**7. Acknowledgement**

This work was supported by a CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAPERGS (Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul), FINEP (Financiadora de Projetos) and MCT (Ministério de Ciência e Tecnologia). A. Cagliari

The Evolutionary History of CBF Transcription Factors:

14485-14491.

*J Biol Chem*, 273, 1430-1434.

metabolisms. *J Bacteriol*, 178, 1842-1849.

eukaryotic genomes. *PLoS Biol*, 2, E55.

angiosperms. *Trends Ecol Evol*, 20, 591-597.

activation potential. *Nucleic Acids Res*, 27, 2578-2584.

CCAAT box-binding proteins. *Cell*, 50, 863-872.

*Acad Sci U S A*, 84, 6249-6253.

*Physiol*, 117, 1015-1022.

*New Phytol*, 183, 557-564.

taxonomic levels. *BMC Evol Biol*, 10, 61.

Gene Duplication of CCAAT – Binding Factors NF-Y in Plants 215

Conant, G.C. and Wagner, A. (2002) GenomeHistory: a software tool and its application to

Conrad, B. and Antonarakis, S.E. (2007) Gene duplication: a drive for phenotypic diversity and cause of human disease. *Annu Rev Genomics Hum Genet*, 8, 17-35. Coustry, F., Maity, S.N., Sinha, S. and de Crombrugghe, B. (1996) The transcriptional activity

Coustry, F., Hu, Q., de Crombrugghe, B. and Maity, S.N. (2001) CBF/NF-Y functions both in

Dang, V.D., Bohn, C., Bolotin-Fukuhara, M. and Daignan-Fornier, B. (1996) The CCAAT

Davis, J.C. and Petrov, D.A. (2004) Preferential duplication of conserved proteins in

De Bodt, S., Maere, S. and Van de Peer, Y. (2005) Genome duplication and the origin of

De Grassi, A., Lanave, C. and Saccone, C. (2008) Genome duplication and gene-family

de Silvio, A., Imbriano, C. and Mantovani, R. (1999) Dissection of the NF-Y transcriptional

Dorn, A., Bollekens, J., Staub, A., Benoist, C. and Mathis, D. (1987a) A multiplicity of

Dorn, A., Durand, B., Marfing, C., Le Meur, M., Benoist, C. and Mathis, D. (1987b)

Duarte, J.M., Wall, P.K., Edger, P.P., Landherr, L.L., Ma, H., Pires, J.C., Leebens-Mack, J. and

Dynan, W.S. and Tjian, R. (1985) Control of eukaryotic messenger RNA synthesis by

Edwards, D., Murray, J.A. and Smith, A.G. (1998) Multiple genes encoding the conserved

Flagel, L.E. and Wendel, J.F. (2009) Gene duplication and evolutionary novelty in plants.

sequence-specific DNA-binding proteins. *Nature*, 316, 774-778.

Conserved major histocompatibility complex class II boxes--X and Y--are transcriptional control elements and specifically bind nuclear proteins. *Proc Natl*

dePamphilis, C.W. (2010) Identification of shared single copy nuclear genes in Arabidopsis, Populus, Vitis and Oryza and their phylogenetic utility across various

CCAAT-box transcription factor complex are expressed in Arabidopsis. *Plant*

evolution: the case of three OXPHOS gene families. *Gene*, 421, 1-6.

of the CCAAT-binding factor CBF is mediated by two distinct activation domains, one in the CBF-B subunit and the other in the CBF-C subunit. *J Biol Chem*, 271,

nucleosomal disruption and transcription activation of the chromatin-assembled topoisomerase IIalpha promoter. Transcription activation by CBF/NF-Y in chromatin is dependent on the promoter structure. *J Biol Chem*, 276, 40621-40630. Currie, R.A. (1998) NF-Y is associated with the histone acetyltransferases GCN5 and P/CAF.

box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon

fully sequenced genomes. *Nucleic Acids Res*, 30, 3378-3386.

received a Ph.D. fellowship from CNPq. and G. Loss a Ph.D. fellowship from CAPES. A. Turchetto-Zolet and F. Maraschin have a PNPD-CAPES fellowship and M. Margis-Pinheiro and R. Margis are recipients of CNPq research fellowships number 308708/2006-7 and 303967/2008-0, respectively.

### **8. References**

received a Ph.D. fellowship from CNPq. and G. Loss a Ph.D. fellowship from CAPES. A. Turchetto-Zolet and F. Maraschin have a PNPD-CAPES fellowship and M. Margis-Pinheiro and R. Margis are recipients of CNPq research fellowships number 308708/2006-7 and

Arents, G. and Moudrianakis, E.N. (1995) The histone fold: a ubiquitous architectural motif

Baxevanis, A.D., Arents, G., Moudrianakis, E.N. and Landsman, D. (1995) A variety of

Beisswanger, S. and Stephan, W. (2008) Evidence that strong positive selection drives

Ben-Naim, O., Eshed, R., Parnis, A., Teper-Bamnolker, P., Shalit, A., Coupland, G., Samach,

Berry, M., Grosveld, F. and Dillon, N. (1992) A single point mutation is the cause of the Greek form of hereditary persistence of fetal haemoglobin. *Nature*, 358, 499-502. Blanc, G. and Wolfe, K.H. (2004) Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes. *Plant Cell*, 16, 1667-1678. Bowers, J.E., Chapman, B.A., Rong, J. and Paterson, A.H. (2003) Unravelling angiosperm

Braybrook, S.A. and Harada, J.J. (2008) LECs go crazy in embryo development. *Trends Plant*

Bucher, P. (1990) Weight matrix descriptions of four eukaryotic RNA polymerase II

Cai, X., Ballif, J., Endo, S., Davis, E., Liang, M., Chen, D., DeWald, D., Kreps, J., Zhu, T. and

Caretti, G., Motta, M.C. and Mantovani, R. (1999) NF-Y associates with H3-H4 tetramers and

Ceribelli, M., Dolfini, D., Merico, D., Gatta, R., Vigano, A.M., Pavesi, G. and Mantovani, R.

Chodosh, L.A., Baldwin, A.S., Carthew, R.W. and Sharp, P.A. (1988) Human CCAAT-

Combier, J.P., Frugier, F., de Billy, F., Boualem, A., El-Yahyaoui, F., Moreau, S., Vernie, T.,

flowering timing in Arabidopsis. *Plant Physiol*, 145, 98-105.

octamers by multiple mechanisms. *Mol Cell Biol*, 19, 8591-8603.

binding proteins have heterologous subunits. *Cell*, 53, 11-24.

microRNA169 in Medicago truncatula. *Genes Dev*, 20, 3084-3088.

between CONSTANS-like proteins and DNA. *Plant J*, 46, 462-476.

Drosophila. *Proc Natl Acad Sci U S A*, 105, 5447-5452.

utilized in DNA compaction and protein dimerization. *Proc Natl Acad Sci U S A*, 92,

DNA-binding and multimeric proteins contain the histone fold motif. *Nucleic Acids*

neofunctionalization in the tandemly duplicated polyhomeotic genes in

A. and Lifschitz, E. (2006) The CCAAT binding factor can mediate interactions

genome evolution by phylogenetic analysis of chromosomal duplication events.

promoter elements derived from 502 unrelated promoter sequences. *J Mol Biol*, 212,

Wu, Y. (2007) A putative CCAAT-binding transcription factor is a regulator of

(2008) The histone-like NF-Y is a bifunctional transcription factor. *Mol Cell Biol*, 28,

Ott, T., Gamas, P., Crespi, M. and Niebel, A. (2006) MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by

303967/2008-0, respectively.

11170-11174.

*Res*, 23, 2685-2691.

*Nature*, 422, 433-438.

*Sci*, 13, 624-630.

563-578.

2047-2058.

**8. References**

The Evolutionary History of CBF Transcription Factors:

embryo development. *Plant Cell*, 15, 5-18.

*Nucleic Acids Res*, 20, 1087-1091.

genes. *Science*, 290, 1151-1155.

*U S A*, 102, 5454-5459.

subfunctionalization. *Genetics*, 154, 459-473.

CCAAT binding factor. *J Biol Chem*, 267, 16574-16580.

1455.

8292.

*Genet*, 20, 287-290.

Gene Duplication of CCAAT – Binding Factors NF-Y in Plants 217

Kondrashov, F.A. and Koonin, E.V. (2004) A common framework for understanding the

Kusnetsov, V., Landsberger, M., Meurer, J. and Oelmuller, R. (1999) The assembly of the

light, cytokinin, and the stage of the plastids. *J Biol Chem*, 274, 36009-36014. Kwong, R.W., Bui, A.Q., Lee, H., Kwong, L.W., Fischer, R.L., Goldberg, R.B. and Harada, J.J.

Lee, H., Fischer, R.L., Goldberg, R.B. and Harada, J.J. (2003) Arabidopsis LEAFY

binding transcription factor. *Proc Natl Acad Sci U S A*, 100, 2152-2156. Li, W.H., Gu, Z., Cavalcanti, A.R. and Nekrutenko, A. (2003) Detection of gene duplications and block duplications in eukaryotic genomes. *J Struct Funct Genomics*, 3, 27-34. Li, W.X., Oono, Y., Zhu, J., He, X.J., Wu, J.M., Iida, K., Lu, X.Y., Cui, X., Jin, H. and Zhu, J.K.

origin of genetic dominance and evolutionary fates of gene duplications. *Trends*

CAAT-box binding complex at a photosynthesis gene promoter is regulated by

(2003) LEAFY COTYLEDON1-LIKE defines a class of regulators essential for

COTYLEDON1 represents a functionally specialized subunit of the CCAAT

(2008) The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. *Plant Cell*, 20, 2238-2251. Li, X.Y., Mantovani, R., Hooft van Huijsduijnen, R., Andre, I., Benoist, C. and Mathis, D.

(1992) Evolutionary variation of the CCAAT-binding transcription factor NF-Y.

