Proteoma_11nov2015 Rubens Nodari

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Comparative proteomic and GMO biohazard do not support the substantial equivalence Rubens Onofre Nodari e Sarah Agapito- Tenfen Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil Graduate Program on Recursos Genéticos Vegetais rubens.nodari@ ufsc.br ASSESSMENT AND REGULATION OF GMOs AND PESTICIDES

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The presentation Context of the Transgenese From Biohazard to Biosafety Comparative proteomics on GM host: a) significant alterations in the genes expression of a variable number of endogenous genes b) Interference of a transgene in the expression of a second one 4 . Epigenetic hypotheses 5 . Conclusions 6. Questions/Discussion

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??? 15 a 10 thousands years ago 1950 Use of the genetic resources 29jun07 Gatherer-Hunter Settlement promotion harvest-saw domestication selection Chemical Agriculture Fertilizers Pesticides Improved Seeds IPR Transgenic Nanobiothecnology Synthetic Life ?

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??? 15 a 10 thousands years ago 1950 Use of the genetic resources 29jun07 Gatherer-Hunter Settlement promotion harvest-saw domestication selection Chemical Agriculture Fertilizers Pesticides Improved Seeds Transgenic Nanobiothecnology Synthetic Life ?

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From Biohazards to Biosafety

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Science, 26 july 1974, p.303

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International Conference on Recombinant DNA Molecules , Asilomar , February 1975 Conferees agreed to address only safety issues at the meeting, leaving ethical discussions for the future. The main guidelines to emerge from the conference stipulated that recombinant DNA research should use only disabled bacteria that could not survive outside the laboratory. The dialogue sparked by Asilomar led to the National Institutes of Health guidelines , considered a landmark of self-regulation by scientists. https:// libraries.mit.edu /archives/exhibits/ asilomar /

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Background for Comparative Proteome 2008 - Introduction of GM maize varieties in the country Proteomic studies, although useful ( Li et al. 2004) , not part of risk analysis (e.g. ; Prescot et al. 2005; Zolla et al. 2008) Experience on Proteomics, Risk Assessment and lab facilities Collaboration with GenØk

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April, 1983 V32, n4 Expression of Nopaline Synthase in Rl Progeny It is not clear from our present data whether the multiple copies of T-DNA in parental tissues are genetically linked, so we cannot predic t what frequency of T-DNA transmission to RI progeny is expected. If all copies of T-DNA were integrated at a single genetic locus, 75% of the progeny from self-pollination would be expected to contain multiple copies of T-DNA. If TDNA is inserted at multiple sites, the transmission rate would be still higher with a distribution of copy numbers. Our finding that nopaline was present in only 24% of the progeny was therefore unexpected .

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Proteins talk to each other – interactome Li et al., 2004.

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αAI from pea induces cross-priming of pea proteins. Pea globulin-, vicilin-4, and lectin -specific IgG1 levels in serum from mice. Western blot analysis of α AI protein in extracts of transgenic peas and the Tendergreen variety of common bean Prescott et al. 2005.Transgenic expression of bean alpha-Amylase inhibitor in peas results in altered structure and immunogenicity J. Agric. Food Chem. 53: 9023-9030. S ignificant post-translational modifications

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Untargeted approach Untargeted profiling usually refers to quantitative surveys of broad classes of molecules . Omics investigation . Proteomics All proteins expressed by a particular cell or organism at a particular time in a defined environment . The proteome is dynamic and you must know where the protein came from, when and under what conditions to evaluate the usefulness of the data . This includes all post- translationally modified variants of a single primary amino acid sequence .

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Campos Novos Chapecó Canoinhas

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Experimental design and sampling Plant Material: event MON810 Hybrid P32R48Y (GM) Hybrid P32R48 (Isogenic non GM)

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Rational for the Statistical analysis

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Enlarged image and 3D view ( right) of the spot 134. Higher expression in transgenic plants (indicated by the arrow). Gels images in a and b are of samples from transgenic plants, and in c and d from non transgenic plants, grown in the same experiment –Campos Novos , SC, Brazil   Protein exclusive to non Transgenic Plant

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Enlarged image and 3D view ( right) of the spot 497. Higher expression in transgenic plants (indicated by the arrow). Gels images in a and b are of samples from transgenic plants, and in c and d from non transgenic plants, grown in the same experiment –Campos Novos , SC, Brazil   Protein exclusive to Transgenic Plant

