Zea mays L.), unilateral cross-incompatibility (UCI) is controlled by Gametophyte factors (Ga), including Ga1, Ga2, and Tcb1; however, the molecular mechanisms underpinning this process remain unexplored. Here, we report the pollination phenotype of an inbred line, 511L, which carries a near-dominant Ga2-S allele. We performed a high-throughput RNA sequencing (RNA-Seq) analysis of the compatible and incompatible crosses between 511L and B73, to identify the transcriptomic differences associated with Ga2-mediated UCI. An in vivo kinetics analysis revealed that the growth of non-self pollen tubes was blocked at the early stages after pollination in 511L, maintaining the UCI barrier in Ga2. In total, 25,759 genes were expressed, of which, 2063 differentially expressed genes (DEGs) were induced by pollination . A gene ontology (GO) enrichment analysis revealed that these genes were specifically enriched in functions involved in cell wall strength and pectic product modification. Moreover, 1839, 4382, and 5041 genes were detected to differentially express under same pollination treatments, including B_G, BG_GG, and BB_GB, respectively. A total of 1467 DEGs were constitutively expressed between the two inbred lines following pollination treatments, which were enriched in metabolic processes, flavonoid biosynthesis, cysteine biosynthesis, and vacuole functions. Furthermore, we confirmed 14 DEGs related to cell wall modification and stress by qRT-PCR, which might be involved in Ga2-S-mediated UCI. Our results provide a comprehensive foundation for the molecular mechanisms involved in silks of UCI mediated by Ga2-S.In maize ( Zea mays L.) is a typical cross-pollinating crop with distinct male and female flowers on the same plant. In general, both selfing and crossing strategies are readily successful in most maize lines; however, certain lines fail to set seeds when pollinated with non-self pollen, hybridizing in only one direction. The factors governing this unilateral cross-incompatibility (UCI) were initially designated Gametophyte factors (Ga) because of the involvement of the gametes in this selective fertilization   and compared with the RNA-Seq data. A relatively higher correlation among the expression levels was obtained by a Pearson correlation coefficient , as shown in To validate the expression level of candidate DEGS revealed by RNA-Seq, we selected four cell wall modification genes, three genes encoding LRKs and seven downregulated stress related genes for cross-incompatibility in 511L and B73 reaction. The gene expression values determined using quantitative real-time PCR (qRT-PCR) and the values were transformed into logArabidopsis thaliana [Lilium longiflorum L.), the pollen tubes grow through a hollow pistil [The entire process of pollination consists of a number of successive steps initiated after pollen deposition on the stigma, including pollen adhesion, hydration, and the germination of the pollen tube. Elongation at the tip is typical for polar pollen tube growth, as pollen tubes first penetrate the intercellular space between the stigma and style, then grow along the nutritious extracellular matrix of transmitting tract tissues towards the ovary. Transmitting tract tissues differ significantly between species, but are thought to consist of specialized cell files associated with the vascular tissue in thaliana  and maizthaliana , while iw pistil .Ga1 and Tcb1, and reported the allelic functions of these loci present in differently compatible maize lines [Ga2-S allele. An in vivo investigation of the dynamic growth of pollen tubes revealed that their growth in plants expressing the ga2 allele was blocked at the style elongation stage  and B73 . Overall, an average of 93% and 86% reads from the B73 and 511L silk tissues, respectively, were mapped to the B73 reference genome, with this difference highlighting the genetic diversity between the B73 and 511L genetic backgrounds. The total number of transcribed genes we identified, 25,759, is similar to that of a previous study in Zheng58 silks, which identified approximately 23,000 genes expressed in different silks after pollination [Over the past decade, transcriptional profiling techniques, including microarrays and RNA-Seq, have been widely used for studying male–female interactions during pollination ,67; howelination . ga2 pollen genotype recognition and rejection. Signal transduction mediated by receptor kinases occurs in a variety of biological processes, such as cell growth, development, and differentiation [Brassica rapa, the papillary cells expressed the S receptor kinase gene (SRK) together with the S locus glycoprotein gene (SLG) to control SI [In flowering plants, RLKs are essential for pollen tube guidance, reception, and the rupture of the pollen tube in the ovule ,70,71; fntiation . In Brasntrol SI ,73. In tntrol SI . In this2+ to strengthen the cell wall. At the same time, the pH of the region surrounding the pectin decreases, which could enhance the enzymatic activity of the polygalacturonases and pectate lyases. These enzymes likely contribute to cell wall loosening and facilitate its extension during pollen tube growth [Arabidopsis thaliana, and the altered expression of VGD1 led to abnormal pollen tube growth [PME48 and QRT1, affected pollen germination and development [PME43 (GRMZM2G128549), PMEI , and cellulose mannan endo-1,4-beta-mannosidase 1  were particularly enriched by ClueGO analysis  and UDP-glycosyltransferase 72B1 (UGT72B1: GRMZM2G162783) were upregulated after pollination in both B73 and 511L  was upregulated in 511L, but not in the B73 silks, suggesting a role for this gene in silk/pollen tube cell wall extension. Moreover, our results indicate that the regulation of pollen tube apical growth during pollen tube elongation was probably controlled by cell wall-related factors derived from the silks. The differences in the expression of genes involved in cell wall modification and remodeling between the ga2 and Ga2-S silks may contribute to the prevention of pollen tube growth during UCI in Ga2-S silks.Plant cell walls provide an essential protective barrier, functioning in development and defense. These complex extracellular structures are mainly composed of cellulose, hemicellulose, and pectins, as well as structural proteins, which are typically assembled into a rigid but flexible and dynamically organized network ,61. Pecte growth . PMEI noe growth ,80. VANGe growth . Similarelopment ,83. In tanalysis . β-Mannaand 511L , which cGRMZM2G1249, PMEI l growth , and dowGa-mediated cross-sterile system provides the potential of avoiding adventitious pollen. Previously, Ga1-S, Tcb1-S, and Ga2-S were reported to facilitate cross-incompatibility in conventional dent and flint maize [ga1 ga1) parental inbred lines were crossed with SDGa25 , and the F1 plants were subsequently backcrossed for multiple generations and self-crossed to establish a homozygous JKN2000 (Ga1-S/Ga1-S) hybrid, which displayed a significantly reduced risk of cross-fertilization [Ga1 in the cross-incompatibility system, it still lacks sufficient genetic resources to prevent outcrossing; therefore, the incorporation of Ga2 will provide an additional strategy to control cross-fertilization.Hybrid maize lines are popular for their increased yields. Controlling the pollination of the parental inbred lines is a key to maximizing the genetic purity of the hybrid seeds, particularly for sweet, waxy, and genetically modified maize. Commercial seed production plots are normally geographically isolated from each other by at least 500 m, and temporally isolated by about 10 days; nonetheless, pollen contamination and outcrossing are still largely unavoidable ,90. The nt maize ,18,91,92lization . DespiteGa2-S silk barrier. Through a comprehensive transcriptome analysis, large numbers of genotype-dependent DEGs were found to be induced by pollination, revealing a complex regulatory network in pollen–pistil communication. Specifically, signal transduction and cell wall metabolism related processes, such as pectin modification and sugar metabolism were most significant responses upon pollination in UCI, including LRR-RLKs, PME/PMEI, polygalacturonases and pectate lyases, UDP-glycosyltransferases, and expansins. Pectin, a major component of the cell wall, likely plays a vital role in the extension of the pollen tube tip. These findings will not only help us to understand the molecular mechanisms underlying Ga2-mediated UCI, but also provide potential genetic resources for controlling pollination during the production of hybrid seeds.In conclusion, we demonstrate that incompatible pollen grains can undergo the early processes of adhesion, hydration, germination, and penetration, however, pollen tube elongation is blocked in the Zea mays L.) inbred line 511L, containing the Ga2-S locus, was obtained from the MaizeGDB stock center [Ga1-S), as well as with the Chinese elite inbred lines Zheng 58, Chang 7-2, L75, Z1, JKN2000F, JKN2000M, Huangzao4, L26, B8-2, Qi319, and B73, was examined. Seed set was also determined by pollinating 30 de-tasseled 511L plants with B73 pollen during the flowering peak . Seed setting was evaluated post-harvest. All pollination tests were conducted in the Experimental Station in Beijing, China . The maize  at 4 °C. The fixed silks were rehydrated in 50% and 25% ethanol for 3–5 min, and washed three times with 0.1 M potassium phosphate buffer. Subsequently, the samples were incubated in 8.0 M sodium hydroxide solution for 2 h and washed as above, with subsequent staining in 0.1% (w/v) hydrosoluble aniline blue (dissolved in 0.1 M potassium phosphate buffer) for 8 h. The pollen tube entry point on the silk surface was regarded as the starting point for growth. Silks with consistent lengths were examined in a relatively dark room and mounted on a slide for microscopic analysis  under UV excitation (wavelengths 340 nm to 488 nm), facilitating the measurement of the pollen tube lengths. In vivo pollen tube growth was examined using the aniline blue staining method ,63, withR2 values higher than 0.85 by QC  were used for subsequent sequencing. To reduce pollen contamination, excess pollen was removed from the silks by washing them three times in distilled water. Non-pollinated silks  were collected as the control group. All tissues were immediately frozen in liquid nitrogen following collection and stored at −80 °C for RNA extraction. Silks were collected for RNA-Seq analysis during the flowering peak, from around 9:00 to 12:00. Fresh, mature pollen of 511L and B73 was collected for use either in self-crossing  or in hybridization . Pollinated silks were collected at 3 hap, when significant differences in pollen tube growth occurred between the compatible and incompatible crosses. Two biological replicates were collected per cross. To ensure the reliability of replicates, only the samples with the Pearson Total RNA was isolated using the TRIzol reagent  . To elimRenfGen_v3) [The raw data (reads) were filtered using the standard Illumina pipeline, with additional filtering steps to identify the adapters. To obtain the clean data (clean reads) with good quality, the reads containing adapter, poly-N, and low-quality reads were abandoned. All data were collected and aligned to the maize reference genome sequence (B73 fGen_v3)  using TofGen_v3) ,96. The fGen_v3) .p-value < 0.05 was considered to reflect a significantly enriched GO term. Pairwise comparisons were performed on the RNA-Seq data using the DESeq R package (1.10.1), to identify DEGs between the silks pollinated with compatible and incompatible pollen . A two-fp-value < 0.05, right-sided hypergeometric test with Benjamini–Hochberg correction of false discovery rate) were included in the analysis. The minimum connectivity of the pathway network was created by initially defined groups based on kappa score threshold ≥0.3. The final groups are fixed or randomly colored and overlaid with the network.Using plugin ClueGO of Cytoscape , DEGs inGAPDH), used as a normalized internal reference, was confirmed by a semi-quantitative PCR reaction using Taq DNA polymerase , and a qRT-PCR with all most equal expression levels in all six samples  by TRIzol reagent (Thermo Fisher Scientific) , and the samples . The rel


