepler's laws of planetary motion are still regarded as basically valid.In the first decade of the seventeenth century Galileo Galilei learned of the invention of the telescope and began to build such instruments, becoming the first person to use a telescope for astronomical observations, and thus discovering craters on the moon, phases of Venus, and the satellites of Jupiter. His observations convinced him of the validity of the Copernican system and resulted in the well-known conflict between Galileo and church authorities. In January 1642 Galileo died, and in December of chat year Isaac Newton was born. Modern science derives largely from the work of these two men.Newton's contributions to science are numerous. He laid the foundations for modem physical optics, formulated the basic laws of motion and the law of universal gravitation, and devised the infinitesimal calculus. Newton's laws of motion and gravitation are still used for calculations of such matters as trajectories of spacecraft and satellites and orbits of planets. In 1846, relying on such calculations as a guide to observation, astronomers discovered the planet Neptune.While calculations based on Newton's laws are accurate, they are dismayingly complex when three or more bodies are involved. In 1915, Einstein announced his theory of general relativity, which led to a set of differential equations for planetary orbits identical to those based on Newtonian calculations, except for those relating to the planet Mercury. The elliptical orbit of Mercury rotates through the years, but so slowly that the change of position is less than one minute of arc each century. The equations of general relativity precisely accounted for this precession; Newtonian equations did not.Einstein's equations also explained the red shift in the light from distant stars and the deflection of starlight as it passed near the sun. However, Einstein assumed chat the universe was static, and, in order to permit a meaningful solution to the equations of relativity, in 1917 he added another term, called a "cosmological constant," to the equations. Although the existence and significance of a cosmological constant is stillbeing debated, Einstein later declared chat this was a major mistake, as Edwin Hubble established in the 1920s chat the universe is expanding and galaxies are receding from one another at a speed proportionate to their distance.Another important development in astronomy grew out of Newton's experimentation in optics, beginning with his demonstration chat sunlight could be broken up by a prism into a spectrum of different colors, which led to the science of spectroscopy. In the twentieth century, spectroscopy was applied to astronomy to gun information about the chemical and physical condition of celestial bodies chat was not disclosed by visual observation. In the 1920s, precise photographic photometry was introduced to astronomy and quantitative spectrochemical analysis became common. Also during the 1920s, scientists like Heisenberg, de Broglie, Schrodinger, and Dirac developed quantum mechanics, a branch of physics dealing with subatomic particles of matter and quanta of energy. Astronomers began to recognize that the properties of celestial bodies, including planets, could be well understood only in terms of physics, and the field began to be referred to as "astrophysics."These developments created an explosive expansion in our knowledge of astronomy. During the first five thousand years or more of observing the heavens, observation was confined to the narrow band of visible light. In the last half of this century astronomical observations have been made across the spectrum of electromagnetic radiation, including radio waves, infrared, ultraviolet, X-rays, and gamma rays, and from satellites beyond the atmosphere. It is no exaggeration to say chat since the end of World War II more astronomical data have been gathered than during all of the thousands of years of preceding human history.However, despite all improvements in instrumentation, increasing sophistication of analysis and calculation augmented by the massive power of computers, and the huge aggregation of data, or knowledge, we still cannot predict future movements of planets and other elements of even the solar system with a high degree of certainty. Ivars Peterson, a highly trained science writer and an editor of Science News, writes in his book Newton's Clock (1993) that a surprisingly subtle chaos pervades the solar system. He states:In one way or another the problem of the solar system's stability has fascinated and tormented asrtonomers and mathematicians for more than 200 years. Somewhat to the embarrassment of contemporary experts, it remains one of the most perplexing, unsolved issues in celestial mechanics. Each step toward resolving this and related questions has only exposed additional uncertainties and even deeper mysteries.Similar problems pervade astronomy. The two major theories of cosmology,general relativity and quantum mechanics, cannot be stated in the same mathematical language, and thus are inconsistent with one another, as the Ptolemaic and Copernican theories were in the sixteenth century, although both contemporary theories continue to be used, but for different calculations. Oxford mathematician Roger Penrose, in The Emperors New Mind (1989), contends that this inconsistency requires a change in quantum theory to provide a new theory he calls "correct quantum gravity."Furthermore, the observations astronomers make with new technologies disclose a total mass in the universe that is less than about 10 percent of the total mass that mathematical calculations require the universe to contain on the basis of its observed rate of expansion. If the universe contains no more mass than we have been able to observe directly, then according to all current theories it should have expanded in the past, and be expanding now, much more rapidly than the rate actually observed. It is therefore believed that 90 percent or more of the mass in the universe is some sort of "dark matter" that has not yet been observed and the nature of which is unknown. Current theories favor either WIMPs (weakly interacting massive particles) or MACHOs (massive compact halo objects). Other similar mysteries abound and increase in number as our ability to observe improves.The progress of biological and life sciences has been similar to that of the physical sciences, except that it has occurred several centuries later. The theory of biological evolution first came to the attention of scientists with the publication of Darwin's Origin of Species in 1859. But Darwin lacked any explanation of the causes of variation and inheritance of characteristics. These were provided by Gregor Mendel, who laid the mathematical foundation of genetics with the publication of papers in 1865 and 1866.Medicine, according to Lewis Thomas, is the youngest science, having become truly scientific only in the 1930s. Recent and ongoing research has created uncertainty about even such basic concepts as when and how life begins and when death occurs, and we are spending billions in an attempt to learn how much it may be possible to know about human genetics. Modern medicine has demonstrably improved both our life expectancies and our health, and further improvements continue to be made as research progresses. But new questions arise even more rapidly than our research resources grow, as the host of problems related to the Human Genome Project illustrates.From even such an abbreviated and incomplete survey of science as this, it appears that increasing knowledge does not result in a commensurate decrease in ignorance, but, on the contrary, exposes new lacunae in our comprehension and confronts us with unforeseen questions disclosing areas of ignorance of which wewere not previously aware.Thus the concept of science as an expanding body of knowledge that will eventually encompass or dispel all significant areas of ignorance is an illusion. Scientists and philosophers are now observing that it is naive to regard science as a process that begins with observations that are organized into theories and are then subsequently tested by experiments. The late Karl Popper, a leading philosopher of science, wrote in The Growth of Scientific Knowledge (1960) chat science starts from problems, not from observations, and chat every worthwhile new theory raises new problems. Thus there is no danger that science will come to an end because it has completed its task, clanks to the "infinity of our ignorance."At least since Thomas Kuhn published The Structure of Scientific Revolutions (1962), it has been generally recognized that observations are the result of theories (called paradigms by Kuhn and other philosophers), for without theories of relevance and irrelevance there would be no basis for determining what observations to make. Since no one can know everything, to be fully informed on any subject (a claim sometimes made by those in authority) is simply to reach a judgment that additional data are not important enough to be worth the trouble of securing or considering.To carry the analysis another step, it must be recognized that theories are the result of questions and questions are the product of perceived ignorance. Thus it is chat ignorance gives rise to inquiry chat produces knowledge, which, in turn, discloses new areas of ignorance. This is the paradox of knowledge: As knowledge increases so does ignorance, and ignorance may increase more than its related knowledge.My own metaphor to illustrate the relationship of knowledge and ignorance is based on a line from Matthew Arnold: "For we are here as on a darkling plain...." The dark chat surrounds us, chat, indeed, envelops our world, is ignorance. Knowledge is the illumination shed by whatever candles (or more technologically advanced light sources) we can provide. As we light more and more figurative candles, the area of illumination enlarges; but the area beyond illumination increases geometrically. We know chat there is much we don't know; but we cannot know how much there is chat we don't know. Thus knowledge is finite, but ignorance is infinite, and the finite cannot ever encompass the infinite.This is a revised version of an article originally published in COSMOS 1994. Copyright 1995 by Lee Loevinger.Lee Loevinger is a Washington lawyer and former assistant attorney general of the United States who writes frequently for scientific c publications. He hasparticipated for many years as a member, co-chair, or liaison with the National Conference of Lawyers and Scientists, and he is a founder and former chair of the Science and Technology Section of the American Bar Association. Office address: Hogan and Hartson, 555 Thirteenth St. NW, Washington, DC 20004.人类从古类人猿进化到当前的状态这个长久的进化过程中的最大成就是有关于人类自身、世界以及宇宙众多知识的获得和积聚。

Mark Scheme (Results)June 2022Pearson Edexcel International Advanced Level In Biology (WBI12) Paper 01Cells, Development,Biodiversity and ConservationEdexcel and BTEC QualificationsEdexcel and BTEC qualifications are awarded by Pearson, the UK’s largest awarding body. We provide a wide range of qualifications including academic, vocational, occupational and specific programmes for employers. For further information visit our qualifications websites at or . Alternatively, you can get in touch with us using the details on our contact us page at /contactus.Pearson: helping people progress, everywherePearson aspires to be the world’s leading learning company. Our aim is to help everyone progress in their lives through education. We believe in every kind of learning, for all kinds of people, wherever they are in the world. We’ve been involved in education for over 150 years, and by working across 70 countries, in 100 languages, we have built an international reputation for our commitment to high standards and raising achievement through innovation in education. Find out more about how we can help you and your students at: /ukJune 2022Log Number P69499APublications Code IAL_2206_WBI12_01All the material in this publication is copyright© Pearson Education Ltd 2022General Marking Guidance•All candidates must receive the same treatment. Examiners must mark the first candidate in exactly the same way as they mark the last.•Mark schemes should be applied positively. Candidates must be rewarded for what they have shown they can do rather than penalised for omissions.•Examiners should mark according to the mark scheme not according to their perception of where the grade boundaries may lie.•There is no ceiling on achievement. All marks on the mark scheme should be used appropriately.•All the marks on the mark scheme are designed to be awarded. Examiners should always award full marks if deserved, i.e. if the answer matches the mark scheme. Examiners should also be prepared to award zero marks if the candidate’s response is not worthy of credit according to the mark scheme.•Where some judgement is required, mark schemes will provide the principles by which marks will be awarded and exemplification may be limited.