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PCA score plots of genetically modified and non-genetically modified near isogenic maize hybrids grown at (a ) Campos Novos and (b) Chapecó Brazil . Campos Novos 20% of the variation GM x non-GM 14% of the variation within biological and technical repli - cates within each genotype Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46

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PCA score plots of genetically modified and non-genetically modified near isogenic maize hybrids grown at (a ) Campos Novos and (b) Chapecó, Brazil . Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46 Chapecó 21% of the variation GM x non-GM O ne technical repetition - skewed the plot towards the left, 24.66 % of the variation

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PCA score plots of (a ) genetically modified and (b ) non -genetically modified near isogenic maize hybrids across location (grown at Campos Novos and Chapecó, Brazil). GM hybrid across locations T he major source of variation ( 34.3 % ) was explained by the different growing environments. Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46

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PCA score plots of (a ) genetically modified and (b ) non -genetically modified near isogenic maize hybrids across location (grown at Campos Novos and Chapecó, Brazil). Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46 Non GM hybrid across locations T he major source of variation ( 30.6% ) was explained by the different growing environments.

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Both locations together Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46 T he major source of variation (30.6%) was explained by the different growing environments. PCA score plots of genetically modified and non-genetically modified near isogenic maize hybrids grown at (a ) Campos Novos and Chapecó together, Brazil . GM Chapecó GM Campos Novos Non GM

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PCA score plots of (a ) genetically modified and (b ) non -genetically modified near isogenic maize hybrids across location (grown at Campos Novos and Chapecó, Brazil). Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46

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Total protein content and number of detected spots genetically modified P32R48YG and non-modified P32R48 maize variety grown under farm conditions in Campos Novos and Chapecó, Brazil. a Values are means of n=9 samples ± standard deviation; b Values are means of n=9 gels ± standard deviation; c Statistically significant at 5%. Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46

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Note: Uncharacterized proteins are not accounted in this table. All protein physiological categories assignments were performed according to KEGG. Number of differentially expressed proteins/transcripts according to their physiological functions and revealed by proteomic and transcriptomic analysis observed by other authors when evaluating genetically modified maize (MON810) under different growing conditions. Proteome Science 2013, 11:46 doi:10.1186/1477-5956-11-46

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Comparative analysis within each field revealed a total of 32 differentially expressed proteins between GM and non-GM samples that were identified and their molecular functions were mainly assigned to: carbohydrate and energy metabolism ( 47% of all identified proteins), stress response and genetic information processing. What are the differences ? All 32 proteins were identified with Mascot scores value greater than 325 using Quadrupole Time-of-Flight (Q-TOF) tandem mass spectrometry analysis (MS/MS) (P < 0.05)

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Proteins related to carbohydrate and energy metabolism Glycolysis and/or Gluconeogenesis , Photophosphorylation , Calvin Cycle, Pyruvate metabolism, and Starch biosynthesis. What are the differences ?

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Why carbohydrate metabolism increased? The explanation for extensive carbohydrate metabolism: heavy energy demand during flowering and grain filling; o verexpressing or expressing a gene in a constitutive way (e.g. a transgene) it would always have the intended effect on the phenotype, strong and constitutive promoters (e.g. CaMV-35S) involve a high energetic cost and it could somehow yield a penalty in transgenic plants.

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Stress response proteins are related to: glutathione metabolism (glyoxylase1 and IN2-1), peroxidases (peroxidase 42 precursor and APx1 ), and pathogenesis -related protein (PR10 ) . Similar results by Coll et al. 2010 and Zolla et al. 2008 O xidative phosphorylation is the source of reactive hydrogen, a poisonous compound for plant tissues. Peroxidases , which are endowed with xenobiotic functions, are of great importance for eliminating H 2 O 2 resulting from oxidative phosphorylation. What are the differences ?

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Genetic information processing proteins APT is a housekeeping enzime . T ranscription of ZmAPT2 in Zea mays were 3.1-fold up-regulated in leaf tumor tissue compared to control tissue of the same age ( Basse , 2005). ZmAPT2 play key roles in plant growth and development , and its detailed functions need to be further studied. What are the differences ?

