In this study, a systematic gene deletion analysis of all putative DHA transporter genes was carried out in Fusarium graminearum, and one DHA1 transporter FgQdr2 was found to be involved in multiple drug resistance, ion homeostasis, and virulence. Further exploration showed that FgQdr2 is mainly localized in the cell membrane; its expression under normal growth conditions is comparatively low, but sufficient for the regulation of drug efflux. Additionally, investigation of its physiological substrates demonstrated that FgQdr2 is essential for the transport of K+, Na+, Cu2+, and the regulation of the membrane proton gradient. For its roles in the FHB disease cycle, FgQdr2 is associated with fungal infection via regulating the biosynthesis of virulence factor deoxynivalenol (DON), the scavenging of the phytoalexin, as well as both asexual and sexual reproduction in F. graminearum. Overall, the results of this study reveal the crucial roles of FgQdr2 in multiple drug resistance, ion homeostasis, and pathogenicity, which advance the understanding of the DHA transporters in pathogenetic fungi.Increased emergence of drug resistance and DON pollution pose a severe problem in Fusarium head blight (FHB) control. While the H Fusarium graminearum, in addition to causing substantial yield losses, is responsible for the serious mycotoxin contaminations of grains. The mycotoxin deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal grains, with a 59% average incidence rate [F. graminearum in wheats, DON is an important virulence factor, being massively produced and crucial for the spread of this pathogen within hosts [Fusarium head blight (FHB) is one of the most devastating diseases on wheat, barley, maize and other important cereal crops worldwide ,2. As onnce rate . During in hosts ,5,6,7,8.in hosts ,11.F. graminearum [F. graminearum strains have been detected in the fields [The best way to prevent DON contamination is to manage FHB in the field during crop cultivation. Currently, due to the lack of highly resistant wheat cultivars, the application of chemical fungicides is still the major approach against minearum . The benminearum ,14,15. Oe fields ,18,19,20e fields ,18,19,20In pathogenic fungi, there are mainly four mechanisms associated with the development of antifungal drug resistance: (i) drug target alteration, (ii) drug target overproduction, (iii) drug inactivation, and (iv) reduced uptake and active extrusion ,21. The + antiporter (DHA) families, including DHA1 and DHA2; the two families differ mainly in the number of transmembrane spans (TMS), with the first having 12, and the second 14 TMS [Saccharomyces cerevisiae has 12 DHA1 and 10 DHA2 transporters [Candida, including Candida albicans, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida tropicalis, Candida guilliermondii, Cryptococcus neoformans, and Aspergillus fumigatus; only 5% of the DHA transporters from these organisms have been characterized so far [As the largest secondary transporter collection, MFS transporters are found in all kingdoms of life, and include millions of sequenced members that are classified into 74 families, with each responsible for a specific substrate type ,29,30. Md 14 TMS ,25. The sporters . Interessporters ,25. For sporters  and mancsporters ,33. ScTpsporters ,34,35. Asporters ,37. AlthF. graminearum, different from the ABC transporters, which have been systematically identified and characterized in our previous study [F. graminearum [F. graminearum, we therefore were interested in investigating the functions of DHA transporters in F. graminearum. In this study, we carried out a systematic gene deletion analysis of all putative DHA transporter genes in F. graminearum and found that among 34 DHA transporters, DHA1 transporter FgQdr2 not only regulates multiple drug resistance and ion homeostasis, but also controls virulence via modulating the production of virulence factor DON, phytoalexin export, and asexual and sexual development. These results provide insights into the increasingly severe problems of DON pollution and antifungal drug resistance in FHB control, broadening the knowledge of DHA transporters in fungi. In us study , only onminearum . As littF. graminearum was used as the parent strain for the transformation experiments. Mycelial growth of the wild-type strain, and the resulting transformants, were assayed at 25 °C on potato dextrose agar . Conidiation in carboxymethyl cellulose (CMC) liquid medium and induction of sexual development on the carrot agar were performed, as described previously [The wild-type strain PH-1 (NRRL 31084) of eviously . Each exF. graminearum using a polyethylene glycol (PEG)-mediated protoplast transformation method were carried out, as described previously [Constructs for gene deletion in eviously . The priFgQDR2-GFP fusion cassette for the complementation, the FgQDR2 fragment containing the native promoter and ORF (without the stop codon) was amplified with primers listed in S. cerevisiae XK1-25 using the alkali-cation yeast transformation kit . The recombined pYF11-FgQDR2-GFP plasmid was extracted from the yeast transformant using a yeast plasmid extraction kit  and then transferred into Escherichia coli strain DH5α for amplification. Then the recombinant plasmid pYF11-FgQDR2-GFP was transformed into the ΔFgQdr2 mutant for complementation via the PEG-mediated protoplast transformation method, and the resulting transformant was designated as ΔFgQdr2-C.To construct the FgQDR2 overexpression strain were constructed via in situ replacement of the native FgQDR2 promoter with gpdA promoter from A. nidulans for constitutive expression. Briefly, a gene replacement cassette carrying the flanking sequences of the FgQDR2 promoter, hygromycin resistance gene (HPH), the coding sequence of the gpdA promoter, the GFP-tag coding sequence was constructed using double-joint PCR. Then, the resulting cassette was transformed into the PH-1 strain. The transformants were examined via PCR assays using primers listed in FgQDR2 ORF upstream of the PCR-confirmed transformants was then sequenced for further examination. Finally, the quantitative reverse transcription PCR (qRT-PCR) and Western blot (WB) analyses were carried out to examine the expression of FgQDR2 under the gpdA promoter.The FgACTIN gene was performed as a reference. The experiment was repeated independently three times.Total RNA was extracted from the mycelia of each sample using the TaKaRa RNAiso Reagent . Each RNA sample was reversely transcribed into cDNA with a HiScript II Q RT Kit . The expression levels of each gene under different culture conditions (as shown in the figure legends) were determined by quantitative real-time PCR with the primers listed in F. graminearum protein extraction was performed, as described previously [eviously . The resw/v) sucrose solution was observed with a Leica TCS SP5 imaging system  after staining with the cell wall-damaging agent calcofluor white (CFW) at 10 μg mL–1. Fluorescence signals were examined with a Zeiss LSM780 confocal microscope . The subcellular location of FgQdr2-GFP was exanimated using the following confocal microscopy settings: laser 488 nm at 50% power, pinhole 90 μm, master gain 580. The morphology of conidia cultured in CMC liquid medium for 4 d and fresh germinating macroconidia in 2%  was injected into a floret of susceptible wheat (Triticum aestivum) cultivar Jimai 22. After 15-d inoculation, infected spikelets in each inoculated wheat head were recorded. For assessing virulence on wheat coleoptiles, the 2 to 3 mm top of the three-day-old coleoptiles was removed, and the wounded top was directly inoculated with a 5 mm diameter fresh mycelial plug. After inoculation, the seedlings were grown in a growth chamber at 25 °C and 95% humidity for later lesion size measurement. To evaluate pathogenicity on corn silks, fresh corn silks were placed on a sterilized filter paper soaked with sterile water. The middle of the corn silks were inoculated with a 5 mm diameter fresh mycelial plug. The infection of the corn silk was measured based on the extent of discoloration after 5 d of incubation at 25 °C. Ten replicates were carried out for each strain. Each virulence experiment was repeated four times.For virulence evaluation on wheat spikelets, a 10 μL aliquot of conidial suspension  culture, then inoculated to TBI liquid medium  at 28 °CS. cerevisiae, C. albicans, C. glabrata, and A. fumigatus were used for BLASTP searches to identify their orthologs in the genome of F. graminearum (http://www.broadinstitute.org/annotation/genome/fusarium_graminearum/MultiHome.html (accessed on 20 September 2022)). A total of 21 putative DHA1 transporters and 13 putative DHA2 transporters were identified. Conserved domain analysis using the SMART program (http://smart.embl-heidelberg.de) showed that the putative DHA1 transporters contain 9-14 TMSs, and the putative DHA2 transporters contain 10-14 TMSs in F. graminearum  exhibited increased sensitivity to prochloraz, tebuconazole, fludioxonil, and iprodione, but not to other fungicides (FGSG_08749 detention mutant (ΔFGSG_08749) exhibited increased sensitivity to prochloraz, but not to the other fungicides  were verified by quantitative reverse transcription PCR (qRT-PCR) assays  assays . The conmembrane A, which prodione B,C. AccoR assays D. Thus, http://www.cbs.dtu.dk/services/NetPhos/ (20 September 2022)), found that FgQdr2 was assumed to be phosphorylated at S260, T264, T282, and S288 (https://swissmodel.expasy.org/ (20 September 2022)), these phosphorylation sites are localized in the LOOP region  [FgTRI1, FgTRI4, FgTRI5, FgTRI6, FgTRI10, and FgTRI101 in ΔFgQdr2 were dramatically decreased by 89.4%, 95.7%, 91.6%, 78.7%, 69.5%, and 81.5%, respectively, as compared to those in the PH-1 and its complemented strains   was dete strains B. These Fusarium [FgQDR2. Phenotypic characterization showed that ΔFgQdr2 was