•When examiners are in doubt regarding the application of the mark scheme to a candidate’s response, the team leader must be consulted.•Crossed out work should be marked UNLESS the candidate has replaced it with an alternative response.QuestionNumberAnswer Additional guidance Mark 1(a) An answer that makes reference to the following point:• group of organs that work together to perform {one / one ormore / specific} functions(1)QuestionNumberAnswer Additional guidance Mark1(b)(i) An answer that makes reference to the following point:• group of (similar) cells working together to perform a(specific) function (1)Question Number Answer Mark1(b)(ii)StructurePropels malegamete towardsfemale gameteModified by theaction of corticalgranulesProduce ATP byrespirationContain linearDNAflagellum⌧ Amitochondria⌧ Cnucleus⌧ Dzona pellucida⌧ B(4)QuestionAnswer Additional guidance Mark Number2(a) An answer that includes the following points:Example of diagram Array•capsule correctly drawn and labelled (1)•(at least) two pili correctly drawn andlabelled (1)(2)QuestionNumberAnswer Additional guidance Mark 2(b) An answer that makes reference to four of the following points:•increasing sodium chloride concentrations (above 10 g dm3) decreases the growth rate (of both bacteria) (1)•(salt concentration between 0-6 g dm3 caused) an initial {increase / constant} rate (1)•more rapid decrease in L. piscium growth rate (1)• B. thermosphacta had a larger growth rate (than L. piscium at all sodium chloride concentrations) (1)• B. thermosphacta was able to continue growing in sodium chloride concentrations above {22-25} g dm3 whereas L.piscium had no growth / L. piscium stopped growing at aconcentration {38-46} g dm3 below the concentration that B.thermosphacta stopped growing (1)•data is more scattered about the line of best fit for B.thermosphacta (than L. piscium) ACCEPT negative correlation (betweensodium chloride concentration andgrowth rate)ACCEPT converseACCEPT converseAccept B. thermosphacta is more {tolerant/ resistant} (than L. piscium)of highersodium chloride concentrationsAccept B. thermosphacta stopped growingat {62-70}g dm3 whereas L. pisciumstopped growing at{22-24}g dm3ACCEPT converse(4)QuestionNumberAnswer Additional guidance Mark3(a)(i) A calculation in which:•calculation of volume of sphere (1)•calculation of volume of hemisphere to nearest whole number(1) Example of calculation:answer between 65416.67 to 65476.19whole number answer between 32708 to 32738Correct answer scores full marks(2)QuestionNumberAnswer Additional guidance Mark 3(a)(ii) An explanation that makes reference to two of the following:•(because) calabash fruits are renewable / more can be grown /will not run out / available to future generations (1)•biodegradable / can be broken down by decomposers (1)•carbon neutral (1) ignore produces less greenhouse gases (2)QuestionAnswer Mark Number3(b)(i)The only correct answer is B oneA is not correct because they provide support to the plantB is not correct because they are not involved in transporting substances(1)C is not correct because they are not involved in transporting substancesQuestionNumberAnswer Additional guidance Mark 3(b)(ii) An answer that makes reference to four of the following:Similarities:•both (fibres) contain cellulose (in the cell wall) (1)•both have tubular structures (1)•both do not contain a nucleus (1)Differences (max 3):•phloem (sieve tubes) have {sieve plates / (perforated) end walls} whereas xylem (vessels) have no {end walls/ sieve plates} (1)•phloem (sieve tubes) {contain cytoplasm / are not hollow} whereas xylem (vessels) {do not containcytoplasm / are hollow} (1)•phloem (sieve tubes) contain no {lignin / secondary thickening} whereas xylem contain {lignin / secondarythickening} (1)•phloem have plasmodesmata whereas xylem have pits (1) ACCEPT piecing together from adjacent sentencesACCEPT both have a cell wallignore organellesACCEPT phloem (sieve tubes) containliving cells whereas xylem (vessels)contain dead cellsACCEPT phloem cell walls are thinnerthan xylem cell walls / converseACCEPT xylem have pits whereas phloemdo not / phloem have plasmodesmata whereas xylem do not (4)Question Number Answer Additional guidance Mark 4(a)(i) An answer that makes reference to the following points:• centrioles and spindle fibres shown (1)• 6 chromosomes being pulled to each pole (1)Example of diagram:(2)Question Number Answer Additional guidanceMark 4(a)(ii) A calculation in which:• calculation of number of degrees per minute (1)• calculation of number of degrees (including unit) (1) Example of calculation:270 ÷ (9 × 60) = 0.5°0.5 × 20 = 10°Correct answer scores full marks(2)QuestionNumberAnswer Additional guidance Mark 4(b)(ii) An explanation that makes reference to the following points:•(egg cells are) haploid (1)•(egg cell chromosomes) have {an altered base sequence / different alleles} (than body cell chromosomes) (1)•due to {(random) mutations (during DNA replication) /crossing over / random assortment / independentassortment} (1) ACCEPT contain {3 / half the number of} chromosomesdo not accept genesACCEPT chromosomes mutations due toerrors in separation of {chromatids / chromosomes}(3)QuestionNumberAnswer Additional guidance Mark 4(b)(i)•location of gene(s) on a(n Indian muntjac) chromosome ACCEPT {nucleotide base pairs / allele} forgeneACCEPT chromatid for chromosome(1)QuestionNumberAnswer Additional guidance Mark 4(c) An answer that makes reference to two of the following points:•(because the parents are) different species (1)•(because {maternal and paternal / 3 and 23}) chromosomes would not pair up (1)•(therefore) cannot make {haploid / sperm / egg} cells (1) ACCEPT because diploid number (ofeither species) could not be restoredignore parents have different number of chromosome pairsACCEPT meiosis cannot occur(2)QuestionNumberAnswer Mark 5(a)(i)The only correct answer is B endemicA is not correct because the correct term is endemicC is not correct because the correct term is endemicD is not correct because the correct term is endemic (1)QuestionNumberAnswer Additional guidance Mark5(a)(ii) A calculation in which:•correct difference (1)•correct percentage change (1) Example of calculation:950-7100 = (-)6150(-6150÷7100) x 100 = (-)87(%)Correct answer scores full marks(2)QuestionAnswerNumber*5(b) Answers will be credited according to candidate’s deployment of knowledge and understanding of the material in relation to the qualities and skills outlined in the generic mark scheme.The indicative content below is not prescriptive and candidates are not required to include all the material indicated asrelevant. Additional content included in the response must be scientific and relevant.•common ancestor {lived on Hawaii islands / had shorter beak than Amakihi / less powerful beak than Palila}•colonisation of new island / geographical isolation•change in the environment / competition for food / (new) selection pressures•genetic variation in population / mutation resulted in new allele(s)•some alleles conferred an advantage therefore bird more likely to survive and reproduce than other birds and pass on those advantageous alleles to next generation•repeated over many generations leading to new species•idea of reproductive isolationAmakihi•Amakihi has a longer beak•(therefore) was able to drink (more) nectar from flowers / tree sap / access more spiders / insects•Amakihi has larger population due to being adapted to live in more habitats / idea that it can access more types of food sourcesPalila•Palila has a {more powerful beak / beak which could crack open the coat (of seed)}•Palila was able to eat (more) seeds / berries(6)Additional guidance Level 0 0 No awardable contentLevel 1 1-2 An explanation may be attempted but with limitedinterpretation or analysis of the scientific information andwith a focus on mainly just one piece of scientificinformation.The explanation will contain basic information, with someattempt made to link knowledge and understanding to thegiven context. evolution of generic new species explained with basic information1 mark – very limited explanation of evolution of a (generic) new species2 marks – more detailed explanation of evolution ofa (generic) new species OR one point about generic explanation plus a basic linkage to either Amakihi or PalilaLevel 2 3-4 An explanation will be given, with occasional evidence ofanalysis, interpretation and/or evaluation of both pieces ofscientific information.The explanation shows some linkages and lines of scientificreasoning, with some structure. Level 1 plus some linkages to either Amakihi or Palila3 marks = basic linkage for one4 marks = detailed linkage for one or basic for bothLevel 3 5-6 An explanation is made that is supported throughout bysustained application of relevant evidence of analysis,interpretation and/or evaluation of both pieces of scientificinformation.The explanation shows a well-developed and sustained lineof scientific reasoning, which is clear and logically structured. Level 2 plus detailed linkage to both Amakihi and Palila5 marks = detailed linkage for one and basic for one6 marks = detailed linkage for bothQuestionNumberAnswer Additional guidance Mark 5(c) An answer that makes reference to the following points:•molecular phylogeny / analysis of (the sequences in) biological molecules (1)•(therefore) the species with the {most similarities / fewest differences} (are the most closely related) (1) e.g. DNA, mRNA, proteins(2)QuestionAnswer Mark Number6(a)The only correct answer is C YA is not correct because W does not contain mitochondriaB is not correct because X does not contain mitochondriaD is not correct because Z does not contain mitochondria (1)QuestionNumberAnswer Additional guidance Mark 6(b)(i) A description that makes reference to the following points:•named organelle involved in protein production (1) •role of organelle described in the production of the enzyme (1) mitochondria / nucleus / ribosomes /{rough endoplasmic reticulum / rER} /Golgi (apparatus/body)e.g.mitochondria – produce ATP for protein synthesis (of enzyme)nucleus – site of transcription of enzymegene / location of gene for enzymeribosomes -site of protein synthesis /where polypeptide is formedrER -site of protein synthesis / where polypeptide is formed / formation of 3° structure / packaging protein into (transport) vesicleGolgi apparatus – modification of{protein / enzyme} / packaging {protein / enzyme} into (secretory) vesicle (2)QuestionNumberAnswer Additional guidance Mark 6(b)(ii)•800 (1)QuestionNumberAnswer Additional guidance Mark 6(c)(i)•image size divided by magnification ACCEPT 4.5±1 ÷200ignore unmanipulated equation (1)QuestionNumberAnswer Additional guidance Mark 6(b)(iii) An explanation that makes reference to four of the following points:•doubling the acrosin activity of the sperm cell increases the percentage of egg cells fertilised by 68% / non-linear increase •(because) higher acrosin activity means the sperm cells can digest through all of the {outer layer / zona pellucida} (of more egg cells) (1)•(allowing) sperm (cells) to {bind to (egg cell) membrane / enter egg (cell)} (1)•(so that) sperm nucleus can fuse with egg (cell) nucleus / fusion (of nuclei) can occur (1)•(low acrosin activity) could result in death of sperm cells before fertilisation could occur (1) ACCEPT greater increase (in percentageof egg cells fertilised) between 2.5 and3(a.u.)ACCEPT 100% fertilisation at 5(a.u.)compared to 32% at 2.5(a.u.)ACCEPT digesting the outer layer {faster/more efficiently} / more digestion occurs Accept converseACCEPT sperm can reach egg (cell)nucleusACCEPT sperm can fuse with egg (cell) ACCEPT converse(4)QuestionNumberAnswer Additional guidance Mark 6(c)(ii) An answer that makes reference to three of the following points:•(when a sperm entered egg cell) cortical granules have fused tocell surface membrane / cortical {reaction/enzymes} resulted inhardening of zona pellucida (1)•as zona pellucida (of some egg cells) are damaged there are areas where it is not {present / hardened} (1)•resulting in polyspermy / {an extra / two} sperm have entered (the egg cell) (1) ACCEPT some zona pellucida is nothardenedreject 3 sperm have entered egg cell(3)Question Number AnswerAdditional guidanceMark7(a)(i)The only correct answer is B oneA is not correct because the first statement is correctC is not correct because the generative nucleus divides to form two haploid male gametesD is not correct because the generative nucleus divides to form two haploid male gametes(1)QuestionNumberAnswer Additional guidance Mark7(a)(ii)•surface area calculated (1)•difference in standard form with units (1)4.128 (µm2)9.02 x 10-1µm2(2)QuestionNumberAnswer Additional guidance Mark 7(b) A description that makes reference to the following points:•differential gene expression (1)•by {epigenetic modification / histone modification / DNA methylation} (1)•{proteins / enzymes} synthesised (from active genes) which (permanently) modify the cell (1)•description of modification to become a sclerenchyma cell ACCEPT only some genes are {active / switched on} / some genes are switchedoffACCEPT {proteins / enzymes} {made / synthesised} produce a {structural /functional / metabolic} changee.g. synthesis of {cellulose/ microfibrils / lignin} / secondary thickening /lignification of cell walls(4)QuestionNumberAnswer Additional guidance Mark 7(c) An answer that makes reference to four of the following points:•meiosis increases genetic variation / crossing over and {random / independent} assortment occur in meiosis (1)•the {genes / alleles / loci} for colour and grain length are linked (1)•(therefore alleles for these traits) will be inherited together / (therefore alleles for these traits) unlikely to be separated during crossing over (1)•the {genes / alleles / loci} for pea pod wrinkles and {colour/grain length} are not linked (1)•(therefore) the alleles for these traits will be inherited independently due to independent assortment (1) ACCEPT meiosis results in haploid cells ACCEPT linkage is when {genes / alleles}are close together on the samechromosomeACCEPT allele for this trait may beseparated from {colour/grain length/other alleles} due to independentassortment(4)QuestionNumberAnswer Additional guidance Mark 8(a) An explanation that makes reference to three of the following points:•fewer young tree shoots eaten (after wolves were reintroduced) (1)•(because) elk would not graze undisturbed when wolves were around (1)•more (young tree shoots) were eaten in forest habitat (than riverside habitat) / fewer (young tree shoots) were eaten in riverside habitat (than forest habitat) (1)•(because) the mature trees provided some {camouflage / protection} (from the wolves) (1) ACCEPT the percentage (of young treeshoots eaten) decreasedACCEPT converseACCEPT because some elk have beeneaten by wolves / fewer elk becausewolves are predators (of elk)ACCEPT {larger/more rapid} decrease (in number of shoots eaten) in riversidehabitatACCEPT (because) elk can hide (from the wolves) in the forest / elk have no {hiding places / protection} from being seen (bythe wolves) by the riverACCEPT predation of elk is higher inriverside (habitat)(3)QuestionNumberAnswer Additional guidance Mark 8(b) A description that makes reference to the following points:•as the number of elk (per km2) increases the average beaver lifespan decreases (1)•{larger/rapid} decrease {initially / as population increases from 30 elk (per km2)} (1)•very little change in average lifespan of beavers when the number of elk increase above {70/80/90} elk per km2 (1) ACCEPT negative correlationACCEPT {exponential / non-linear}decreaseACCEPT the beaver lifespan plateauswhen the number of elk increase above{70/80/90} elk per km2(3)QuestionAnswer Additional guidance Mark Number8(c)(i) An answer which includes the following points:•(count) number of species (1)•{area / size} of habitat (1) ACCEPT per unit area (2)QuestionAnswerNumber*8(d) Answers will be credited according to candidate’s deployment of knowledge and understanding of the material in relation to the qualities and skills outlined in the generic mark scheme.The indicative content below is not prescriptive and candidates are not required to include all the material indicated asrelevant. Additional content included in the response must be scientific and relevant.Basic•increasing numbers of wolves (until 2003) / decrease in number of wolves (from 2007 to 2010)•overall increase in wolves / 20 more wolves in 2010 (than in 1995)•the numbers of elk decreased as they were hunted by the wolves•overall decrease in numbers of elk (from 1995 to 2010)•the number of cottonwood shoots rapidly increased (after 2003)•the number of beaver colonies increased from 1 (in 1999) to 12 (in 2009)Linkage•the numbers of cottonwood shoots increased {when the numbers of elk decreased / as they weren’t being eaten}•beavers and elk have similar niches / beavers were outcompeted for young tree shoots by elk•beaver {numbers/colonies} started to increase when {elk population size decreased / more shoots were available}•discussion of causes of decrease in wolf population e.g. decrease in elk food source•introduction of wolves increases biodiversitySustained•{more ponds / new habitat} increase {number of species / species richness / biodiversity}•more trees (woodland and riverside habitats) increase {number of species / species richness / biodiversity}•biodiversity increases due to additional species in Yellowstone National Park e.g. wolf(6)ExpertAdditional guidanceLevel 0 0 No awardable contentLevel 1 1-2 Demonstrates isolated elements of biological knowledgeand understanding to the given context with generalisedcomments made.Vague statements related to consequences are made withlimited linkage to a range of scientific ideas, processes,techniques and procedures.The discussion will contain basic information with someattempt made to link knowledge and understanding to thegiven context.Information from one sectionLevel 2 3-4 Demonstrates adequate knowledge and understanding byselecting and applying some relevant biological facts /concepts.Consequences are discussed which are occasionallysupported through linkage to a range of scientific ideas,processes, techniques and procedures.The discussion shows some linkages and lines of scientificreasoning with some structure. Information from two sections linkage of 2 conceptsLevel 3 5-6 Demonstrates comprehensive knowledge andunderstanding by selecting and applying relevant biologicalfacts / concepts.Consequences are discussed which supported throughoutby sustained linkage to a range of scientific ideas, processes,techniques and procedures.The discussion shows a well-developed and sustained line ofscientific reasoning which is clear and logically structured. Level 2 plusDiscussion of how biodiversity would be increasedPearson Education Limited. Registered company number 872828with its registered office at 80 Strand, London, WC2R 0RL, United Kingdom。

C hapter 18 C onstruction of shRNA Expression Plasmidsfor Silkworm Cell Lines Using Single-StrandedDNA and B st DNA PolymeraseHiromitsu TanakaAbstractT ransfection of short hairpin RNA (shRNA) expression plasmids is conventionally performed for gene-speci fic knockdown in cultured mammalian and insect cells. Here, I describe a simple method to synthesize an inverted repeat DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded inverted repeat DNA and B st DNA polymerase. The shRNA expression plasmids constructed by this method were con fir med to promote ef fic ient RNA interference knockdown in silkworm cell lines. This method may be useful for constructing a relatively large number of shRNA expression plasmids.K ey words:s hRNA expression plasmids ,B st DNA polymerase ,S ingle-stranded inverted repeat DNA , S ilkworm cell line1.IntroductionP osttranscriptional silencing by RNA interference (RNAi) is widelyused as a technique for suppressing the expression of speci fic genesin many organisms (1–4). A conventional procedure for inducingRNAi knockdown in cultured mammalian and insect cells is thedirect transfection of 21–23 nucleotides (nt) of small interferingRNA (siRNA) (5, 6)or over-expression of short hairpin RNA(shRNA) composed of 19–29 nt of stem regions and 4–23 nt ofloop sequences by the transfection of an RNA polymerase III-dependent promoter-driven shRNA expression plasmid (7, 8).Knockdown by transfection of an shRNA expression plasmid hassome advantages over knockdown by siRNA transfection (9). First,the RNAi effect may be more stable because of the sustainedproduction of shRNA. Second, the transfected cells can be selectedby antibiotics when the shRNA expression plasmid possesses Debra J. Taxman (ed.), siRNA Design: Methods and Protocols, Methods in Molecular Biology,vol. 942,DOI 10.1007/978-1-62703-119-6_18, © Springer Science+Business Media, LLC 2013347348H. Tanakaantibiotic-resistance genes. Furthermore, inducible shRNAexpression is available. In general, shRNA expression plasmids canbe generated by two methods. One method is the insertion of adouble-stranded inverted repeat (IR) DNA that is obtained byannealing of two complementary oligonucleotides into a parentvector ( 9) . The second method is a polymerase chain reaction (PCR)-based strategy in which the promoter sequence serves as the template( 10) . We developed another simple method to create IR DNA in the parent vector using a single-stranded DNA possessing a short hairpinstructure and B st DNA polymerase, which has strand displacementactivity (11 ) . This method comprises the following steps: (a) linear-ization of the plasmid with the 5 ¢ end of one terminus dephosphory-lated by treating stepwise with one restriction enzyme, alkalinephosphatase, and then a second restriction enzyme; (b) ligation of ahairpin oligonucleotide to one end of the linear plasmid; (c) execu-tion of the strand displacement reaction by B st DNA polymerase; and(d) self-ligation of the linear plasmid (Fig.1 ). This method reduces the cost of unique oligonucleotides compared with the conventionalmethod. Therefore, it is useful for constructing relatively large num-bers of shRNA expression plasmids. We further demonstrated thatthe shRNA expression plasmid constructed by this method effec-tively induces target-speci fi c RNAi a silkworm cell line ( 11) . 1. S ynthesized oligonucleotides: 53 mer; 21 mer of IR structure separated by 11 mer of spacer DNA. These oligonucleotides maybe obtained from most custom oligonucleotide-synthesizingfacilities and companies. For a discussion of the oligonucleotidedesign, see N otes 1 – 5 . Store at −20°C.2. 10× M buffer: 100 mM Tris–HCl (pH 7.9), 100 mM MgCl 2 , 500 mM NaCl, and 10 mM DTT. Store at −20°C.3. U ltrapure water: Milli Q grade; sterilized by autoclaving.1. P arent plasmid for constructing an shRNA expression plasmid: We used pIEx-4-BmU6M, which contains the enhancer and promoter region between S ma I and N co I . Multicloning sitesbetween the N co I and D ra I II sites of pIEx-4 were substitutedby 467 bp of the promoter region of the B ombyx mori U6-2small nuclear RNA gene (12 ) and the sequence “5 ¢ - C CATGG C TGCAG A GGCCT T TTTCACTAAGTG-3 ¢ ” (underliningindicates the N co I site; bold letters indicate the S tu I site),respectively.2. R estriction endonucleases: N co I and S tu I at 10 U/ m L . Storeat −20°C.2.Materials2.1.OligonucleotideAnnealing 2.2.Constructionof shRNA ExpressionPlasmids34918 shRNA Construction for Silkworms Using Bst DNA Polymerase 3. 10× H buffer: 500 mM Tris–HCl (pH 7.9), 100 mM MgCl 2 , 1 M NaCl, and 10 mM DTT. Store at −20°C.4. A lkaline phosphatase: 10 U/ m L . Store at −20°C.5. C IA: 24:1 (v/v) mixture of chloroform and isoamyl alcohol.6.P henol/chloroform: 1:1 (v/v) mixture of Tris–HCl (pH 8.0) buffered phenol and CIA. Store at 4°C.7. E thanol: 100% and 70% (v/v) solution.5’ P 3’ OHNco I digestion Alkaline phosphatase treatmentStuOH 3’OH 5’LigationOH 5’Bst DNA polymerase treatment5’ OH3’ OH T4 polynucleotide kinase treatmentSelf-ligationa b F ig. 1. T he structure of pIEx-4-BmU6M and the procedure for construction of an shRNA expression plasmid. ( a ) Diagram of pIEx-4-BmU6M. The nucleotide sequences possessing the S tu I recognition site ( S tu I ) and a T cluster were inserted into the N co I and D ra I II sites of pIEx-4 (Novagen); the enhancer and promoter region between the S ma I and N co I sites of pIEx-4 was replaced by 467 bp of a promoter region of B ombyx mori U6-2 small nuclear RNA gene ( b lack box ). Gray box indicates the terminator region from the A utographa californica nucleopolyhedrovirus-derived immediate early 1 gene. ( b )Strategy to create the IR DNA in pIEx-4-BmU6M. A short hairpin oligonucleotide is ligated with the S tu I -digested blunt end of linear pIEx-4-BmU6M. B st DNA polymerase extends the 3 ¢ end of the N co I -digested terminus and 3 ¢ end at the nick followed by the displacement of the 5 ¢ portion of the hairpin oligonucleotide. Kinase reaction and self-ligation yield a circular shRNA expression plasmid.350H. Tanaka8. 3 M Sodium acetate (pH 5.2): Sterilized by autoclaving. 9. T E buffer: 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA. Sterilized by autoclaving. 10. 10× M buffer: 100 mM Tris–HCl (pH 7.9), 100 mM MgCl 2 , 500 mM NaCl, and 10 mM DTT. 11. D NA Ligation Kit Mighty Mix: Available from Takara Bio. Store at −20°C. 12. 50× TAE: 2 M Tris–acetate, 50 mM EDTA. 13. A garose gels: Electrophoresis grade agarose in 1× TAE. 14. W izard SV Gel and PCR Clean-Up System: Available from Promega. 15. B st DNA polymerase large fragment and 10× ThermoPol Reaction Buffer: B st DNA polymerase at 8 U/ m L and 10× ThermoPol Reaction Buffer at 200 mM Tris–HCl (pH 8.8), 100 mM KCl, 100 mM (NH 4 ) 2 S O 4 , 20 mM MgSO 4 , and 1% Triton X-100. Available from New England Biolabs. Store at −20°C. 16. 10 mM dNTP mixture: A mixture in water that contains 10 mM of each deoxyribonucleoside triphosphate. Store at −20°C. 17. T 4 polynucleotide kinase and 5× kinase buffer: T4 polynucle-otide kinase at 10 U/ m L and 5× buffer at 50 mmol/L Imidazole–HCl (pH 6.4), 18 mM MgCl 2 , 5 mM DTT, 6% (w/v) PEG6000. Store at −20°C. 18. 