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Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8 n.a . = Not applied PLANT MATERIAL

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2D- DIGE - two dimensional differential gel electrophoresis analysis, quantitative Real-Time PCR experiments (RT- qPCR ) to determine differences in the proteome and transcription levels of transgenes between stacked and single events. 2D - DIGE provides a platform for controlling variation due to sample preparation, protein separation and difference detection by fluorescent labeling and the co -migration of treatment and control samples in the same gel. 2D- DIGE + RT- qPCR

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C ircumvents many of the issues associated with traditional 2D-PAGE, such as: low reproducibility, limited dynamic range, and unsuitable of Coomassie staining for quantitative analysis. A llows for more accurate and sensitive quantitative proteomics studies. Difference gel electrophoresis (DIGE ) Kathryn et al 2004

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Transgene transcripts normalized relative expression levels measured by delta-delta Cq method and Pffafl [[19]correction equation. The epsps , cry1A.105 and cry2Ab2 transgenes were quantified from stacked versus single transgenic maize events grown under controlled conditions at V3 stage. Experiment 1 (A) and under the same conditions in Experiment 2 (B). Samples are means of three pools, each derived from five different plants. ‘RR’ samples are transgenic maize seedlings from MON-ØØ6Ø3-6 event, ‘ Bt ’ samples are from MON-89Ø34-3 event, and ‘ RRxBt ’ samples are transgenic maize seedlings from MON-89Ø34-3 x MON-ØØ6Ø3-6 event. Bars indicate standard deviation and statistically significant values (P < 0.05) are represented by ‘*’. Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8

Slide39:

Average reduction of transcripts 31 % epsps 41% cry1A . 105 29% cry2Ab2 Transgene /transgene interactions - homologous DNA sequences ( Fagard & Vaucheret 2000) Homologous -dependent gene silencing post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) ( Matzke & Matzke 1998, Dong et al 2001, Weld et al.2001 , Kohli et al. 2003 , Cogoni & Macino 1999) Homologous P35S promoters control the epsps and cry1A.105 transgenes present in the stacked line. Reduced transgene expression might also be related to the high energetic demand of the cell. Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8

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Monsanto’s application to CTNBio ( Brazil) of MON-89Ø34-3 x MON-ØØ6Ø3-6 D iscrepancies for Cry2Ab2 protein levels (leaves) determined by ELISA assay, in stacked versus single events, grown under farm conditions in three locations: MON -89Ø34- 3 51 , 24 and 24 ug.g−1 MON-89Ø34-3 x MON-ØØ6Ø3- 6 33, 26 and 38 ug.g− 1 Large variation (standard deviation values (up to 19) and small sampling size ( N = 4 ) likely explain the lack of statistical significance . Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8

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Field-evolved resistance to Bt toxins in GM crops was first reported in 2006 for S. frugiperda in Puerto Rico ( Storer et al 2010). The causes ? lack of compliance of growers that may not strictly adhere to the requirements for planting refuge areas ( Gassmann et 2011, Kruger et al 2011), too low or variable to consistently kill the TO, heterozygous resistant insects [44,46,48,49]. g enotype, seasonal and spatial variation of Cry toxin content in GM ( Showalter et al 2009; Nguyen & Jehle 2009), The latters can be consequences of gene silencing or transcript reduction ! Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8

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The declines in the concentration of one toxin in a pyramid could also invalidate the fundamental assumption of the pyramid strategy (i.e., the killing of insects resistant to one toxin by another toxin), and thus accelerate evolution of resistance . S ignificant exponential increase in the frequency of alleles conferring Cry2Ab resistance in Australian field populations of H. punctigera since the adoption of a second generation, two-toxin Bt cotton ( Downes et al. 2010 ) Why does it matter? Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8

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Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8 Figure 2 PCA score plots of proteome data of genetically modified stacked and single events, non-genetically modified near-isogenic variety, and landrace maize variety. Proteome data was obtained by 2D-DIGE analysis from leaf material of maize plants grown under controlled conditions. PC1 and PC2 (A) and PC1 and PC3 (B) show the results of ‘RR’ samples (transgenic maize seedlings from MON-ØØ6Ø3-6 event, filled squares), ‘ Bt ’ samples (MON-89Ø34-3 event, filled circles), ‘ RRxBt ’ samples (transgenic maize seedlings from MON-89Ø34-3 x MON-ØØ6Ø3-6 event, filled triangles), ‘CONV’ samples (conventional non-transgenic near isogenic maize variety, blank triangles), and ‘landrace’ ( Pixurum 5 landrace variety, blank squares).