hypersensitive to BOA and 2-AP  and its degradation product 2-AP (2-aminophenoxazin-3-one) . PreviouFusarium . To furtand 2-AP C,D, indiF. graminearum [F. graminearum during the FHB disease cycle.Asexual and sexual reproduction plays a critical role in the infection cycle of homothallic fungus minearum . To furtF. graminearum is one of the most devastating diseases on wheat, barley, maize, and other important cereal crops worldwide [+ antiporter (DHA) family of the MFS [F. graminearum was carried out. Surprisingly, among the 34 DHA transporters, only FgQdr2 exhibited severe deficiency in multiple drug resistance and growth , as the first DHA reported in pathogenic yeast, was also identified as playing important roles in the resistance of antifungal drugs, including quinidine, miconazole, tioconazole, clotrimazole, and ketoconazole. Notably, neither ScQdr2 nor CgQdr2 confer resistance to triazoles [F. graminearum, S. cerevisiae, and C. glabrata in the spectrum of antifungal drugs transported by Qdr2  upon the induction of clotrimazole [C. glabrata to confer chemical stress. In F. graminearum, the confocal microscope examination of the ΔFgQdr2::Qdr2OE-GFP strain showed that the FgQdr2 was localized along the plasma membrane (C. glabrata and S. cerevisiae [FgQDR2 (F. graminearum. Considering that the functions of approximately one-third of proteins are regulated via phosphorylation/dephosphorylation in eukaryotes [FgQDR2 was upregulated in the presence of massive H+ (Although Qdr2 has been characterized as the efflux pump of a wide variety of drugs, the molecular mechanisms behind its functions remain elusive and controversial ,23,27. Tmembrane A, indicarevisiae . However [FgQDR2 D. Accord [FgQDR2 B,C, indikaryotes , we thenssive H+ C, indicaS. cerevisiae, ScQdr2 was proposed to have a role in potassium homeostasis [S. cerevisiae showed that ScQdr2 is associated with the transport of sodium, manganese, calcium, and copper [F. graminearum, FgQdr2 acts as a key factor in osmotic adjustment by balancing potassium homeostasis, and it is also involved in sodium and copper transport, but not in divalent cations (Besides working as drugs efflux pumps, ScQdr2 also plays a prominent role in maintaining ion homeostasis to provide the first layer of sophisticated regulation at the boundary between an organism and its environment. In eostasis , while Ceostasis . Additiod copper . However cations A,B, suggC. albicans [F. graminearum (Owing to its significance in aiding the movement of ions and solutes through the membranes via facilitating diffusion, symport, or antiport, MFS is crucial for multiple physiological processes including cell growth, reproduction, and even virulence in pathogenetic fungi ,53. For albicans . Consistminearum . Overall


Crohn’s disease is characterized of dysregulated inflammatory and immune reactions. The role of the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome in Crohn’s disease remains largely unknown.The microarray-based transcriptomic data and corresponding clinical information of GSE100833 and GSE16879 were obtained from the Gene Expression Omnibus (GEO) database. Identification of in the NLRP3 inflammasome-related genes and construction of LASSO regression model. Immune landscape analysis was evaluated with ssGSEA. Classification of Crohn’s-disease samples based on NLRP3 inflammasome-related genes with ConsensusClusterPlus. Functional enrichment analysis, gene set variation analysis (GSVA) and drug-gene interaction network.The expressions of NLRP3 inflammasome-related genes were increased in diseased tissues, and higher expressions of NLRP3 inflammasome-related genes were correlated with generally enhanced immune cell infiltration, immune-related pathways and human leukocyte antigen (HLA)-gene expressions. The gene-based signature showed well performance in the diagnosis of Crohn’s disease. Moreover, consensus clustering identified two Crohn’s disease clusters based on NLRP3 inflammasome-related genes, and cluster 2 was with higher expressions of the genes. Cluster 2 demonstrated upregulated activities of immune environment in Crohn’s disease. Furthermore, four key hub genes were identified and potential drugs were explored for the treatment of Crohn’s disease.Our findings indicate that NLRP3 inflammasome and its related genes could regulate immune cells and responses, as well as involve in the pathogenesis of Crohn’s disease from transcriptomic aspects. These findings provide in silico insights into the diagnosis and treatment of Crohn’s disease and might assist in the clinical decision-making process. It is eIn the past two decades, substantial progress has been achieved in the field of treatment for Crohn’s disease . The advThe etiology of Crohn’s disease remains largely unknown, and consequently, clarifying the mechanisms of pathogenesis is important. Inflammasomes are well-known to involve in inflammatory disorders in various systems . The NODIn this study, we aimed to provide a comprehensive overview of NLRP3 inflammasome-related genes and pathways in Crohn’s disease by a computational approach. Machine learning was applied to classify the patients based on the NLRP3 inflammasome-related genes. The correlations with immune landscape and potential drugs were further investigated.22.1homo sapiens NLRP3 inflammasome-related genes were acquired from the publication by Ju et al.  database. In total, the samples obtained from dataset GSE100833 includes 159 samples derived from inflamed areas of CD patient undergoing surgical bowel resection, and 168 samples derived from normal, non-inflamed bowel located more than 10 cm away from patients undergoing surgical bowel resection for sporadic colon cancer, and the samples with Crohn’s disease were all isolated from areas with moderate to severe inflammation . Suppleu et al. . Briefly2.2https://string-db.org), and an interaction score > 0.4 was considered as statistically significant, and hiding individual target protein nodes  was performed by the GSVA package  to asses2.5P < 0.05, |log2FC| > 1)  . The DEGFC| > 1) .2.6Based on the DEGs, the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses between the two subgroups were performed using the clusterProfiler package . GSVA wa2.7P < 0.05.Statistical analysis was performed by R (version 4.1.0) and SPSS (version 25.0) software. T-test or Wilcoxon rank-sum test was used to analyze the continuous variables according to the normality. Pearson chi-square test was used to examine the differences of categorical variables. All significant thresholds were set at a two-sided 33.1ARRDC1-AS1, CARD8, GSDMD, ATAT1,CD36,CPTP,DHX33,EIF2AK2,GBP5,NLRC3,PYDC2,SIRT2,TLR4,TLR6,USP50,APP,CASP1,HSP90AB1,MEFV,NFKB1,NFKB2,NLRP3,P2RX7,PANX1,PSTPIP1,PYCARD,RELA,SUGT1,TXN,TXNIP, were demonstrated in The locations of 30 NLRP3 inflammasome-related genes, including 3.2CARD8, CASP1, GBP5, HSP90AB1, MEFV, NFKB1, NFKB2, NLRC3, NLRP3, PANX1, PSTPIP1, RELA, TLR4 and TLR6) in the inflamed samples compared to non-inflamed samples in GSE100833 .A total of 30 NLRP3 inflammasome-related genes were included in our study. However, there are 5 NLRP3 inflammasome-related genes for which some expression data is lacking. As a result, 25 NLRP3 genes having expression data were chosen for further analysis, and 14 DEGs were identified (3.3P < 2.2e-16) between CARD8 and GBP5 expressions (P < 2.2e-16) between TLR4 and PANX1 expressions . The coefficient of each gene was demonstrated in Univariate logistic regression demonstrated that 16 DEGs were associated with Crohn’s disease -related genes were increased in the disease group  demonstrated good distinction of the two clusters  were further selected as the hub genes. Using the DGIdb database, four key NLRP3 inflammasome-related genes  were further selected as potential druggable targets for Crohn’s disease treatment -gene expressions. and the Crohn’s disease are associated with enhanced infiltration and activation of immune cells and immune-related signaling. Therefore, our findings indicate that NLRP3 inflammasome and its related genes could regulate immune cells and responses, as well as involve in the pathogenesis of Crohn’s disease.APP, HSP90AB1, NFKB1 and TLR4 were identified. Amyloid-beta precursor protein (APP) is the precursor of amyloid β before sequential proteolytic cleavage (Four key hub genes including cleavage , and amycleavage . Heat shcleavage . Nuclearcleavage , 48. Tolcleavage . The fouin silico data, and further studies based on cell and animal models could be performed in the future. In addition, current publicly available datasets are limited in the diversity of CD lesion locations, and therefore validation of different locations of the intestine in CD cohorts could be performed in future studies. Furthermore,the prognostic value and prediction of therapeutic responses based on the NLRP3 inflammasome-based signature could be further assessed with available information.Several limitations exist for our study. Our study mainly involves 5In conclusion, our study identified the dysregulations of NLRP3 inflammasome-related genes and their associations with the immune landscape, subtypes of patients, phenotype-related hub genes, and potential drugs of Crohn’s disease. Our findings provide a comprehensive overview of NLRP3 inflammasome-related genes in the etiology and promising possibilities for the treatment of Crohn’s disease. Further investigations on the specific mechanisms and validations are warranted.The original contributions presented in the study are included in the article/HW, RZ acquired the data, performed the analysis and wrote the manuscript. XQ and KC acquired the data and performed the analysis. ZZ, KG, YX, QY, and RJ participated in data analysis. FL, QL, WS and HC were involved in study design and supervision. FL, WS and HC acquired the funding. All authors contributed to the article and approved the submitted version.