CCAAT boxes: role of Q-rich domains and histone fold helices. *J Mol Biol*, 285, 1441-

R.L., Goldberg, R.B. and Harada, J.J. (1998) Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. *Cell*, 93, 1195-1205. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. and Richmond, T.J. (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. *Nature*, 389, 251-260. Lynch, M. and Conery, J.S. (2000) The evolutionary fate and consequences of duplicate

Liberati, C., di Silvio, A., Ottolenghi, S. and Mantovani, R. (1999) NF-Y binding to twin

Lotan, T., Ohto, M., Yee, K.M., West, M.A., Lo, R., Kwong, R.W., Yamagishi, K., Fischer,

Lynch, M. and Force, A. (2000) The probability of duplicate gene preservation by

Maere, S., De Bodt, S., Raes, J., Casneuf, T., Van Montagu, M., Kuiper, M. and Van de Peer,

Maity, S.N. and de Crombrugghe, B. (1992) Biochemical analysis of the B subunit of the

Maity, S.N., Sinha, S., Ruteshouser, E.C. and de Crombrugghe, B. (1992) Three different

Maity, S.N. and De Crombrugghe, B. (1996) Purification, characterization, and role of CCAAT-binding factor in transcription. *Methods Enzymol*, 273, 217-232.

Y. (2005) Modeling gene and genome duplications in eukaryotes. *Proc Natl Acad Sci*

heteromeric CCAAT-binding factor. A DNA-binding domain and a subunit interaction domain are specified by two separate segments. *J Biol Chem*, 267, 8286-

polypeptides are necessary for DNA binding of the mammalian heteromeric

Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.L. and Postlethwait, J. (1999)

Forsburg, S.L. and Guarente, L. (1989) Identification and characterization of HAP4: a third

Gallagher, C.E., Matthews, P.D., Li, F. and Wurtzel, E.T. (2004) Gene duplication in the

Gu, Z., Nicolae, D., Lu, H.H. and Li, W.H. (2002) Rapid divergence in expression between duplicate genes inferred from microarray data. *Trends Genet*, 18, 609-613. Gusmaroli, G., Tonelli, C. and Mantovani, R. (2001) Regulation of the CCAAT-Binding NF-Y

Gusmaroli, G., Tonelli, C. and Mantovani, R. (2002) Regulation of novel members of the Arabidopsis thaliana CCAAT-binding nuclear factor Y subunits. *Gene*, 283, 41-48. Harrison, P.M., Echols, N. and Gerstein, M.B. (2001) Digging for dead genes: an analysis of

Harrison, P.M., Hegyi, H., Balasubramanian, S., Luscombe, N.M., Bertone, P., Echols, N.,

He, X.L. and Zhang, J.Z. (2005) Gene complexity and gene duplicability. *Current Biology*, 15,

Hinman, V.F. and Davidson, E.H. (2007) Evolutionary plasticity of developmental gene regulatory network architecture. *Proc Natl Acad Sci U S A*, 104, 19404-19409. Holland, P.W., Garcia-Fernandez, J., Williams, N.A. and Sidow, A. (1994) Gene duplications

Huang, H.S., Chen, C.J. and Chang, W.C. (1999) The CCAAT-box binding factor NF-Y is

Jordan, I.K., Wolf, Y.I. and Koonin, E.V. (2004) Duplicated genes evolve slower than

Keeling, C.I., Weisshaar, S., Lin, R.P. and Bohlmann, J. (2008) Functional plasticity of

Kim, I.S., Sinha, S., de Crombrugghe, B. and Maity, S.N. (1996) Determination of functional

Kimura, M. (1989) The neutral theory of molecular evolution and the world view of the

required for the expression of phospholipid hydroperoxide glutathione peroxidase

paralogous diterpene synthases involved in conifer defense. *Proc Natl Acad Sci U S*

domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule. *Mol Cell Biol*, 16,

and the origins of vertebrate development. *Dev Suppl*, 125-133.

singletons despite the initial rate increase. *BMC Evol Biol*, 4, 22.

in human epidermoid carcinoma A431 cells. *FEBS Lett*, 455, 111-116. Hurley, I., Hale, M.E. and Prince, V.E. (2005) Duplication events and the evolution of

subunits in Arabidopsis thaliana. *Gene*, 264, 173-185.

genome. *Nucleic Acids Res*, 29, 818-830.

segmental identity. *Evol Dev*, 7, 556-567.

*Genetics*, 151, 1531-1545.

135, 1776-1783.

*Res*, 12, 272-280.

*A*, 105, 1085-1090.

neutralists. *Genome*, 31, 24-31.

4003-4013.

1016-1021.

Preservation of duplicate genes by complementary, degenerative mutations.

component of the CCAAT-bound HAP2/HAP3 heteromer. *Genes Dev*, 3, 1166-1178.

carotenoid biosynthetic pathway preceded evolution of the grasses. *Plant Physiol*,

the characteristics of the pseudogene population in the Caenorhabditis elegans

Johnson, T. and Gerstein, M. (2002) Molecular fossils in the human genome: identification and analysis of the pseudogenes in chromosomes 21 and 22. *Genome*

The Evolutionary History of CBF Transcription Factors:

deacetylases. *J Biol Chem*, 278, 8385-8394.

factors. *Curr Opin Plant Biol*, 3, 423-434.

duplication. *J Mol Evol*, 56, 718-729.

*Genome*, 47, 868-876.

*Acids Res*, 26, 3800-3805.

*Genet Dev*, 6, 715-722.

270.

factor NF-Y. *J Biol Chem*, 278, 1336-1345.

mixed models. *Bioinformatics*, 19, 1572-1574.

grp78/BiP promoter. *Mol Cell Biol*, 15, 2263-2274.

expression. *Nat Struct Mol Biol*, 11, 812-815.

Gene Duplication of CCAAT – Binding Factors NF-Y in Plants 219

Paterson, A.H., Freeling, M., Tang, H. and Wang, X. (2010) Insights from the comparison of

Peng, Y. and Jahroudi, N. (2002) The NFY transcription factor functions as a repressor and activator of the von Willebrand factor promoter. *Blood*, 99, 2408-2417. Peng, Y. and Jahroudi, N. (2003) The NFY transcription factor inhibits von Willebrand factor

Remenyi, A., Scholer, H.R. and Wilmanns, M. (2004) Combinatorial control of gene

Riechmann, J.L. and Ratcliffe, O.J. (2000) A genomic perspective on plant transcription

Rieping, M. and Schoffl, F. (1992) Synergistic effect of upstream sequences, CCAAT box

Rodin, S.N. and Riggs, A.D. (2003) Epigenetic silencing may aid evolution by gene

Romier, C., Cocchiarella, F., Mantovani, R. and Moras, D. (2003) The NF-YB/NF-YC

Ronquist, F. and Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under

Roy, B. and Lee, A.S. (1995) Transduction of calcium stress through interaction of the human

Sandman, K., Krzycki, J.A., Dobrinski, B., Lurz, R. and Reeve, J.N. (1990) HMf, a DNA-

Schlueter, J.A., Dixon, P., Granger, C., Grant, D., Clark, L., Doyle, J.J. and Shoemaker, R.C.

Schmidt, E.E. and Davies, C.J. (2007) The origins of polypeptide domains. *Bioessays*, 29, 262-

Serra, E., Zemzoumi, K., di Silvio, A., Mantovani, R., Lardans, V. and Dissous, C. (1998)

Shan, H., Zahn, L., Guindon, S., Wall, P.K., Kong, H., Ma, H., DePamphilis, C.W. and

Shi, Q., Gross, K.W. and Sigmund, C.D. (2001) NF-Y antagonizes renin enhancer function by blocking stimulatory transcription factors. *Hypertension*, 38, 332-336. Sidow, A. (1996) Gen(om)e duplications in the evolution of early vertebrates. *Curr Opin*

concerted gene duplications. *Mol Biol Evol*, 26, 2229-2244.

promoter activation in non-endothelial cells through recruitment of histone

elements, and HSE sequences for enhanced expression of chimaeric heat shock

structure gives insight into DNA binding and transcription regulation by CCAAT

transcription factor CBF with the proximal CCAAT regulatory element of the

binding protein isolated from the hyperthermophilic archaeon Methanothermus fervidus, is most closely related to histones. *Proc Natl Acad Sci U S A*, 87, 5788-5791.

(2004) Mining EST databases to resolve evolutionary events in major crop species.

Conservation and divergence of NF-Y transcriptional activation function. *Nucleic*

Leebens-Mack, J. (2009) Evolution of plant MADS box transcription factors: evidence for shifts in selection associated with early angiosperm diversification and

plant genome sequences. *Annu Rev Plant Biol*, 61, 349-372.

genes in transgenic tobacco. *Mol Gen Genet*, 231, 226-232.

