Creation of humans with desirable features. This has led to rising interest in the forward approach, which will be taken into consideration by dedicating the last part of the article to this topic. The .gov means its official. , Yeast genome-wide drug-induced haplo-insufficiency screen to determine drug mode of action, Bain, J, McLauchlan, H, Elliott, M, and Cohen, P (2003). Genetic engineering. Cons of Genetic Engineering / Disadvantages of Genetic Engineering. These findings suggest a model reminiscent of the centromere-microtubule interaction in eukaryotic cells. Pseudomonas putida KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. The first official genetic manipulation happened in 1972 by Paul Berg when he combined the DNA from a monkey virus with the lambda virus. , Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity, Nobel Lectures, Physiology or Medicine 1901-1921, Fenteany, G, Standaert, R F, Lane, W S, Choi, S, Corey, E J, and Schreiber, S L (1995). Discusses applications of genetic engineering such as cloning in plant cells and cloning in mammalian . (Genetic Engineering Approval Committee), which will make decisions regarding the validity of GM research and the safety of introducing GM-organisms for public services. The BLS estimates that a biological technician (such as a genetic engineer with a bachelor's degree) earned a median of $43,800 per year or $21.06 per hour doing the routine work of running lab experiments designed by the research scientists. For example, genetic engineering can be used to produce plants that have a higher nutritional value or can tolerate exposure to herbicides. SnapFect works via a bio-orthogonal reaction. Moreover, the effect of small molecules is usually equally strong in all cells treated, while methods like antibody microinjection and RNAi are highly variable. Chemical engineers develop economical commercial processes to convert raw materials into useful products. Epub 2015 Aug 7. about navigating our updated article layout. However, tools for studying, engineering, and modulating protein complexes and biosynthetic enzymes in this organism are largely underdeveloped. 4. , Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening, Khodjakov, A, Copenagle, L, Gordon, M B, Compton, D A, and Kapoor, T M (2003). Engineering digitizer circuits for chemical and genetic screens in human cells . to identify the target of the actin polymerization inhibitor pirl1 (Peterson et al., 2006). Epub 2008 Apr 24. A recent example of the powerful combination of phenotypic screen and genetics possible in this system is provided by the group of Peter Roy who set up a phenotype-based screen to identify calcium channel inhibitors in the nematode Caenorhabditis elegans (Kwok et al., 2006). Fig.3A].3A]. Despite recent progress, this organism lacks sufficient engineering tools to fully leverage its native strengths for application in metabolic engineering (Jin et al., 2020). A heterozygous mutation in one copy of the FKBP12 alleles (marked with an asterisk) impairs the interaction between FKBP12 and rapamycin. The phenotype generated by genetic manipulation can, therefore, often be different from the result of a chemical genetic experiment (Knight and Shokat, 2007). Importantly, this fraction was also able to enhance PIP2-induced actin polymerization in the absence of pirl1 suggesting that the observed complementation was specific and not mediated by an unspecific binding partner of pirl1. The term genetic engineering is generally used to refer to methods of recombinant DNA technology, which emerged from basic research in microbial genetics. Chemical Genetic Engineering- Chemical genetic engineering prepares genes for applied genetic engineering activities by classifying and separating genes by genetic mapping, genetic coding, and gene interaction. To reduce the side effects caused by the free phenolic group of salicylic acid Eichengrn and Hoffmann acetylated the free hydroxyl functional group yielding acetylsalicylic acid. , Selective inhibition of c-MycMax dimerization and DNA binding by small molecules, Chemical genetics: where genetics and pharmacology meet, Kunz, J, Henriquez, R, Schneider, U, Deuter-Reinhard, M, Movva, N R, and Hall, M N (1993). provide an elegant example where the cellular target of a compound derived from a phenotype-based approach was identified by biochemical methods. However, as mentioned before, modulating the activity of a protein might result in a phenotype different from the one induced by removing the protein from the system (Knight and Shokat, 2007). The fear for unintended selection and any unwanted transfer of genes. This process is genetic engineering in much simpler terms than we think of it today. Finally, we would like to mention that chemical genetic screens have already been performed in vertebrates. The strong correlation between