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Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8 Figure 2 PCA score plots of proteome data of genetically modified stacked and single events, non-genetically modified near-isogenic variety, and landrace maize variety. Proteome data was obtained by 2D-DIGE analysis from leaf material of maize plants grown under controlled conditions. PC1 and PC2 (A) and PC1 and PC3 (B) show the results of ‘RR’ samples (transgenic maize seedlings from MON-ØØ6Ø3-6 event, filled squares), ‘ Bt ’ samples (MON-89Ø34-3 event, filled circles), ‘ RRxBt ’ samples (transgenic maize seedlings from MON-89Ø34-3 x MON-ØØ6Ø3-6 event, filled triangles), ‘CONV’ samples (conventional non-transgenic near isogenic maize variety, blank triangles), and ‘landrace’ ( Pixurum 5 landrace variety, blank squares). B

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Proteins can be toxins or allergens They may be converted into allergens through post -translational modifications They may be converted into toxins through post -translational modifications They may only demonstrate adverse effects in the context of the GMO or through special uses of the GMO Why does it matter?

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What is a suitable comparator? “The current concept that for GM plants containing a single event the choice of comparator must be the conventional counterpart which will be a non-GM genotype with a genetic background as close as possible to the GM plant .”

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What is a suitable comparator? Increase complexity Bt11 x MIR162 c ry1Ab x pat x vip 3Aa20 x manA Bt11 x MIR162 x GA21 cry1Ab x pat x vip 3Aa20 x manA x mepsps * * 6 copies, being 5 distinct among themselves Is there a such comparator for all the interactions ?

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Properties of a Landrace as comparator : The presence of a landrace variety allows: to consider the extent of proteomic variation related to different maize genetic backgrounds, disclose differences in GM lines that might fit within the variation observed in non- modified hosts. However: is not a requirement of international guidelines addressing the issue for comparative assessments of the environmental and health risk analysis of GM plants. What is a suitable comparator? Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8

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Properties of a Landrace as comparator in our work: PC1 × PC3 revealed clear separation for landrace samples, explaining 15.6% of the variation in the full dataset. The landrace variety did not account for the majority of the variation in the dataset. There was no variation between biological replicates within each plant variety. What is a suitable comparator? Agapito-Tenfen et al. BMC Plant Biology 2014 14:346 doi:10.1186/s12870-014-0346-8

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Mass spectral identification of differentially expressed proteins

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Jablonka & Lamb, 2006; ilustrações de Anna Zeligowski. Waddington ’ s pictures of epigenetic landscapes. His legend for the upper picture began “ Part of an epigenetic landscape. The path followed by the ball, as it rolls down towards the spectator, corresponds to the developmental history of a particular part of the egg. ” The lower picture had the legend “ The complex system of interactions underlying the epigenetic landscape. The pegs in the ground represent genes; the strings leading from them the chemical tendencies which the genes produce . The modeling of the epigenetic landscape, which slopes down from above one ’ s head towards the distance, is controlled by the pull of these numerous guyropes which are ultimately anchored to the genes. ”

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DNA mRNA Protein Genomic Transcriptomic Proteomic Metabolomic Phenomic rRNA Ribosomes tRNA Transport snRNA , siRNA , miRNA , hnRNA , others Gene Regulation Interactome ?

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”… leaves us where science almost always leaves us: unsure . … ” Les Real, Disease Ecologist at Emory University, Atlanta, Georgia. Science 303: 298-299, 2004 Ebola Outbreaks May Have Had Independent Sources Gretchen Vogel

Conclusions:

Comparative proteome can help to identify significant differences between GM and non-GM organisms; Risk analysis should take into account the uncertainties; The risk should also consider epigenetics; Science alone is not sufficient, because decision making process is not only based on science. Conclusions

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Merci rubens.nodari@ufsc.br

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Even though substantial equivalence between GMO and its counterpart has been largely used by regulatory agencies across the word, independent scientific studies do not give support as safety criteria. Among those scientific results, comparative proteomics analysis provided the identification of major and significant alterations in the expression of a variable number of endogenous genes after the insertion of transgene(s). In addition, new proteins have been produced by host species after transgene insertion. Moreover, in stacked transgenic events, the regulatory sequences of one transgene can affect the expression of the other, in addition to the troublesome in the expression of the host genes. More intriguingly, the major differences in biohazards posed by GMO in comparison to its counterpart, so far robustly proven, is not taken into account by the substantial equivalence procedure. Thus, it is time to rethink the use of that procedure to decide if a GMO is safe or not safe to human health and environment.