Sugars are the major source of energy in living organisms and play important roles in osmotic regulation, cell signaling and energy storage. SWEETs  are the most recent family of sugar transporters that function as uniporters, facilitating the diffusion of sugar molecules across cell membranes. In plants, SWEETs play roles in multiple physiological processes including phloem loading, senescence, pollen nutrition, grain filling, nectar secretion, abiotic  and biotic stress regulation. In this review, we summarized the role of SWEET transporters in plant development and abiotic stress. The gene expression dynamics of various SWEET transporters under various abiotic stresses in different plant species are also discussed. Finally, we discuss the utilization of genome editing tools (TALENs and CRISPR/Cas9) to engineer SWEET genes that can facilitate trait improvement. Overall, recent advancements on SWEETs are highlighted, which could be used for crop trait improvement and abiotic stress tolerance. Photosynthetic organisms synthesize sugars during photosynthesis, a primary source of carbon and energy in cells . Synthes+/sugar antiporters . These two products are used by sucrose-phosphate synthase to produce sucrose phosphate, which is then converted to sucrose by sucrose-phosphate phosphatase , indicating that SWEET17 may be engaged in sucrose-mediated axillary bud outgrowth via the auxin transport pathway [Sugars are involved in shoot branching and bud outgrowth ,103,104.d growth . Simulta pathway .AtSWEET8/RPG1 (Ruptured pollen grain 1) is expressed in microsporocyte and tapetum. Pollen grains of atsweet8 mutants are aborted and sterile, suggesting its involvement in anther and pollen development [AtSWEET13/RPG2 partly restores the male fertility of atsweet8 at the late reproductive stages, which is also expressed in the anther during microsporogenesis, indicating functional redundancy among SWEETs. However, the double mutant of rpg1:rpg2 was fully sterile and was unable to restore [AtSWEET11 and OsSWEET11/Os8N3/Xa13 also produced defective pollen grains and reduced male fertility in Arabidopsis and rice, respectively [AtSWEET1, PwSWEET1, and AtSWEET5/VEX1 are expressed at different stages of pollen development, which indicates their role in pollen development [SWEET genes are expressed at different stages of pollen development. In Arabidopsis, elopment . Further restore . Knockouectively ,108,109.elopment ,110. To cope with different abiotic constraints, plants tightly regulate the vacuolar storage and transport of sugars. For instance, sugar accumulation occurs in vacuoles to minimize freezing stress ,111. AddAtSWEET4, AtSWEET13, AtSWEET14 and AtSWEET15 were induced in Arabidopsis and OsSWEET12, OsSWEET15 and OsSWEET16 were induced in rice  treated Arabidopsis plants. This suggests that plants use distinct mechanisms to cope with drought and PEG-induced osmotic stress. Prolonged drought increases the root to shoot ratio , which i in rice . Likewisbidopsis . An incrbidopsis  reportedDianthus Spiculifolius gene DsSWEET12 have longer roots, high fructose, and glucose with a lower sucrose content and higher tolerance to osmotic and oxidative stresses compared to wild type plants [Malus domestica SWEET gene MdSWEET17 showed a higher accumulation of fructose and enhanced drought tolerance [SWEET10b under drought stress was also reported in potato [Several other examples demonstrate the role of SWEETs in drought tolerance . Arabidoe plants . Transgeolerance . It is wolerance . The totolerance . A signin potato .AtSWEET17, localized in the vacuolar membrane, was markedly upregulated during lateral root (LR) development under drought stress [N3 (LOC101510607), SWEET1-like (LOC101515250), and SWEET4 (LOC101488443)) were reported to be upregulated in chickpea genotypes [GmSUC2, GmSWEET6, and 15) was upregulated in the leaves and roots [OsSWEET13 and OsSWEET15 were induced in response to drought stress. The higher expression of these two genes was due to the binding of an ABA-responsive TF (OsbZIP72) to their promoter sequences. This modulates sucrose transport and distribution in response to drought stress, thus maintaining sugar homeostasis in response to drought stress [TaSWEET2a1-6B, TaSWEET2a2-6D, and TaSWEET2b2-3A), four members of clade III , and five members of clade IV . However, these genes are expressed in a clade-specific manner, i.e., the members of clade I show downregulation, clade III show upregulation, and clade IV showed upregulation [CsSWEET1a, 2a, 2c, 3a, 7a, 7b and 10a) were induced under drought stress [In a recent study, another sugar transporter t stress . In anotenotypes . In soybnd roots . In ricegulation . Similart stress . HoweverAtSWEET1, AtSWEET4, AtSWEET13 and AtSWEET15 were induced, and AtSWEET2, AtSWEET10 and AtSWEET17 were suppressed in Arabidopsis. In rice, OsSWEET14 and OsSWEET16 were induced, and OsSWEET3b, OsSWEET4 and OsSWEET5 were suppressed  were up-regulated after heat stress [B. rapa, BrSWEET1 was expressed after 2 h of heat stress, while BrSWEET11 was expressed after 8 h of heat [GhSWEET4, 5, 10e, 49 and 55 showed induced expression under heat stress [SWEET1, 2, 3, 4, 6, 14, 15, 16, and 17) showed a differential expression after 6 h of heat stress at the seedling stage. However, these genes expressed in a clade-specific manner, i.e., the members of clade II, III, and IV showed downregulation (except TaSWEET15a-7D), while the members of clade I showed both upregulation and downregulation [In wheat, 22 sugar transporters were up-regulated and 19 were suppressed under heat stress . In Brast stress . In B. r of heat . In cottt stress ,73. In wgulation . StudiesAtSWEET15/SAG29 are up-regulated under cold stress [AtSWEET1, AtSWEET2b, AtSWEET4, AtSWEET13 and AtSWEET15 was induced in Arabidopsis and OsSWEET7c and OsSWEET14 were induced in rice  under various stresses, indicating its role in stress tolerance to multiple stresses [Brassica oleracea reported the downregulation of BoSWEET11b, 11c, 12b, 16a, and 17 after chilling stress, possibly resulting in the accumulation of glucose and fructose and an enhanced chilling tolerance [Cold stress induces sugar accumulation in plants, and several SWEET genes such as d stress . The exp in rice . Cold st vessels . Additioolerance . AtSWEETolerance . In tea, vacuole , while tnditions ,138. Furstresses . A genomolerance . Similarolerance ,127. TheSWEET15 (clade III member) is mainly involved in the sucrose transportation. Our analysis showed that AtSWEET1, AtSWEET2, AtSWEET4, AtSWEET14 and AtSWEET15 were induced in Arabidopsis and OsSWEET1b, OsSWEET7c and OsSWEET15 were induced in rice . The FLN2 knockout generated by CRISPR/Cas9 was hypersensitive to salinity stress and showed the disruption of the sugar metabolism, inhibition of Rubisco activity, and downregulation of sucrose synthesis and transportation genes. In FLN2 knockout lines, SWEET11 and SWEET14 were down-regulated after salt stress and indicated that FLN2 protein enhanced salinity tolerance in plants via influencing the sugar metabolism [OsSWEET13 and 15) were also involved in the modulation of sucrose transport in response to salinity stress [Medicago truncatula, banana, revealed differential expression of SWEET genes under salinity stress  are involved in the transportation of various forms of gibberellins (GA) [sweet13;14 double mutant shows a phenotype related to the GA response, i.e., delayed anther dehiscence, and exogeneous GA application restored this phenotype. This experiment supports that SWEETs are involved in GA transport in plants. Similarly, in pea, the interaction between cytokinins, SWEET and cell wall invertase (CWIN) led to the formation of multiple shoots during pathogen infection [The SWEET members have also been demonstrated to transport hormones other than sugars. The two Arabidopsis SWEET genes (ins (GA) . The Aranfection . These tXanthomonas oryzae (Xoo) transcriptionally activate rice disease-susceptibility (S) genes, including SWEET genes. Thus, genome-editing techniques could be used to enhance disease resistance by deleting effector-binding elements (EBEs) in the promoter region of S genes. For example, TALEs  from different