Apelin receptor(s) (APLNR) are suggested to mediate the actions of apelin and Elabela (ELA) peptides in many physiological processes, including cardiovascular development and food intake in vertebrates. However, the functionality of APLNR has not been examined in most vertebrate groups. Here, we characterized two APLNRs APLNR1, APLNR2) in chickens and red-eared sliders, and three APLNRs in zebrafish , which are homologous to human APLNR. Using luciferase-reporter assays or Western blot, we demonstrated that in chickens, APLNR1 (not APLNR2) expressed in HEK293 cells was potently activated by chicken apelin-36 and ELA-32 and coupled to Gi-cAMP and MAPK/ERK signaling pathways, indicating a crucial role of APLNR1 in mediating apelin/ELA actions; in red-eared sliders, APLNR2 (not APLNR1) was potently activated by apelin-36/ELA-32, suggesting that APLNR2 may mediate apelin/ELA actions; in zebrafish, both APLNR2a and APLNR2b were potently activated by apelin-36/ELA-32 and coupled to Gi-cAMP signaling pathway, as previously proposed, whereas the novel APLNR3a was specifically and potently activated by apelin. Similarly, an apelin-specific receptor (APLNR3b) sharing 57% sequence identity with zebrafish APLNR3a was identified in Nile tilapia. Collectively, our data facilitates the uncovering of the roles of APLNR signaling in different vertebrate groups and suggests a key functional switch between APLNR1 and APLNR2/3 in mediating the actions of ELA and apelin during vertebrate evolution. APLN) gene, and have been shown to be capable of binding APLNR with high affinities  has been identified as a class A (rhodopsin-like) G protein-coupled receptor (GPCR) and originally named APJ, which shares a relatively high amino acid sequence identity 31%) with angiotensin II receptor (AT1), but displays no specific binding toward angiotensin II peptide % with an. A laterfinities . It is rfinities –8. Sincefinities . For insfinities –11. In hfinities , 13. Morfinities –14. The finities , 16, blofinities , 18, flufinities , angiogefinities , neuroenfinities , heart dfinities –24, foodfinities , 26, drifinities , 28, pitfinities , 29, metfinities , and neufinities . Similarelopment , 33, myoelopment , 35, furtebrates .in vitro studies support that ELA can bind to the two APLNRs (APLNRa and APLNRb) in zebrafish and cause receptor internalization    has been identified in zebrafish , 38. Zeblization , 38. Morelopment –39. All elopment –42. Likeelopment  and angielopment . Loss ofcidence) , 44. Int pathway .Despite the identification of APLNR(s) and its two ligands  in mammals and zebrafish, the structure, functionality and tissue expression of the APLNRs in many vertebrate groups including birds have not been fully characterized. Therefore, in our present study, we aimed to characterize APLNR in several representative vertebrate species including chickens, turtles and zebrafish. Strikingly, we identified two APLNRs (APLNR1 and APLNR2) in chickens and turtles, and three functional APLNRs in zebrafish, including a novel apelin-specific receptor (APLNR3), which has not been reported in any vertebrate before. Undoubtedly, our study establishes a molecular basis to elucidate the roles of APLNRs and their ligands in different vertebrate groups.Taq DNA polymerase were purchased from Takara . Primers used in this study were synthesized by TSINGKE  and listed in Supplementary Table Chicken apelin-36, apelin-13, ELA32a (with an intra-molecular disulfide bond), ELA32b (without a disulfide bond), and ELA-11, and zebrafish apelin-36 and ELA-36/ELA-22 (without a disulfide bond) peptides were synthesized (>95% purity) by GL Biochem  and their structures were verified by mass spectrometry. Restriction enzymes and Gallus gallus), zebrafish (Danio rerio), Nile tilapia (Oreochromis niloticus), and red-eared sliders (Trachemys scripta elegans) purchased from local suppliers were sacrificed and various tissues were collected. Zebrafish embryos at consecutive developmental stages  were also collected. All these animal experiments were conducted in accordance with the Guidelines for Experimental Animals issued by the Ministry of Science and Technology of People's Republic of China. The experimental protocol used in this study was approved by the Animal Ethics Committee of College of Life Sciences, Sichuan University .Adult chickens  was performed using MMLV Reverse Transcriptase  from 2 μg total RNA. These RT samples were then used in PCR amplification of target genes, or subjected to quantitative real-time PCR assay (qPCR) of gene expression in chicken or zebrafish tissues on the CFX96 Real-time PCR Detection System , as described in our recent studies  and dissolved in DEPC-treated H studies , 47.APLN and ELA, rapid amplification of 5′- or 3′-cDNA ends (RACE) was performed to amplify cDNAs of both genes from brain tissue using SMART-RACE cDNA amplification Kit  according to the manufacturer's instruction. The amplified PCR products were cloned into pTA2 vector  and sequenced . The gene structure of APLN and ELA was then determined by comparing their cDNA sequences with genomic sequences from the chicken genome database .To determine the gene structure of chicken APLNR cDNA (NM_005161) as a reference, we search its homologous gene(s) in the genome of several representative vertebrate species, including chickens, turtles, Xenopus tropicalis, zebrafish, Nile tilapia, spotted gars, tetraodons, takifugu rubripes (http://www.ensembl.org). The amino acid sequences of APLNR(s) were either predicted according to their genomic sequences or retrieved from GenBank. A phylogenetic tree of APLNRs was constructed using the Neighbor-Joining Method supplied in MEGA6 software (http://www.megasoftware.net).Using human APLNRs from chicken  genome database (http://www.ensembl.org/). Then the open reading frame (ORF) of each APLNR was amplified from the animal  heart tissue with the use of high-fidelity KOD DNA polymerase . The PCR products were cloned into pcDNA3.1 (+) expression vector  and sequenced . Using genomic DNA extracted from human embryonic kidney 293 (HEK293) cells as the template, human APLNR ORF was also amplified by PCR and cloned into pcDNA3.1 (+) expression vector.Primers were designed based on sequence information of 2 and routinely sub-cultured every 3 days.Human embryonic kidney 293 (HEK293) cells were maintained in DMEM supplemented with 10% (vol/vol) fetal bovine serum , 100 U/ml penicillin G and 100 μg/ml streptomycin  in a 90-cm culture dish . Cells were cultured at 37°C in a humidified atmosphere with 5% COAccording to our previously established method, the functionality of APLNRs from chickens, red-eared sliders, zebrafish, Nile tilapia and humans was examined in HEK293 cells by a pGL3-CRE-luciferase reporter or pGL4-SRE-luciferase reporter system, which have been shown to be capable of monitoring receptor-activated (-inhibited) cAMP or MAPK signaling pathways –51. In bHEK293 cells cultured in a 60-mm culture dish  with 70% confluence were transfected by 1 μg of receptor expression plasmid using jetPRIME reagent according to manufacturers' instruction . After the 24-h transfection, HEK293 cells were sub-cultured into a 24-well plate, serum-deprived for 12 h and then treated by peptides for 10 min. These cells were washed with cold PBS and lysed by RIPA Lysis Buffer  on ice. Western blot was used to examine ERK1/2 phosphorylation and β-actin levels in cell lysates.The luciferase activity in each treatment group was expressed as the relative fold changes as compared with their respective control groups. The data were analyzed by non-linear regression followed by one-way ANOVA using GraphPad Prism 5 . To validate the results, all experiments were repeated at least twice.APLN and ELA were predicted to exist in birds, their full-length cDNAs have not been determined in any avian species. In this study, we cloned the full-length cDNAs of APLN and ELA from the chicken heart. Chicken APLN cDNA is 878 bp in length (KX017222) and predicted to encode a 78-amino acid (a.a.) precursor, which shows 42-73% a.a. sequence identities with preproapelin of other vertebrate species, including humans, turtles, X. tropicalis, spotted gars, and zebrafish. As in mammals, chicken apelin precursor is likely to produce multiple forms of mature apelin peptide, such as apelin-36 (36 amino acids) and apelin-13 (13 amino acids), after removal of its signal peptides and proteolytic processing at the dibasic residues  encodes a precursor of 54 amino acids, which shows 52–87% a.a. identities with that of ducks, zebra finches, American alligators, Chinese soft-shelled turtles, X. tropicalis, spotted gars, coelacanths, zebrafish, mice, rats, and humans. Like chicken apelin, cELA precursor also contains a signal peptide at its N-terminus, thus it is predicted to generate a 32-a.a. ELA peptide (ELA-32) after removal of its signal peptide. The mature chicken ELA-32 shows 63–97% a.a. sequence identities with ELA from other vertebrate species examined  as the query sequence, we searched the genome database of chickens and western painted turtles. Interestingly, we identified two APLNR-like genes in chickens and turtles. Based on these sequence information, we designed primers to amplify and clone the cDNAs of the two APLNRs from chickens and red-eared sliders, confirming the existence of both genes in these species. According to their evolutionary origin, we named them as APLNR1 and APLNR2, respectively.Using human APLNR1 cDNA is predicted to encode a 370-a.a. receptor (KU887746), which shares high a.a. sequence identities with human APLNR (64%). Chicken APLNR2 