2 mM ATP: Store at −20°C. 19. C ompetent E scherichia coli : We successfully used both the Sure2 Supercompetent Cells (Stratagene) and DH5 a (Takara Co. Ltd) strains. Store at −80°C. 20. 2× YT agar plate: To make 1 L, add 16 g of polypeptone, 10 g of yeast extract, 5 g of NaCl, and 15 g of agar to 900 mL of water. Fill to 1 L with water and autoclave. After cooling, add ampicillin to a fi n al concentration of 100 m g /mL. Pour into plates and store the plates at 4°C. 1. F orward and reverse primers: Dilute each synthetic oligonucle-otide to 10 m M with water. Store at −20°C. 2. 10× PCR buffer: 100 mM Tris–HCl (pH 8.3), 500 mM KCl,and 15 mM MgCl 2 . Store at −20°C.3. T aq polymerase (5 U/m L): Store at −20°C.4. 2× YT medium: 1.6% polypeptone, 1.0% yeast extract, and85 mM NaCl. Sterilize by autoclaving.2.3.Con firmationof Insert Sizeby Colony PCR35118 shRNA Construction for Silkworms Using Bst DNA Polymerase 1. O ligonucleotides were suspended in water to a concentration of 100 pmol/ m L . 2. M ix 32 m L of oligonucleotide solution (100 pmol/ m L ), 32 m L of 10× M buffer, and 40 m L of water in a 0.2 mL tube. 3. H eat at 95°C for 5 min and gradually cool to 30°C (1–2°C/min). Annealed oligonucleotides should form a hairpin structure. 4. S tore at −20°C if the annealed oligonucleotides are not to be used immediately. T he construction method using pIEx-4-BmU6M ( 11 ) is as follows: 1. D igest 10 m g of pIEx-4-BmU6M with 25 units of N co I at37°C for 1–12 h in a 400 m L reaction volume containing 40 m L of 10×H buffer. Heat DNA at 65°C for 5 min to inactivate the enzymes.2. A dd 2 m L of alkaline phosphatase and incubate the solution at37°C for 30 min.3. E xtract the reaction solution with phenol/chloroform andthen CIA. Add 1 mL of absolute ethanol and 40 m L of 3 M sodium acetate to the upper phase solution. Centrifuge for 12,000 × g at 4°C for 10 min. Discard the supernatant, wash the pellet in 70% ethanol, and recentrifuge for 5 min. Dissolve the pellet with 357 m L of water and then add 40 m L of 10× M buffer and 2 m L of S tu I . Incubate at 37°C for 1–12 h. Extract the reaction solution with phenol/chloroform and then CIA. Precipitate the reactant DNA with ethanol. The pellet is dis-solved with TE buffer at a concentration of 0.25 m g / m L . The product can be stored at −20°C.4. M ix 1.5 m L of linear plasmid, 1 m L of annealed oligonucle-otide, 5 m L of DNA Ligation Kit Mighty Mix, and 2.5 m L of water. Incubate at 16°C for 30 min.5. L oad 10 m L of ligated DNA solution onto a 1% agarose gel in1× TAE gel running buffer. After electrophoresis is performed, remove the desired bands from the gel. See N ote 6 .6. R ecover DNA from the gel slice using a Wizard SV Gel andPCR Clean-Up System according to the instruction manual. Finally, elute DNA with 50 m L of water.7. M ix 43 m L of recovered DNA, 5 m L of 10× ThermoPolReaction Buffer, 1 m L of 10 mM dNTP mixture, and 1 m L ofB st DNA polymerase. Incubate at 50°C for 2 min, and then move to 62.5°C for 30 min.8. E xtract the reaction solution with phenol/chloroform and then CIA. Precipitate the reactant DNA with ethanol. Dissolve3.Methods3.1.OligonucleotideAnnealing3.2.Constructionof shRNA ExpressionPlasmids352H. Tanakathe pellet with 43 m L of water. Add 11.3 m L of 5× kinase buffer, 1 m L of 2 mM ATP, and 1 m L of T4 polymerase kinase. Incubate at 37°C for 30 min.9.E xtract the reaction solution with phenol/chloroform and then CIA. Precipitate the reactant DNA with ethanol. Dissolve the pellet with 20 m L of water.10. M ix 5 m L of reactant solution and 5 m L of DNA Ligation KitMighty Mix. Incubate at 16°C for 30 min.11. T ransform 10 m L of ligation reaction into 100 m L of a compe-tent strain of E . coli . Plate the appropriate amount of cells onto 2× YT agar plates. Incubate at 37°C overnight.1. P repare the PCR reaction mixture. For 500 m L , mix 50 m L of 10× PCR buffer, 10 m L of 10 mM dNTP mixture, 10 m L of each of forward and reverse primers (10 m M ), and 5 m L of Taqpolymerase. Then, bring to 500 m L with water. Dispense 10 m L of the PCR reaction mixture to each 0.2 mL PCR tube.2.P ick each colony with a sterile toothpick, and swirl it into the PCR reaction mixture in a tube.3.P lace each PCR tube in a thermal cycler. Heat at 95°C for 2 min, and then subject to 35 cycles as follows:4.R un on a 1.5–2% agarose gel to analyze the insert size (See N otes 7 and 8 ).5.C ulture positive colonies in 2 mL of 2× YT medium at 37°C overnight.6. P repare a plasmid from the cultured E . coli , and con fi r m thatthe nucleotide sequence of the inserted DNA is correct.1.G C contents in the stem region should be less than 55%; the extension by B st polymerase may not be completed if the GC contents are higher.2.S tretches of four or more T nt should not be included in the stem region because RNA polymerase III may terminate transcription by recognizing it as a terminator in the transfected cells.3.F or the spacer sequence of oligonucleotides, we used “5-GTGT GCTGTCC-3 ¢ ,” which was derived from human microRNA 3.3.Con firmationof Insert Size by Colony PCR 4.Notes35318 shRNA Construction for Silkworms Using Bst DNA Polymerasemir26b and has been reported to be an effective spacer sequencein mammalian and D rosophila cell lines (13, 14). We con fir medthat the shRNA possessing this spacer sequence also effectivelyknocked down the target gene in silkworm cells. Furthermore,fir med that a randomly designed spacerwe con“5 ¢-AGTCCAACAGG-3 ¢” functioned ef fic iently in the silk-worm cell lines.4. s hRNA with 19 or 17 nt stem regions were inef fic ient in silk-worm cell lines (11). Therefore, the length of the stem regionshould be at least 21 nt.5. I n general, RNAi ef fic iency in the cells is known to depend onthe sequence of the stem region, and only approximately 30%of random siRNAs have been reported to show highly effectiveRNAi in cultured mammalian cells (15). However, in ourexperiment, six of eight shRNA expression plasmids—eachhaving randomly designed nucleotide sequences at the stemregion—suppressed the expression of the reporter gene bymore than 95% in silkworm cells (11). Another two constructsalso showed 75–80% reductions. These results suggest thatsequence preference in silkworm cell lines is much lower thanthat in mammalian cell lines.6. A garose gel electrophoresis should be performed after ligationof a hairpin oligonucleotide to the linear plasmid to removefree hairpin oligonucleotides (Fig. 2).7. I R DNA-inserted plasmids can be easily distinguished fromempty plasmids by colony PCR (Fig. 3). However, incompleteIR DNA is sometimes inserted into the vector. Therefore,con fir mation of the nucleotide sequence of each plasmid isnecessary.8. A n examination of nine constructions using oligonucleotideswith 21 nt of stem regions and 11 nt of the spacer sequence“5 ¢-GTGTGCTGTCC-3 ¢” revealed that 20–70% of the trans-formed clones contained correctly sized inserts by colony PCR.The ef fic iency of creating an expected DNA insert in the plas-mid would be dependent on the nt sequences of the stemregion. Analysis of nt sequences revealed that 68% of therecombinant clones possessing correctly sized inserts had cor-rect nt sequences, and that one additional nt was created in80% of the clones at the junction between the N co I site and theoligonucleotide (11).AcknowledgmentsT his work was supported by a grant from Promotion of BasicResearch Activities for Innovative Biosciences (PRO-BRAIN).354H. TanakaReferencesF ig. 2. A garose gel electrophoresis to separate the short hairpin oligonucletotide-ligated plasmid and free oligonucleotides. The short hairpin oligonucleotide-ligated plasmid ( a rrow) is recovered by Wizard SV Gel and the PCR Clean-Up System. M; 1 kb DNA ladder.F ig. 3. C on fir mation of insert size in pIEx-4-BmU6M by colony PCR. Colony PCR was per-formed in a total volume of 10 m L that contained 200 nM of each forward “5 ¢-TGTAAAGTCGAGTGTTGTTGTA-3¢” and reverse “5 ¢-CAAAACCCCACACCAACAAC-3¢”primer. In this experiment, an oligonucleotide, “5 ¢-TCATTCCTGAAGACAGCTGAG¢,” was used for construction of an shRNA expres-sion plasmid. Two different sizes of bands were detected. The band that was ampli fie d from empty plasmids (Lines 3 and 5) was 221 bp long, and the band that was ampli fie d from shRNA plasmids was 275 bp long (Lines 1, 2, 4, 6, 7, 8, 9, 10, 11). Nucleotide sequences of these plasmids were con fir med. C; pIEx-4-BmU6M. M; 100 bp DNA ladder.1. A grawal N, Dasaradhi PV, Mohmmed A,Malhotra P, Bhatnagar RK, Mukherjee SK (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67: 657–6852. D awe RK (2003) RNA interference, transposons,and the centromere. Plant Cell 15:297–301 3. F ire A, Xu S, Montgomery MK, Kostas SA,Driver SE, Mello CC (1998) Potent and speci fic genetic interference by double-stranded RNA inC aenorhabditis elegans. Nature 391:806–8114. H annon GJ (2002) RNA interference. Nature418:244–251355 18 shRNA Construction for Silkworms Using Bst DNA Polymerase5. M cManus MT, Sharp PA (2002) Gene silencingin mammals by small interfering RNAs. Nat Rev Genet 3:737–7476. H ammond SM, Bernstein E, Beach D, HannonGJ (2000) An RNA-directed nuclease mediatespost-transcriptional gene silencing in D rosophilacells. Nature 404:293–2967. B rummelkamp TR, Bernards R, Agami R(2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–5538. D ykxhoorn DM, Novina CD, Sharp PA (2003)Killing the messenger: short RNAs that silencegene expression. Nat Rev Mol Cell Biol 4:457–4679. S ano M, Kato Y, Akashi H, Miyagishi M, Taira K(2005) Novel methods for expressing RNA interference in human cells. Methods Enzymol392:97–11210. C astanotto D, Scherer L (2005) Targeting cel-lular genes with PCR cassettes expressing shortinterfering RNAs. Methods Enzymol 392:173–18511. T anaka H, Fujita K, Sagisaka A, Tomimoto K,Imanishi S, Yamakawa M (2009) shRNAexpression plasmids generated by a novel methodef fic iently induce gene-speci fic knockdown in asilkworm cell line. Mol Biotechnol 41:173–179 12. H ernandez G Jr, Valafar F, Stumph WE (2007)Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase speci fic ity. Nucleic Acids Res 35:21–3413. M iyagishi M, Sumimoto H, Miyoshi H,Kawakami Y, Taira K (2004) Optimization of an siRNA-expression system with an improved hairpin and its signi fic ant suppres-sive effects in mammalian cells. J Gene Med 6:715–72314. W akiyama M, Matsumoto T, Yokoyama S(2005) D rosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interfer-ence in Schneider 2 cells. Biochem Biophys ResCommun 331:1163–117015. U i-Tei K, Naito Y, Takahashi F, Haraguchi T,Ohki-Hamazaki H, Juni A, Ueda R, Saigo K (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32:936–948。

分解学术英语教程2 答案【1 】Unit 1 Multidisciplinary EducationKeys to the ExercisesApproaching the Topic1. 1) The aim of college education is to produce individuals who are well on their way to become experts in their field of interest.2) The growing importance of producing professionals who have the skills to work with people from a diverse set of disciplines.3) First, through an interdisciplinary approach; Second, through a multidisciplinary approach.4) College education should produce individuals who may later become expert who areinterdisciplinary problem solvers.2. 1) f2) d3) a4) e5) g6) m7) j8) k9) l10) i11) b12) h13) c4. (1) offered (2) stresses (3) ability (4) different (5) approach(6) increasingly (7) graduates (8) enter (9) positions (10) Employment6. 1) Multidisciplinary studies.2) They both believe that current college education should lay emphasis on multidisciplinarystudies, which is a prerequisite to producing future expert who are interdisciplinary problem solvers.3) Open.4) Open.5) Open.Reading about the Topic3. 1) The students have brought to MIT their individual gifts, such as their own intellect, energy, ideas, aspirations, distinctive life experience and point of view, etc.2) They represent the geographic and symbolic center of MIT.3) Names of intellectual giants.4) Leonardo da Vinci was a painter, scientist, engineer, sculptor, inventor, city planner andarchitect.4.Set 1: 1) c2) e3) d4) h5) a6) g7) f8) bSet 2: 1) e2) a3) h4) b5) c6) f7) d8) g5.(b) Para. A (b) Para. B (a) Para. C (c) Para. D(f) Para. E (e) Para. F (d) Para. G (g) Para. A6. 1) Because for him, the simplicity he appreciated in nature became his ultimate standard indesign.2) First was da Vinci’s complete disregard for the accepted boundaries between different f ieldsof knowledge. The second facet of da Vinci’s character was his respect for and fascination with nature. The third quality of da Vinci’s character was an enthusiastic demand forhands-on making, designing, practicing and testing, and for solving problems in the real world.3)“There is a good chance that you will never again live and work in a community with as many different cultures and backgrounds as MIT.”(Para. F)4) Because by doing so, the students can engage themselves in new intellectual adventures so as to use their time at MIT to its fullest potential.5) It means that “They took the initiative to search for the deepest answers, instead of sitting back and letting things happen to them.”7. Set 1: 1) h2) d3) a4) g5) f6) e7) b8) cSet 2: 1) c2) g3) d4) a5) h6) f7) e8) b8. 