Xoo strains targets different SWEET genes (OsSWEET11/12/13/14) [OsSWEET14 were edited using TALENs in a susceptible rice cv. Kitake and the mutated lines were found to be resistant towards AvrXa7 and PthXo3 strains [OsSWEET12 was induced by the infection of a Xoo strain transformed with dTALEs, hence providing susceptibility [Manihot esculenta), by utilizing dTALE that complements TAL20Xam668 mutant phenotypes, it was shown that MeSWEET10a is the primary virulence gene that is the target of TAL20Xam668 [Genome editing has modernized biology and can facilitate the targeted modifications of genomes . Zinc-Fi2/13/14) ,152,153. strains ,152. Fortibility . Additio20Xam668 .OsSWEET11 and OsSWEET14, which are involved in resistance to bacterial blight caused by Xoo. In another study, the CRISPR/Cas9 approach was utilized to edit OsSWEET13, an S-gene of the pathotype PthXo2 in rice [OsSWEET11 and 14) by CRISPR/Cas9 system. Interestingly, the mutation was introduced into the rice cultivar Kitaake, containing the recessive resistance allele of Xa25/OsSWEET13 [OsSWEET11/Os8N3 showed a decreased sucrose concentration in the mutant plants’ embryo sacs, which lead to aberrant grain filling. These results suggest that OsSWEET11/Os8N3 is involved in sucrose transportation during the early phase of caryopsis development [Similarly, CRISPR/Cas9 technology has been implemented to edit rice SWEET genes ,162,163  in rice . The brosSWEET13 ,159,164.elopment . Future This review highlights the role of SWEET sugar transporters in phloem-loading, symplastic sucrose transport during, pollen nutrition, nectar secretion, grain filling, biotic and abiotic stress regulation, and transport of GAs. The functional characterization of SWEETs under various developmental and stress conditions has been well documented in Arabidopsis. However, in crop plants, functional characterization studies of SWEET transporters have just begun. Gene editing tools such as TALENs or CRISPR/Cas9 can be crucial in this context and have been used to study SWEET gene function under biotic stresses, but less functional studies exist in the case of abiotic stress regulation . Their c


The Delta and Omicron variants of SARS-CoV-2 are currently responsible for breakthrough infections due to waning immunity. We report phase I/II trial results of UB-612, a multitope subunit vaccine containing S1-RBD-sFc protein and rationally designed promiscuous peptides representing sarbecovirus conserved helper T cell and cytotoxic T lymphocyte epitopes on the nucleocapsid (N), membrane (M), and spike (S2) proteins.n = 3,875, 18–85 years old). We evaluated interim safety and immunogenicity of phase I until 14 days after the third (booster) dose and of phase II until 28 days after the second dose.We conducted a phase I primary 2-dose (28 days apart) trial of 10, 30, or 100 μg UB-612 in 60 healthy young adults 20 to 55 years old, and 50 of them were boosted with 100 μg of UB-612 approximately 7 to 9 months after the second dose. A separate placebo-controlled and randomized phase II study was conducted with 2 doses of 100 μg of UB-612 , including Delta and Omicron, and a strong booster-recalled memory immunity with high cross-reactive neutralizing titers against the Delta and Omicron VoCs.UB-612 has presented a favorable safety profile, potent booster effect against VoCs, and long-lasting B and broad T cell immunity that warrants further development for both primary immunization and heterologous boosting of other COVID-19 vaccines.ClinicalTrials.gov: NCT04545749, NCT04773067, and NCT04967742.UBI Asia, Vaxxinity Inc., and Taiwan Centers for Disease Control, Ministry of Health and Welfare. The combined effects of SARS-CoV-2 neutralization-escape variants with high transmissibility by asymptomatic persons , 2 and bhttps://doi.org/10.1172/JCI157707DS1). Individuals infected with the Delta and Omicron variants can carry up to 1,000 times more virus in their nasal passages than those infected with other variants  viral antigen. Antigenic variants Delta and Omicron have accounted for greater than 95% of all current infection cases  of sarbecoviruses and can be recognized by individuals who have recovered from COVID-19  (NCT04545749), an interim 14-day phase I extension study with a 100 μg booster (n = 50) (NCT04967742), and an interim 56-day placebo-controlled phase II primary 2-dose study of UB-612 with a 100 μg dose  (NCT04773067) that confirms the reproducibility of B cell and robust, broad, Th1-predominant T cell immunity. The 100 μg dose used in the phase I extension and phase II trials was selected as optimal in the initial phase I dose-ranging study.Here we report the results of 3 clinical trials, which include a 196-day phase I primary 2-dose series (28 days apart) of 10, 30, or 100 μg of UB-612 in healthy adults (1/2t of 187 days) and durable antigen-specific T cell responses. While inducing a modest level of neutralizing titer after 2 doses, a single booster dose prompted striking neutralizing antibodies against the original strain (hereafter referred to as the WT strain) isolated in Wuhan, China  associated with an unusually high cross-neutralization effect against the live Delta variant  and Omicron  strains, which rivalled titers observed with the most effective vaccines up to now and was predictive of greater than 90% efficacy  who received 2 doses (28 days apart) of UB-612 at 10, 30, or 100 μg; and the 84-day extension booster vaccination following the primary series, where 50 participants were enrolled to receive 1 additional 100 μg booster between 7.6 and 9.6 months after the second shot for the 10 μg (n = 17), 30 μg (n = 15), and 100 μg (n = 18) groups. The boosted participants were followed for 14 days for assessment of safety and immunogenicity in this interim report, and subsequently monitored until 84 days after booster.The characteristics of the open-label phase I trial participants  includedThe phase II trial was of a randomized and observer-blind design; participant characteristics are shown in In the 196-day primary series and up to 14 days after booster, neither vaccine-related severe adverse events  nor dose-limited increase in incidence or severity was recorded. The solicited local and systemic AEs reported within 7 days in all vaccination groups  were milThere were no vaccine-related SAEs. Both local and systemic AEs were mild and transient, and were self-limited in a few days.Overall, 2,546 participants reported solicited local AEs, of which 2,386 (72.0%) were from UB-612 and 160 (28.9%) from the placebo group after 1 and 2 doses . These lP > 0.05) (There was no significant difference in the incidence of solicited systemic AEs between UB-612 vaccine and placebo groups across age strata ( > 0.05) . Solicit50 against WT of 4,643, 3,698, and 3,992, respectively  were assessed for their cross-reactive neutralizing antibody titers against pseudo-SARS-CoV-2 and VoCs, including Omicron, as shown in 50 against WT, Omicron, Alpha, Gamma, and Beta were 12,778, 2,325, 9,300, 13,408, and 4,974, respectively, when compared with the WT , with modest respective GMFRs of 5.5, 1.4, 1.0, and 2.6  relative to the WT strain.The pVNT50 against WT observed at 14 to 28 days after dose 2, as compared with the lower-dose 10 and 30 μg groups  for the anti-WT neutralizing VNT50 over days 42 to 196 . The neutralizing antibody VNT50 GMT slowly declined, with a 1/2t of 187 days (Prior to boosting (days 255–316), none of the 18 participants (0%) in the 100 μg group with VNT187 days .n = 20) from the primary series of phase I trial of the 100 μg UB-612 dose group   across participants of all ages (18–85 years old) in phase II were from 88.6% for the elderly to 96.4% for the young adults (On day 57 (4 weeks after the second dose), across participants of all ages (18 to 85 years), the anti–S1-RBD titer with a GMT of 518.8  and the  of 87.2 . The you of 103) , while tg adults .n = 39 for young adults 18–65 years old; n = 9 for elderly adults ≥65 years old) were subjected to an ad hoc live virus assay analysis in 2 independent laboratories . The results were concordant and revealed that immune sera could neutralize 2 key SARS-CoV-2 prototypes with a similar VNT50: 