cDNA encodes a receptor of 365 a.a. (KU887747), which shows low amino acid identity (43%) with human APLNR. However, like human APLNR, both cAPLNR1 and cAPLNR2 contain seven transmembrane domains, a DRY/ERY motif near the end of the 3rd transmembrane domain, and a cysteine pair for disulfide bond formation known to be critical for receptor conformation  and APLNR2  share high a.a. sequence identities with chicken APLNR1 (73%) and APLNR2 (67%), respectively. In addition, like chicken APLNR1 and APLNR2, they also share relatively high structural similarity with human APLNR  and contain many conserved structural motifs characteristic of class A GPCR, such as 7 transmembrane domains and an ERY motif  and APLNRb , respectively . Thus, we re-named zebrafish APLNRa and APLNRb as APLNR2a and APLNR2b respectively in this study. Interestingly, the third receptor gene we found encodes a novel APLNR-like receptor of 408 a.a. (KU887750), which shows low a.a. identities with human APLNR (36%), and zebrafish APLNR2a/APLNR2b (41%). Hence, this novel receptor was designated as APLNR3a.In this study, we also searched the genome database of zebrafish and identified three ectively , 35, 52.APLNR2a  and APLNR2b  were identified in Nile tilapia. In addition, we also identified and cloned a novel APLNR-like gene in tilapia. This gene encodes a receptor of 493 a.a. (KU887751), which shows relatively high a.a. sequence identity (57%) to zebrafish APLNR3a, but a low degree of identity to human APLNR (34%), zebrafish APLNR2s (~38%), and chicken APLNR1/2 (35–37%)  and X. tropicalis , ELA-32b (without the disulfide bond) and ELA-11] Figure . The rec50: 0.18 ± 0.10 nM) > ELA32a (EC50: 0.59 ± 0.13 nM) ≈ ELA32b (EC50: 0.87 ± 0.23 nM) > apelin-13 (EC50: 7.86 ± 2.73 nM) > ELA-11 (EC50: 42.1 ± 18.96 nM). As a positive control, all peptides could dose-dependently inhibit forskolin-induced luciferase activities of HEK293 cells expressing human APLNR  expressed in HEK293 cells could be potently activated by apelin-36 and ELA-32 Table , suggest50: 1.88 ± 0.54 nM), but not by ELA peptide, suggesting that APLNR3a may function as a receptor specific to apelin. Like zebrafish APLNR3a, tilapia APLNR3b could also be potently activated by apelin-36 (EC50: 1.36 ± 0.96 nM), but not by ELA-32, suggesting that APLNR3b may also function as an apelin-specific receptor , and APLNR2b (APLNRb) could be activated by chicken apelin-36, apelin-13, ELA-32a, and ELA-32b with high potencies, whereas ELA-11 is much less potent Figure ; Table 1Using the pGL4-SRE-luciferase reporter system Figure , we demoAPLN, ELA, APLNR1, and APLNR2 , we examined the mRNA expression of  Figures , in 13 cAPLN, ELA, and APLNRs are reported to play crucial roles in cardiovascular development during embryogenesis  is highly expressed in the brain and heart and weakly in other tissues examined. APLNR2b  is highly expressed in the heart, liver and spleen, moderately in the brain and testes, and weakly in other tissues. APLNR3a is highly expressed in the spleen and heart, and weakly in other tissues examined , but increases rapidly at 12 hpf stage (~15-fold increase vs. 10 hpf), and then drops sharply at later stages (24 and 48 hpf).Since APLNR3a is a novel apelin-specific receptor, thus, the mRNA expression of In this study, we identified and characterized two APLNRs in chickens and turtles. Moreover, we identified three functional APLNRs in zebrafish, including a novel apelin-specific receptor. To our knowledge, this study represents the first to systematically characterize all APLNRs in several representative vertebrate species, including chickens and zebrafish. Undoubtedly, our data will aid to elucidate the physiological roles of APLNRs and their ligands in different classes of vertebrates.APLN, ELA, APLNR1, and APLNR2 from chicken heart. Like human APLN, chicken APLN cDNA consists of three exons, including a non-coding exon at its 3′-UTR   Figure . It is r) Figure , 55. This Figure .cAPLN, chicken ELA cDNA contains three exons and encodes a precursor of 54 a.a.   may exist in ELA-32 peptide and it may be required for its full biological activity. However, our functional study prove that the absence of this disulfide bond does not impair the biological activity of ELA-32, in view of the comparably high potencies of both forms of ELA-32 in activating human APLNR, chicken APLNR1 and zebrafish APLNR2s  Figure . Two cysIn this study, we identified two APLNRs in chickens. cAPLNR1 is orthologous to human APLNR and shows structural conservation with its mammalian counterparts, including a DRY motif characteristic of class A GPCR family and a serine/threonine-rich C-terminus critical for receptor internalization and desensitization , 57. ApaAPLNRs homologous to human APLNR were identified in other non-mammalian vertebrates including turtles, coelacanths, spotted gars, zebrafish and tilapia, and we designated them as APLNR1, APLNR2, and APLNR3, respectively. Synteny analyses further revealed that APLNR1, APLNR2, and APLNR3 are localized in three separate syntenic regions conserved in vertebrates  which occurred around 350 million years ago (mya) (APLNR2 (APLNR2a and APLNR2b) and APLNR3 (APLNR3a/APLNR3b) in teleosts. Our hypotheses are further supported by the phylogenetic analysis, in which APLNRs from different vertebrate species were clustered into three separate clades, namely the APLNR1 clade, APLNR2 clade (APLNR2a/APLNR2b), APLNR3 clade (APLNR3a/APLNR3b), and APLNR1 and APLNR2 clades show a much closer evolutionary relationship to each other  may have caused the varying numbers of APLNRs in different vertebrate species  tebrates . The relgo (mya)  may haver Figure . Althoug Figures . ClearlyIn this study, we found that chicken APLNR1 can be potently activated by apelin-36 and ELA-32 with high potencies and its activation results in the inhibition of the cAMP signaling pathway, indicating that like human APLNR , 41, cAPIt is reported that in mammals, apelin-13 is a potent ligand for APLNR . Similarin vivo.Although chicken APLNR2 shows 48% a.a. sequence identity to chicken APLNR1, it could only be activated by chicken apelin-36 or ELA-32 at high concentrations (≥100 nM) far beyond the physiological range. This finding implies that APLNR2 may play a less significant role in mediating apelin/ELA actions in chickens APLNR2 exists in X. tropicalis genome, while APLNR1 is likely lost, as evidenced by our synteny analysis  specific to apelin in tilapia. To our knowledge, this study represents the first to report an apelin-specific receptor in vertebrates. The high structural and functional similarity of APLNR3a and APLNR3b also supports our hypothesis that t Figure .cAPLNR1 is widely expressed in all chicken tissues examined   , 41. In elopment , 38. In o apelin , 62.cAPLNR1, cAPLNR2 mRNA is widely expressed in chicken tissues, including the brain and heart. Similar wide tissue expression pattern of APLNR2 has also been reported in adult bullfrogs  are abundantly expressed in zebrafish spleen  and regulate the movement of mesoendodermal cells, heart development, and angioblast migration (37–39). Unlike APLNR2s and ELA, APLNR3a is highly expressed during segmentation period (10–24 hpf) of zebrafish embryos. This finding, together with the expression of APLN (not ELA) at the later stage  can be activated by apelin-36 and ELA-32 potently, indicating that it is a major functional receptor common for both peptides in chickens. In sharp contrast, in turtles and zebrafish, APLNR2(s) can be activated by apelin-36 and ELA-32 potently, supporting the notion that it is the major functional receptor(s) common for both peptides, while APLNR1 is likely lost, or non-functional in these lineages. Strikingly, a novel apelin-specific receptor (APLNR3a/APLNR3b) was identified in zebrafish and tilapia. The identification and functional characterization of APLNRs in model vertebrates not only provides a clear molecular basis for interpreting the actions of APLNR in different classes of vertebrates, but also suggests a functional switch between APLNR1 and APLNR2/3 in mediating apelin and ELA actions during vertebrate evolution Figure .The major content of this work was presented in the 11th International Symposium in Avian Endocrinology , p70 (Abstract).JZ, YZ, CW, YiW, CF, JiL, WF, RY, and GZ conducted the experiments. JZ, JuL, and YaW designed and drafted the manuscript. All authors read and approved the final manuscript and joined the analysis and interpretation of data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salvia species find widespread application in food and pharmaceutical products owing to their large polyphenol content. The main polyphenols in Chinese Salvia species are phenolic acids and flavonoids, which exhibit anti-oxygenation, anti-ischemia-reperfusion injury, anti-thrombosis, anti-tumour, and other therapeutic effects. However, there are few peer-reviewed studies on polyphenols in Chinese Salvia species, especially flavonoids. This review is a systematic, comprehensive collation of available information on the biosynthesis, chemistry, and pharmacology of Chinese Salvia species. We believe that our study makes a significant contribution to the literature because this review provides a detailed literary resource on the currently available information on various polyphenolic components of Chinese Salvia species, including their bioactivities and structures. In addition, the study provides information that would encourage further investigation of this plant material as a natural resource with potential for a broad range of applications in various industries, such as the food and pharmaceutical industries. Salvia is a dominant genus of the Lamiaceae family, which is extensively distributed in China (84 species). The primary phytochemical constituents of Chinese Salvia species include sesquiterpenes , diterpenoids , triterpenoids  [Salvia species are polyphenolic compounds (phenolic acids and flavonoids). Phenolic acids have diverse structures  and are present in high concentrations, which has important taxonomic significance for Chinese Salvia species [Salvia species, such as anti-oxygenation [Salvia species play an important role in the food [The genus n China 8 species.n-3-one) . The mai species . In addigenation , anti-isgenation  effects.the food ,11, pharthe food ,13, and the food .Salvia species [Salvia species because of the nutritional potential of polyphenols.Polyphenols are a class of important secondary metabolites with multiple phenolic hydroxyl groups, and include flavonoids, phenolic acids, stilbenes, and tannins (hydrolysable and condensed) , which a species . There hSalvia species. Moreover, it highlights the biosynthetic pathways of polyphenols.This review focuses on the chemical and pharmacological properties of polyphenols in Chinese Salvia species are mainly produced by the phenylpropanoid metabolic pathway [The biosynthetic pathways of polyphenols include the