Maity, S.N. and de Crombrugghe, B. (1998) Role of the CCAAT-binding protein CBF/NF-Y

Mantovani, R. (1998) A survey of 178 NF-Y binding CCAAT boxes. *Nucleic Acids Res*, 26,

Mantovani, R. (1999) The molecular biology of the CCAAT-binding factor NF-Y. *Gene*, 239,

Marland, E., Prachumwat, A., Maltsev, N., Gu, Z. and Li, W.H. (2004) Higher gene

Masiero, S., Imbriano, C., Ravasio, F., Favaro, R., Pelucchi, N., Gorla, M.S., Mantovani, R.,

Matuoka, K. and Yu Chen, K. (1999) Nuclear factor Y (NF-Y) and cellular senescence. *Exp*

Matuoka, K. and Chen, K.Y. (2002) Transcriptional regulation of cellular ageing by the

McNabb, D.S., Xing, Y. and Guarente, L. (1995) Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding. *Genes Dev*, 9, 47-58. Miyoshi, K., Ito, Y., Serizawa, A. and Kurata, N. (2003) OsHAP3 genes regulate chloroplast

Muro, A.F., Bernath, V.A. and Kornblihtt, A.R. (1992) Interaction of the -170 cyclic AMP

Myers, R.M., Tilly, K. and Maniatis, T. (1986) Fine structure genetic analysis of a beta-globin

Nelson, D.E., Repetti, P.P., Adams, T.R., Creelman, R.A., Wu, J., Warner, D.C., Anstrom,

Nicole, M.C., Hamel, L.P., Morency, M.J., Beaudoin, N., Ellis, B.E. and Seguin, A. (2006)

Nowak, M.A., Boerlijst, M.C., Cooke, J. and Smith, J.M. (1997) Evolution of genetic

Ober, D. (2010) Gene duplications and the time thereafter - examples from plant secondary

Olesen, J.T. and Guarente, L. (1990) The HAP2 subunit of yeast CCAAT transcriptional

Papp, B., Pal, C. and Hurst, L.D. (2003) Dosage sensitivity and the evolution of gene families

MAP kinases and MAP kinase kinases. *BMC Genomics*, 7, 223.

model for the HAP2/3/4 complex. *Genes Dev*, 4, 1714-1729.

response element with the adjacent CCAAT box in the human fibronectin gene

D.C., Bensen, R.J., Castiglioni, P.P., Donnarummo, M.G., Hinchey, B.S., Kumimoto, R.W., Maszle, D.R., Canales, R.D., Krolikowski, K.A., Dotson, S.B., Gutterson, N., Ratcliffe, O.J. and Heard, J.E. (2007) Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. *Proc*

MAP-ping genomic organization and organ-specific expression profiles of poplar

activator contains adjacent domains for subunit association and DNA recognition:

CCAAT box-binding factor CBF/NF-Y. *Ageing Res Rev*, 1, 639-651.

duplicabilities for metabolic proteins than for nonmetabolic proteins in yeast and E.

Colombo, L. and Kater, M.M. (2002) Ternary complex formation between MADSbox transcription factors and the histone fold protein NF-YB. *J Biol Chem*, 277,

in transcription. *Trends Biochem Sci*, 23, 174-178.

1135-1143.

26429-26435.

*Cell Res*, 253, 365-371.

biogenesis in rice. *Plant J*, 36, 532-540.

promoter. *J Biol Chem*, 267, 12767-12774.

*Natl Acad Sci U S A*, 104, 16450-16455.

redundancy. *Nature*, 388, 167-171.

in yeast. *Nature*, 424, 194-197.

metabolism. *Plant Biol (Stuttg)*, 12, 570-577.

promoter. *Science*, 232, 613-618.

coli. *J Mol Evol*, 59, 806-814.

15-27.

The Evolutionary History of CBF Transcription Factors:

Arabidopsis. *Science*, 290, 2114-2117.

*Acad Sci U S A*, 97, 6579-6584.

279, 279-289.

143, 1590-1600.

2971-2984.

Gene Duplication of CCAAT – Binding Factors NF-Y in Plants 221

Thirumurugan, T., Ito, Y., Kubo, T., Serizawa, A. and Kurata, N. (2008) Identification,

Vision, T.J., Brown, D.G. and Tanksley, S.D. (2000) The origins of genomic duplications in

Wagner, A. (2000) Decoupled evolution of coding region and mRNA expression patterns

Wapinski, I., Pfeffer, A., Friedman, N. and Regev, A. (2007) Natural history and evolutionary principles of gene duplication in fungi. *Nature*, 449, 54-61. Warpeha, K.M., Upadhyay, S., Yeh, J., Adamiak, J., Hawkins, S.I., Lapik, Y.R., Anderson,

Wenkel, S., Turck, F., Singer, K., Gissot, L., Le Gourrierec, J., Samach, A. and Coupland, G.

Wolberger, C. (1998) Combinatorial transcription factors. *Curr Opin Genet Dev*, 8, 552-559. Woollard, A. (2005) Gene duplications and genetic redundancy in C. elegans. *WormBook*, 1-6. Xiong, A.S., Peng, R.H., Zhuang, J., Gao, F., Zhu, B., Fu, X.Y., Xue, Y., Jin, X.F., Tian, Y.S.,

Yamamoto, A., Kagaya, Y., Toyoshima, R., Kagaya, M., Takeda, S. and Hattori, T. (2009)

Yazawa, K. and Kamada, H. (2007) Identification and characterization of carrot HAP factors

Yokoyama, S. and Yokoyama, R. (1989) Molecular evolution of human visual pigment

Zahn, L.M., Leebens-Mack, J., DePamphilis, C.W., Ma, H. and Theissen, G. (2005) To B or

Zanetti, M.E., Blanco, F.A., Beker, M.P., Battaglia, M. and Aguilar, O.M. (2010) A C subunit

interacting with seed-specific ABRE-binding factors. *Plant J*, 58, 843-856. Yang, J., Xie, Z. and Glover, B.J. (2005) Asymmetric evolution of duplicate genes encoding the CCAAT-binding factor NF-Y in plant genomes. *New Phytol*, 165, 623-631. Yang, X., Tuskan, G.A. and Cheng, M.Z. (2006) Divergence of the Dof gene families in

chloroplast genome. *Biotechnol Adv*, 27, 340-347.

duplication. *Plant Physiol*, 142, 820-830.

of the angiosperms. *J Hered*, 96, 225-240.

symbiosis. *Plant Cell*, 22, 4142-4157.

genes. *Mol Biol Evol*, 6, 186-197.

*Bot*, 58, 3819-3828.

characterization and interaction of HAP family genes in rice. *Mol Genet Genomics*,

after gene duplication: implications for the neutralist-selectionist debate. *Proc Natl*

M.B. and Kaufman, L.S. (2007) The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis. *Plant Physiol*,

(2006) CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis. *Plant Cell*, 18,

Zhao, W. and Yao, Q.H. (2009) Gene duplication, transfer, and evolution in the

Arabidopsis NF-YB subunits LEC1 and LEC1-LIKE activate transcription by

poplar, Arabidopsis, and rice suggests multiple modes of gene evolution after

that form a complex with the embryo-specific transcription factor C-LEC1. *J Exp*

Not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution

of the plant nuclear factor NF-Y required for rhizobial infection and nodule development affects partner selection in the common bean-Rhizobium etli

Siefers, N., Dang, K.K., Kumimoto, R.W., Bynum, W.E.t., Tayrose, G. and Holt, B.F., 3rd

Simillion, C., Vandepoele, K., Van Montagu, M.C., Zabeau, M. and Van de Peer, Y. (2002)

Singh, K.B. (1998) Transcriptional regulation in plants: the importance of combinatorial

Sinha, S., Maity, S.N., Lu, J. and de Crombrugghe, B. (1995) Recombinant rat CBF-C, the

Sinha, S., Kim, I.S., Sohn, K.Y., de Crombrugghe, B. and Maity, S.N. (1996) Three classes of

Steidl, S., Papagiannopoulos, P., Litzka, O., Andrianopoulos, A., Davis, M.A., Brakhage,

Sterck, L., Rombauts, S., Vandepoele, K., Rouze, P. and Van de Peer, Y. (2007) How many

Stoltzfus, A. (1999) On the possibility of constructive neutral evolution. *J Mol Evol*, 49, 169-

Tasanen, K., Oikarinen, J., Kivirikko, K.I. and Pihlajaniemi, T. (1992) Promoter of the gene

Taylor, J.S., Van de Peer, Y. and Meyer, A. (2001) Genome duplication, divergent resolution

Taylor, J.S. and Raes, J. (2004) Duplication and divergence: the evolution of new genes and

Testa, A., Donati, G., Yan, P., Romani, F., Huang, T.H., Vigano, M.A. and Mantovani, R.

Struhl, K. and Moqtaderi, Z. (1998) The TAFs in the HAT. *Cell*, 94, 1-4.

and speciation. *Trends Genet*, 17, 299-301.

old ideas. *Annu Rev Genet*, 38, 615-643.

*Biol Chem*, 280, 13606-13615.

Soltis, P.S. (2005) Ancient and recent polyploidy in angiosperms. *New Phytol*, 166, 5-8.

641.

1628.

203.

181.

11513-11519.

13627-13632.

*Biol*, 16, 328-337.

control. *Plant Physiol*, 118, 1111-1120.

aestivum. *Plant Mol Biol*, 65, 77-92.

(2009) Tissue-specific expression patterns of Arabidopsis NF-Y transcription factors suggest potential for extensive combinatorial complexity. *Plant Physiol*, 149, 625-

The hidden duplication past of Arabidopsis thaliana. *Proc Natl Acad Sci U S A*, 99,

third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3. *Proc Natl Acad Sci U S A*, 92, 1624-

mutations in the A subunit of the CCAAT-binding factor CBF delineate functional domains involved in the three-step assembly of the CBF-DNA complex. *Mol Cell*

A.A. and Hynes, M.J. (1999) AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR. *Mol Cell Biol*, 19, 99-106. Stephenson, T.J., McIntyre, C.L., Collet, C. and Xue, G.P. (2007) Genome-wide identification

and expression analysis of the NF-Y family of transcription factors in Triticum

genes are there in plants (... and why are they there)? *Curr Opin Plant Biol*, 10, 199-

for the multifunctional protein disulfide isomerase polypeptide. Functional significance of the six CCAAT boxes and other promoter elements. *J Biol Chem*, 267,

(2005) Chromatin immunoprecipitation (ChIP) on chip experiments uncover a widespread distribution of NF-Y binding CCAAT sites outside of core promoters. *J*

**Part 3**

**Examining Bundles of Genes**

**Part 3**

**Examining Bundles of Genes**

222 Gene Duplication

Zhang, J.Z. (2003) Evolution by gene duplication: an update. *Trends in Ecology & Evolution*,

Zwicker, J. and Muller, R. (1997) Cell-cycle regulation of gene expression by transcriptional

18, 292-298.

repression. *Trends Genet*, 13, 3-6.