329 against WT obtained in Taiwan and 308 against the USA WA1/2020 strain in the United States  against the S1-RBD–ACE2 interaction  were lar50 data . The 10050 data , with an50 data . Thus, t50 findings (Spearman’s r = 0.9012) (50 results by the cytopathic effect (CPE) assay  , thus coE) assay . FurtherE) assay  were 21650 findings (Spearman’s r = 0.9073). A good correlation existed also between the anti–S1-RBD antibody titers and the WHO International Reference–based Binding Antibodies Unit (BAU/mL), with similar boosting patterns  ELISpot  were collected from vaccinees, with aliquots of 250,000 PBMCs plated into each well and stimulated with 10 μg/mL (each stimulator) for evaluation by interferon-γ ELISpot . The higol alone , demonst+ ELISpot responses for the 100 μg dose group remained at approximately 50% of the peak responses, which decreased from 254 to 121 SFU/106 cells with RBD plus Th/CTL peptide pool restimulation, or from 173 to 86.8 with Th/CTL peptide pool restimulation only. This observation suggests that the UB-612 vaccine–elicited T cell responses after 2 vaccine doses persisted for at least 6 months. This is in concert with the persistence of neutralizing antibodies noted earlier  with S1-RBD plus Th/CTL restimulation, 322 with Th/CTL restimulation, and 181 with Th/CTL peptide pool without UBITh1a (P < 0.0001). In contrast with IFN-γ, the IL-4 responses were far lower: 13.6, 7.5, and 5.4, respectively  were evaluated . Substanfindings , lower I+ T cells expressing the cytotoxic markers CD107a and granzyme B were observed, accounting for 3.5%, 2.1%, and 1.8% of circulating CD8+ T cells after restimulation with S1-RBD plus Th/CTL, Th/CTL, and Th/CTL pools without UBITh1a, respectively. Overall, UB-612 elicited Th1-oriented immunity with a robust CD8+ CTL response, which would be favorable for clearance of the viral infection, and the restimulation results indicated that Th/CTL peptides, which include non-spike N and M structural proteins, are the principal factor responsible for the T cell immunity.CD8Most of the authorized COVID-19 vaccines use the S protein as the immunogen. The UB-612 vaccine product uses the most important functional region, the RBD of the S protein and combines it with promiscuous Th and CTL epitope peptides from the N, M, and S proteins that are highly conserved across all VoCs, including Delta and Omicron, and recognized by individuals who have recovered from prior SARS-CoV-2 infection.UB-612 is designed to not only induce neutralizing antibodies intended to block initial virus entry into human cells, but also to induce a broad T cell immunity that could eliminate virus-infected cells and a fast post-booster recall of memory immune cells upon reinfection or revaccination, should the vaccinated immunity wane overtime. Virus-specific humoral B cell and T cell responses act synergistically to protect the host from viral infection and disease severity. In the phase I primary series, UB-612 demonstrated induction of a durable neutralizing antibody response, with a long half-life of 187 days  and a suThere have been reports on homologous booster vaccination by other vaccine platforms –47. Whil50 measured 14 or 28 days after booster was shown to range from 122 to 6,039, with the associated differential fold increases (GMFIs) from 1.7 to 37 when compared with the respective “peak responses” in the primary vaccination series. The counterpart GMFIs of the “pre-boosting responses” were found to range from 10.3 to 92.9. The UB-612 vaccine in the 100 μg dose group produced an anti-WT VNT50 of 3,992, representing a 37- and a 65-fold increase over the peak primary response of 108 and over the pre-boosting response of 61.5, respectively  ranged from 1.2 to 3.6. After boosting, UB-612 elicited an unusually high anti-Delta neutralizing VNT50 of 2,358  is consistent with that observed in the primary series of the phase I trial, where UB-612 retained a remarkable 83% (1.2-fold reduction) based on the Delta versus WT VNTrsus 255 , and in rsus 308 . OverallA limited study of UB-612 sera from the phase I primary series showed a preservation of notable neutralizing antibodies in vaccinee sera against Alpha B.1.1.7 with 62% retained (1.6-fold reduction), Gamma P.1 with 42% retained (2.4-fold reduction), while that against Beta B.1.351 was weaker, with 23% retained (4.3-fold reduction) . In the 50) observed 14 days after booster of the 100 μg group (n = 18) prompted us to assess cross-reactive neutralizing antibody titers (pVNT50) against pseudo-SARS-CoV-2 Omicron (BA.1 variant) and other VoCs, compared with WT pseudovirus .The high anti-Delta neutralizing antibody titer  has been shown to be more efficient at stimulating high antibody responses and vaccine effectiveness than homologous boosting , 49, UB-The Delta variant (B.1.617.2) contains at least 10 mutations in the S protein . Only 2 50 of 2,325  and has accelerated viral clearance . UB-612 cination . These ocination .Overall, in the combined 3 clinical trials of the phase I primary series, an extended booster third-dose vaccination , and theOf special note, the 5 precision-designed T cell epitope peptides represent the Th and CTL epitopes from sarbecovirus regions of the N, M, and S2 proteins . These eSafety is a major concern, especially for vaccines that will be given to billions of people that may require intermittent or even annual vaccinations. Adenoviral vectors and mRNA are innovative technologies that have only been used widely in the context of the COVID-19 pandemic. These vaccines are associated with local and systemic reactogenicity that may become more severe after repeat dosing. In addition, they have been associated with rare but serious AEs, including myocarditis, pericarditis, Guillain-Barré syndrome, and thrombosis-thrombocytopenia .UB-612 has not yet been deployed widely enough to reveal rare AEs. Its composition  suggests that it should have a good safety profile. So far, with approximately 4,000 people vaccinated, UB-612 has been shown to be very well tolerated and with acceptable reactogenicity upon repeated dosing. With the current sample size for safety, an upper bound of 0.08% is established for the 95% confidence interval for the incidence of an unobserved AE.While UB-612 has demonstrated induction of a profound virus-neutralizing immunity against the Delta and Omicron variants, we understand the limitation of the small sample size of participants and the lack of booster data for the elderly and high-risk groups who have decreased immunity. An additional booster vaccination in our extended study of a phase II trial  is ongoing to further demonstrate UB-612’s benefit in offering potent B and T cell immunity against multiple VoCs, including Delta and Omicron.n = 20/group). Over 6 months following completion of the primary series, 50 participants were enrolled in the extension study to receive a booster dose of 100 μg UB-612, with an interim analysis at 14 days and were also monitored until 84 days after booster. The booster dose of 100 μg was selected based on the favorable results of the phase I primary series.The safety and immunogenicity of the UB-612 vaccine were evaluated in an open-label phase I study, conducted at China Medical University Hospital, Taiwan  and an 84-day extension study to evaluate a third booster dose  . The priThe phase II, placebo-controlled, randomized, observer-blind, multicenter study , conducted at 12 study sites in Taiwan, planned to enroll approximately 3,850 male or female adults 18+ to 85 years old  who wereThe principal investigators at the study sites agreed to conduct the study according to the specifics of the study protocol and the principles of Good Clinical Practice (GCP), and all the authors assured accuracy and completeness of the data and analyses presented. The protocols were approved by the ethics committee at the site and all participants provided written informed consent. Full details of the trial design, inclusion and exclusion criteria, conduct, oversight, and statistical analyses are available in the study protocols in the UB-612 used in the phase I and II trials is a multitope vaccine designed to activate both humoral and cellular