shikimic acid and phenylpropanoid metabolism pathways . The pol pathway ,23,24, a pathway . Phenyla pathway ,27. The  pathway ,29.Salvia species are caffeic acid derivatives, which are mostly formed by esterification of caffeic acid with danshensu [The phenolic acids of anshensu ,30. Caffanshensu , and is anshensu . The preanshensu . Studiesanshensu . Salviananshensu ,35,36. Tanshensu .Salvia species is shown in Moreover, the phenylpropanoid metabolic pathway is an important upstream pathway for producing flavonoids  ,38,39,40Salvia species are phenolic acids, flavonoids, and anthocyanins [The polyphenolics in Chinese ocyanins . Phenoliocyanins .Salvia species [Caffeic acid and danshensu are the structural units of phenolic acids in  species ,43. Phen species ,44,45,46Rosmarinus officinalis L. in 1958 [Salvia species [Salvia species [Salvia species and represents the main active constituent [Salvia yunnanensis C. H. Wright. They are hexamers of caffeic acid and regarded as products of the combination of caffeic acid and rosmarinic acid. Yunnaneic acid B contains two units of yunnaneic acid C, while yunnaneic acid A is a combination of yunnan acid C and D [Caffeic acid monomers mainly consist of caffeic acid, danshensu, protocatechuic acid, and protocatechuic aldehyde. Caffeic acid is the basic compound of caffeic acid monomers . Danshen in 1958 . It has  species . Salvian species ,36. Caff species ,3. Struc species ,47,49. L species . They ma species ,36,47. S C and D ,51.Salvia species [Presently, three compounds of phenolic acid salts, namely sodium danshensu, magnesium lithospermate B, and ammonium-potassium lithospermate B, have been detected in  species . Magnesi species , anti-li species , and ant species .Salvia species [Salvia plebeia R. Br. and Salvia miltiorrhiza Bunge [Salvia cavaleriei Levl. to be 14.69% [In addition to phenolic acids, other phenolic compounds, such as flavonoids and anthocyanins, are also present in  species ,101,102. species ,38. Currza Bunge . Furthere 14.69% .Salvia species contain anthocyanin and tannin compounds. The compounds salvianin and monardalins are obtained from Salvia coccinea L. and Salvia splendens Ker-Gawl., which are good natural spices [Salvia species are shown in Moreover, l spices ,106. TheSalvia species with an active function in promoting health. Phenolic acids are rich in phenolic hydroxyl groups. Accordingly, representative compounds have been reported to possess a wide variety of activities, including anti-oxygenation [Phenolic acids are the main active polyphenols in Chinese genation , anti-isgenation , and antgenation  effects.Oxidative damage is mainly caused by substances, such as free radicals, present inside and outside the cell. The inability to remove a large amount of free radicals in a timely manner causes dynamic imbalances in the redox system of the body. The antioxidant effects of phenolic acids involve three processes: free radical scavenging, inhibiting free radical generation, and anti-lipid peroxidation ,122,123.−1, rosmarinic acid, caffeic acid, and danshensu were as effective as quercetin (positive control), which scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals in a concentration-dependent manner. In contrast, ferulic acid was less effective [2−, DPPH, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) than the other constituents. Among them, the half-maximal inhibitory concentration (IC50) of danshensu and salvianolic acid B were 94 and 102 μg·mL−1, respectively, and there was no obvious difference in the scavenging ability of hydroxyl radicals [Phenolic acids possess radical scavenging activity. A study showed that, at 0–0.7 mmol·Lffective . MoreoveSalviamiltiorrhiza leaves, had strong antioxidant activity. Among them, salvianolic acid B was positively correlated with DPPH scavenging activity (correlation coefficient (r) = 0.693, P < 0.05), reducing ability , and inhibition of linoleic acid oxidation . It could be regarded as a new possible resource of natural phenolic antioxidants [The antioxidant activities of rosmarinic acid and salvianolic acid B in plant extracts were determined using DPPH radical scavenging, superoxide radical quenching, a β-carotene-linoleic acid system, and reductive potential assays. The data indicated that salvianolic acid B and rosmarinic acid, which were abundantly found in oxidants .−1, 54.5%), protocatechuic aldehyde (50.1%), chlorogenic acid (46.9%), caffeic acid , ferulic acid , danshensu , and 3-hydroxycinnamic acid (32.2%). However, the protective effects of various constituents on hydrogen peroxide (H2O2)-induced liver lipid peroxidation in mice showed the following values: rosmarinic acid , caffeic acid (98.9%), protocatechuic aldehyde (98.3%), chlorogenic acid (90.0%), ferulic acid (83.2%), danshensu (54.3%), and 3-hydroxycinnamic acid (42.1%). At the same time, the total radical reducing activities of the seven phenolic acids were determined to be consistent with the above results [Studies have shown that the order of inhibition of spontaneous lipid peroxidation in liver tissue of mice by the following constituents is rosmarinic acid (minimum inhibitory concentration (MIC): 12.5 μg·mL results .Salvia species are water-soluble components that are active against cardiovascular diseases. The pathophysiological mechanism of myocardial/cerebral ischemia-reperfusion injury is complex, and phenolic acids act against it through mechanisms such as regulation of active oxygen metabolism, inhibition of inflammatory reaction, apoptosis, and calcium overload [The phenolic acids in overload ,8.−1·kg−1) against myocardial ischemia-reperfusion injury were determined by measuring the concentration and apoptotic index of the plasma level of myocardial enzymes (cardiac troponins (CTn) I and creatine kinase-MB (CKMB)), malondialdehyde (MDA), endothelin (ET), nitric oxide (NO), and superoxide dismutase (SOD), and histological changes of the heart. The effects were superior in the salvianolic acid B high-dose group (60 mg·d−1·kg−1). The results showed that salvianolic acid B significantly increased the plasma CTn I, CKMB, MDA, and ET contents; decreased NO and T-SOD contents; reduced the infarct size; and improved myocardial ultrastructure changes. The study showed that salvianolic acid B protected against conditions such as myocardial ischemia-reperfusion injury by regulating active oxygen metabolism, reducing oxidative stress, and myocardial apoptosis [Salvianolic acid B was found to effectively reduce myocardial ischemia-reperfusion injury. The experiment established an ischemia-reperfusion model by ligating the left circumflex artery in Sprague–Dawley (SD) rats. The effects of different doses of salvianolic acid B  was downregulated, tissue inhibitor of metalloproteinase-1 (TIMP-1) was upregulated, and damage to the blood–brain barrier was reduced. Salvianolic acid A inhibits activation of cerebral nuclear factor (NF)-κB p65 and reduces inflammatory response. Salvianolic acid A could be used as an effective drug to alleviate cerebral ischemia-reperfusion injury [2+ influx [Salvianolic acid A protected rats against cerebral ischemia-reperfusion injury by inhibiting the expression of matrix metalloproteinase (MMP)-9 and inflammatory response. Cerebral ischemia-reperfusion injury significantly upregulates the expression of MMP-9, leading to severe damage of the blood–brain barrier. Furthermore, it activates inflammatory response, produces oxygen free radicals, or releases lysosomal enzymes to damage tissue. Studies have shown that the level of MMP-9 in salvianolic acid A-treated groups  only antagonizes the action of platelet P2Y12 receptors, while salvianolic acid A and C (both 100 µmol·L−1) are P2Y1 and P2Y12 receptor inhibitors [Studies have shown that salvianolic acids in hibitors .−1) repressed ADP-induced platelet aggregation, P-selectin expression, ATP release, and Ca2+ mobilization. Furthermore, it attenuates the activation of p38, ERK, integrin αIIbβ3, and JNK. It could also increase the expression level of cAMP [Caffeic acid can inhibit platelet-mediated thrombosis. High activation of platelets is one of the major causes of thrombosis. The adhesion and aggregation of platelets are enhanced in the activated state. The results demonstrated that caffeic acids have beneficial effects on aberrant platelet activation-related diseases. Caffeic acid (25–100 μmol·L of cAMP .−1) had better anti-thrombotic and antiplatelet therapeutic efficacy than other constituents. It mainly contributed to intensely and selectively suppressing the expression of cyclooxygenase (COX)-2 and balancing the ratio of thromboxane A2 (TXA2)/prostacyclin (PGI2) [Danshensu (15–60 mg·kgn (PGI2) .Liver damage is a common pathological manifestation of various liver diseases, and further progression leads to different degrees of hepatic cell necrosis, fatty liver, liver cirrhosis, and other liver diseases. Phenolic acids are potential anti-liver damage compounds. Liver protection can be achieved through the following mechanisms: elimination of free radicals, inhibition of lipid peroxidation, and inhibition of inflammatory cytokine expression ,135.−1·kg−1) protects liver cells by preserving lysosomal membrane integrity through scavenging ROS and enhancing lysosome-associated membrane protein 1 (LAMP1) expression. LAMP1 acts as a barrier to soluble hydrolase, preventing cell damage and death caused by the release of luminal contents into the cytoplasm. H2O2 attack significantly decreases LAMP1 expression, thereby disrupting the integrity of hepatocyte lysosomal membrane, leading to cell damage and death. Liver cells are protected by the anti-inflammatory and anti-oxidant properties of salvianolic acid B [Salvianolic acid B (15 or 30 mg·dc acid B .−1) significantly reduced ConA-induced ALT and AST activity. In addition, it reduced hepatotoxic cytokine levels, such as tumour necrosis factor (TNF)-α and interferon (IFN)-γ; ameliorated the increased NF-κB level and cleaved caspase-3; and reversed B-cell lymphoma-extra-large (Bcl-xL) expression. Notably, pretreatment with salvianolic acid A obviously upregulated the expression of SIRT1, which could alleviate acute hypoxic injury and metabolic liver disease. The study showed that the increase in SIRT1 was closely related to the p66 isoform (p66shc) of the growth factor adapter Shc. Other studies have shown that salvianolic acid A-alleviated ConA-induced hepatitis may be inhibited by the SIRT1-mediated p66shc pathway [Salvianolic acid A protects against acute hepatic injury induced by concanavalin A (ConA) in mice. The results of the liver function indicators serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) showed that salvianolic acid A (15 or 25 mg·kg pathway .S.miltiorrhiza (0.06–1 mg·mL−1) has the same hepatoprotective effect as salvianolic acid A does, and the mechanism may be related to the reduction of extracellular histone and related cytokines. A water extract of S.miltiorrhiza contained danshensu (8.2–130.5 μmol·L−1) and salvianolic acid B (3.3–53.5 μmol·L−1) as the main compounds that protected against liver injury. However, rosmarinic acid, protocatechuic aldehyde, and salvianolic acid A had no protective effects [In addition, studies have shown that the water extract of  effects .Phenolic acids have multitarget anti-tumour effects; however, their mechanisms are more complicated. They prevent the invasion of tumour cells by inducing apoptosis of tumour cells, regulating immune function, reversing multidrug resistance of cancer cells, targeting tumour microtubules to inhibit their proliferation and division, and inhibiting metastasis of cancer cells ,140.