scientific progress and the advancements made in drug discovery is nicely exemplified by the timeline of breakthroughs related to acetylsalicylic acid and its usage as a drug (Vane and Botting, 2003): Antiquity: first mentions of willow bark extract as an effective antipyretic and analgesic. could demonstrate that dihydromotuporamine C, a compound in preclinical studies that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism (Baetz et al., 2004). Gene therapy seeks to alter genes to correct genetic defects and thus prevent or cure genetic diseases. Chemical and Genetic Engineering Strategies to Improve the Potency of Pharmaceutical Proteins and Enzymes Authors: Dimitris Platis Institute of Child Health - Greece Nikolaos Labrou. However, most of the known antimitotic compounds target the same protein, namely tubulin. While it is a very versatile tool that effectively complements the toolbox of biologists, chemical genetics is not a universal solution for any biological problem and success strongly depends on the right choice of the biological question to be answered. FOIA The rapamycin-FKBP12 complex inactivates its target TOR, a protein kinase family regulating cell cycle progression in response to nutrient availability. Identification of domains influencing assembly and ion channel properties in alpha 7 nicotinic receptor and 5-HT3 receptor subunit chimaeras. Rapamycin is a membrane-permeable small molecule that binds to and inhibits FKBP12. Obviously, only small organisms can be kept and are able to survive in multi-well plates. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. We think that setting up core facilities providing libraries and screening infrastructure, which is happening in many institutions now, will be very helpful in this context. Biol. By correlating the segregation pattern of Nemadipine-A resistance with the segregation of single nucleotide polymorphisms (Wicks et al., 2001) they discovered that Nemadipine-A resistance was always linked to mutations within the egl-19 gene locus, which encodes the only L-type calcium channel 1-subunit in C. elegans. genetic engineering, the artificial manipulation, modification, and recombination of DNA or other nucleic acid molecules in order to modify an organism or population of organisms. The term was coined in the 1990s, when combinatorial chemistry was developed as a fast method to synthesize large compound libraries [Mitchison (1994) Towards a pharmacological genetics, Chem. Chemical GE includes genes interaction, gene mapping and gene coding (Greene 12). Obviously, only small organisms can be kept and are able to survive in multi-well plates. "Glyphosate is the most widely used chemical weedkiller in human history because of genetic engineering," said Dave Murphy, the founder of Food Democracy Now, an advocacy group that tests. This genetic engineering meaning includes any organisms in which humans have altered the organism's DNA. Since farming began, humans have been selectively breeding different plants, doing so provided more food and better food for all. Hydrothermal carbonization of biomass: A summary and discussion of chemical mechanisms for process engineering. Genetic code expansion for the incorporation of unnatural amino acids (unAAs) into proteins can . Accessibility The new PMC design is here! In both traditional and chemical genetics, the reverse approach starts with the manipulation of a known protein or its function, while the forward approach starts with random manipulation to obtain a defined phenotype and the responsible protein is identified in a second step. It starts in the lab, much like science, yet progresses through the design and implementation of a full . To work as a genetic engineer, you will need a bachelor's degree at a minimum. Disclaimer, National Library of Medicine The https:// ensures that you are connecting to the This target identification approach is based on the assumption that the inhibitory effect of the small molecule can be suppressed by adding the target protein in high concentrations. Additionally, it is a common strategy to confirm that the compound-induced phenotype mimics the one caused by RNAi-mediated depletion of the assumed target protein. The https:// ensures that you are connecting to the Careers, CORRESPONDENCE: Thomas U. Mayer: mayer@biochem.mpg.de. For the future, it will be important to elucidate how the compound increases VEGF levels in D. rerio. Epub 2005 Oct 17. By screening for spontaneous yeast mutants resistant to the antifungal effect of rapamycin they identified TOR1DRR1 and TOR2DRR2 (target of rapamycindominant rapamycin resistant) as the cellular targets of rapamycin. The following year American microbiologist Hamilton O. Smith purified so-called type II restriction enzymes, which were found to be essential to genetic engineering for their ability to cleave