1) She wanted to describe for the new students three of his characteristics that particularly f it with the value of MIT.2) Because by doing so, the students can encounter the most stimulating minds and inspiringrole models, experience a life in a community with diversif ied cultures and backgrounds and participate in various new intellectual adventures, so that they can get the most out of their MIT education.3) The three of Da Vinci’s characteristics will be the heritage of MIT to be inherited by thestudents. She hoped that the new students would follow Da Vinci as well as a great manyextraordinary MIT teachers as their role models to use their time to its fullest potential.4) Multidisciplinary thinking is a mode of thinking that goes beyond disciplinary boundaries inorder to gain new ideas and fresh perspectives.9. 1) Human ingenuity will never devise any inventions more beautiful, nor more simple, nor more to the purpose than Nature does. (Para. A)2) For Da Vinci, the simplicity he appreciated in Nature became his ultimate standard in design. (Para. B)3) Be as determined in your curiosity as Leonardo da Vinci — and you will use your time atMIT to its fullest potential. (Para. F)4) MIT is a place of practical optimism and of passionate engagement with the most importantproblems of the world. (Para. G)5) I had long since observed that people of accomplishment rarely sat back and let thingshappen to them. (Para. H)10. Many scientists and engineers at MIT pursue simplicity in their design and development oftechnologies.Exploring the Topic4. 1) It is believed that a multidisciplinary approach to scientific education is of vital importance.2) Second, a multidisciplinary emphasis is believed to be a prerequisite to training individuals.3) It cannot be denied that these f irms are participating in turning out the future thinkers.4) How about examining our problems about science and technology from a liberal artsperspective.5) Surprisingly, however, our universities and colleges fail to switch from the conventionaldivisions and departmental sections to daily extracurricular multidisciplinary work.5. Reading 1 begins with a contrast —“College education has always had the responsibility to ... However, ... we also see the growing importance of producing ...”. The introduction of Reading 2 is informative as well as interesting, which arouses the readers’ interest to go on reading. Integrated Exercises2. (1) ultimate (2) spirit (3) feed (4) approach (5) property (6) represent(7) discipline (8) aspiration (9) inspire (10) perspective (11) inherit (12) generate3.(6) inherit (7) celebrity (8) speculated (9) representative (10) anatomical4. (1) D(2) A(3) C(4) B(5) D(6) A(7) B(8) C(9) A(10) C5. (1) Many celebrated researchers around the world are collaborating to develop a new vaccine.(2) The scientists’ experiment generated an unexpected outcome.(3) If the systems are restructured, their effectiveness will be ultimately integrated into theglobal economy.(4) The doctors speculate that he died of a stroke caused by a blow on the head.(5) The murder trial attracted considerable public attention.(6) The aspiration for college education inspires people in remote areas to work hard.(7) He inherited his parents’ fortune after their death.(8) He disregarded his father’s advice and left college.(9) In this address, he asked the youngsters, who embody the spirits of the nation, to join thecampaign.(10) The special diet incorporates many different fruits and vegetables.7. (1) Whoever run the red light shows a complete disregard for public safety.(2) Success, as he explained, was nothing more than a consistent pursuit of art and good luck.(3) The new product has benef ited from research work at the crossroads between biological and medical studies.(4)It was amazing that his idea echoed well the great philosopher’s belief, which he claimed not to have heard about before.(5) The one-month intense training program prepared the team members well for possibleemergencies.(6) The audience was deeply impressed with the vigor and power of the speech delivered by theenvironmentalist.(7) This traveling experience will provide you with a rare opportunity to sample a different way of life.(8) Using the limited time to its fullest potential is one of the must-have/required skills in adapting to the fast-paced modern life.(9) More and more countries are bringing robots to bear on their various problems.(10) These students are encouraged from a very early age to follow their own boundless interests well beyond the boundaries of conventional belief in obedient learning.8.A. (1) B(2) C(3) B(4) B(5) DB. As multidisciplinary design has become a trend in the industry, there is a need for moreemphasis on multidisciplinary perspectives. Educational institutions should take their role intraining individuals who can function in a collaborative environment and be prepared to facemultifaceted projects that they may not have been exposed to. However, our universities andcolleges fail to shift from traditional divisions and departmental sections to multidisciplinarywork being practiced on a daily basis outside the classroom.C. 1) F2) T3) F4) T5) TD. (1) what learning is about (2) be inquisitive (3) learn a new subject(4) analyze a new problem (5) teacher-taught (6) master-inspired(7) self-learner (8) the trap of dogma (9) no single simple answer(10) black and white (11) critical thinking (12) tolerant and supportive(13) a new thesis topic (14) flexibility (15) style of leadershipUnit 2 The Scientific MethodKeys to the ExercisesApproaching the Topic1. 1) The Scientific Method is a body of techniques for investigating phenomena and acquiringknowledge, as well as correcting/integrating previous knowledge. It involves gatheringobservable, empirical and measurable evidence, the collection of data through observationand experimentation, and the formulation and testing of hypotheses.2)Scientists put forward hypotheses to explain what is observed. They then conduct experiments to test these hypotheses. The steps taken in the experiment must be capable of replication andthe results emerge as the same. What is discovered may lead to a new hypothesis.3) Scientists are human and can be unintentionally biased; total objectivity is impossible.4) Scientists are human and can be unintentionally biased. Science uses our senses and our senses can be mistaken. We can never understand something as it really is because our very presence affects what is being studied.5) ① Science is both a body of knowledge and a process.② Science is exciting.③ Science is useful.④ Science is ongoing.⑤ Science is reliable.⑥ Science is a community endeavor.2. 1) c2) g3) e4) f5) a6) d7) h8) k9) b10) i11) j4. (1) aspects (2) process (3) satisfy (4) technologies (5) puzzle(6) collection (7) evidence (8) ensure (9) diversity (10) professional6. 1) Science.2) It brings to mind many different pictures: white lab coats and microscopes, a scientist peering through a telescope, the launch of the space shuttle, and so on.3) Science can discover the laws to understand the order of nature.4) Because it relies on a systems of checks and balances, which helps ensure that science movestowards greater accuracy and understanding, and this system is facilitated by diversity within the scientific community, which offers a range of perspectives on scientific ideas.5) Open.Reading about the Topic3. 1) The modern scientific method is characterized by confirmations and observations which “verified”the theories in question, but some genuinely testable theories, when found to befalse, are still upheld by their admirers, which rescues the theory from refutation only at theprice of destroying, or at least lowering, its scientific status.2) A theory which is not refutable by any conceivable event is non-scientific.3) Their theories were constantly verified by their clinical observations. They always fitted andwere always confirmed.4) Light must be attracted by heavy bodies (such as the sun).5) There is the risk involved in a prediction: the theory is incompatible with certain possibleresults of observation — in fact with results which everybody before Einstein would haveexpected.4. Set 1: 1) c2) a3) d4) b5) f6) e7) h8) gSet 2: 1) b2) e3) a4) f5) d6) c7) h8) g5. Para. A (b) Para. B (c) Para. C (e)Para. D (e) Para. E (a) Para. F (d)6. 1) Observations, hypotheses, and deductions, then conclusions.2) You will need to research everything that you can f ind about the problem.3) You shouldn’t change the hypothesis. Instead, try to explain what might have been wrongwith your original hypothesis.4) An important thing to remember during this stage of the scientific method is that once youdevelop a hypothesis and a prediction, you shouldn’t change it, even if the results of yourexperiment show that you were wrong.5) Because there is a chance that you made a miscue somewhere along the way.7. Set 1: 1) c2) a3) d4) b5) f6) e7) h8) gSet 2: 1) e2) g3) a4) f5) c6) b7) d8) h8. 1) Observation, as the f irst stage of the scientific method, is a way of collecting informationfrom any possible sources, which can serve as a foundation in verifying a theory. In thisprocess, one should expect an event which could refute the theory. Only through beingrefuted by new observations which are incompatible with the theory could it be falsified, which ref lects its truescientific virtue.2) A hypothesis is a possible solution to a problem, based on knowledge and research, while atheory is a hypothesis confirmed by the research findings. Every theory cannot be applied toevery situation; otherwise, it is not a good theory.3) It is always possible to verify nearly every theory, but that would rescue the theory fromrefutation at the price of destroying, or at least lowering its scientific status.4) To falsify a theory is more valuable, because a theory which is not refutable by any conceivable event is non-scientific.9. 1) Because of this personal experience and an interest in the problem, you decide to learn more about what makes plants grow. (Para. B)2) The experiment that you will design is done to test the hypothesis. (Para. D)3) Through informal, exploratory observations of plants in a garden, those with more sunlight appear to grow bigger. (Para. H)4) The judges at your science fair will not take points off simply because your results don’t match up with your hypothesis. (Para. K)5) You cannot prove the hypothesis with a single experiment, because there is a chance that you made a miscue somewhere along the way. (Para. Q)10. Observation, the initial stage of the research, requires a thorough understanding of a research project you have chosen by collecting adequate information from various sources, and isfollowed by the next stage known as hypothesis, an uncomplicated statement that defineswhat you think the outcome of your experiment will be.Exploring the Topic4. 1) Science does not include explanations based on no empirical evidence.2) The human nature of science, however, renders it unlikely to be free of personal prejudices,misapprehensions, and bias.3) The scope of science encompasses the whole universe and natural world.4) Science is a process of deciding whether the acquired evidence may prove what is mostlikely to be correct currently.5) It is not possible to prove a hypothesis with a single experiment, as chances are that amistake was made somewhere in the process.Integrated Exercises2. (1) additional (2) illustrate (3) interpret (4) conduct (5) previous (6) involve(7) design (8) verify (9) reflect (10) collect (11) research(12) support3.(6) exposure (7) constitutive (8) emphatic (9) confirmation (10) identity4. (1) A(2) B(3) A(4) D(5) A(6) B(7) C(8) A(9) D(10) B5. (1) This observation motivated Newton to develop a theory of gravity.(2) Other scholars attempt to approach the subject from an economical perspective.(3) Participating in the activity will provide one with an initial taste of the objectives ofsociology.(4) Scientists insisted there was a rational explanation for the strange phenomenon.(5) To most young people, higher education is nothing but a process of acquiring knowledge.(6) The study demonstrates the necessity of taking a much broader view in the matter.(7) The new car’s design successfully integrates art and technology.(8) China actually encountered the identical stages of its development in the early 1990s to the West.(9) The virus can spread to a document or application between computers and render thecomputer useless.(10) If the sustainable development of small economies is facilitated, their effectiveness will be ultimately integrated into the global economy.7. 