−1) significantly reduced the cell viability of U80 cells in a dose- and time-dependent manner. At the same time, it enhanced the production of intracellular ROS and induced apoptosis of U57 cells. The anti-tumour activity of salvianolic acid B in vivo was observed in a nude mouse xenograft model. Therefore, salvianolic acid B seems to be safe and effective, and this natural component could be developed into a potential therapeutic agent for glioma [Salvianolic acid B inhibits the growth of human glioma U80 cells, which may be involved in p38-activation-mediated ROS production. These experimental results indicate that salvianolic acid B , which are based on caffeic acid with compounds formed from two to four or more caffeic acid units. Most of the biological activities, such as anti-oxidant, anti-ischemia-reperfusion, and anti-thrombosis effects are attributed to phenolic acids. Nevertheless, in the study of the pharmacological mechanism of phenolic acids, detailed experiments have been conducted only on common compounds, such as rosmarinic acid, salvianolic acid A, and salvianolic acid B, whereas other compounds are less studied.In recent years, the antioxidant properties of polyphenolic compounds have encouraged their widespread used as supplements in food or pharmaceutical products. Salvia species are apigenin, acacetin, and luteolin. However, few studies have been conducted on flavonoids, including their phytochemistry and pharmacological effects. This is a shortcoming in the study of chemical constituents and pharmacological effects of Chinese Salvia species. The flavonoids in Chinese Salvia species should be analysed using modern analytical techniques, such as liquid chromatography-tandem mass spectrometry to supplement current knowledge on the chemical composition of phenolic acids.The flavonoids found in Chinese Salvia species. The medicinal and economic values of phenolic acids make it particularly important to increase the content of phenolic acids in Chinese Salvia species. Presently, only the synthesis of upstream rosmarinic acid has been partially elucidated in the biogenic pathways of phenolic acids. The source pathways of other phenolic acids, such as salvianolic acid B downstream of rosmarinic acid, have not yet been elucidated. Therefore, elucidation of the conversion pathway of phenolic acid, particularly identifying key enzymes involved in transformation, and in-depth study of the biotransformation mechanism would promote the production and development of new phenolic acids.Investigating the biosynthesis of polyphenols is a challenging task in Chinese Salvia species in food and medicine, it is necessary to explore polyphenols more comprehensively. In this review, we systematically summarized the biosynthesis, phytochemistry, and pharmacology of Chinese Salvia species, which provide a basis for its further development and utilization as a resource.Owing to the extensive use of Chinese


A 49-year-old male presented to his physician with three weeks of dyspnea, dry cough, and fever. He did not respond to antibiotics and corticosteroids. He presented to the emergency department with worsening symptoms, where blood work revealed severe anemia, leukocytosis, thrombocytopenia, and 61% blasts on peripheral smear. Bone marrow biopsy showed acute myeloid leukemia (AML). While the results of other studies were awaited, treatment was begun with 7+3 induction (cytarabine and daunorubicin). Karyotyping returned positive for the BCR-ABL1 fusion gene (Philadelphia chromosome), near-tetraploidy, and 5q deletion. Follow-up bone marrow biopsy revealed residual disease (12% blasts). Re-induction was initiated with 5+2 cytarabine and daunorubicin with the addition of dasatinib. Subsequent bone marrow biopsies revealed minimal residual disease and BCR-ABL on polymerase chain reaction (PCR). The patient was placed on dasatinib maintenance and later switched to nilotinib. This case demonstrates the simultaneous presence of rare cytogenetic abnormalities in AML. It also discusses the successful utilization of tyrosine kinase inhibitors (TKIs) in the treatment of BCR-ABL-positive AML, as there are no established guidelines. Cytogenetic analysis has become the cornerstone of diagnosis, treatment, and prognostication in various malignancies. BCR-ABL rearrangement corresponding to tq34;q11) translocation, commonly known as Philadelphia chromosome, is a hallmark of chronic myeloid leukemia (CML) and frequently associated with acute lymphoblastic leukemia (ALL) 1 translo-6. TetraA 49-year-old male with no significant past medical or family history presented to his primary care physician with three weeks of dry cough, exertional dyspnea, low-grade fever, night sweats, fatigue, and generalized weakness. He was initially treated empirically with azithromycin and prednisone but failed to respond. He subsequently presented to the emergency department for a re-evaluation of worsening symptoms. Physical examination was significant for tachycardia, tachypnea, and pallor. Complete blood count showed severe anemia with significant leukocytosis and thrombocytopenia. Peripheral blood smear showed 61% blasts. Routine infectious workup, including chest X-ray, blood culture, sputum gram stain, and culture were negative. These findings prompted workup for hematological malignancy.The results of the initial workup are as follows:*Complete blood count (CBC) on presentation: White blood cells (WBCs) 33,300/μl, hemoglobin 6.5 g/dl, hematocrit 19.9%, platelets 85,000/μl*LDH: 888 units/L*Peripheral blood smear: Anemia with aniso-poikilocytosis, reduced platelets, increased WBCs with 61% blasts*Flow cytometry of peripheral blood: 63% large blasts positive for CD117, HLA-DR, CD34, CD13, TdT, and CD45 (moderate); partial expression of CD33, CD22, and CD11b; negative for CD14, CD56, MPO, CD19, CD20, surface light chains, CD3, CD4, CD5, and CD7; immunophenotype most consistent with AML.*Initial bone marrow biopsy findings ; partial expression of CD22 and CD11b; largely negative for CD14, CD56, CD19, CD20, CD3, CD4, CD5, and CD7 22%; immunophenotype most consistent with AML.*Immunohistochemistry: Blasts positive for CD34, lysozyme, CD117, and myeloperoxidase (MPO).*Karyotyping/chromosome analysis: “95~98, XXYY, del(5)(q31q35), +6, tx2, -16, +17, +19, +22, +22, +der(22)t[cp18]/46,XY(2); abnormal karyotype, male.”*Eighteen of the 20 mitotic cells were near-tetraploid, had loss of one copy of chromosome 16, an additional copy of chromosomes 6, 17, and 19, two additional copies of chromosome 22, a deletion in the long arm of chromosome 5, two copies of translocation between the long arms of chromosomes 9 and 22, resulting in two Philadelphia (Ph) chromosomes, and an additional Philadelphia (Ph) chromosome.*Mutation analysis: Negative for NPM1, IDH1/IDH2, c-KIT, CEBPA, FLT3 ITD/TKD mutation.*Fluorescence in-situ hybridization (FISH): Trisomy, tetrasomy, and pentasomy of chromosome 7, 8, and 20 consistent with a polyploid neoplastic clone; negative for RUNX1T1/RUNX1 (ETO/AML1), KMT2A (MLL), PML/RARA, CBFB rearrangements; negative for monosomy 5 and 7, trisomy 8, 7q deletion, and PTPRT deletion.Bone marrow biopsy on Day 21 revealed residual disease with 12% blasts and the biochemical presence of BCR-ABL on quantitative PCR (0.5406%). The patient underwent re-induction with 5+2 cytarabine and daunorubicin, this time with the addition of dasatinib (BCR-ABL tyrosine kinase inhibitor). He was continued on prophylactic allopurinol, acyclovir, and voriconazole. Follow-up bone marrow biopsy was negative for any morphological evidence of blasts but quantitative PCR for BCR-ABL was positive (.0745%), indicating minimal residual disease (MRD). The patient was placed on oral dasatinib maintenance therapy and later switched to nilotinib. This was because our patient had developed a parapneumonic effusion but it was decided to avoid a drug like dasatinib, which may cause pleural effusions as an adverse effect.This case report encompasses a period of eight months from the time of diagnosis. For AML, given the presence of MRD, as demonstrated by positive quantitative BCR-ABL, the patient will be continued indefinitely on daily nilotinib maintenance therapy. This will be followed by quantitative serum PCR for BCR-ABL every four weeks and bone marrow biopsy every 12 weeks or earlier if clinically warranted. Of note, the patient developed necrotizing pneumonia with empyema during the course of treatment, which was treated with antibiotics and chest tube placement. The empyema recurred, therefore, thoracoscopic decortication was done later. He is not currently a candidate for allogeneic stem cell transplantation (ASCT), given the clinical condition and frailty. However, it may be pursued once he recovers from the pulmonary complications.According to the 2017 European Leukemia Net (ELN) risk stratification by genetics, BCR-ABL positivity, complex karyotype (like near-tetraploidy), and 5q deletion are all placed in the ‘adverse’ risk category . The preAML with Philadelphia chromosome poses a challenge in distinguishing it from CML with blast crisis. As in our patient, the absence of any antecedent hematological abnormality and lack of basophilia or splenomegaly are clues that favor de-novo AML versus CML transformation.There has been some improvement in the prognosis with the use of TKIs, increasing the median overall survival from nine months to 18 months . Isolate5q deletion is the most frequent cytogenetic aberration in MDS and can be seen in high-risk AML . Two disTetraploidy and near-tetraploidy (T/NT) is a rare finding in AML occurring in 0.7%-1.2% of adult AML patients . AlthougAlthough the BCR-ABL fusion gene is pathognomonic for CML and frequently associated with ALL, it may also play a role in the treatment course of AML. TKIs are effective adjuncts for treating BCR-ABL positive AML. However, monotherapy with TKIs is not substantiated for induction or consolidation. Therefore, age and risk factor-appropriate chemotherapy and ASCT should still be used as the first-line treatment until more robust guidelines are created. TKIs may be useful as single agents in continuous maintenance therapy and as a bridge to other first-line therapies in AML such as ASCT.