responses . For SARThe phase I trial was initiated with a sentinel group of 6 participants to receive the low 10 μg dose, followed by the remaining 14 participants if without vaccine-related grade 3 or higher adverse reaction. The same procedure was extended for the 30 and 100 μg dose groups. Additional follow-up visits were scheduled for all participants on days 14, 28, 35, 42, 56, 112, and 196. Study participants were scheduled for visits 14 and 84 days after the booster. Electronic diaries were provided to the participants to be completed for the 7-day period after each injection to record solicited local reactions at the injection site  and solicited systemic reactions . Severity was graded using a 5-level (0 to 4) scale from none to life-threatening. In addition, participants recorded their axillary temperature every evening starting on the day of the vaccination and for the 6 subsequent days. Safety endpoints included unsolicited AEs reported for up to 14 days after booster in this interim phase I extension report. Complete details for solicited reactions are provided in the study protocols in the The primary safety endpoints of the phase II trial were to evaluate the safety and tolerability of all participants receiving study intervention from days 1 to 57 (28 days after the second dose). Vital signs were assessed before and after each injection. Participants were observed for 30 minutes after each injection for changes in vital signs or any acute anaphylactic reactions. After each injection, participants had to record solicited local and systemic AEs in their self-evaluation electronic diary for up to 7 days while skin allergic reactions were recorded in their electronic diary for up to 14 days. Safety endpoints included unsolicited AEs reported for days 1 to 57 in this interim phase II report. Complete details for solicited reactions are provided in the study protocols in the The study protocols are provided in the https://downloads.regulations.gov/FDA-2020-D-1137-0019/attachment_1.pdf) and WHO guidelines on clinical evaluation of vaccines .As the phase I and its extension studies were not powered for formal statistical comparisons of between-dose and between-phase vaccination, we report descriptive results of safety and immunogenicity. Immunogenicity results for GMT are presented with the associated 95% confidence intervals. Statistical analyses were performed using SAS version 9.4 (SAS Institute) or Wilcoxon’s signed-rank test. Spearman’s correlation was used to evaluate the monotonic relationship between non-normally distributed data sets. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. For the phase II study, the sample size of our trial design meets the minimum safety requirement of 3,000 study participants in the vaccine group, as recommended by the US FDA and WHO: US FDA Emergency Use Authorization for vaccines to prevent COVID-19  was established to monitor data safety and trial conduct. An interim analysis was triggered because the following conditions were met: all participants had completed the second dose of study intervention by 1 month (28 days), and half of participants had completed the second dose of study intervention by 2 months.Additional methods related to immunogenicity assessment of B and T cell immunity including immunogenicity, virus-neutralizing antibody titers against SARS-CoV-2 WT and VoCs, neutralizing antibody titers against WT Wuhan-HU-1 and VoCs  by pseudovirus luciferase assay, inhibition of S1-RBD binding to ACE2 by ELISA, anti–S1-RBD binding IgG antibody by ELISA, and T cell responses by ELISpot and ICS are provided in the Phase I, phase II, and phase I extension studies were approved by Taiwan Food and Drug Administration (TFDA) and the Committee of Institutional Review Board (IRB) from all clinical trial sites in Taiwan with approved letters included in the CYW, FL, SD, and WJP conceptualized and designed the vaccine. SD, ZL, HKK, WJP, and HTW were responsible for vaccine manufacturing and quality control. CYW, HKK, WJP, FG, and TPM were responsible for vaccine development. YHS, CTC, JHH, KPH, HL, CYW, HKK, TK, DGH, and TPM contributed to the protocol design of the study. KPH, YHS, CTH, YJL, MCL, YCY, PLL, HCT, CH Lee, ZYS, CEL, CH Liao, FYC, HC Cheng, FDW, HTW, HL, JHH, and HC Chiu contributed to the implementation of the clinical studies, and acquired and interpreted the clinical data. CTC conducted the statistical analysis. HKK, KLH, JC, MSW, YTY, MHJ, HY Shih, HY SHen, YRL, PYC, YLL, JJL, CCL, YCC, MKM, and CVH were responsible for assay development and validation, laboratory testing and data collection, and preparation of respective reports. CYW, YHS, HKK, HL, WJP, HJY, YHH, MH, and BSK had full access to and verified all the data in the study and take responsibility for the integrity and accuracy of the data analysis. BSK and CYW drafted, edited, and prepared the manuscript. CCK provided manuscript revision and scientific comments. All authors reviewed and approved the final version of the manuscript. CYW had final responsibility for the decision to submit for publication.


Homo sapiens , Sapajus apella, and Cebus capucinus  using fluorescence in situ hybridisation (FISH). The choice of platyrrhine species are due to the fact they are taxa characterised by a high level of rearrangements; for this reason, they could be a useful model for the study of LINE-1 and chromosome evolution. LINE-1 accumulation was found in the two Cebidae at the centromere of almost all acrocentric chromosomes 16–22 and on some bi-armed chromosomes. LINE-1 pattern was similar in the two species but only for chromosomes 6, 8, 10, and 18, due to intrachromosomal rearrangements in agreement with what was previously hypothesised as through g banding. LINE-1 interstitial accumulation was found in humans on the 1, 8, 9, 13–15, and X chromosomes; on chromosomes 8, 9, and 13–15, the signal was also at the centromeric position. This is in agreement with recent and complete molecular sequence analysis of human chromosomes 8 and some acrocentric ones. Thus, the hypothesis regarding a link between LINE-1 and centromeres as well as a link with rearrangements are discussed. Indeed, data analysis leads us to support a link between LINE-1 and inter- and intrachromosomal rearrangements, as well as a link between LINE-1 and structural functions at centromeres in primates.This work focuses on the distribution of LINE-1 (a Long Interspersed Nuclear Element) in primates and its role during evolution and as a constituent of the architecture of primate genomes. To pinpoint the LINE-1 repeat distribution and its role among primates, LINE-1 probes were mapped onto chromosomes of  The human genome is composed of 1–2% coding regions, while 98–99% are noncoding regions; the latter are composed of variable sequences which are highly repetitive and not easily understandable, also termed the ‘dark matter’ of the genome. Mobile elements, also known as transposable elements (TEs), are recognised among the variety of repetitive elements in genomes. These sequences are quite abundant in the complex genomes of animals such as primates, and the first account has estimated they make up about 26% of the human genome . With imBased on the mechanism of their mobility, these transposable elements can be divided into two main classes; elements of Class I jump from one site to another through an RNA intermediate, while elements of Class II move directly without intermediaries; class I, or retrotransposons, mobilise in genomes via a “copy-and-paste” mechanism directed by reverse transcription of an RNA intermediate. This class is typically subdivided into Long Terminal Repeat (LTR) and non-LTR retrotransposons ,3. In paThe majority of LINEs are truncated or defective copies that were originally produced by a relatively small number of full-length, retrotransposition-competent copies . Within Callimico goeldii and Platyrrhini primates [LINE-1 insertions are polymorphic (presence/absence) in the primate genome; their absence is considered the ancestral condition, and when they are present, they are identical by descent since the probability of convergence is very low ,10,11. Fprimates  and to