**12**

*India*

**L-** *Myo***-Inositol 1-Phosphate**

Manoj Majee and Harmeet Kaur

**Synthase (MIPS) in Chickpea:**

**Gene Duplication and Functional Divergence**

*National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi*

Gene duplication is one of the key driving forces in the evolution of genes and important features of genomic architecture of living organisms including plants. Moreover, much of the plant diversity may have arisen largely due to duplication, followed by divergence and adaptive specialization of the pre existing genes (Ohno,1970; Zhang, 2003; Flagel & Wendel,2009). Current impetus on genomic sequence data provides substantial evidence for the profusion of duplicated genes in all organisms surveyed. Functional divergence after gene duplication can possibly result in two alternative evolutionary fates: i) neofunctionalization where one copy acquires an entirely new function whereas the other copy maintains the original function. ii) Subfunctionalization, in which each copy adopts part of the task of their parental gene (Ohno,1970; Nowak et al., 1997; Jenesen,1976; Orgel,1977;Hughes,1994). However, subfunctionalization is reported as a more prevalent outcome than neofunctionalization in nature. In any case, functional divergence of such paralogous proteins is found to be the key force shaping molecular network in organisms (Ohno, 1970). Recent studies also suggest that duplicate genes diverge mostly through the partitioning of gene expression as in subfunctionalization (Force et al.,1999; Wagner,2000; Gu et al.,2002). In addition, subfunctionalization can also take place at the protein function level leading to functional specialization, when one of the duplicated genes becomes better at performing one of the original functions of the progenitor gene (Hughes, 1994; Gu et

Myo-inositol-1-phosphate synthase (MIPS;EC<IP_ADDRESS>) is an evolutionary conserved enzyme which catalyzes the rate limiting step in well conserved inositol biosynthetic pathway and is extremely widespread in living organisms including plants (Loewus & Murthy, 2000; Majumder et al., 2003). The evolution of MIPS gene/ protein among the prokaryotes seems to be more divergent and complex than amongst the eukaryotes, however they preserve a

Many of the plant species are known to contain more than one copy of gene encoding MIPS and are hypothesized to arise through gene duplication. Expression studies of multiple gene encoding MIPS have revealed the possibility of specialized role for individual enzyme isoforms. Previously, two genes encoding MIPS have been identified and characterized from chickpea by Kaur *et al.* A comparative study of two divergent genes (*CaMIPS1* & *CaMIPS2*)

conserve core catalytic domain among the MIPS proteins (Majumder et al., 2003).

al.,2002; Conant &Wolfe, 2008; Hughes, 1999; Zhang et al., 2002).

**1. Introduction**


*2.3. E*ff*ects of Reaction Conditions on DGAS Transglycosylation*

Table 1 shows the conversion yields of IFAs to transglycosylated IFA glucosides according to the concentrations of donor and acceptors as well as the amount of DGAS. Glycitein reacted at lower concentrations than daidzein and genistein because of its lower solubility [38]. Regardless of IFA and sucrose concentrations, the conversion yield of each IFA used in this study increased as the amount of DGAS used increased, whereas the conversion yield of each IFA generally decreased with increasing IFA concentration. In transglycosylation reactions, a large amount of enzymes ensures not only a fast reaction rate, but also an increased reaction yield [33]. However, at a low donor concentration (0.1 M) and high acceptor concentration (20 mM), the conversion yields for daidzein and genistein were the lowest during transglycosylation for the highest amount of enzymes (5.0 U). The ratio of donor to acceptor suggests that the conversion yield is affected even if a sufficient amount of enzyme is supplied. Similar to the results of this study (Table 1), previous studies reported that the conversion efficiency increased when the concentration of the donor was higher than that of the acceptor [31,39,40]. The enzyme-modified glycosylation of lipophilic compounds is known to be complex because of the low solubility of lipophilic compounds in aqueous systems [41]. To increase the conversion yield despite low solubility, various solvents such as ionic liquids have been used in enzyme reactions for flavonoid glucoside synthesis [13,41]. In this study, we used dimethyl sulfoxide (DMSO) as the solvent to dissolve all IFAs (acceptors). However, cloudiness was observed during the enzyme reaction when the amount of acceptor was increased in an aqueous system containing a donor, acceptor, buffer, and enzyme (data not shown). Acceptor solubility was found to be the most important factor determining the lowest conversion yields at the highest acceptor conditions (glycitein, 4 mM; daidzein and genistein, 20 mM) (Table 1). Therefore, it is reasonable to assume that the reason glycitein had the lowest conversion yield was its relatively lower solubility than that of daidzein and genistein.

**Table 1.** Conversion yields of soy isoflavone aglycones after *Deinococcus geothermalis* amylosucrase (DGAS)-treated transglycosylation according to concentrations

 Data are expressed as means ± standard deviations (*n* = 3). Means with different superscripted letters indicate that for the same amount of DGAS, each isoflavonesignificantly by the Tukey–Kramer honestly significant difference test (*p*<0.05).

 of

The conversion yields of daidzein tended to be similar to those of genistein for the various reaction conditions used in this study. Glycitein had a lower conversion yield than genistein and daidzein (Table 1). Transglycosylation has been reported to occur at the hydroxyl group of the acceptor, resulting in the formation of an *O*-glycosidic bond between the acceptor and donor [35]. Likewise, a correlation between the number of hydroxyl groups and reactivity after the transglycosylation of hydroxyflavonoids with DGAS was observed [27]. The average conversion yields of daidzein and genistein were approximately 96.7% and 94.8%, respectively, at the highest amount of enzyme (5.0 U), lower acceptor concentrations (0.2–5.0 mM), and higher donor concentrations (1.0–2.0 M). In general, the highest conversion yield is achieved when sufficient enzymes and sucrose are provided for transglycosylation [33]. However, excess sucrose has been reported to reduce conversion yields [32]. Therefore, it is essential to have an adequate ratio of donor and acceptor as well as a sufficient amount of enzyme to attain higher conversion yields.


*Review* **Polyphenolic Flavonoid Compound Quercetin Effects in the Treatment of Acute Myeloid Leukemia and Myelodysplastic Syndromes**

**Cristiane Okuda Torello \* , Marisa Claudia Alvarez and Sara T. Olalla Saad \***

Hematology and Transfusion Medicine Center—Hemocentro, University of Campinas, Campinas 13083-878, Brazil<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>(C.O.T<EMAIL_ADDRESS>(S.T.O.S.);

Tel.: +55-(19)-35218734 (C.O.T.); +55-(19)-35218733 (S.T.O.S.)

**Abstract:** Flavonoids are ubiquitous groups of polyphenolic compounds present in most natural products and plants. These substances have been shown to have promising chemopreventive and chemotherapeutic properties with multiple target interactions and multiple pathway regulations against various human cancers. Polyphenolic flavonoid compounds can block the initiation or reverse the promotion stage of multistep carcinogenesis. Quercetin is one of the most abundant flavonoids found in fruits and vegetables and has been shown to have multiple properties capable of reducing cell growth in cancer cells. Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) therapy remains a challenge for hematologists worldwide, and the outcomes for patients with both disorders continue to be poor. This scenario indicates the increasing demand for innovative drugs and rational combinative therapies. Herein, we discuss the multitarget effects of the flavonoid quercetin, a naturally occurring flavonol, on AML and MDS.

**Keywords:** polyphenolic compounds; quercetin; hematological disorders; anti-proliferative effect; apoptosis; autophagy; epigenetic modifications

### **1. Introduction**

Flavonoids are ubiquitous groups of polyphenolic compounds present in natural products and plants such as fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine [1]. These natural substances have been shown to present promising chemopreventive and chemotherapeutic properties with multiple target interactions and multiple pathway regulation against various human cancers. Polyphenolic flavonoid compounds can block the initiation or reverse the promotion stage of multistep carcinogenesis [2]. Flavonoids are usually classified into six main subgroups: flavone (baicalein, apigenin), flavonol (fisetin, quercetin), flavanone (eriodyctiol, hesperidin), flavanol (catechin, epicatechin), anthocyanidin (cyaniding, apigeninidin), and isoflavone (daidzein, prunetin) (Figure 1).

Quercetin (3,30 ,40 ,5,7-pentahidroxiflavonol) is one of the most abundant flavonoids (Figure 2) belonging to the flavonol subgroup, found in fruits, vegetables, tea, and red wine [3,4]. Quercetin is also found in medicinal plants including *Allium cepa* (Liliaceae), *Allium fistulosum* (Amaryllidaceae), *Apium graveolens* (Apiaceae), *Asopargus officinalis* (Aspargaceae), *Camellia sinensis* (Theaceae), *Capparis spinosa* (Capparaceae), *Calamus scipionum* (Calamoidaceae), *Centella asiatica* (Apiaceae), *Coriandrum sativum* (Apiaceae), *Ginkgo biloba* (Ginkgoaceae), *Hypericum perforatum* (Hyperiaceae), *Hypericum hiricinum* (Clusiaceae), *Lactuca sativa* (Asteraceae), *Moringa oleifera* (Moringa), *Morus alba* (Moraceae), *Nasturtium officinale* (Brassicaceae), *Solanum lycopersicum* (Solanaceae), *Vitis vinifera* (Vitaceae), and *Sambucus canadensis* (Adoxaceae) [5–7]. Quercetin has anti-inflammatory, anticancer, cardiovascular protection, antiviral, antidiabetic, immunomodulatory, antiallergy, and antihypertensive therapeutic properties, in addition to gastroprotective effects. Quercetin is a yellow-colored

**Citation:** Torello, C.O.; Alvarez, M.C.; Olalla Saad, S.T. Polyphenolic Flavonoid Compound Quercetin Effects in the Treatment of Acute Myeloid Leukemia and Myelodysplastic Syndromes. *Molecules* **2021**, *26*, 5781. https:// doi.org/10.3390/molecules26195781

Academic Editors: Stefano Castellani and Massimo Conese

Received: 26 August 2021 Accepted: 20 September 2021 Published: 24 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

crystalline insoluble solid substance and has a bitter taste. Quercetin is slightly soluble in alcohol, aqueous alkaline solutions, and glacial acetic acid [5]. Quercetin is a yellow-colored crystalline insoluble solid substance and has a bitter taste. Quercetin is slightly soluble in alcohol, aqueous alkaline solutions, and glacial acetic acid [5]. Quercetin is slightly soluble in alcohol, aqueous alkaline solutions, and glacial acetic acid [5].

anticancer, cardiovascular protection, antiviral, antidiabetic, immunomodulatory, antiallergy, and antihypertensive therapeutic properties, in addition to gastroprotective effects.

anticancer, cardiovascular protection, antiviral, antidiabetic, immunomodulatory, antiallergy, and antihypertensive therapeutic properties, in addition to gastroprotective effects. Quercetin is a yellow-colored crystalline insoluble solid substance and has a bitter taste.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 2 of 12

*Molecules* **2021**, *26*, x FOR PEER REVIEW 2 of 12

**Figure 1.** Structure of the major classes of flavonoids. **Figure 1.** Structure of the major classes of flavonoids. **Figure 1.** Structure of the major classes of flavonoids.