1) We could not attend a conference without hearing some talks about change and challenge.2) Things seem highly optimistic in the light of numerous reports, especially from countrydistricts.3) I am in favor of the argument that urbanization should be controlled properly.4) Something slowly began to dawn on me — I still loved what I did.5) My computer does not work because it was rendered paralyzed by some viruses.6) Experts are working on the plan in question. And they’ll come to an answer.7) A national curriculum framework is logically incompatible with pupil-centered learning.8) For many women success is often achieved at the price of their married life.9) Many attempts had been made before I successfully entered a key university.10) There is a good chance that it will turn fine tomorrow.8. A. (1) B(2) A(3) C(4) A(5) BB. Understanding scientific method is critical to your scientific endeavor. The scientificmethod is a series of steps that serve as guidelines for scientific efforts, and a tool thathelps scientists solve problems and determine answers to questions in a logical format.There are two forms of scientific method: the experimental method and the descriptivemethod. The former employs numerical data and graphs, used in physical sciences, while the latter gathers Information through visual observation and interviewing, employed inzoology and anthropology. The scientific method involves five steps, namely, identifying a problem, researching the problem, formulating a hypothesis, conducting an experiment and reaching a conclusion.C. (1) The process of science, in contrast to the linear steps of the simplified scientific method,is iterative.(2) Science circles back on itself so that useful ideas are built upon and used to learn evenmore about the natural world.(3) Gregor Mendel showed that inheritance is particulate that information is passed along indiscrete packets that cannot be diluted.(4) Any point in the process leads to many possible next steps, and where that next stepleads could be a surprise.(5) Science may involve many different people engaged in all sorts of different activities indifferent orders and at different points in time.D. (1) natural world (2) investigations (3) basic question(4) information (5) Experiments (6) detailed understanding(7) built upon (8) deepen and extend (9) in the process(10) testing (11) observation (12) new direction(13) in different orders (14) represent (15) less importantUnit 3 Ancient China’s Contribution to ScienceKeys to the ExercisesApproaching the Topic1. 1) Needham is the world’s famous Sinologist and author of Science and Civilization inChina.2) The European people just take these inventions for granted. All originated in China buthave long since been adopted by the West.3) They helped to inspire the European agricultural and industrial revolutions.4) It has won five literary awards in America and been translated into 43 languages.5) His book, The Spirit of Chinese Invention, was approved by the Chinese Ministry ofEducation for use in connection with the national secondary curriculum in China.2. 1) f 2) k 3) c 4) a 5) o 6) n 7) g8) e9) d10) m 11) i12) h13) j14) l15) b4. (1) credit (2) considerable (3) befriended (4) breakthroughs (5) thoroughly(6) flown (7) academic (8) embark (9) suggested (10) staff6. 1) The overlooked great breakthroughs in ancient China.2)Dr. Needham argued that a proper book on the history of Chinese science and technologywould have a wide bearing on the general history of thought and ideas.3) He helped to bring due credit to China’s overlooked contribution to scientific innovation.4) Yes, he does. Because he believed that a proper popular book would have a wide bearing onthe general history of thoughts and ideas, which could not be possible if the book was too academic.Reading about the Topic3. 1) Both Westerners and Chinese people are ignorant of the fact that the West imported a lot ofinventions from ancient China.2) Because more than half of the basic inventions and discoveries upon which the“modernworld”rests come from China.3) Because they take many great achievements for granted, and even the Chinese themselves lost sight of the truth, so their western inheritors wouldn’t trouble themselves to know thetruth.4) Because it is always more satisfying to the ego to think that they have reached theirpresent position alone and unaided, and that they are the proud masters of all abilities and all crafts.4. Set 1: 1) d2) e3) a4) b5) c6) g7) f8) hSet 2: 1) d2) g3) e4) h5) b6) a7) c8) f5. Para. A (e)Para. B (b)Para. C (f)Para. D (a)Para. E (a)Para. F (a) Para. G (c) Para. H (g) Para. I (h) Para. J (d)6. 1) The three inventions transform-ed completely the modern world and mark-ed it off fromthe ancient and the Middle Ages.2) The European agricultural revolution, which laid the basis for the Industrial Revolution,came about only because of the importation of Chinese ideas and inventions.3) The truth that half of the basic inventions and discoveries originated from China needs tobe imparted to schoolchildren. The purpose is to let them know the truth and then tobridge the chasm between the East and the West.4) The bureaucratic organization of China in its earlier stages strongly helped science togrow; only in its later ones did it inhibit further growth, and in particular prevented abreakthrough which has occurred in Europe.5) The author points out the reasons why China was developed in the past but backward atpresent and why the West was underdeveloped in the past but advanced at present.7. Set 1: 1) c2) g3) h4) b5) f6) d7) a8) eSet 2: 1) c2) d3) g4) e5) a6) b7) f8) h8. 1) The two readings both list a series of great inventions and discoveries that originated inancient China. Reading 1 tends to be factual, while Reading 2 is more critical of the factthat the Chinese are ignorant of their ancient achievements and the Westerners simply take them for granted.2) The argument in Reading 2 is more reasonable and acceptable since the author uses a lot ofexamples and examines the question from both the Chinese and Western perspectives to illustrate his point.3) Reading 2 holds more obvious negative attitudes towards Westerners.4) It would be better if the nations and the peoples of the world had a clearer understandingof each other, allowing the mental gap between East and West to be bridged. (Reading 2) The discoveries and inventions made in Europe in the seventeenth century and thereafter depended so much in so many cases on centuries of previous Chinese progress in science, technology and medicine. (Reading 3)9. 1) He regarded the origins of these inventions as “obscure”and he died without everknowing that all of them were Chinese. (Para. B)2) Chauvinistic Westerners, of course, always try to minimize the indebtedness of Europe to China in the ancient and the Middle Ages, but often the circumstantial evidence iscompelling. (Para. C)3) In many cases we simply cannot identify the channels through which knowledge wasconveyed from East to West. (Para. C)4) Modern science which developed in the seventeenth century was a mathematization ofhypotheses about nature, combined with experimentation. (Para. D)5) One factor which must have great relevance here is the circumstance that the feudalism of Europe and China were fundamentally different. (Para. E)10.The feudalism of China differed greatly from that of Europe in that its bureaucraticorganization promoted the growth of science in ancient China but inhibited its furtherdevelopment later on.Exploring the Topic4. 1) Increasingly being bewitched by the advanced European technology, the Chinese haveforgotten their own achievements.2) A book like that would be absolutely non-academic; it would nevertheless have afar-reaching influence on the general history of thought and ideas.3) The lesson to be drawn from the history of agriculture can best illustrate the ignorance ofthe egoistic westerners.4) The Chinese and Westerners are equally surprised when they realize that modernagriculture, modern shipping and even the essential design of the steam engine alloriginated from China.5) A clear understanding among the nations and the peoples of the world would be welcomedto bridge the gap between East and West.5. 2) The deafening noise, and the glare of the engine fire, would have a bad effect on nerves.Further, being moved through the air at a high speed would do great injury to delicatelungs. The sudden plunging of a train into the darkness of a tunnel, and the equally sudden rush into full daylight, would cause great damage to the eyesight.3) What was it that enabled them to become great or successful? Were they born withsomething special? Or did their greatness have more to do with timing, devotion and,perhaps, an uncompromising personality? The answer is a never surrender attitude. If great achievers share anything, it is an unrelenting drive to succeed. There is a tendency to think that they are endowed with something super-normal.Integrated Exercises2. (1) insight (2) expertise (3) obscure (4) backward(5) undertake (6) ignorant (7) acknowledge (8) essential(9) minimize (10) shatter (11) fading (12) illustration3.(6) indebted (7) backwards (8) irrelevant (9) unparalleled (10) illusionary4. (1) C(2) D(3) A(4) B(5) A(6) A(7) C(8) D(9) B(10) D5. (1) Examples will be drawn from literature and popular media to illustrate the range ofleadership and non-leadership behaviors and competencies.(2) You’ll never be able to eliminate interruptions altogether but you can do a lot to minimizethem.(3) There is evidence that the movie reinforces negative stereotypes about women.(4) The violence to property will do nothing to facilitate that investigation.(5) Determination and effort enable-d the young man to acquire success.(6) The project was held back by budget restraints.(7) We will continue to press governments in the region to undertake political reforms.(8) This level of economic growth is unprecedented and unique.(9) This policy could isolate the country from the other permanent members of the UnitedNations Security Council.(10) The profound economic effect would accumulate day by day, and much of it might bereversible.7. (1) It must be realized that China experienced a great transformation in the last century.(2) However, it is rather questionable whether the majority of Americans know the truthabout China and Chinese people.(3) All of the information can be conveyed by simple graphs.(4) It is essential that our children absorb this lesson into their outlook on the world.(5) It is a lesson that all of us should take to heart.(6) We must never lose sight of the fact that many inventions originated in China.(7) Many of us take it for granted that technology is the top priority in economicdevelopment.(8) How was it that you had the right information at the right place and at the right time?(9) I can think of no better illustration of the importance of higher education than the fact thatmany university graduates have become the leaders in various f ields.(10) The demand for a raise ref lects as much a desire for the recognition of their success as for more money.8. A. (1) C(2) B(3) D(4) D(5) CB. China’s ancient great inventions and discoveries, as the forerunners of some of themodern technologies, both enhance the quality of human life and change Chinese history of science. The most signif icant ones are papermaking, gunpowder, compass and printing.Paper, one of the most widely used and indispensable materials, led to subsequentinnovations like paper currency, woodblock printing and ceramic movable type printing.The most important invention of gunpowder triggered a series of related discoveries like fireworks, land mine-s,naval mine-s, exploding cannonballs, multistage rocket-s, etc. The compass, originally in a crude form, was followed by a magnetic device and a magneticneedle for navigation in waters.C. (1) He accidentally stumbled upon f ireworks by mixing 3 routine kitchen ingredients —saltpeter, sulfur and charcoal and ignited them.(2) The fireworks came to be used for auspicious occasions like wedding-s, religiousceremonies and to celebrate victories and achievements, and even as rocket fuel.(3) He was called the founder of f ire crackers.(4) The gun powder tubes were found to be strong enough to launch arrows and this is howthe rocket was conceptualized and used against the Mongolians in a Kai keng battle.(5) He wanted to see how these rockets could be used for transportation.。