a specific site within the DNA (as opposed to type I restriction enzymes, which cleave DNA at random sites). Engineering a carbon-concentrating mechanism into E. coli. Genetic engineering became possible only when scientists had discovered exactly what is a gene . , Acetylation of prostaglandin synthase by aspirin, Rupasinghe, C N, and Spaller, M R (2006). In 1827, Emanuel Merck introduced morphine, a naturally occurring alkaloid, as the first commercially available pure drug in large-scale production. In general, it is possible to perform a screen in systems ranging from pure proteins to whole organisms. , Pharmacological inhibitors of glycogen synthase kinase 3, Discovery of protein phosphatase inhibitor classes by biology-oriented synthesis, Cyclin dependent kinases and cell cycle control, An inhibitor of Bcl-2 family proteins induces regression of solid tumours, Natural products and combinatorial chemistry: Back to the future, Peterson, J R, Lebensohn, A M, Pelish, H E, and Kirschner, M W (2006). . , Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map, Zeng, X, Tamai, K, Doble, B, Li, S, Huang, H, Habas, R, Okamura, H, Woodgett, J, and He, X (2005). This may involve changing a single base pair (A-T or C-G), deleting a region of DNA or adding a new segment of DNA. [Development of antituberculous drugs: current status and future prospects]. were able to dissect the role of MreB in bacterial cell division. The earliest drugs, which have been used empirically for centuries, were mostly natural compounds that were discovered by chance. The third type of genetic engineering is Analytical GE which involves computer modeling. Using a fluorescence-based assay, the authors succeeded in the identification of pirl1, a micromolar inhibitor of the phosphatidylinositol-bisphosphate (PIP2)-induced actin polymerization in Xenopus extract. government site. Epilepsy Curr. Chemical engineering . The transformed bacterium is converted into somatostatin synthesising factory. to a sheep. 2022 Apr 29;16:882366. doi: 10.3389/fncir.2022.882366. The diversity of ion channel tools accessible from this approach will be useful for examining the relationship between neuronal activity and animal behavior, as well as for cell biological and physiological applications requiring chemical control of ion conductance. 2022 Mar 25;11(7):1110. doi: 10.3390/cells11071110. These screens can be done in a high-throughput mode, using 96 well-plates, where each well contains cells treated with a unique compound. We combined chemistry and protein engineering to enable the systematic creation of a toolbox of ligand-gated ion channels (LGICs) with orthogonal pharmacologic selectivity and divergent functional properties. Chem. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In this technique, a recombinant DNA is constructed and inserted into the host genome using a vector. Molecular matchmaking for neural control. Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue. 8600 Rockville Pike Bethesda, MD 20894, Web Policies Christopher J Magnus 1, Peter H Lee, Deniz Atasoy, Helen H Su, Loren L Looger, Scott M Sternson. The red triangle indicates the area of possible combinations of screening system and phenotype. Gene transfer, genetic engineering and rDNA are now known to us, we are using them to perform different genetic and genomics experiments. will also be available for a limited time. , Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation, Quality, not quantity: The role of natural products and chemical proteomics in modern drug discovery, Photoaffinity study of the cellular interactions of ilimaquinone, Drug Discovery and Development: Technology in Transition, Raught, B, Gingras, A C, and Sonenberg, N (2001). were not able to identify the target, they could demonstrate that zebrafish embryos treated with the identified compound have elevated levels of the angiogenic cytokine VEGF. Despite the importance of proteins as therapeutic agents, they have a number of disadvantages in comparison to small-molecule drugs, including immunogenicity and antigenicity, poor efficacy and oral bioavailability as well as, in many cases, short serum half-lives. Applied GE is a type of genetic modification which includes cloning and transgenesis. The mentioned problems, however, should not leave the impression that chemical genetics requires alot of resources and is successful only in exceptional cases. Of the 308 identified bioactive compounds they focused on one compound, named Nemadipine-A. Our results show how concerted chemical and genetic engineering of a complex ligand-binding interface can be used to develop pharmacologically selective actuators and small-molecule effectors for construction of a LGIC toolbox. A reverse chemical genetic screen with a known target might be more successful if a biased library is used, containing compounds