Sesquiterpene lactones constitute a major class of bioactive natural products. One of the naturally occurring sesquiterpene lactones is costunolide, which has been extensively investigated for a wide range of biological activities. Multiple lines of preclinical studies have reported that the compound possesses antioxidative, anti-inflammatory, antiallergic, bone remodeling, neuroprotective, hair growth promoting, anticancer, and antidiabetic properties. Many of these bioactivities are supported by mechanistic details, such as the modulation of various intracellular signaling pathways involved in precipitating tissue inflammation, tumor growth and progression, bone loss, and neurodegeneration. The key molecular targets of costunolide include, but are not limited to, intracellular kinases, such as mitogen-activated protein kinases, Akt kinase, telomerase, cyclins and cyclin-dependent kinases, and redox-regulated transcription factors, such as nuclear factor-kappaB, signal transducer and activator of transcription, activator protein-1. The compound also diminished the production and/expression of proinflammatory mediators, such as cyclooxygenase-2, inducible nitric oxide synthase, nitric oxide, prostaglandins, and cytokines. This review provides an overview of the therapeutic potential of costunolide in the management of various diseases and their underlying mechanisms. This compound was first isolated from costus (Saussurea lappa Clarke) root and then isolated from various other plant species. [Drug development from natural sources, particularly from plants, has long been the mainstay in medical management of various human ailments. A wide variety of non-nutritive plant constituents, commonly known as phytochemicals, are being used as therapy for many disease processes, including, but not limited to, infections, diabetes, heart diseases, neurological disorders, and cancer. In fact, it is estimated that about 40% of all medicines are natural compounds or their semisynthetic derivatives . One of species. . The bio2O2)-induced ROS production in rat pheochromocytoma (PC12) cells [Oxidative stress resulting from cellular redox imbalance leads to many diseases, such as diabetes, atherosclerosis, and cardiovascular diseases . The antcytoma PC cells [1N-acetylglucosaminidase (NAG) activity in mice [Persistent tissue inflammation plays an important role in the pathogenesis of various infectious and noninfectious diseases, such as rheumatoid arthritis, Alzheimer’s disease, and arteriosclerosis . Costuno in mice . One of  in mice . Chen et in mice . Costuno in mice . Likewis in mice . Moreove in mice . Costuno in mice . In addi in mice .+ T cell activation and proper differentiation into T helper (Th) cells are important for establishing an adaptive immune response against foreign pathogens. However, an excessive activation of Th cells leads to inflammation and autoimmune diseases [+ T cells were induced to differentiate, costunolide markedly reduced the differentiation into a population of Th1, Th2, and Th17 subsets. Costunolide also inhibited the expression level of Th subset-polarizing master genes such as T-bet, GATA3, and RORγt. The compound reduced the level of CD4+ T cell activation marker CD69 and attenuated T cell proliferation by blocking phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK [Heme oxygenase-1 (HO-1) has been reported to mediate anti-inflammatory and cytoprotective activity . Pae anddiseases . When CDp38 MAPK . Chemokines play an important role in inducing various allergic and inflammatory skin diseases, such as atopic dermatitis, psoriasis, and eczema. Keratinocytes are known to respond to various chemokines, such as chemokine (C-C motif) ligand (CCL)-17 , CCL-22 , CCL-5 (synonym RANTES), and IL-8, which are involved in precipitating atopic dermatitis . CostunoOsteoporosis, a disease of the bone generally characterized by excessive bone resorption due to poor osteoblastic and enhanced osteoclastic activity, is very common among the elderly population. Although few therapeutic interventions, such as the use of calcium and vitamin D3, parathyroid hormone analogs, bisphosphonates, and monoclonal antibodies, are current clinical recommendations, there is emerging need of developing new drugs . Several2O2-induced apoptosis of PC12 cells by reducing intracellular ROS, stabilizing mitochondrial membrane potential (MMP), and decreasing the caspase-3 activity (2O2-induced cell death by blocking the phosphorylation of p38 MAPK and ERK [Parkinson disease (PD) is one of the neurodegenerative diseases characterized by reduced dopaminergic (DAergic) neuronal transmission in the substantia nigra (SN). One of the key regulators of DAergic nerve transmission, especially the regeneration of synaptic vesicles, and the storage, metabolism, and release of DA at nerve endings is α-synuclein (ASYN), which is transcriptionally regulated by nuclear receptor related-1 (Nurr1). In PD patients with Nurr1 mutations, Nurr1 expression was decreased and ASYN expression was increased. Whereas the expression of the Nurr1 gene is essential for the development and maintenance of nigral DAergic neurons, overexpression of ASYN causes selective degeneration and toxicity of DAergic neurons. Nurr1 also participates in DA metabolism by regulating vesicular monoamine transporter type 2 (VMAT2) and dopamine transporter (DAT) . Ham et activity . Moreove and ERK .Mycobacterium tuberculosis H37Rv (M. tuberculosis) [Mycobacterium avium (M. avium) [Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) [H. pylori [Trichophyton mentagrophytes, T. simum, T. rubrum, Epidermophyton floccosum, Scopulariopsis sp., Aspergillus niger, Curvulari lunata, Magnaporthe grisea, and Candida albicans [Botauttis cinereal, Colletotrichum acutatum, Colletotrichum fragariae and Colletotrichum gloeosporioides [C. echinulata [Several studies have demonstrated the antimicrobial activity of costunolide . The antculosis)  and Myco. avium)  in fluor. pylori , which ialbicans . Costunoorioides , and C. hinulata . The anthinulata . Lipoteichoic acid (LTA)-induced acute lung injury (ALI) in mice is a model to represent experimental pneumonia. Treatment with costunolide significantly reduced LTA-induced inflammatory cell infiltration and lung tissue damage by decreasing the production of various cytokines and chemokines. Moreover, the compound inhibited LTA-induced iNOS expression in mouse bone marrow-derived macrophages by blocking phosphorylation of TAK1, p38 MAPK, and ERK, without affecting the activation of NF-κB . Thus, cThe cosmetic use of herbal products, especially for preventing hair loss or promoting hair growth, have long been practiced throughout the world. The herbal therapies used as hair growth promoters are expected to have low toxicity, be easy to use, low cost, and have high patient compliance. As the physiological and biochemical pathways in hair follicle dermal papillary cells (hHFDPCs) are unfolded, the mechanistic basis of hair growth promotion by many natural products is being explored ,45. It hThe search for anticancer agents from natural sources, especially plants, has led to the discovery of many clinically useful drugs. Extensive investigation of the anticancer effects of costunolide have shown that the compound induces apoptosis and inhibits proliferation of various cancer cells in vitro, and suppresses angiogenesis and metastasis. The following section will shed light on the biochemical processes and molecular targets of costunolide in exerting its anticancer effects. Costunolide decreased the proliferation of various cancer cells including those of the colon, breast, prostate, liver, gastric, and blood cancer cells ,49,50,51WAF1 expression was independent of p53 activation [INK4c, p21CIP1/Waf-1, and p27KIP1. However, the compound did not affect the proliferation of normal mammary epithelial (MCF-10A) cells [WAF1 and reduced expression of cyclin B1 and CDK2 by costunolide led to the G2/M phase arrest in K562 cells. According to this study, the compound enhanced imatinib-induced apoptosis in K562 cells via modulation of B cell receptor (Bcr)/Abl and STAT5 signaling pathways. In another study, these authors reported that costunolide sensitized K562 cells to doxorubicin via inhibition of the PI3K/Akt activity [Several other studies have reported that costunolide inhibits various tumor cell proliferation by blocking G2/M phase of the cell cycle and modulating the effect of cyclins and cyclin-dependent kinases (Cdk) ,53,54. Ttivation . Roy andA) cells . Likewisactivity . In anotactivity . Incubatactivity . Moreoveactivity . 