Chapter 12Immunochromatographic Lateral Flow Strip TestsGaiping Zhang, Junqing Guo, and Xuannian WangSummaryThe immunochromatographic lateral flow strip test is a one-step test that facilitates low-cost, rapid iden-tification of various analytes at the point of care. We have developed lateral flow strip tests for the specific qualitative or semiquantitative detection of antigens, antibodies, and haptens, such as drug residues. Here, we describe in detail the preparation of three examples of the strip tests for detection of (a) the infectious bursal disease virus; (b) Trichinella specific antibodies, and (c) Clenbuterol residues in urine samples.Key words: Immunochromatography, Lateral flow strip test, Infectious bursal disease virus, Trichinella, Clenbuterol.1. IntroductionThe membrane-based immunochromatographic lateral flowstrip test (ILFST) represents a well-established and appropriatetechnique for a variety of point-of-care and field-use applica-tions (1, 2), which was initially developed as a convenient test forpregnancy (3). As the technique eliminates the need for trainedpersonnel and expensive equipment, ILFST has become a popularplatform for the development of rapid tests since their introduc-tion in the late 1980s. The technique is widely used for thespecific qualitative or semiquantitative detection of many classesof analytes including antigens, antibodies, haptens, and evenoligonucleotides (4–10). Some of the more common ILFSTcurrently available commercially are those for pregnancy, infectionsincluding Streptococcus, Chlamydia, human immunodeficiencyvirus (HIV), and Hepatitis C virus (HCV).Avraham Rasooly and Keith E. Herold (eds.), Methods in Molecular Biology: Biosensors and Biodetection, Vol. 504© Humana Press, a part of Springer Science + Business Media, LLC 2009DOI:10.1007/978-1-60327-569-9_12169170 Zhang, Guo, and WangILFST is based on the principles of immunochromatography.A test strip typically consists of porous materials in four zonescontaining different reagents: a sample application pad, a conju-gate pad containing colloidal gold conjugate, a detection mem-brane containing narrow absorbed bands of proteins as test andcontrol lines, and an absorbent pad (Fig. 1). For the convenienceof storage and handling, the porous materials are laminated with asemirigid material of appropriate mechanical strength. The topis partially covered with a thin plastic material so as to leavea portion of the sample pad exposed for sample application (4).When applied to the sample pad, the liquid sample migratesby capillary diffusion through the conjugate pad, rehydrates thegold conjugate, and the analyte interacts with the conjugate.The complex of gold conjugate and analyte then moves onto themembrane toward the capture target, where it becomes immo-bilized and concentrated, producing a distinct signal in the formof a sharp red line. A second line, the control line, may also beformed on the membrane by the trapping of excess gold conju-gate, indicating the test is complete (11).Fig. 1. The lateral flow strip structure: (A ) Schematic representation of the lateral flow strip. A lateral flow strip typically consists of four zones of a sample pad, a conjugate pad, a membrane containing the test and control lines, and an absorbent pad on a backing plate. (B ) Lateral (top ) and over (bottom ) views of a lateral flow strip. The outmost layers of the ends of the strip are plastic cover films.Sample padCover film Cover filmMembrane Conjugate pad Absorbent pad Backing plate ABImmunochromatographic Lateral Flow Strip Tests 171The two predominant formats used in ILFST are the sand-wich and competitive reaction schemes (12). These can be bestexplained graphically as shown in Fig. 2. The test strip for humanchorionic gonadotropin (hCG) is a typical sandwich assay, andthose for drugs of abuse are competitive or inhibition assays. Inserum assays, specific antibodies are detected as an indicator ofFig. 2. Principles of the lateral flow strip test. (A ) Sandwich format: This format is usedwhen testing for larger analytes with multiple binding sites, such as viruses and anti-bodies. In the case of detecting antigens, target analytes in the sample effluent arerecognized by the antibody conjugate forming analyte–antibody complexes and boundto the immobilized antibody on the test line while the excess conjugate is trapped byanti-mouse IgG antibody on the control line, formation of two red colored lines on themembrane indicates a positive result. In the case of detecting antibodies, targets inthe sample effluent are bound with conjugated antigens forming analyte–antigen com-plexes and immobilized by SPA or anti-immunoglobulin antibodies on the test line, whilethe excess conjugate is trapped by antibodies that recognize the conjugated antigen onthe control line, formation of two red colored lines on the membrane indicates a positiveresult. (B ) Competitive format: This is used most often when testing for small moleculeswith a single antigenic determinant. In this format, free analytes in the sample solutioncompete with the immobilized analytes on the test line to bind colloidal gold conjugatedantibodies at a defined concentration. The dense of the test line is dependent on theconcentration of free analytes present in the test samples.ABPositiveNegative: Conjugated antibody/antigen;: Blotted antibody/SPA;: Analyte: Conjugated antibody;: Blotted antibody;: Blotted or free Hapten172 Zhang, Guo, and Wangvarious disease states. Here, we describe three types of ILFSTfor the detection of pathogens, specific antibodies, and haptensusing infectious bursal disease virus (IBDV) (5), Trichinella (7),and Clenbuterol (CL) (9) as examples.The general manufacturing process for production of teststrips includes the preparation of colloidal gold conjugates, appli-cation of reagents onto the membrane and pads, lamination ofthe membrane, conjugate pad, sample pad, and absorbent padonto a support backing, cutting the prepared master cards intostrips of defined length and width, and strip packaging (Fig. 3).1. The XYZ 3000 platform with BioJet Quanti 3000 and AirJetQuanti 3000 dispensers (BioDot Inc., Irvine, CA) is used formembrane blotting and conjugate dispensing.2. OV2000 Forced Air Drying Oven (BioDot Inc.) is used fordrying the blotted membrane and conjugate pad.3. LM5000 Clamshell Laminator (BioDot Inc.) is used forassembly of the master card of test strips.4. CM4000 Guillotine Cutter (BioDot Inc.) is used to cut theassembled master cards into strips.2. Materials2.1. Devices Fig.3. Outline of a lateral flow strip manufacturing process.Pretreatment Reagents Membrane blottingDryFiberglassDry Membrane system Sample padStrip packagingImmunochromatographic Lateral Flow Strip Tests 1731. Nitrocellulose Membrane: The Hi-Flow Plus membrane HF180 (Millipore Corp., Bedford, MA; Cat. No. SA3J044106) is used (see Note 2).2. Fiberglass (Millipore; Cat. No. GFCP203000) is used for making sample and conjugate pads.3. Filter paper (Millipore; Cat. No. FP10102500) is used for absorbent pads.4. Double-sided adhesive tapes (G&L Precision Die Cutting, Inc. San Jose, Cat. No. GL-187) are used for making adhesive layers on the backing plate.5. Plastic films with different colors are used for strip covers.6. Plastic boards are used for strip backing plate.1. Hydrogen tetrachloroaurate (1% (w/v) ): 0.1 g of HAuCl 4 (Sigma-Aldrich, St. Louis, MO; Cat. No. G4022) is dissolved in 10 mL of triple-distilled water and stored in a brown bottle.2. Trisodium citrate (1% (w/v) ): 0.5 g of Na 3C 6H 5O 7·2H 2O (Fluka, Buchs, Switzerland; Cat. No. 71402) is dissolved in 50 mL of triple-distilled water.3. NaCl (1 M): 5.8 g of NaCl is dissolved in 100 mL of triple-distilled water.4. K 2CO 3 (0.2 M): 2.8 g of K 2CO 3 (Fluka; Cat. No. 60108) is dissolved in 100 mL of triple-distilled water.5. Sodium borate (20 mM): 0.76 g of Na 2B 4O 7·10H 2O (Fluka; Cat. No. 71997) is dissolved in 100 mL of triple-distilled water.6. Phosphate buffered saline (PBS): 8 g NaCl, 0.2 g KCl, 1.44 g Na 2HPO 4, 0.24 g KH 2PO 4 are dissolved in 1,000 mL of triple-distilled water.1. Conjugate reagent: The anti-IBDV monoclonal antibody (mAb) is purified by a protein A column from ascitic fluids of mice carrying the specific hybridoma (5), and stored at −20°C in 1 mL aliquots.2. Test line reagent: A second anti-IBDV mAb IgG recognizing a different epitope of the viral protein is similarly purified from ascitic fluid (5), and stored at −20°C in 1 mL aliquots.3. Control line antibody: The goat anti-mouse IgG is isolated from sera of goats immunized with mouse IgG and stored at −20°C in 1 mL aliquots.1. Conjugate reagent: The excretory-secretory (ES) antigens are purified from the supernatants of cultured Trichinella muscle larvae (7), and stored at −70°C in 1 mL aliquots.2.2. Materials(see Note 1)2.3. StockSolutions2.4. Reagents2.4.1. ILFST for Detection ofIBDV Antigens2.4.2. ILFST for the Detec-tion of Anti-TrichinellaAntibodies174 Zhang, Guo, and Wang2. Test line reagent: Protein A, Staphylococcus aureus (SPA, Merk,Darmstadt, Germany; Cat. No. 539202) is dissolved in PBS(pH 7.2) at 10 mg/mL, stored at −20°C in 1 mL aliquots.3. Control line reagent: The goat anti-ES IgG is isolated fromsera of goats hyperimmunized with ES antigens (7) and storedat −20°C in 1 mL aliquots.1. Conjugate reagent: The anti-CL mAb IgG is purified from ascitic fluid (9), and stored at −20°C in 1 mL aliquots.2. Test line reagent: CL (Sigma-Aldrich; Cat. No. C5423) is conju-gated to bovine serum albumin (BSA, Amresco Inc., Solon,Ohio, Cat. No. 0332) and used as the test line reagent(BSA-CL) (9). Briefly, 5 mg of CL dissolved in 4 mL of0.01 M HCl (cooled to 0–5°C) is mixed with 10 mg sodiumnitrite. After stirring for 6 h at 4°C, 20 mg BSA predissolvedin 2 mL of 0.1 M phosphate buffer (pH 8.6) is added andthe mixture stirred for a further 6 h at 4°C. The solution iskept at room temperature (RT) overnight and then conju-gates are purified by filtration on Sephadex G-50 or dialyzedagainst PBS.3. Control line reagent: The goat anti-mouse IgG is isolated byprotein A column as above from sera of goats immunized withmouse IgG and stored at −20°C in 1 mL aliquots.1. Add 1 mL of 1% (w/v) HAuCl 4 solution in 100 mL of triple-distilled water to a clean 500 mL Erlenmeyer flask on a stirring hot plate and bring the solution to a boil (see Note 3).2. To the boiling solution, quickly add 1 mL of 1% (w/v) of trisodium citrate under constant stirring.3. After the color of the solution has changed from blue to darkred (within 3 min), continue to boil the solution for a further2 min.4. When the solution cools to RT, add triple-distilled water upto the original volume, stopper the flask to prevent evapora-tion, air circulation, and entry of contaminants and store thesample away from light.5. Scan the optical density of the red solution between 500 and600 nm, and the absorption maximum should be at 525 nm,indicating that the gold particles have an average diameter of40 nm (see Note 4).6. The colloidal gold suspension can be stored at RT in the darkfor several months.2.4.3. ILFST for Detectionof Clenbuterol Residues 3. Methods3.1. ILFST for Detec-tion of IBDV Antigens3.1.1. Preparation of Col-loidal Gold SuspensionImmunochromatographic Lateral Flow Strip Tests 1751. Adjust the pH of the colloidal gold suspension to 9.0 with 0.2 M K 2CO 3 (see Note 5).2. Dialyze the anti-IBDV mAb IgG against 100 volumes of 20 mM sodium borate for 24 h at 4°C with three changes of solution over this period.3. Add 2 mL of the IgG solution at the predetermined opti-mum concentration (see Note 6) into 10 mL of the colloidal gold solution (pH 9.0), rapidly mix, and then incubate for 20–40 min at RT.4. Add 1/10 volume of 10% (w/v) BSA in 20 mM sodium borate. Mix rapidly upon addition and incubate for 10–15 min at RT (see Note 7).5. Centrifuge for 30 min at 15,000 × g at 4°C and carefully discard the supernatant.6. Resuspend the pellet in 20 mM sodium borate containing 1% (w/v) BSA.7. Centrifuge again, discard supernatant, and resuspend the pellet in 20 mM sodium borate containing 1% (w/v) BSA.8. Wash one more time and finally resuspend the conjugate in 1 mL of 20 mM sodium borate containing 1% (w/v) BSA.1. Cut nitrocellulose membranes into 2.5 × 30 cm 2 tapes (seeNote 8).2. Dilute the other anti-IBDV mAb IgG and goat anti-mouseIgG to 1 mg/mL in PBS (pH 7.2) for the test and controllines, respectively (see Note 9).3. Put membrane tapes on the XYZ 3000 platform (Fig. 4). Dis-pense the solutions onto the membranes at 1 μL/cm as test3.1.2. Conjugationof IgG with Colloidal Gold3.1.3. Membrane BlottingFig. 