selected for the structure of the protein or derived from inhibitors of other proteins of the same family, e.g., adenosine triphosphate (ATP) analogues to screen for inhibitors of protein kinases. Biochemical fractions were assayed for their ability to restore actin polymerization in the presence of the small molecule pirl1. Therefore, efficient collaborations are often mandatory for a project to be successful. Chemical engineering uses principles of chemistry, biology and economics to efficiently use, produce, design, transport and transform products, energy and materials. In these initial screens, all recessive mutations were mapped to FKBP12 and all dominant mutations were mapped to TORDRR. Strategies for Targeting Neural Circuits: How to Manipulate Neurons Using Virus Vehicles. Effect of heterozygous mutations in the FKBP12 and TOR genes, respectively. (3) Assess compound effectivity in animal models and, if successful, use it in clinical trials. , Synthetic small molecules that control stem cell fate, Driscoll, M, Dean, E, Reilly, E, Bergholz, E, and Chalfie, M (1989). The task of target identification can be lessened by simplifying the screening system. One very useful model organism here is the zebrafish (Danio rerio), which has a long tradition in developmental biology. Following structure-activity relationship studies, they identified one bioactive compound, named TWS 119, which was sufficiently potent to identify its binding partner via affinity chromatography (chemical proteomics). Br J Pharmacol. 18201840: isolation of salicylic acid from the willow bark, 1874: commercial organic synthesis and distribution by Heyden Chemical Company, 1853: Charles Frederic Gerhardt synthesizes for the first time acetylsalicylic acid. Although Peterson et al. While screening in complex organisms enables us to study problems that were out of reach using cell lines or unicellular organisms, target identification can often be very difficult. , Gridlock, a localized heritable vascular patterning defect in the zebrafish, Wicks, S R, Yeh, R T, Gish, W R, Waterston, R H, and Plasterk, R H (2001). HHS Vulnerability Disclosure, Help For example, corn has been modified by food engineers to be resistant to specific bugs. In 1973, Herbert Boyer and Stanley Cohen were the first to . Genetic engineering can be applied to any organism, from a virus? There are three major types of genetic engineering. The last piece of the puzzle to make chemical genetics useful for biological research consisted of the biochemical and molecular genetic tools necessary for target (receptor) identification and characterization. In fact, Ehrlich even attempted to analyze the drug-receptor interaction systematically and performed screens for drugs effective against trypanosomes, the pathogen causing sleeping disease. Obtain a bachelor's degree. Previously, these assays were mainly focused on enzymes because their activity could be easily measured and it was assumed that proteins lacking any enzymatic activity are hardly targetable by small molecules. Anderson, C, Bartlett, S J, Gansner, J M, Wilson, D, He, L, Gitlin, J D, Kelsh, R N, and Dowden, J (2007). This site needs JavaScript to work properly. DNA fingerprinting. Bassackwards: Construction Spanish and Other Signs of the Times LGICs constructed for neuronal perturbation could be used to selectively manipulate neuron activity in mammalian brains in vivo. A successful example of chemical genetics applied in bacteria is provided by the group of Lucy Shapiro who was interested in the function of MreB, the prokaryotic homologue of actin, in the process of chromosome segregation (Gitai et al., 2005). While in higher organisms, these concepts are already well established [many of them using small molecules (Peterson and Mitchison, 2002)], in bacteria, the cytoskeleton has been discovered only recently (Gitai, 2005) and the systematic mechanistic analysis of bacterial cell division is just beginning. Genetic Engineering task sheet Solutions.pdf. PMC Target identification strategies which at least partially worked in zebrafish include the use of compound libraries already containing a linker molecule for immobilization on a solid matrix, which greatly facilitates chemical proteomics approaches (Khersonsky et al., 2003), and the educated guess approach (Anderson et al., 2007). Corrections? The resulting high CO 2 concentration achieved within the carboxysome maximizes the activity of Rubisco and therefore increases overall CO 2 uptake ( Figure 1A ). FOIA Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis. Chemical Genetic Engineering: It is the examination of the capacity of proteins and signs transduction pathways in cells by the screening of compound libraries of little atoms. Genetic engineering can be defined as manipulation of an organism's genes with the help of biotechnology. Genetic