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Agapito-Tenfen et al.: Comparative proteomic analysis of genetically modified maize grown under different agroecosystems conditions in Brazil. Proteome Science 2013 11:46. Agapito-Tenfen et al.: Effect of stacking insecticidal cry and herbicide tolerance epsps transgenes on transgenic maize proteome. BMC Plant Biology 2014 14:346. Barton KA, Binns AN, Matzke AJ, Chilton MD. 1983. Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell.32(4):1033-43 . Basse CW: Dissecting defense-related and developmental transcriptional responses of maize during ustilago maydis infection and subsequent tumor formation. Plant Physiol 2005, 138:1774–1784. Berg, P., Baltimore, D., Boyer, H.W., Cohen, S.N., Davis, R.W., Hogness , D.S., Nathans, D., Roblin , R., Watson, J.D., Weissman , S. and Zinder , N.D. Potential Biohazards of Recombinant DNA Molecules. Science 26 July 1974: 303 . Coll A, Nadal A, Rossignol M, Puigdomènech P, Pla M: Proteomic analysis of MON810 and comparable non-GM maize varieties grown in agricultural fields. Transgenic Res 2011, 4:939–949.

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Downes S, Parker T, Mahon R: Incipient resistance of Helicoverpa punctigera to the Cry2Ab Bt toxin in Bollgard II (R) cotton. PLoS One 2010, 5:9. Gassmann AJ, Petzold -Maxwell JL, Keweshan RS, Dunbar MW: Field-evolved re- sistance to Bt maize by western corn rootworm. PLoS One 2011, 6:e22629. Jablonka , E.; Lamb, M.J. Evolution in Four Dimensions: Genetic, Epigenetic, Behavioral, and Symbolic Variation in the History of Life (Life and Mind: Philosophical Issues in Biology and Psychology). London: MIT Press, 462p, 2006. Com ilustrações de Anna Zeligowski . Kathryn S Lilley & David B Friedman (2004) All about DIGE: quantification technology for differential-display 2D-gel proteomics, Expert Review of Proteomics, 1:4, 401-409 Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P: Transgene integration, organization and interaction in plants. Plant Mol Biol 2003, 52:247–258 . Kruger M, Van Rensburg J, Van den Berg J: Resistance to Bt maize in Busseola fusca (Lepidoptera: Noctuidae ) from Vaalharts . S Afr Environ Entomol 2011, 40:477–483 Li, S., Armstrong, C.M., Bertin , N., Ge , H., Milstein, S., Boxem , M., Vidalain , P.O., Han, J.D., Chesneau , A., Hao , T., et al. (2004). A map of the interactome network of the metazoan C. elegans . Science 303, 540–543.

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Prescott VE, Campbell PM, Moore A, Mattes J, Rothenberg ME, Foster PS, Higgind TJV, Hogan SP. Transgenic expression of bean alpha-Amylase inhibitor in peas results in altered structure and immunogenicity J. Agric. Food Chem. 53: 9023-9030, 2005 Storer NP, Babcock JM, Schlenz M, Meade T, Thompson GD, Bing JW, Huckaba RM: Discovery and characterization of field resistance to Bt maize : Spodoptera frugiperda ( Lepidoptera : Noctuidae ) in Puerto Rico. J Econ Entomol 2010, 103:1031–1038. Showalter AM, Heuberger S, Tabashnik BE, Carrière Y, Coates B: A primer for using transgenic insecticidal cotton in developing countries. J Insect Sci 2009, 9:22. Nguyen HT, Jehle JA: Expression of Cry3Bb1 in transgenic corn MON88017. J Agric Food Chem 2009, 57:9990–9996. Trtikova M, Wikmark OG, Zemp N, Widmer A, Hilbeck A (2015) Transgene Expression and Bt Protein Content in Transgenic Bt Maize (MON810) under Optimal and Stressful Environmental Conditions. PLoS ONE 10(4): e0123011. Zolla L et al. Proteomics as a complementary tool for identifying unintended side effects occurring in transgenic maize seeds as a result of genetic modification. Journal of Proteome Research 7: 1850-1861, 2008

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Agricultural Frontier continuous forward...

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Brazil - Consumption of active ingredients of pesticides per hectare (1 ha = 2.471 acres) Source: http:// www.sidra.ibge.gov.br / bda / tabela /protabl2.asp?c=771 k/ha    Soybean Cotton Maiz

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