**Figure 2.** Structure of quercetin. Quercetin contains the basic structure of flavonoids: diphenylpropane (C6–C3–C6) with hydroxyl groups attached to the rings. The official nomenclature according to IUPAC is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one. Other names include 5,7,3′,4′-flavon-3-ol, sophoretin, meletin, quercetine, xanthaurine, quercetol, quercitin, quertine, and flavin meletin. Chemical formula: C15H10O7. **Figure 2.** Structure of quercetin. Quercetin contains the basic structure of flavonoids: diphenylpropane (C6–C3–C6) with hydroxyl groups attached to the rings. The official nomenclature according to IUPAC is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one. Other names include 5,7,3′,4′-flavon-3-ol, sophoretin, meletin, quercetine, xanthaurine, quercetol, quercitin, quertine, and flavin meletin. Chemical formula: C15H10O7. **Figure 2.** Structure of quercetin. Quercetin contains the basic structure of flavonoids: diphenylpropane (C6–C3–C6) with hydroxyl groups attached to the rings. The official nomenclature according to IUPAC is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one. Other names include 5,7,30 ,40 -flavon-3-ol, sophoretin, meletin, quercetine, xanthaurine, quercetol, quercitin, quertine, and flavin meletin. Chemical formula: C15H10O7 .

Quercetin biosynthesis occurs though the phenylpropanoid metabolic pathway. Firstly, phenylalanine is converted to cinnamic acid; this reaction is catalyzed by the enzyme phenylalanine ammonialyase (Figure 3). Cinnamic acid undergoes the action of the enzyme cinnamate 4-hydroxylase to produce *p*-coumaric acid. This carboxylic group of synthesized *p*-coumaric acid undergoes ligation with CoA and produces 4-coumaroyl-CoA; this reaction is catalyzed by the enzyme p-coumarate:CoA ligase. Naringenin chalcone is produced by the enzyme chalcone synthase from one molecule of 4-coumaroyl-CoA and three molecules

of malonyl-CoA to form the essential A and B rings of the flavonoid skeleton (i.e., C6–C3– C6). The chalcone isomerase synthesizes naringenin (a flavanone); this reaction enables the construction of the heterocyclic C ring. Flavanone 3β-hydroxylase undergoes hydroxylation of naringenin and produces dihydrokaempferol. Flavonol 30 -hydroxylase undergoes the hydroxylation reaction on dihydrokaempferol to produce dihydroquercetin. Finally, the activity of the enzyme flavonol synthase on dihydroquercetin catalyzes the biosynthesis of quercetin [8,9]. CoA and three molecules of malonyl-CoA to form the essential A and B rings of the flavonoid skeleton (i.e., C6–C3–C6). The chalcone isomerase synthesizes naringenin (a flavanone); this reaction enables the construction of the heterocyclic C ring. Flavanone 3βhydroxylase undergoes hydroxylation of naringenin and produces dihydrokaempferol. Flavonol 3'-hydroxylase undergoes the hydroxylation reaction on dihydrokaempferol to produce dihydroquercetin. Finally, the activity of the enzyme flavonol synthase on dihydroquercetin catalyzes the biosynthesis of quercetin [8,9].

Quercetin biosynthesis occurs though the phenylpropanoid metabolic pathway. Firstly, phenylalanine is converted to cinnamic acid; this reaction is catalyzed by the enzyme phenylalanine ammonialyase (Figure 3). Cinnamic acid undergoes the action of the enzyme cinnamate 4-hydroxylase to produce *p*-coumaric acid. This carboxylic group of synthesized *p*-coumaric acid undergoes ligation with CoA and produces 4-coumaroyl-CoA; this reaction is catalyzed by the enzyme p-coumarate:CoA ligase. Naringenin chalcone is produced by the enzyme chalcone synthase from one molecule of 4-coumaroyl-

*Molecules* **2021**, *26*, x FOR PEER REVIEW 3 of 12

Quercetin has high bioavailability due to its aglycone structure, i.e., without a sugar group. The daily intake of quercetin in the diet is estimated to be 5–40 mg/day [2]; however, these values may be as high as 200–500 mg/day in individuals who consume large amounts of fruits and vegetables rich in quercetin (apples, onions, and tomatoes) [10]. In food, quercetin is found in a glycosylated pattern with bioavailability, which depends on the type of glycoside present in the molecule. After absorption, quercetin is metabolized in different organs, such as the intestine, colon, liver, and kidneys, where the molecule is conjugated to methyl, sulfate, and glucuronic acid [11]. The total amount of quercetin available in the plasma deriving from the diet is usually in the nanomolar concentration range (<100 nM); however, upon quercetin supplementation, this availability may increase to the high nanomolar or low micromolar range. For example, after 28 days of sup-Quercetin has high bioavailability due to its aglycone structure, i.e., without a sugar group. The daily intake of quercetin in the diet is estimated to be 5–40 mg/day [2]; however, these values may be as high as 200–500 mg/day in individuals who consume large amounts of fruits and vegetables rich in quercetin (apples, onions, and tomatoes) [10]. In food, quercetin is found in a glycosylated pattern with bioavailability, which depends on the type of glycoside present in the molecule. After absorption, quercetin is metabolized in different organs, such as the intestine, colon, liver, and kidneys, where the molecule is conjugated to methyl, sulfate, and glucuronic acid [11]. The total amount of quercetin available in the plasma deriving from the diet is usually in the nanomolar concentration range (<100 nM); however, upon quercetin supplementation, this availability may increase to the high nanomolar or low micromolar range. For example, after 28 days of supplementation with 1 g/day of quercetin, concentrations of up to 1.5 µM have been observed [12,13].

plementation with 1 g/day of quercetin, concentrations of up to 1.5 μM have been observed [12,13]. In vivo studies reported that low doses of 50–500 mg/kg per day of quercetin do not cause relevant adverse effects or carcinogenic effects in animals [13]. In humans, a phase I clinical study recommended a dose of 1400 mg/m2, i.e., 2.5 g for subjects weighing 70 kg, administered via intravenous infusions for 3 weeks at weekly intervals. High doses exceeding 50 mg/kg (i.e., 3.5 g/70 kg) produced renal toxicity without signs of nephritis or obstructive uropathy [14]. Other studies found no adverse effects associated with oral administration of quercetin in a single dose of up to 4 g or after one month with 500 mg twice In vivo studies reported that low doses of 50–500 mg/kg per day of quercetin do not cause relevant adverse effects or carcinogenic effects in animals [13]. In humans, a phase I clinical study recommended a dose of 1400 mg/m2 , i.e., 2.5 g for subjects weighing 70 kg, administered via intravenous infusions for 3 weeks at weekly intervals. High doses exceeding 50 mg/kg (i.e., 3.5 g/70 kg) produced renal toxicity without signs of nephritis or obstructive uropathy [14]. Other studies found no adverse effects associated with oral administration of quercetin in a single dose of up to 4 g or after one month with 500 mg twice daily [15]. In 1999, the International Agency for Research on Cancer concluded that quercetin is not classified as carcinogenic to humans. In the United States and Europe, supplements of quercetin are commercially available, and the beneficial effects of quercetin supplements were reported from clinical trials [16].

Quercetin has been shown to have multiple properties capable of reducing cell growth in cancer cells due to a variety of biological activities including antioxidant, antiproliferative, apoptotic, and autophagic capacities.

Acute myeloid leukemia (AML) is an aggressive and malignant disorder of hematopoietic stem cells characterized by the clonal expansion of abnormally differentiated blasts of

myeloid lineages, having unfavorable prognosis and short life expectancy [17]. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic neoplasms characterized by aberrant myeloid differentiation, ineffective hematopoiesis, refractory cytopenia, and increased rates of AML progression [18].

AML and MDS therapy remains a challenge for hematologists worldwide, and the outcomes of patients with both disorders remain poor. In MDS, the approval of azacitidine, decitabine, and lenalidomide over the last decade represented a major advance for this disease, despite the limited efficacy of these agents and most patients progressing within 2 years. Stem cell transplantation remains the only curative therapy; however, this option is associated with restricted efficacy and toxicity, and the lack or loss of response following standard therapies is related to dismal outcomes [18]. In AML, despite approximately 50% of patients successfully recovering from chemotherapy and bone marrow transplantation, elderly people are not capable of undergoing aggressive therapies, which demonstrates the urgent need for innovative drugs and rational combination therapies [19].

Herein, we discuss the multitarget effects of the flavonoid quercetin, a naturally occurring flavonol, on acute myeloid leukemia and myelodysplastic syndrome (Table 1).

**Table 1.** Summary of activities for quercetin in AML and SMD.

### **2. Multitarget Effects of the Flavonoid Quercetin on Acute Myeloid Leukemia and Myelodysplastic Syndrome**

#### *2.1. Antiproliferative Activity*

Quercetin inhibits the growth and proliferation of cell lines from different types of cancers (prostate, lung, breast, colon, and neoplasms) [20,37–40], and several mechanisms have been proposed to explain these effects. These studies have identified the ability of this compound to interact with specific regulatory proteins such as cyclins A, B, D and E; cyclin-dependent kinases (CDKs); and CDKs inhibitors (p21 and p27). Depending on the cell type, quercetin is able to block the G2/M or G1/S transition of the cell cycle.