Engineering is an area that has gotten Beyond Pesticides' attention in light of the pesticide paradigm that is being pushed via genetically engineered food crops. Science. Accessibility Thus, the first pharmacological inhibitor of GSK3 was identified, which was followed by the discovery of a plethora of highly potent inhibitors with varying specificity profiles from both natural sources and synthetic libraries (Meijer et al., 2004). Advances in the pharmacology of lGICs auxiliary subunits. (Fig.3).3). While every effort has been made to follow citation style rules, there may be some discrepancies. Thus, the more complex the screening conditions are, the more likely it is that the identified compounds meet the required criteria of, e.g., specificity, membrane permeability and stability. A way to minimize the effort required to apply chemical genetics to biological problems is to use small molecules with identified targets. Recently, chemical genetics has also started to embrace multicellular organisms as a screening system especially in the field of developmental biology. This development resulted in the first commercial success with the registration of imatinib (Gleevec) by Novartis, which is effective in chronic myeloid leukaemia (CML). The term genetic engineering initially referred to various techniques used for the modification or manipulation of organisms through the processes of heredity and reproduction. Molecular tuning of fast gating in pentameric ligand-gated ion channels. Discovery and structure-activity relationship of quinuclidine benzamides as agonists of alpha7 nicotinic acetylcholine receptors. ( A ), PSAM/IPD chimeric ion channels for neuron activation and silencing. Clearly, this is a challenge for both chemists and biologists and requires the discovery of sophisticated methods to synthesize complex molecules, to perform elaborate small molecule screens and finally to demonstrate specificity of the identified bioactive compounds. Although this work is promising, functional tests to prove relevance of the identified targets are missing. The term genetics pays tribute to the analogy between chemical genetics and the classic genetic approach, where manipulations at the gene level are used to draw conclusions about the function of the corresponding protein. The site is secure. As mentioned before, other well established methods exist to modulate protein activity both in vitro and in vivo, so how can chemical genetics help to gain new insights and how does it complement or improve the existing tools? Would you like email updates of new search results? (, Selective interactions between ligands and mutated 7-5HT3 chimeric ion channels. Enter your email address to receive updates about the latest advances in genomics research. However, they are less suitable for experiments requiring precise timing and fast reversibility of the perturbation. Sci. Although the application of small molecule screens to multicellular organisms is a rather novel approach, recent publications already indicate the impact chemical genetics will have on this aspect of biology in the future. HHS Vulnerability Disclosure, Help While seemingly innocuous, CRISPR sequences are a crucial component of the immune systems [3] of these simple life forms. , Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components, Peterson, J R, and Mitchison, T J (2002). Genetic engineering based on recombination was pioneered in 1973 by American biochemists Stanley N. Cohen and Herbert W. Boyer, who were among the first to cut DNA into fragments, rejoin different fragments, and insert the new genes into E. coli bacteria, which then reproduced. The following examples support this claim: mechanical engineers produce toothpaste, while IT engineers refine the fuel used in automobiles. Med. ADA (adenosine deaminase) deficiency is a disease like combined immune deficiency which killed the bubble boy David in 1984. October 19, 2022 September 8, 2022 by Alexander. Would you like email updates of new search results? Subsequent in vitro assays revealed that pirl1 inhibits CDC42RhoGDI but not Arp23, which is acting downstream of CDC42RhoGDI. Epub 2009 Aug 25. applied a combination of two phenotype-based screens and in vitro assays to select for antimitotic molecules that do not target tubulin (Mayer et al., 1999). The addition of compounds to the . Biofuels, Bioprod. FEBS J. Recent progress in biomolecular engineering. , Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin, The mechanism of action of colchicine. As an example, biologists have now learned how to transplant the gene that produces light in a firefly into tobacco plants. The DREADDful Hurdles and Opportunities of the Chronic Chemogenetic Toolbox. By adding A22 at different stages of the cell cycle the authors demonstrated that the activity of MreB is dispensable for DNA replication in