50 of 25 µM, and induced apoptosis, which was mediated through induction of Bax, decreased the expression of Bcl-2 and cleavage of caspase-3 and 9. Moreover, the compound induced autophagy as evidenced by the elevated expression of LC3 II and Beclin 1 [Costunolide induced mitochondria-mediated apoptosis as evidenced by the inhibition of Bcl-2, induction of Bax, and release of cytochrome c in human prostate (PC3 and DU-145) , leukemiBeclin 1 . N-acetyl cysteine abrogated costunolide-induced ER stress and apoptosis [N-acetyl cysteine, suggesting the involvement of ROS in costunolide-induced retardation of tumor cell growth. Furthermore, costunolide treatment of mice transplanted with colon cancer cells inhibited tumor growth and decreased TrxR1 activity and ROS levels [Apoptosis may result from continued ER stress that activates unfolded protein response (UPR) signaling pathways. Costunolide activated the ionositol requiring enzyme (IRE)-1α, a resident ER membrane protein, which further activated JNK by recruiting adapter molecules TRAF2 and ASK1 in cultured lung adenocarcinoma cell line A549 cells . Costunopoptosis . A similpoptosis . Recent S levels .Extrinsic mechanisms of apoptosis induction by costunolide have also been reported. The induction of apoptosis in estrogen receptor-negative human breast cancer (MDA-MB-231) cells by costunolide involves the activation of Fas, caspase-8, caspase-3, and the degradation of PARP . CostunoTelomeres, which maintain genomic integrity in normal cells, are shortened upon each cell division, thus leading to chromosomal instability, cellular senescence, and aging. However, the length of the telomeres is maintained by high levels of telomerase enzyme present in tumor cells, thereby allowing cancer cells to be immortal. Therefore, telomerase has been considered as a possible target for cancer treatment . It has The persistent growth and spread of a tumor require a constant supply of nutrients and oxygen to the cancer cells. The formation of new blood vessels, a process known as angiogenesis, is therefore an essential step in tumor invasion and metastasis. The discovery of angiogenesis inhibitors  helps reduce the morbidity and mortality from various cancers. A key angiogenic molecule is vascular endothelial growth factor (VEGF), which by binding with VEGF receptors (VEGFR) on vascular endothelial cells promotes formation of new blood vessels .In a murine cannulated sponge implant angiogenesis model, administration of costunolide to Swiss albino mice implanted with polyester polyurethane sponges used as a framework for fibroblast tissue growth reduced the levels of VEGF and hemoglobin content in fibrovascular tissue, suggesting the antiangiogenic property of the compound . Jeong eCancer metastasis refers to the spread of tumor cells from their site of origin to other distant parts of the body. Metastasis consists of multistep processes including tumor cell spread, extracellular matrix (ECM) degradation, tumor cell invasion in ECM, angiogenesis, and secondary metastatic tumor growth . CostunoCostus speciosus inhibited α-glucosidase activity with an IC50 value of 67.5 μg/ml and attenuated α-amylase activity with an IC50 value of 5.88 mg/ml, which is lower than the reference compound acarbose [Costus speciosus, this study indicates the potential of costunolide in managing glycemic control. A subsequent study demonstrated that costunolide significantly reduced blood glucose level, glycosylated hemoglobin (HbA1c), serum total cholesterol, triglyceride, and LDL cholesterol level in streptozotocin (STZ)-induced diabetic rats [An in vitro assay has revealed that the methanol extract of leaves of acarbose . Since ctic rats . Moreovetic rats . Since omax) and time required to attain highest plasma level of the molecule (Tmax) after oral administration of costunolide to Wistar rats were found as 0.024 ± 0.004 mg/L and 9.0 ± 1.5 h, respectively. The half-life (t1/2) and area under the curve (AUC) were 4.97 h and 0.33 ± 0.03 mg·h/mL, respectively [max and Tmax were 19.84 ng/mL and 10.46 h, respectively, and the half-life (t1/2) and AUC were 5.54 h and 308.83 ng·h/mL, respectively [max, Tmax, t1/2, and AUC as 0.106 ± 0.045 μg/mL, 8.00 h, 14.62 ± 3.21 h, and 1.23 ± 0.84 μg·h/mL, respectively [max as 12.28 ± 1.47 μg/mL, and the half-life (t1/2) and AUC were detected as 1.16 ± 0.06 h and 3.11 ± 0.13 μg·h/mL, respectively [Pharmacokinetic studies are an integral part of the drug discovery process. The understanding of the absorption, distribution, metabolism, and elimination of the drug-to-be is an essential step in new drug development. There have been several studies reporting the pharmacokinetic profile of costunolide. The maximum plasma concentration (Cectively . Howeverectively . Accordiectively . The larectively . These r50) and ensure safety of the compounds is of paramount importance in further progressing the development of costunolide as a drug candidate. Although costunolide has been examined extensively for its therapeutic potential in various animal models as discussed in the previous sections of this review, acute and chronic toxicity studies are scarce. A recent study demonstrated that the compound induced apoptosis in normal Chinese hamster ovarian cells by inducing clastogenic and genotoxic effects as evidenced by micronuclei formation and chromosomal breaks . Thus, mSesquiterpene lactones form a large, structurally diverse group of natural products found almost universally in plants. Extensive investigation of the therapeutic potential of sesquiterpene lactones has yielded important candidates for pharmaceutical development . Costuno


Myeloproliferative neoplasms represent a heterogenous group of disorders of the hematopoietic stem cell, with an intrinsic risk of evolution into acute myeloid leukemia. The frequency of leukemic evolution varies according to myeloproliferative neoplasms subtype. It is highest in primary myelofibrosis, where it is estimated to be approximately 10–20% at 10 years, following by polycythemia vera, with a risk of 2.3% at 10 years and 7.9% at 20 years. In essential thrombocythemia, however, transformation to acute myeloid leukemia is considered relatively uncommon. Different factors are associated with leukemic evolution in myeloproliferative neoplasms, but generally include advanced age, leukocytosis, exposure to myelosuppressive therapy, cytogenetic abnormalities, as well as increased number of mutations in genes associated with myeloid neoplasms. The prognosis of these patients is dismal, with a medium overall survival ranging from 2.6–7.0 months. Currently, there is no standard of care for managing the blast phase of these diseases, and no treatment to date has consistently led to prolonged survival and/or hematological remission apart from an allogeneic stem cell transplant. Nevertheless, new targeted agents are currently under development. In this review, we present the current evidence regarding risk factors, molecular characterization, and treatment options for this critical subset of myeloproliferative neoplasms patients. BCR-ABL1-negative myeloproliferative neoplasms (MPNs) are clonal disorders of the hematopoietic stem cell, mainly characterized by hyperproliferative bone marrow with varying degrees of reticulin/collagen fibrosis, extramedullary hematopoiesis, abnormal peripheral blood count, and constitutional symptoms. They include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) [The BCR-ABL1-negative MPNs. This setting now represents the principal clinical challenge in these diseases.The major causes of morbidity and mortality in these patients are most commonly represented by thrombo-hemorrhagic events and less frequently by infectious complications, and/or transformation to blast phase, often termed secondary acute myeloid leukemia (AML) or blast-phase MPN (MPN-BP) ,3. The tBCR-ABL1-negative MPN patients, where the cumulative incidence of MPN-BP was 0.038 for ET, 0.068 for PV, and 0.142 for PMF [The frequency of leukemic evolution varies according to MPN subtype. It is highest in PMF, where it is estimated to be approximately 10–20% at 10 years, following by PV, with a risk of 2.3% at 10 years and 7.9% at 20 years ,5,6. On The prognosis of these patients is dismal, with a medium overall survival (OS) ranging from 2.6–7.0 months . CurrentIn this review, we present the current evidence regarding molecular characterization and treatment options for this subset of MPN patients.BCR-ABL1-negative MPNs vary according to the specific MPN subtype, they generally include advanced age, leukocytosis, exposure to myelosuppressive therapy, cytogenetic abnormalities, as well as an increased number of mutations in genes associated with myeloid neoplasms.Even though risk factors for leukemic evolution in 9 L [9 L), and red blood cell (RBC) transfusion need were also associated with an increased risk of leukemic transformation in PMF, with an incidence at 7.4 × 100 persons per year in RBC-transfused patients vs. 1.5 × 100 persons per year in non-transfused patients (p < 0.001) [In particular, independent risk factors for leukemic transformation in PMF included peripheral blood (PB) blast >3% and platelet count <100 × 109 L . Using t< 0.001) ,16. Trea< 0.001) , or C-re< 0.001) .Unfavorable karyotype together with thrombocytopenia were then identified as being the most important risk factors for leukemic evolution in PMF . The lat9 L [With regards to PV, historical treatments, such as P32, chlorambucil, or pipobroman, have been clearly demonstrated to be associated with a higher risk of leukemic transformation ,22. Othe9 L ,25, and 9 L  have als9 L ,23, desp9 L), and age as independent risk factors for leukemic transformation in this subset of MPN patients. In detail, the risk of leukemic transformation was low at 0.4% if both the aforementioned risk factors were absent, and was significantly higher at 4.8% and 6.5% in the presence of one or both risk factors, respectively (p < 0.001). Interestingly, many important retrospective case series have supported the absence of any convincing evidence for drug leukemogenicity in ET [Concerning ET, Gangat et al.  identifity in ET , even thAs reported above, a complex/monosomal karyotype represents an important risk factor for leukemic evolution, as a favorable karyotype is infrequent in MPN-BP.JAK2, CALR, or MPL mutations) [Concerning the molecular profile, if driver mutations are important in MPN pathogenesis, they also have a critical prognostic role in terms of leukemic transformation. It is best recognized for PMF patients, where a higher risk has been associated with the so-called triple-negative molecular status , defining a high-molecular risk (HMR) category [CBL, RUNX1, CEBPA, SH2B3, and KIT, as interindependent risk factors for OS and LFS [ASXL1, SRSF2, RUNX1, and IDH2, these were identified as adverse variants or mutations based on their effect on OS, LFS, or MF-free survival [TP53, EZH2, SRSF2, and IDH2 variants or mutations as being associated with a higher risk of leukemic transformation [However, it is now clear that achinery . Using acategory . In detacategory ,30,31,32survival . More reormation .BCR-ABL1-negative MPNs have been included. Accordingly, the finding of 10–19% of blasts in the PB and/or in the bone marrow (BM), as well as the immunohistochemical detection of an increased number of CD34+ cells with cluster formation and/or an abnormal endosteal location in the BM [In the updated version of the WHO