eDespite being considered “junk DNA” at the beginning, researchers have obtained evidence that LINE-1 elements make significant contributions to genome evolution; they are involved in DNA stability, maintaining genome integrity including DNA packaging, centromere stability, or plasticity ,14,15,16Despite the demonstrated importance of TE sequences, the reason for the lack of large-scale comparative studies for LINE-1 transposition in primates is due to the high content of LINEs in the primate genomes; the reference genome sequences are still incomplete or there are assembly errors, especially for the nonhuman primate genomes, due to the complex nature of the repetition of these sequences ,26. WithH. sapiens , S. apella, and C. capucinus   permit us to discuss LINE-1 evolution in a comparative perspective in light of previously published cytogenomic data. Furthermore, the sequential DAPI/CMA3 staining, FISH of LINE-1, and post-FISH C banding of chromosomes permits the evaluation of the possible correlation between heterochromatin and LINE-1 preferential insertion sites and, in a phylogenetic framework, allowed us to show many interspersed and centromeric LINE-1 signals and to hypothesis their possible role and function. LINE-1s have been mapped in many mammals ,28, inclS. apella and C. capucinus (Cebidae), from Catoctin Zoo, Thurmont MD, USA and the Laboratory of Genomic Diversity of the National Cancer Institute, Frederick, MD, USA. Fibroblast cells were grown for 72 h in alphaMEM culture medium , 5% Antibiotics Penicillin/Streptomicin, 15% FBS, 10% amniomax (Gibco).Metaphases were obtained from fibroblast cell cultures from a male sample of H. sapiens were grown in RPMI culture medium, following standardised protocols to obtain metaphases. Lymphoblasts of a male sample of Cells harvesting was performed after 3 h incubation of colcemid 10 μL (10 μg/mL Gibco) followed by hypotonic treatments 0.075 M KCl for 20 min at 37 °C following a protocol from Small et al. .S. apella and C. capucinus species were compared with previously published R- or G-banded karyotypes [S. apella was previously recognised as Cebus apella  according to the basic DNA extraction protocol. LINE-1-like repeat sequences called LINE-1 have been amplified by polymerase chain reaction (PCR); each probe was amplified from the species own DNA; the universal set of primers, developed for the PCR of LINE-1 repeats in mammals, have been used: L1R, 5′-ATTCTRTTC CAT TGG TCT A-3′ and L1F 5′-CCA TGC TCATSGAT TGG -3′ ,41.2, 50 mM KCl. PCR reactions were performed using an Applied biosystems SimplyAmp  with the following cycling parameters: 30 cycles each of 94 °C, 30 s; 52.5 °C, 30 s; 72 °C, 30 s, following a 2 min denaturation at 94 °C. A bright band of about 400 pb was visualised on 1% agarose gel. The PCR products were directly labelled through nick translation using 11-dUTP-Fluorescein (green) (Invitrogen) for H. sapiens and dUTP-cy5 (red) (Amersham) for the Cebidae species.Genomic DNA was amplified in 50 μL PCR-reactions: five units of Taq GOLD DNA Polymerase (Invitrogen), the template DNA, 500 nM of each primer, 200 μM each of dATP, dCTP, dTTP, and dGTP in 10 mM TRIS-HCl, pH 8.3, 1.5 mM MgClFISH was performed following previously described protocols using LINE-1 probes obtained by PCR ,43. The H. sapiens. Furthermore, using the hub_2004795_RepeatMasker available through USCS, we downloaded annotation tracks of LINE-1-related repeated elements for the analysed species.The genomic browser UCSC has been used to extrapolate data on LINE-1 localisation in After FISH, the metaphases were analysed under a Zeiss Axio2 epifluorescence microscope. Images were captured using a coupled Zeiss digital camera. At least ten methaphase spreads were analysed for each sample. Chromosomes were classified according to the proposed nomenclature .LINE-1 probe mapping revealed bright signals on the metaphases of the three species analysed, with a different accumulation pattern d,e,f. SeC. capucinus and S. apella were in agreement with previously published ones [S. apella (pairs 1–10), eight pairs in C. capucinus , and fourteen and sixteen acrocentric chromosomes, respectively, thus differing over chromosome pairs 8 and 10 that are submetacentric in S. apella and acrocentric in C. capucinus.Karyotypes were reconstructed accordingly , using thed ones ,36,37, wDAPI/CMA3 staining was helpful for identifying chromosomes and preferential sites of LINE-1 insertion . The posS. paella  and Lemuriformes (lemurs). Haplorrhini is further subdivided into the Platyrrhini , Catarrhini, composed of Cercopithecidae , and Hominoidea (apes and humans) . Here, tS. apella and C. capucinus (Cebidae), and of H. sapiens (Hominoidea), were expanded to better analyse LINE-1′s distribution and role among primate species’ genomes. The LINE-1 pattern was compared with those previously published for NWMs [Cebidae species analysed in the present work, we found an accumulation of LINE-1 elements displaying a nonrandom distribution by accumulating primarily in CMA-3 and C- positive bands at centromeres or pericentromeric regions   . This rebicolour , S. myst rosalia , Aotus nlite DNA  are presApart from signals at centromeres, noncentromeric LINE-1 signals were found along chromosomal arms on the X chromosome and on autosomes in the two analysed NWM species  in euchrC. capucinus and S. apella [From an evolutionary perspective, LINE-1 signals found along chromosomes at noncentromeric regions, through a comparison with the supposed human chromosomal homologies reported for . apella  . The first attempts to use FISH to map a LINE-1 probe on the human genome was for an incomplete sequence; this early work reported signals with a banding pattern on AT regions [H. sapiens and other mammals [Among primates, Catarrhini are OWMs, including  regions , but com mammals .In this work, apart from mapping LINE-1 probe distribution using FISH on representative species of anthropoid primates, molecular cytogenomic data including sequence and FISH analysis of LINE-1 in primate genomes were reviewed. LINE-1 originated well before the origin of primates (at least 170 mya). Three classes of LINEs have been recognised at about 70 mya; however, already at about 40 mya in Anthropoid primates, only one class remains—LINE-1 ,50,51. IHomo and Pan lineages, indicating that LINE-1 amplification may have changed rapidly during primate evolution [Saimiri and Saguinus genera through sequence comparisons in NWMs, but results in the Ateles lineage are in conflict [The LINE-1 elements amplified during primate radiation ,53,54 arvolution . In partconflict . Pan troglodytes [Gorilla gorilla [Recent comparative studies have analysed raw LINE-1 elements uniquely owned by each primate genome showing different rates of accumulation. In particular, raw numbers of LINE-1 elements have been estimated for chimpanzees (5913), orangutans 21,711), crab-eating macaques (782), rhesus macaques (3016), green monkeys , and humans, based on the most updated reference sequences . Other dglodytes  and Gori gorilla , while h11, crab- gorilla , even in gorilla . C. capucinus/S. apella GSC_monkey_1.0 Dec. 2019 (GCF_009761245.1) and H. sapiens/GCF_000001405.39_GRCh38.p13, Feb 2019, were analyzed. In the NWMs, a kind of L1ME3G#LINE/L1 with a count of 1,423,257 was found  permits the study of highly repetitive genomic regions such as centromeres ,66; this genomes ,68. This genomes .Saimiri and Saguinus [The present work shows a rich content of LINE-1 for the two NWM species analysed, in agreement with previous molecular cytogenetic data in other Cebidae ,22,38; iSaguinus ,60.LINE-1 distribution in the NWM species analysed can be summarised as follows: LINE-1 signals are at the junction of some human syntenic associations, which is in agreement with previous molecular cytogenetic analysis ; this loLINE-1 signals are at the centromeric position, as already seen in other Platyrrhini ; FISH haDespite all this evidence, further multidisciplinary approaches, including the comparison of sequence analysis and FISH mapping in many other species, are needed to test previous hypotheses and better define the precise role and function of LINE-1.