The CDK activity regulator p21 is an important cell growth arrest mediator that inhibits cell entry into the G1 phase in response to DNA damage and hampers the reentry of G2 cells into the S phase by blocking cyclin E-CDK2 mediated retinoblastoma

(Rb) phosphorylation [41]. Rb is a direct substrate of CDKs and multiple mechanisms are known to potentially inhibit Rb phosphorylation, including proteasomal degradation [42] and inhibition of upstream Ras/RAF/MAPK or PI3K/Akt pathways [43,44].

Quercetin in vitro blocks cell-cycle progression at the G1 phase in P39 cells, a myeloid cell line derived from MDS-chronic myelomonocytic leukemia. Quercetin decreased the levels of CDK2, CDK6, cyclin D, cyclin E, cyclin A, and the phosphorylation of Rb accompanied by induction of p21 and p27 in these cells. In accordance, quercetin induced arrest in the G1 phase of the cell cycle in immunodeficient mice xenografted with P39 cells. The treatment of these mice with quercetin once every four days by intraperitoneal injection at 120 mg/kg body weight decreased the expression of phosphorylated Rb and the cyclin D and cyclin E proteins. Quercetin treatment also resulted in pronounced induction of p21 in xenografts [21]. In addition, pronounced ERK 1/2 and JNK phosphorylation in P39 cells and in P39 xenografts was induced. P39 cells treated with a combination of quercetin and selective inhibitors of ERK 1/2 and/or JNK (PD184352 or SP600125, respectively) decreased cells in the G1 phase, supporting the idea that ERK and JNK activation plays an important role in quercetin-induced G1 phase arrest in P39 cells [21].

Further studies have demonstrated that quercetin suppressed cell proliferation in the HL-60 cell line, derived from a 36-year-old woman with acute promyelocytic leukemia. Quercetin-induced G0/G1-phase arrest occurred [22,23] when expressions of cyclin-dependent kinases CDK2 and CDK4 were inhibited, and the CDK inhibitors, p16 and p21, were induced [23]. Quercetin also displayed antiproliferative activity against the HL-60 xenografts. After 3 weeks of quercetin treatment, a pronounced decrease in the protein levels of CDK2, CDK6, cyclin D, cyclin E and cyclin A were found. Quercetin treatment also resulted in Rb-phosphorylation loss [24].

The antiproliferative effect of quercetin was also demonstrated in the human promonocytic U937 cell line [20]. Quercetin induced antiproliferation and arrest of G2/M phase in these cells. An increase in the level of cyclin B in contrast to decreased levels of cyclin D, cyclin E, E2F1, and E2F2 was found in quercetin-treated U937 cells. Removal of quercetin from the cultures stimulated U937 cells to synchronously re-enter the cell cycle decreased the expression level of cyclin B, and increased the expression level of cyclin D and cyclin E, demonstrating that quercetin promotes reversible G2/M phase arrest. Quercetin further caused DNA fragmentation and increased the sub-G1 population in U937 cells [20].

#### *2.2. Apoptotic Activity*

The apoptotic effect of quercetin may result in the activation of multiple pathways in different types of cancer cell lines [25]. In vitro, quercetin led to pronounced apoptosis in the MDS-chronic myelomonocytic leukemia P39 cell line by modulating apoptotic cell death and stimulating the intrinsic and extrinsic apoptosis pathway [21]. Quercetin induced upregulation of the pro-apoptotic protein Bax and suppression of antiapoptotic proteins BcL-2, BcL-xL, and McL-1 in P39 cells. Furthermore, a decline in Rho123 fluorescence intensity and a release of cytochrome c from the mitochondria to the cytoplasm suggested that intracellular events related to P39 cell death caused by quercetin treatment are related to mitochondrial dysfunction and changes in the mitochondrial membrane potential (∆ψm). In addition, significant enhancements in cleaved caspase-9, caspase-8, and caspase-3 were observed after quercetin treatment in P39 cells. Improvement in active caspase-8 and increased expression of FasL indicate that the extrinsic or death receptor pathway of apoptosis is activated by quercetin in P39 cells. The results obtained in vivo confirm these findings, since quercetin treatment resulted in the reduction in P39 cell viability and decreased tumor volume of P39 subcutaneously xenografted in immunodeficient mice. Quercetin caused apoptosis activation and decreased the expression of BcL2, BcL-xL, and McL-1 followed by increased Bax expression in P39 xenografts, as well as enhanced cleaved caspase-3 in P39 xenografts [21].

The apoptotic effect of quercetin was also studied in NOD/SCID mice xenografted with HL-60 AML cells. Quercetin reduced the expression of antiapoptotic proteins BcL-

2, BcL-xL, and McL-1, but increased the expression of the proapoptotic protein BAX in xenografts. Induction of caspase-3 activation was further observed after quercetin treatment [24]. Yuan et al. [22] showed that quercetin caused apoptosis in HL-60 cells by decreasing the protein expression of PI3K and Bax, inhibiting Akt phosphorylation, decreasing the levels of BcL-2, and increasing the activation of caspase-2 and caspase-3 [22]. Quercetin induced apoptosis in HL-60 AML cells by increasing the activation of caspase-9, caspase-3, and cleaved poly ADP-ribose polymerase (PARP). Cleaved PARP protein levels were also increased in tumors obtained from HL-60 xenografts [23]. Niu et al. [26] demonstrated that quercetin induced apoptosis in HL-60 cells in a caspase-3-dependent manner by regulating the expression of downstream apoptotic proteins Bcl-2 and Bax [26]. Quercetin further potentiated the apoptosis induced by the glycolytic inhibitor 2-deoxy-dglucose (2-DG) in HL-60 cells. Cotreatment with quercetin and 2-DG elicited the opening of mitochondria pore transition, which preceded the triggering of apoptosis [27]. Quercetin induced the activation of ERK, and the inhibition of ERK by an ERK inhibitor abolished the quercetin-induced cell apoptosis in HL-60 cells, as confirmed by the activation of caspase-8, caspase-9, caspase-3, PARP cleavage, and mitochondrial membrane depolarization in these AML cells. In vivo experiments corroborated these findings by demonstrating that quercetin reduced tumor growth through activating the ERK pathway and the subsequent cell apoptosis in HL-60 xenografts [28].

The ability of quercetin to induce apoptosis via the mitochondrial pathway was also demonstrated in human promonocytic U937 cells: loss of mitochondrial membrane potential prompted the release of cytochrome c in the cytoplasm with the activation of caspase-3 [20]. Cheng et al. [29] reported that the administration of quercetin led to pronounced apoptosis in the human leukemia cell line U937 by Mcl-1 downregulation, Bax conformational change, and mitochondrial translocation that triggered cytochrome c release. To prove quercetin's effects, knockdown of Bax by siRNA was found to reverse quercetin-induced apoptosis and abolish the apoptosis and activation of caspase. Knockdown of Mcl-1 by siRNA improved Bax activation and translocation, and cell death induced by quercetin. Furthermore, in vivo administration of quercetin mitigated tumor growth and increased TUNEL-positive apoptotic cells in tumor sections of U937 xenografts. Quercetin also increased the expression of the pro-apoptotic protein Bax and inhibited the antiapoptotic McL-1 protein in xenografts [29]. Quercetin in combination with shHSP27 synergistically induced apoptosis by decreasing the Bcl2-to-Bax ratio in U937 cells [30]. The apoptosis induced in U937 cells occurred by downregulation of Mcl-1 acting directly or indirectly on mRNA stability and protein degradation [31]. Quercetin was also reported to cause the induction of PARP cleavage in the three AML cell lines: THP-1, MV4-11, and U937 [28]. Quercetin induced cell death by downregulating VEGRF2 and PI3K/Akt signaling; these pathways were related to quercetin's action on mitochondria and BCL2 proteins [36]. Increased VEGRF2 is related to chemotherapy resistance in human leukemia cells, and VEGRF2 reduction sensitizes AML cells to chemotherapy treatment, leading to an increase in mitochondrial mass and oxidative stress [45]. These findings reveal quercetin as a promising compound targeting VEGRF2. These data suggest that the ability of quercetin to induce apoptosis (both intrinsically and extrinsically) into cancer cells establishes quercetin, without a doubt, as a promising molecule in the field of oncology.

#### *2.3. Autophagic Activity*

Quercetin demonstrated autophagic activity on both acute myeloid leukemia and myelodysplastic syndrome cell lines. Quercetin was found to induce autophagy and cell death [46] and, apparently, autophagy activation is an attempt to rescue cells from induced apoptosis [47]. This flavonol may induce sirtuins [48], reactive oxygen species (ROS) production [49], and JNK 1/2; Mek/ERK [50], all of which are capable of influencing the regulation of the autophagic process. Another target is represented by PI3K pathway proteins, which may positively or negatively affect autophagic regulation. In addition, quercetin assists in the accumulation of hypoxia-induced factor (HIF)-1 alpha, which

represses mTOR signaling and induces the expression of BNIP3/BNIP3 ligand, supporting the rupture of the Beclin-1/BcL-2 (BcL-xL) complex for activation of Atg7 and Atg12-Atg5 and the cleavage of LC3 [47]. Conversely, quercetin may further inhibit autophagic flow. This result was obtained by the inhibition of PI3K class III and the NF-κB pathway (which may inhibit or induce autophagy depending on the context) [51]. Another interesting property of quercetin is that, when in combination with other substances, it may potentiate autophagic and apoptotic effects [52].