classification of tumors of the hematopoietic and lymphoid tissues , specifin the BM , indicatBlast-phase MPNs commonly involved the myeloid lineage, being the lymphoid lineage only rarely involved. The morphological and cytogenetic characteristics of MPN-BP have been reported to be different from primary (de novo) AML. According to the French-American-British (FAB) classification of AML, erythroleukemia (FAB-M6) D,E and mBCR-ABL1-negative MPN have been collected. Inclusion criteria included: increased PB (≥2%) and/or BM (≥5%) blasts, presence of dysplastic features, persistent leukocytosis (≥15 × 109 L) or monocytosis (≥1 × 109 L), and extreme thrombocytosis (≥1000 × 109 L). On follow-up, 22 (22%) patients developed AP and 19 (19%) BP. Forty-seven patients (41%) expired after a median follow-up of 11 months from disease progression, as compared to 2/40 (5%) control patients (p < 0.0001). Furthermore, there was no significant difference in OS between patients with AP and other types of progression. Accordingly, a review of the blasts threshold to define AP of BCR-ABL1-negative MPNs could be proposed [Interestingly, the percentage of blasts suggesting MPN progression is going to be investigated in a cooperative European and American effort, also involving our group. In detail, 114 patients with a diagnosis of proposed .KRAS mutation in codon 12 or 13 with a low allelic burden. On this basis, the development of monocytosis during PMF has been proposed as an AP of the disease [9 L), sustained for more than 3 months, together with dysplastic features involving one or more myeloid lineages [However, types of progression other than blast percentage increase have been described in MPNs. In particular, a cohort of 10 PMF patients developed absolute monocytosis during the disease course. It arose at a median interval of 42 months from diagnosis (range: 1-180), persisting for a median period of 23 months (range: 2–57). Among these patients, five died after developing monocytosis (range: 20–188 months), and two experienced worsening disease with transfusion dependence. Interestingly, four of nine patients analyzed showed  disease . Clearly9 L) developed at or around the time of evolution in post-polycythemic MF, was associated with a worse outcome: four patients out of 10 died after developing leukocytosis and one experienced worsening disease. In addition, when compared with a control group of post-polycythemic MF patients (n = 23) who never developed persistent leukocytosis, the former showed a shorter OS, suggesting that persistent leukocytosis could be associated with an overall more aggressive course of the disease [JAK2 and BCR-ABL1 status or cytogenetic evolution. Furthermore, the mutational status of CSF3R, SETBP1, and SRSF2, genes associated with other chronic myeloid neoplasms with neutrophilic leukocytosis, was investigated, but no mutation was detected.Considering instead PV patients, in a previous study involving the same cooperative group, absolute neutrophilic leukocytosis (q21.3q26.2)/t or i(17)(q10). In addition, the cytogenetic profile was similar between post-PMF and post-PV/ET MPN-BP .JAK2-mutated chronic phase disease transformed into JAK2-mutated MPN-BP in some patients, whereas in other cases the JAK2 mutation was not detected further [JAK2-mutated ancestral clone, or chronic phase MPN could be biclonal from its outset.According to what has been previously reported, analysis of paired samples in chronic phase MPN vs. MPN-BP has clearly demonstrated that more than one signaling pathway is associated with leukemic transformation. In addition,  further ,42. AccoFLT3, NPM1, and DNMT3A are predominate, MPN-BP is frequently associated with mutations in IDH1, IDH2, TET2, SRSF2, ASXL1, and TP53 [TP53 somatic mutations were present for many years at a low allelic burden in the chronic phase of the disease, with loss of heterozygosity resulting in clone expansion and AML transformation [The mutational profile of MPN-BP is different from that of de novo AML. Indeed, in contrast to the latter, in which mutations in and TP53 ,44,45,46ormation .BCR-ABL1–negative MPNs in accelerated or blast phase of the disease have been associated with a poor response to therapy and severely shortened survival [survival ,48,49.Conventional antileukemic therapy has limited efficacy in this setting for patients, and current therapeutic strategies for MPN-BP and AP rarely offer more than palliative benefit ,49,50,51At present, ASCT is the best therapeutic option, but initially requires intensive chemotherapy to reduce the disease burden to become eligible. However, in most patients, ASCT is not feasible, mainly due to advanced age, significant comorbidities, and poor performance status, and consequently fewer than 10% of patients undergo a transplant .Patients ineligible for ASCT are treated with supportive therapy and non-intensive chemotherapy, like hypomethylating agents , becauseThis approach included antibiotics, RBC, and/or platelet transfusions, and oral chemotherapy with hydroxyurea for prevention of leukostasis. However, none of these regimens has been shown to be able to produce an effective response, and the median OS with these approaches is only 2.0 months (range 0.0–20.1 months) .Effectively-targeted chemotherapeutic agents that are currently available include two hypomethylating agents that have been approved by the US FDA for MPN-BP. These are 5-azacytidine (azacytidine) and 5-aza-2′deoxycitidine (decitabine). The rationale for their use is based on the observation that hypermethylation of p15/p16 gene promoter sites has been reported in patients with MPN-BP, but not in the chronic phase of these diseases . These g2 subcutaneously for 7 days every 28 days, for up to 6 cycles, consecutively.Azacitidine is administrated at a dosage of 75 mg/mIn a study of 26 azacitidine-treated patients, a 38% response rate (median time to response: 9 months) with an OS of 8 months was reported. Interestingly, the authors observed better responses in post-ET, as opposed to post-PV, MPN-BP, though with no significant OS difference . AnotherTherefore, it can be concluded that hypomethylating agents should be considered for patients who are ineligible for ASCT . In addi2 intravenously over 1 h for 5 days every 28 days, for up to 6 cycles, consecutively. This hypomethylating agent has been used also in MF to alleviate splenomegaly and anemia [The use of decitabine has demonstrated efficacy with a median survival beyond 9 months . It is ad anemia ,60.Decitabine compares favorably with either supportive care or intensive induction chemotherapy. However, it requires support with both RBC and platelet transfusions, owing to the common adverse effect of myelosuppression.JAK2 inhibitor ruxolitinib has also shown promising activity [Combination therapy of decitabine and the activity ,56. In pactivity . This asactivity .Despite these encouraging findings, the response duration of hypomethylating agents is short, and therefore, other therapies need to be evaluated as well.p = 0.005) reported in the relevant phase III trial of CPX-351 involved older patients and high-risk AML, including therapy-related AML, and with antecedent myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia [Among the most promising ones, CPX-351 is a liposome formulation of cytarabine and daunorubicin which are encapsulated in a fixed 5:1 molar ratio. This delivery system improved drug concentration in BM and its uptake into blasts, determining superior antileukemic efficacy in vivo ,65. In aleukemia . Howeverleukemia .JAK inhibitors is synergistically lethal against AML cells sensitive to JAK inhibitors, whereas combined therapy with BETi and heat shock protein 90 inhibitors exerts synergistic lethality against AML cells which are resistant to JAK inhibitors [Another possible therapeutic target is now represented by the family of bromodomain and extraterminal domain (BET) proteins. They are chromatin reader proteins that contain N-terminal, double-tandem bromodomains that bind to the acetylated lysine on the nucleosomal histones and transcription factors . Their ihibitors .IDH2 (enasidenib), CD33 (gemtuzumab ozogamycin), or BCL-2 (venetoclax) [Other agents which are currently under investigation include histone deacetylase inhibitors, target therapy against etoclax) .Histone deacetylase inhibitors, including panobinostat, have been evaluated in small studies of MPN patients, showing an initial clinical improvement and a good safety profile ,73.IDH inhibitor-associated differentiation syndrome [Enasidenib effectively suppressed 2-hydroxyglutarate production, thus releasing myeloid blasts from differentiation block . As an osyndrome .Gemtuzumab ozogamycin initially received FDA approval for relapsed AML in May 2000, but was soon retired due to a lack of benefit when added to standard therapy in a phase III confirmatory study . It was The BCL-2 inhibitor venetoclax was only partially effective as monotherapy in relapsed/refractory AML (19% CR/CRi) . NeverthBCR-ABL1-negative MPNs, with a frequency varying according to the specific MPN subtype. It is highest in PMF, following by PV and finally by ET.Leukemic evolution represents a critical complication in the natural history of Many different risk factors for leukemic evolution has been recognized, among them, a prominent role must be attributed to biological findings. In particular, a complex/monosomal karyotype represents an important risk factor, as a favorable karyotype is infrequent in MPN-BP. In addition, several recurrent gene mutations, including those involving epigenetic modifiers and spliceosome machinery, are involved in this phase of the disease.The conventional antileukemic therapy has limited efficacy in this setting of patients and current therapeutic strategies rarely offer more than a palliative benefit. Nevertheless, based on new molecular acquisitions, new targeted agents are currently under development. In this context, participation of these patients in clinical trials should be strongly encouraged.