Caulobacter crescentus but essential for correct segregation of the ori loci (origin of replication). Since appropriate mutants that mimic human disease already exist, this could be used to identify new drug candidates for treatment of arrhythmia. Med. Finally, we want to emphasize that the ultimate goal of chemical genetics is to provide biologists with a toolbox of specific small molecules for every protein in a cell. PMID: 18691050 All these methods have proven to be extremely helpful, as they possess a high degree of specificity. Smita Rastogi and Neelam Pathak. Epub 2022 Sep 8. Compounds can also be used across species and in different systems (e.g., in vitro, tissue culture cells, or multicellular organisms), if the target protein is well conserved, whereas mutants have to be generated one by one, which is a tedious and time-consuming process. Mutant TOR protein unable to bind rapamycin-FKBP12, however, is dominant in conferring resistance against rapamycin. That means that rapamycin is effective as long as cells express at least a minimal amount of wildtype FKBP12 (corresponding to a heterozygous FKBP12 mutation), while it is not effective if there is a minimal amount of TORDRR, which cannot bind to the rapamycin-FKBP12 complex and thus stays active (corresponding to a heterozygous TORDRR mutation). There have been many examples of elegant chemical genetic solutions to biological problems in the past, long before this term even existed. Also, they have known exercises or essentially assorted synthetic constructions. 1Chemical Genetics, Independent Research Group, Department of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany. Let us know if you have suggestions to improve this article (requires login). Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins. Caenorhabditis elegans has been shown to be a powerful tool for target identification already two decades ago, e.g., with the identification of tubulin as a target of nematocides (Driscoll et al., 1989) and was also useful for the characterization of the target proteins of drug candidates (Fitzgerald et al., 2006) and drugs like Fluoxetine (Prozac) (Choy and Thomas, 1999). A new generation of chemical engineering-trained entrepreneurs are forming innovative new businesses, no doubt influenced by the fact that chemical engineers have served as CEOs of such leading global businesses as 3M, DuPont, Intel, General Electric, Union Carbide, Dow Chemical, Exxon, BASF, Gulf, and Texaco. It is a broad reaching field impacting everything we touch, feel, breath, eat, and do. Although many strategies have been suggested and some of them have been shown to work, successful projects beyond proof-of-concept work are still scarce. , MreB actin-mediated segregation of a specific region of a bacterial chromosome, An efficient proteomics method to identify the cellular targets of protein kinase inhibitors, Gross, E R, Hsu, A K, and Gross, G J (2004). Can chemical engineering work in biotechnology? Demonstration of two forms of iron in the holoenzyme, Obtaining and screening compound collections: a users guide and a call to chemists, Hoyt, M A, Totis, L, and Roberts, B T (1991). Historically, most compounds used in chemical genetics and drug development were natural products or derivatives thereof. Updates? However, the observation that rapamycin derivatives inhibit FKBP12 but do not immunosuppress argued against the idea that the inhibition of FKBP12 accounts for the immunosuppressive effect of rapamycin. Gene deletion and RNAi eliminate the protein of interest and thus both characteristics that define protein function are affected: its (catalytic) activity and its potential relevance as a part of protein complexes. FOIA , Small molecules, big impact: a history of chemical inhibitors and the cytoskeleton, Peterson, R T, Shaw, S Y, Peterson, T A, Milan, D J, Zhong, T P, Schreiber, S L, MacRae, C A, and Fishman, M C (2004). By now, monastrol is a well established tool in cell biology that provided insights into the process of spindle formation, chromosome capture, and the activation of the spindle assembly checkpoint, which would have been hardly if at all amenable to traditional techniques (Kapoor et al., 2000; Kapoor and Mitchison, 2001; Khodjakov et al., 2003). target identificationvalidation including specificity tests. If these genetic manipulations suppress the phenotype mediated by the small molecule one can take it for granted that the assumed target is the relevant binding partner of the identified compound at the given concentration. B. Bookshelf The basic outline of a chemical genetic project is as follows: Conceptually, the most important part is the design of the screening strategy, since it has fundamental implications for the entire screening procedure and outcome of the screen [Fig.
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