Quercetin triggered autophagy both in vitro and in vivo in P39 cells in the MDSderived leukemia cell line [21]. High acidic vesicular organelle formation was detected in quercetin-treated P39 cells. Quercetin increased the expressions of light chain 3 (LC3)-II, PI3K, Beclin-1, Atg5-Atg12, and Atg7 in P39 cells. The inhibition of autophagy in P39 cells using chloroquine (endossomal acidification inhibitor) triggered apoptosis but did not alter the modulation in the G1 phase, suggesting that quercetin-induced autophagy plays a protective role against apoptotic cell death in P39 cells but is not associated with the cell cycle [21]. In accordance with the in vitro results, quercetin increased the expressions of PI3K, Beclin-1, Atg5- Atg-12, and Atg7 in P39 xenografts. Increased cleaved LC3 was further detected in the tumor sections of xenografts by immunohistochemistry. Quercetin additionally dephosphorylated Akt and mTOR in P39 cells and P39 xenografts, underlining the functional importance of Akt–mTOR signaling in quercetin-mediated protective autophagy in P39 cells [21]. Quercetin was described to affect both mTOR activity and activation of PI3K/Akt signaling pathway, giving quercetin the advantage of functioning as a dual-specific mTOR/PI3K inhibitor. Akt-mTOR signaling is a frequently hyperactivated pathway in cancers and, as a key regulator of homeostasis, controls the essential pathways leading to cell growth, protein synthesis, and the autophagic process [53].

The induction of autophagy by quercetin on HL-60 AML xenografts was additionally described [24]. Quercetin increased the expressions of PI3K, Beclin-1, ATG7, and ATG12- ATG5 in HL-60 xenografts, and caused the pronounced induction of the LC3-I to LC3-II switch [24]. Chang et al. [23] reported the effect of quercetin on autophagy both in vitro and in vivo in the HL-60 AML cell line. Increased expression of LC3-II, decreased expression of p62, and formation of acidic vesicular organelles were found after quercetin treatment [23]. Decreased protein expression of PI3K and phosphorylated Akt were reported in the HL-60 cell line after quercetin treatment [22].

#### *2.4. Antioxidant Activity*

Quercetin is considered one of the most prominent dietary antioxidants [11]. The antioxidant effect of quercetin has been extensively described [54]. Quercetin is a potent scavenger of ROS, including superoxide (O2−), and reactive nitrogen species (RNS) such as nitric oxide (NO) and peroxynitrite (ONOO–). These antioxidative capacities of quercetin are attributed to the presence of two pharmacophores within the molecule that have the optimal configuration for free radical scavenging: the catechol group in the B ring and the OH group at position 3 of the AC ring [11].

During antioxidative activities, quercetin becomes oxidized into oxidation products (QQ: ortho-quinone) and reacts with glutathione (GSH) to form 6-glutathionyl quercetin (6- GSQ) and 8-GSQ (Figure 4). Oxidation products such as semiquinone radicals and quinones have been well-described to display various toxic effects in vitro only due to their ability to arylate protein thiols [11]. The antioxidative mechanism of quercetin occurs primarily with the O2 bond yielding the ortho-quinone and H2O2 radicals. Quercetin further reacts with H2O2 in the presence of peroxidase, decreasing H2O2 levels. In addition, the antioxidant capacity of quercetin depends on the availability of intracellular glutathione (GSH).

The antioxidative aspect of quercetin was associated with the amelioration of adverse side effects derived from cancer therapy, which is considered a suitable strategy for the prevention and treatment of cancer [55]. This property of quercetin was demonstrated in both acute myeloid leukemia and myelodysplastic syndrome.

scavenger of ROS, including superoxide (O2−), and reactive nitrogen species (RNS) such as nitric oxide (NO) and peroxynitrite (ONOO–). These antioxidative capacities of quercetin are attributed to the presence of two pharmacophores within the molecule that have the optimal configuration for free radical scavenging: the catechol group in the B ring and

During antioxidative activities, quercetin becomes oxidized into oxidation products (QQ: ortho-quinone) and reacts with glutathione (GSH) to form 6-glutathionyl quercetin (6-GSQ) and 8-GSQ (Figure 4). Oxidation products such as semiquinone radicals and quinones have been well-described to display various toxic effects in vitro only due to their ability to arylate protein thiols [11]. The antioxidative mechanism of quercetin occurs primarily with the O2 bond yielding the ortho-quinone and H2O2 radicals. Quercetin further reacts with H2O2 in the presence of peroxidase, decreasing H2O2 levels. In addition, the antioxidant capacity of quercetin depends on the availability of intracellular glutathione

the OH group at position 3 of the AC ring [11].

(GSH).

**Figure 4.** Antioxidative mechanism of quercetin. Quercetin is oxidized into oxidation products (QQ: ortho-quinone) and reacts with glutathione (GSH) to form 6-glutathionyl quercetin (6-GSQ) and 8- GSQ. **Figure 4.** Antioxidative mechanism of quercetin. Quercetin is oxidized into oxidation products (QQ: ortho-quinone) and reacts with glutathione (GSH) to form 6-glutathionyl quercetin (6-GSQ) and 8-GSQ.

The antioxidative aspect of quercetin was associated with the amelioration of adverse side effects derived from cancer therapy, which is considered a suitable strategy for the Quercetin decreased ROS levels in P39 MDS-derived leukemia cells induced by terbutylhydroperoxide [21]. The antioxidative activity of quercetin in P39 cells could be explained by its capacity to interact with important antioxidant cellular defense systems, such as the Nrf2/Keap complex. This complex is connected with apoptotic pathways through the regulation of proteins from the Bcl-2 family. Keap1 appears to restrain and destabilize Bcl-2 and decrease Bcl-2:Bax heterodimers, facilitating cancer cells apoptosis [56].

Quercetin further reduced ROS and NO in LPS-stimulated human THP-1 acute monocytic leukemia cells [32]. In contrast, quercetin was shown to induce the cell death of HL-60 AML cells in vitro and in vivo through the induction of intracellular oxidative stress following activation of ERK. The authors demonstrated that mitochondrial superoxide and intracellular peroxide levels in HL-60 AML cells and HL-60 xenografts were higher after quercetin treatment [28]. In another study, quercetin caused disparate effects on ROS generation in HL60 cells, and did not affect GSH levels [27]. In a human acute promyelocytic leukemia NB4 cell line, quercetin increased the levels of Nrf2 in the cytosol, reducing them in the nucleus [33].

#### *2.5. Epigenetic Modulation*

An important feature of quercetin was recently reported: the ability to affect epigenetic regulators. Studies evaluating the epigenetic effects of quercetin in MDS are not available.

In AML, quercetin induced cell death in HL-60 and U937 leukemia cell lines by targeting the epigenetic regulators of pro-apoptotic genes [34]. The results showed that quercetin treatment abolishes DNA methyltransferase (DNMT) 1 and DNMT3a expressions partly due to STAT-3 regulation. Downregulation of class I histone deacetylases (HDACs) by quercetin was also detected. Treatment of cells with MG132, a proteasome inhibitor, in combination with quercetin prevented the degradation of class I HDACs compared to cells treated with quercetin alone, indicating increased proteasome degradation of class I HDACs by quercetin. Demethylation of the pro-apoptotic BCL2L11 and DAPK1 genes in a dose- and time-dependent manner was observed after quercetin treatment. Moreover, quercetin caused a global increase in acetylated histone 3 and histone 4, resulting in threeto ten-fold increases in the promoter regions of DAPK1, BCL2L11, BAX, APAF1, BNIP3, and BNIP3L. The increase in the mRNA levels of all these genes induced by quercetin corroborates these findings [34]. Briefly, the induction of apoptosis by quercetin appears to be related to the influence of this flavonoid on DNA demethylation activity, HDAC inhibition, and the enrichment in H3ac and H4ac in the promoter regions of genes involved in the apoptosis pathway, leading to their transcription activation.

Induction of FasL-related apoptosis by quercetin was described by transactivation through activation of c-jun/AP-1 and promotion of histone H3 acetylation in human HL-60 leukemia cells [35].

#### **3. Conclusions**

The natural polyphenolic flavonoid compound quercetin has emerged as a molecule possessing multiple properties (Figure 5). Quercetin has been proven to be an excellent antioxidant that also possesses antiproliferative, apoptotic, and autophagy capacities in AML in vitro and in vivo, in addition to exerting effects on epigenetic modulation. The effects on MDS are poorly understood as few studies have been performed. Clinical studies evaluating quercetin's effects on AML and MDS patients are not available. However, we conclude that quercetin has the potential to induce cell death in human AML and MDSderived leukemia cell lines, both in vitro and in vivo, and that this capacity is related to a mechanism involving multitarget cooperation. The ability of this flavonoid to interfere with different mechanisms involved in cancer development indicates quercetin as an alternative for acute myeloid leukemia and myelodysplastic syndrome. *Molecules* **2021**, *26*, x FOR PEER REVIEW 10 of 12 to a mechanism involving multitarget cooperation. The ability of this flavonoid to interfere with different mechanisms involved in cancer development indicates quercetin as an alternative for acute myeloid leukemia and myelodysplastic syndrome.

**Figure 5.** Multitarget effects of quercetin on acute myeloid leukemia and myelodysplastic syndrome. **Figure 5.** Multitarget effects of quercetin on acute myeloid leukemia and myelodysplastic syndrome.

**Author Contributions:** Conceptualization, C.O.T. and S.T.O.S; writing—original draft preparation, C.O.T.; writing—review and editing, C.O.T., M.C.A., and S.T.O.S; supervision S.T.O.S. All authors

**Acknowledgments:** The authors would like to thank Raquel S. Foglio for the English review. The

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manu-

script; or in the decision to publish the results.

**Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable. **Data Availability Statement:** Not applicable.

have read and agreed to the published version of the manuscript.

de São Paulo (FAPESP), grant numbers 2017/21801-2 and 14/08939-7.

figures were produced using BKchem 0.13.0 and ChemDraw 19.0.0 software.