4. XYZ 3000 platform with BioJet Quanti 3000.Syringe pumps Syringe pumpsBiojet Quanti 3000MembraneXYZ3000 Platform176 Zhang, Guo, and Wangand control lines in parallel using the BioJet Quanti 3000. The test and control lines are located at the center of the membrane with a space of 0.5 cm.4. Dry the bloted membranes at 42°C for 30 min in the OV2000 Forced Air Drying Oven.5. Seal the membranes in a plastic bag in the presence of desic-cants and store at 2–8°C (see Note 10).1. Cut fiberglass into 1.5× 30 cm 2 strips (see Note 11).2. Add 1 mL of the conjugate in 2 mL of 20 mM sodium borate (pH8.0) containing 2% (w/v) BSA, 3% (w/v) sucrose, 0.6 M NaCl, 0.2% (v/v) Tween 20, and 0.1% (w/v) sodium azide.3. Put the fiberglass strips on the XYZ 3000 platform. Dispense the conjugate onto the fiberglass at 15 μL/cm using the AirJet Quanti 3000.4. Dry the conjugate pads at 50°C for 30 min using the OV2000 Forced Air Drying Oven.5. Seal the strips with desiccants in a plastic bag and store at 2–8°C (see Note 12).1. Cut fiberglass into 1.5 × 30 cm 2 strips (see Note 11).2. Soak the strips in PBS (pH 7.2) containing 0.1 M NaCl, 0.2% (v/v) Tween 20, and 0.1% (w/v) sodium azide (see Note 13).3. Dry the strips at 50°C for 30 min using the OV2000 Forced Air Drying Oven.4. Seal the sample pads with desiccant in a plastic bag and store at RT.1. Cut filter paper in 2.5 × 30 cm 2 strips.2. Seal the absorbent pads with desiccant in a plastic bag and store at RT.1. Put double-sided adhesive tapes on one side of the support cards (see Note 14).2. Cut the adhesive board into 7.5 × 30 cm 2 strips to make adhe-sive cards.1. Using a LM5000 Clamshell Laminator or manually, the precut materials are assembled into a lateral flow strip master card(Fig. 5).2. As illustrated in Figs. 1 and 5, the blotted membrane isstamped in the middle of the adhesive backing card. The con-jugate and sample pads are affixed sequentially next to themembrane with a 1–2 mm overlap at the sample end, and the3.1.4. Preparationof Conjugate Pads3.1.5. Preparationof Sample Pads3.1.6. Preparationof Absorbent Pads3.1.7. Preparationof Adhesive Cards3.1.8. Assemblyof Master CardImmunochromatographic Lateral Flow Strip Tests 177absorbent pad to distal end of the membrane with 1–2 mmoverlap (see Note 15).3. Cover the sample and conjugate pads at the sample end, andthe absorbent pad at the distal end with different color films,respectively.1. Cut the assembled master card into 0.3 cm strips using a CM4000 guillotine cutter.2. Seal the test strips with desiccants in a plastic package andstore at 2–8°C (see Notes 10 and 12).1. Simply break up the bursal tissue in a whirl pack with finger massage, and dilute in PBS or water.2. Dip the IBDV antigen test strip into the solution for 10–20 sat RT.3. Take the strip out and place horizontally for 1–2 min toobserve the result.4. If both the test and control lines turn red, the sample isrecorded as positive, indicating the presence of IBDV anti-gens in the sample. When the control line but not thetest line is colored, it is considered as negative. No coloredband appears on the membrane indicates an improper testingprocedure or deterioration of the strip, and the test should berepeated using a new strip (Fig. 6A ).The protocol is similar to that described above. Differences are clarified as below.3.1.9. Cutting and Packag-ing 3.1.10 Detectionof IBDV Antigens 3.2. ILFST for Detec-tion of TrichinellaAntibodies Fig. 5. Assembly of the lateral flow strip master card. (A ) Components for the strip lamination: 1, white cover film with arrows (sample end); 2, sample pad; 3, conjugate pad; 4, blotted membrane; 5, absorbent pad; 6, blue cover film (distal end); 7, adhesive backing plate. (B ) The blotted membrane, conjugate, sample and absorbent pads are put in the cor-respondent places on the adhesive backing plate, sequentially as described in the text. (C ) A master card. The sampleand distal ends are covered with the white and blue films, respectively.178 Zhang, Guo, and Wang1. Adjust the pH of the stock colloidal gold solution to 8.5 with 0.2 M K 2CO 3.2. Dialyze the ES antigens against 20 mM sodium borate at 4°C.3. Prepare the ES-conjugate as described in Subheading 3.1.2.1. Dilute the SPA and goat anti-ES IgG solutions to 1 mg/mL inPBS (pH7.2).2. Filter the solutions using 0.22 μm filters.3. Dispense the SPA and goat anti-ES IgG solutions as test andcontrol line reagents, using the BioJet Quanti 3000 on 2.5 ×30 cm 2 nitrocellulose membranes as described in Subheading3.1.3.1. Dilute blood or serum samples of human, pigs, cats, or dogs in PBS or water at 1:100.2. Dip the ES-antibody test strip into the solution for 10–20 sat RT.3. Take the strip out and place horizontally for 1–2 min toobserve the result.3.2.1. Conjugationof ES Antigens withColloidal Gold 3.2.2. Membrane Blotting 3.2.3. Detectionof Trichinella Antibodies Fig. 6. Detection of analytes using the lateral flow strips: (A ) Result of IBDV antigen detection; (B ) Result of anti-ES anti-body detection; (C) Result of CL residue detection.4. If both test and control lines appear in red, the sample is recorded as positive, indicating the presence of antibodies specific to ES antigens. Appearance of only the control line but not the test line is considered as negative. No colored line appearing on the membrane indicates an improper testing procedure or deterioration of the strip, and the test should be repeated using a new strip (Fig. 6B ).The protocol is similar to that described in Subheading 3.1, and differences are clarified as below. In this case, anti-CL mAb IgG is conjugated with colloidal gold and the hapten conjugate, BSA-CL, is used on the test line. A positive standard sample of the hapten is required for the quantitive detection.1. Adjust the pH of the stock colloidal gold to 9.0 with 0.2 M K 2CO 3.2. Dialyze anti-CL mAb IgG against 20 mM sodium borate at 4°C.3. Prepare the CL antibody conjugate as described in Subheading 3.1.2.1. Dilute the BSA-CL and goat anti-mouse IgG to 1 mg/mL in PBS (pH7.2) for application as the test and control line capture reagents, respectively.2. Filter the solutions using 0.22 μm filters.3. Dispense the test and control line solutions using the BioJet Quanti 3000 on 2.5 × 30 cm 2 nitrocellulose membranes as described in Subheading 3.1.3.1. Dissolve 10 mg CL in 10 mL PBS (pH 7.2).2. Dilute the CL solution with PBS to the final concentration of 1 ng/mL as the positive standard.3. PBS is used as the negative standard.1. Dip CL test strips into urine samples and standard solutions for 10–20 s at RT, respectively.2. Take the strips out and place horizontally for 5 min to observethe result.3. Both control and test lines develop on the strip to whichapplied the negative standard. The test line is invisible andthe control appears on the strip to which applied the positivestandard. When testing urine samples, if the test line developequal intensity to that seen with the negative standard, thesample is recorded as negative indicating no detectable levelof CL. If the intensity of the test line is weaker than thatof the negative standard, the sample is considered as positive3.3. ILFST for Detec-tion of CLResidues3.3.1. Conjugationof Anti-CL mAb IgG withColloidal Gold3.3.2. Membrane Blotting3.3.3. Preparation ofStandard ReferenceSolutions3.3.4. Detection of CLResidues in Urine Samplesand indicates the presence of CL residues at a concentra-tion of less than 1 ng/mL; An invisible test line indicatesthat the concentration of CL residues at 1 ng/mL or higher.No colored bands appearing on the membrane indicates animproper testing procedure or deterioration of the strip, andthe test should be repeated using new strips (Fig. 6C).4. Notes1. Materials for the lateral flow strips including membranes,fiberglass, and filter paper are commercially available fromseveral manufactures, such as Millipore Corp., Schleicher &Schuell (S&S, Keene, NH), Whatman, Inc. (Clifton, NJ), andPall Biosciences (East Hills, NY) etc.2. The membrane is the most important material in a lateral flowstrip. Protein-binding capacity and capillary flow rate are themost critical parameters to be considered for the membraneselection, and these are determined by polymer composition,pore size, porosity, thickness, etc. Membrane manufacturersgenerally offer a wide variety of material types and pore sizesand trial investigations should be undertaken to find the optimalmembrane for a specific use. In practice, nitrocellulose mem-branes with the pore sizes of 5–15 μm and supplied as supportedand unsupported forms are the most commonly used (13).3. Containers for colloidal gold must be cleaned thoroughly. It isbest that all glassware be sliconized and autoclaved before eachuse. It is recommended that all solutions be filtered througha 0.22 μm filter before use. Caution: The flask will be hot. Besure to wear thermal gloves while handling.4. The size of gold colloidal particles is directly dependenton the amount of trisodium citrate used in its preparationprocess, and decreases with increasing amounts of sodiumcitrate (Table 1). The procedure developed by Frens (14)is most commonly used to produce 40-nm gold particles,which are considered to be optimal for lateral flow tests.High quality gold particles should be monodisperse andspherical, and should include less than 5% uneven shapes(15). Colloidal gold solutions from reputable suppliers canbe used to ensure that compliance with stringent qualitycontrol procedures is met.5. In general, a protein maximally adsorbs on the gold surfaceat the isoelectric point (pI) of the molecule or 0.5 pH unitshigher (16). Therefore, the pH value of colloidal gold solutionshould be adjusted slightly higher than pI value of proteinsprior to conjugation.6. The optimum ratio of protein concentration to colloidal goldsolution is determined prior to conjugation by the follow-ing steps: (1) Serially twofold dilute antibody solution with25 μL of distilled water in microplate wells; (2) Add 125 μLof colloidal gold solution to each well; (3) Incubate for 5 minat RT; and (4) Add 125 μL of 1 M NaCl solution to eachsample. The color of samples gradually changes from bril-liant red to blue as the concentration of protein decreases.The highest dilution of the solution with no change of colorcontains the optimum protein for colloidal gold labeling.7. Following the conjugation of the specific protein withcolloidal gold, the conjugate must then be stabilized witha suitable agent, such as BSA, gelatin, polyethylene glycol(PEG), or casein. The stabilizer can reduce nonspecific inter-actions by blocking any sites on the colloidal surface thatare not occupied by the specific protein, and helps provide amore-stable suspension (11).8. There are manufacturers, such as Millipore, S&S, providingcut membranes in defined width for lateral flow strips.9. The blotting solutions should be filtered through 0.22 μmfilters to prevent clogging of the dispensers.10. It is important to prevent rehydration of the blottedmembrane.11. There are manufacturers, such as Millipore, S&S, providingcut fiberglass in defined width and length for lateral flowstrips.12. It is important to keep the conjugate pad dry. If the pad isexposed to high humidity, water can complex with the sugarmolecules, converting them to syrup and delaying particleresolubilization (13).13. The concentrations loaded into the pad will be determinedby the bed volume of the material, the volume of sampleanalyzed in the strip, and the variability of the samples (13).Table 1Sizes of gold particles correlated with addition of trisodium citrateVolumes (mL) of 1% (w/v) trisodium citrate added in 100 mL of HAuCl 4 solution0.300.450.70 1.00 1.50 2.0014. The backing materials used in test-strip assembly aregenerally polyester, styrene, or polyvinyl chloride (PVC) withan adhesive layer on one side. The pressure-sensitive adhe-sive has proven to be a reliable means of bonding togetherthe component materials. Selection of an appropriate typeof adhesive that offers the best compromise between bondstrength and the extent of adhesive migration is importantto improve processing and increase the shelf-life of strips.Adhesive migration especially during long periods of shelfstorage can result in blocked pores, hydrophobic patches,and material rewetting problems that may interfere withthe performance of the test (17). There are commercialbacking cards.15. It is very important that the extent of the overlaps be consis-tent so that the flow dynamics are uniform on all of the stripsmanufactured.This work was supported by grants from the National BasicResearch Program of China (Grant No. 2005CB523203) and theChinese High Technology Research and Development Project(Grant No. 2001AA249031). The authors thank Dr. Norman A.Gregson at King’s College, University of London for his criticalreview of this manuscript.AcknowledgmentsReferences1. Paek, S. H., Lee, S. H., Cho, J. H., and Kim,Y. S. (2000) Development of rapid one-step immunochromatographic assay. Methods 22, 53–602. O’Farrell, B., and Bauer, J. (2006) Develop-ing highly sensitive, more-reproducible later-al-flow assays. Part 1: New approaches to old problems. IVD Technol. 7(6), 41–503. May, K. (1991) Home tests to monitor fertility.Am. J. Obstet. Gynecol. 165, 2000–20024. Huang, C., and Fan, E. (1998) One-stepimmunochromatographic device and method of use. U.S. Pat. 5,712,1725. Zhang, G. P., Li, Q. M., Yang, Y. Y., Guo,J. Q., Li, X. W., Deng, R. G., Xiao, Z. J., Xing, G. X., Yang, J. F., Zhao, D., Cai, S.J., and Zang, W. M. (2005) Development of a one-step strip test for the diagnosis of chicken infectious bursal disease. Avian Dis.49, 177–1816. Ketema, F., Zeh, C., Edelman, D. C., Saville,R., Constantine, N. T. (2001) Assessment of the performance of a rapid, lateral flow assay for the detection of antibodies to HIV.J. Acquir. Immune Defic. Syndr. 27, 63–70 7. Zhang, G. P., Guo, J. Q., Wang, X. N., Yang,J. X., Yang, Y. Y., Li, Q. M., Li, X. W., Deng, R. G., Xiao, Z. J., Yang, J. F., Xing, G. X., and Zhao, D. (2006) Development and eval-uation of an immunochromatographic strip for trichinellosis detection. Vet. Parasitol.137, 286–2938. Mapes, J. P. (2002) Indirect label assay devicefor detecting small molecules and method of use thereof. U.S. Pat. 6, 376,1959. Zhang, G. P., Wang, X. N., Yang, J. F., Yang,Y. Y., Xing, G. X., Li, Q. M., Zhao, D., Chai, S. J., and Guo, J. Q. (2006) Development of an immunochromatographic lateral flow test strip for detection of β-adrenergic agonist。

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