**Author Contributions:** Conceptualization, C.O.T. and S.T.O.S.; writing—original draft preparation, C.O.T.; writing—review and editing, C.O.T., M.C.A. and S.T.O.S.; supervision S.T.O.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant number 303405/2018-0; and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant numbers 2017/21801-2 and 14/08939-7.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Raquel S. Foglio for the English review. The figures were produced using BKchem 0.13.0 and ChemDraw 19.0.0 software.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


*3.3. RNA-Based Delivery*

Similar to recombinant alphavirus particle delivery, RNA replicon administration has proven efficient in vaccine development [34]. For example, a single intramuscular injection of 0.1 μg SFV-LacZ replicon RNA generated antigen-specific antibody and CD8+ T cell responses in immunized mice [37]. Immunization with SFV-LacZ RNA prior to challenges by colon tumors provided protection in mice. Moreover, the therapeutic vaccination of animals with pre-existing tumors resulted in prolonged survival. Interestingly, the levels of antigen production for RNA replicons in vitro were not significantly higher than those observed for conventional DNA vaccines, but in vivo the enhanced efficacy correlated with a caspase-dependent apoptotic cell death. In another approach, a SIN RNA replicon expressing the rabies virus glycoprotein gene was applied for immunization studies with 10 μg of SIN-Rab-G RNA in comparison to a conventional rabies DNA vaccine and the commercial cell culture vaccine Rabipur [38]. The SIN-Rab-G RNA immunization elicited similar cellular and humoral IgG responses in comparison to the rabies DNA vaccine. Moreover, the alphavirus RNA vaccine provided similar protection to the rabies DNA vaccine against challenges with the lethal rabies virus CVS strain.

In addition to naked RNA delivery, alphavirus vectors have also been subjected to nanoparticle encapsulation procedures [39]. An in vivo expression comparison of 1 × 106 IU recombinant VEE particles, 1 μg of naked replicon RNA, 1 μg of replicon RNA encapsulated in lipid nanoparticles (RNA/LNPs), 10 μg of conventional plasmid DNA, and 10 μg of replicon DNA expressing firefly luciferase was carried out in mice 7 days after bilateral intramuscular administration. The luciferase levels were similar for RNA/LNPs and VEE particles, but significantly higher than for naked replicon RNA, replicon DNA, and plasmid DNA. The immunogenicity of delivery modes was evaluated by heterologous expression of the respiratory syncytial virus fusion protein (RSV-F) after intramuscular administration. The F-specific IgG response to 1 μg RNA/LNPs was equivalent to that of 1 × 106 IU of VEE particles. In contrast, plasmid DNA/LNPs at a dose of 0.1 μg and 20 μg of electroporated plasmid DNA elicited much lower IgG titers. RNA/LNPs, replicon RNA, VEE particles and an RSV-F subunit vaccine were evaluated for protection against viral challenges after intranasal RSV challenges in cotton rats. All replicon RNA vaccines protected animals for RSV challenges reducing the viral load more than 1000-fold in the lungs. The RNA/LNPs (1 μg) elicited similar responses as VEE particles. However, the recombinant F subunit vaccine formulated with alum showed the highest potency. In another study, naked RNA from SFV replicon (rSFV-NP) and poliovirus (rDELTA1-E-NP) vectors expressing the influenza type A virus nucleoprotein (NP) were intramuscularly administered in C57BL/6 mice [40]. Both rSFV-NP and rDELTA1-E-NP elicited antibodies against the influenza virus NP, but CTL responses against the immunodominant H-2D(b) epitope NP366 was only obtained with the SFV replicon RNA. Furthermore, reduced virus load was demonstrated for rSFV-NP after challenges with a mouse-adapted influenza A/PR/8/34 virus in immunized mice. The protective potential for RNA replicon immunization was similar to what has previously been achieved for plasmid DNA immunizations.


**3.1 Improving impact assessment**

Almost a decade ago, it was argued that no impact assessment method had been developed for biomimicry and that current ways of impact assessment followed a "reduced unsustainability" paradigm instead of following an "achieving sustainability" paradigm [4]. This "achieving sustainability" paradigm is not entirely opposed to the conventional way of performing impact assessment. However, these paradigms differ from each other as follows [4]:

Having a beneficial impact, here, refers to the impact on the environment contributing to the regeneration of that environment toward a sustainable state [4]. Internalizing such a new paradigm helps enlighten how the impacts are assessed, highlighting the product benefits to sustainability's environmental, social, and economic aspects.

#### *Including Nature-Based Success Measurement Criteria in the Life Cycle Assessment DOI: http://dx.doi.org/10.5772/intechopen.110401*

Recent studies found in the literature present potential to materialize such a newparadigm-based tool based on biomimetics [46], on biomimicry [40], and on cradleto-cradle (C2C, also known as regenerative design approach) [15]. These studies followed the ten biomimicry principles [45]. **Table 1** presents a comparison of these three approaches.

Moreover, Terrier et al. [46], in 2019, proposed an assistance tool to provide a quantitative performance tool for biomimetic-based designs. This tool has been developed as a complement to the ISO 1845 standard. In this tool, the ten principles of biomimicry are grouped into three dimensions of biomimetics design: Efficiency and frugality, preservation and resilience, and circularity and systemic approach. Whether this tool is based on biomimicry or biomimetics, or the authors used these terms to refer to the same, needs to be clarified. However, this tool only assesses the environmental impact of the biomimetic design based on the midpoint approach in the LCIA, not including quantifications for other aspects of sustainability, that is, economy and society. Such a tool for impact assessment of biomimetic design still follows the "reduced unsustainability" paradigm due to the questions and metrics employed [46].

Furthermore, Peralta et al. [15], in 2021, proposed an upgrade to the cradle-tocradle approach (a form of biomimicry) for sustainable designs. Since for the cradleto-cradle approach there are not yet reported normative and guidelines, considering only environmental and social aspects qualitatively without any available tool, the proposed upgrade intends to assess both positive and negative impacts by dividing the assessment into three levels, that is, evaluating the LCI at micro, meso, and macro levels. At the micro level, the calculations are based on midpoint indicators; thus, they do not account for the entire context. At the meso level, the quality of the resources (or inputs) is evaluated. Finally, at the macro level, future effects are considered, including calculations for optimal design, allowing more sustainable product versions. The latter indicates that this proposed upgrade (or methodology) finalizes with assessing the product's progress, which coincides with the "reduction of unsustainability" paradigm. Despite this, this proposed methodology advances the field by providing a quantitative tool for complementing the cradle-to-cradle approach [15].

Finally, later in the same year, among the interest in considering sustainability, a different approach based on biomimicry may have potential application toward the "achieving sustainability" paradigm. This approach, referred to as the "Biocircular model" by Beermann and Austin [40], is a conceptual nature-inspired approach built upon the sustainability consideration discrepancies among sustainable construction projects. Following the problem-based approach [47, 48], this Biocircular model is founded upon various biomimicry principles, combined with the circular economy and sustainability, leading to four supporting qualitative valuations, that is, active (A), behavior (B), housing (H), and share (S), that helps include sustainability as a target to the problem considered. Here, a complete qualitative analysis is presented for the six phases of construction projects, ending with the delivery phase, which was then supported by surveying experts in the field. Thus, as a qualitative approach that looks for the sustainability target in each phase of the construction projects individually, it coincides with the new paradigm previously mentioned. However, no quantitative criteria are provided [40]. Besides, existing indicators were contrasted with the Biocircular model approach, and none fulfilled the four supporting valuations. In contrast, four existing qualitative indicators did fulfill the supporting valuations: Reuse of construction elements [49], reuse of excavation materials for backfill [49], use of local material to reduce emissions [49], and water reuse system [50]. In a sense, this also follows the cradle-to-cradle principles.

*The color indicates potentially promising.\* Provide a detailed way for calculation (quantitative). \*\*Qualitative.*

#### **Table 1.**

*Evaluation of recent potential nature-inspired tools for impact assessment toward sustainability against the "achieving sustainability" paradigm.*

## **3.2 Biomimicry, circularity, and sustainability in buildings**

Now, a brief look into the applications of the arguments previously presented from the point of view of the built environment, specifically to sustainable construction projects such as net-zero-energy (NZE) buildings or green buildings.

*Including Nature-Based Success Measurement Criteria in the Life Cycle Assessment DOI: http://dx.doi.org/10.5772/intechopen.110401*

**Figure 6.** *Principles of sustainable buildings and biomimetic design applicable to the life cycle of a building.*

Although the BiomiMETRIC tool [46] has an environmental dimension, in conjunction with the principles of sustainable construction [41], principles applicable to the LCA stages of a building were identified and can be visualized in **Figure 6**. While it is true that achieving sustainability requires going beyond the environmental impact, it is notorious that LCA can maximize its scope and approach, where the use of a second life through circularity in materials and components can have great benefits [39]. In the principles for biomimetic design, circularity is complemented by systematization, and beyond environmental preservation, resilience is addressed [46], which is a more developed degree of adaptation.

Another approach not included in the sustainable principles but found in the Green Building Rating Systems (GBRT) [42] is efficiency and optimization, one of the most highlighted points when applying a biomimetic methodology to solve a problem. It should be emphasized that another challenge within the principles and their application is to maintain consistency between the different phases. In GBRT, the environmental dimension predominates with the number of indicators, while the construction and end-of-life phases have the fewest indicators [42].

In **Table 2**, matching principles were identified between [46] and [41] in order to obtain pinnacles, that is, biological entities that attend or act according to the established principles, applying the biomimetic methodology of "Living envelope" [51] complemented by the AskNature tool [52] from the Biomimicry Institute.

Based on the pinnacles found by [40], the inspirational capabilities in nature are illustrated:

#### **Table 2.**

*Pinnacles identified that meet the principles of biomimetics and sustainable construction according to area.*

In addition to the principles mentioned in **Figure 6**, design and management principles, such as quality and cooperation, were added in **Table 2** to broaden the areas of principles that do not apply to the life cycle itself but are present in its formulation, strategy, and control, and which are increasingly becoming a necessity in early design [53]. Other principles regarding decentralization, such as diversity, redundancy, and independence, were not considered, as they can be included at the qualitative level of planning and management [54].