Cyclic nucleotides  play a major role in normal and pathologic signaling. Beyond receptors, cyclic nucleotide phosphodiesterases; (PDEs) rapidly convert the cyclic nucleotide in its respective 5′-nucleotide to control intracellular cAMP and/or cGMP levels to maintain a normal physiological state. However, in many pathologies, dysregulations of various PDEs (PDE1-PDE11) contribute mainly to organs and tissue failures related to uncontrolled phosphorylation cascade. Among these, PDE4 represents the greatest family, since it is constituted by 4 genes with multiple variants differently distributed at tissue, cellular and subcellular levels, allowing different fine-tuned regulations. Since the 1980s, pharmaceutical companies have developed PDE4 inhibitors (PDE4-I) to overcome cardiovascular diseases. Since, they have encountered many undesired problems, (emesis), they focused their research on other PDEs. Today, increases in the knowledge of complex PDE4 regulations in various tissues and pathologies, and the evolution in drug design, resulted in a renewal of PDE4-I development. The present review describes the recent PDE4-I development targeting cardiovascular diseases, obesity, diabetes, ulcerative colitis, and Crohn’s disease, malignancies, fatty liver disease, osteoporosis, depression, as well as COVID-19. Today, the direct therapeutic approach of PDE4 is extended by developing allosteric inhibitors and protein/protein interactions allowing to act on the PDE interactome. Nevertheless, but interestingly, PDEs dysregulation plays a major role in many known, as well as unsolved, pathologies ,7,8,9,103′,5′- cyclic AMP (cAMP) plays a major role in the physiological control as well as in the pathophysiological control of tissue and cellular responses  Figure  2]. Its. Its2]. The PDEs represent a multigene superfamily, selectively hydrolyzing cAMP and /or cGMP . They arThe PDE4 was first described as a PDE hydrolyzing selectively cAMP and thereafter was characterized by its specific sensitivity to the antidepressant compound rolipram (ZK 62711) and consequently named rolipram inhibited-cAMP-PDE (ROI-cAMP-PDE). Thus, differentiating it from the specific cAMP-PDE isolated from human platelets, named cGI-PDE, since this cAMP-PDE is inhibited by cGMP ,13. In a1 and R2 adenine nucleotides , is the most extensive family, coded by four genes, namely PDE4A to PDE4D. Each gene encodes multiple protein products generated by alternative splicing and/or by the use of multiple promoters (see 2+-binding motifs (Hx3HxnE/D) and an Mg2+-binding motif, related to metal-ion phosphohydrolases [The PDE4 family, which exclusively hydrolyses the cAMP  and the high-affinity binding site (HARBS) ,26,27,28Interestingly, it has been shown that pregnancy induces a modulation of the PDE4-conformers ratio in human myometrium, designing LPDE4 as potential pharmacological targets for the design of new tocolytic treatments . In cont®) to treat COPD [®), an oral PDE4 inhibitor, has received FDA approval to treat patients with active psoriatic arthritis and plaque psoriasis [Rolipram and Ro 20–1724 acting in the µmolar range were first identified as highly selective competitive inhibitors of PDE4 [eat COPD . Lastly,soriasis . It is isoriasis . Thus, tsoriasis , re-openNowadays, there are different approaches under development targeting PDE4–protein interactions  allowingConsequently, there are multiple pharmacological means to interact at the level of PDE4 changes inducing new specific and localized therapeutic approaches allowing less undesired effects. PDE4 participates in the control of cardiovascular function at the level of vessels and heart. m < PDE4 Km, and PDE3 Vmax/Km > PDE4 Vmax/Km) as illustrated in PDE4 has been characterized in human, rat, and bovine aortas . It is cMoreover, it should be noted that PDE4 is mainly present in human endothelial cells ,44,45,46In particular, PDE4 plays a role in the regulation of endothelial permeability . NotablyThe cAMP in cardiac muscle is the second intracellular messenger mediating the positive inotrope effects of β-agonists . Thus, PStudies performed on guinea pig cardiac ventricle showed that PDE4 might be implicated differently than PDE3 in the regulation of cardiac contraction, since in opposite to PDE3 inhibitor, PDE4 inhibitor did not per se increase cardiac inotropy, although PDE3 and PDE4 are both cytosolic and membrane bound ,62. HoweThus, we firstly showed in canine and human, purified cardiac microsomal fractions, that PDE3 is associated with the microsomal membranes enriched in vesicles derived from T-tubule and junctional SR membranes; meanwhile, PDE4 is mostly associated with the enriched fraction of sarcolemma membranes, revealing different subcellular compartmentations for PDE3 and PDE4 . To go fA study using PDE4D KO mice, clearly demonstrated that PDE4D deficiencies in the ryanodine receptor (RyR) complex promote heart failure and arrhythmias . Further2+ and cardiac contractility, independently of L-type Ca2+ current, allowing for precise pharmacological control of cardiac contractility [v1.2 signaling complex, the inactivation of the pde4d gene had no consequence on basal or β-AR stimulated ICa,L. In another way, studies performed in KO mice reveal that PDE4B plays also a key role during β-AR stimulation of cardiac function. As a result, PDE4B, by limiting the amount of Ca2+ entering the cell through the L-type Ca2+ channels, prevents Ca2+ overload and arrhythmias [PDE4D represents the main PDE4 isoform in the human heart and its expression is decreased in idiopathic dilated cardiomyopathy as well as in rats during early angiotensin II-induced cardiac hypertrophy, although total cAMP-PDE activity was increased . In thisactility . This coactility . Surprishythmias .2+ overload and arrhythmias. Interestingly, UCR1C is a new, long isoform PDE4 activator that alleviates cardiomyocyte hypertrophy [Altogether, decreases in PDE4D activity, which represents the main PDE4 isoform in human cardiac tissue, might favor idiopathic dilated cardiomyopathy and its deficiency in the RyR complex promotes heart failure and arrhythmias, whereas PDE4B prevents Caertrophy . Lastly,ertrophy .Obesity is considered the fifth highest cause of death worldwide by the World Health Organization and is associated with pathological conditions and diseases associated with obesity, including hyperlipidemia, heart diseases such as coronary artery disease (CAD) and myocardial infarction, stroke, type 2 diabetes (T2D), hypertension, cancers, low-grade and chronic inflammation, fatty liver disease, osteoarthritis, respiratory problems, and neurodegenerative diseases . Obesity is defined by the body mass index (BMI). It is calculated as weight in kilograms divided by height in meters squared, rounded to one decimal place. Obesity in adults (>20 years old) was defined as a BMI greater than or equal to 30. The prevalence of obesity was 39.8% and affected about 93.3 million US adults in 2015~2016 . Therefore, it was pointed out that caloric restriction might extend lifespan and that the metabolic effect of resveratrol, a mimetic of caloric restriction, might be mediated by inhibiting cAMP phosphodiesterases . In thatThus, the involvement of PDE4 in obesity has been demonstrated since, under submaximal β-adrenoceptor stimulation of brown adipocytes, the PDE4 inhibitor alone may increase lipolysis . TherefoA study of the Saudi population found that PDE4D polymorphism (rs295978) was not associated with a risk of obesity and did not affect the blood lipid profile . NeverthAltogether, PDE4 regulation seems to be implicated in obesity at the level of PDE4B and PDE4D, and an allosteric inhibitor of PDE4D would be effective in the loss of fat mass without changing cognitive function.The global prevalence of diabetes among 20- to 79-year-olds is estimated to be about 8.8% in 2015, or 415 million, and approximately 10.4% in 2040, or 642 million . DiabeteRecently, TAK-648, a PDE4 inhibitor, with demonstrated preclinical anti-diabetic properties, has been developed for human T2D (dose prediction using HbA1c results from a db/db mouse study), opening the way for new therapeutic approaches in diabetes . InteresCrohn’s disease is a chronic inflammatory condition of the gastrointestinal tract. It can affect any part of the gastrointestinal (GI) tract, but ulcerative colitis affects only the colon. Additionally, while Crohn’s disease can affect all layers of the bowel wall, ulcerative colitis (UC) only affects the lining of the colon. Both have been together classified as inflammatory bowel diseases (IBD). In the United States, it is currently estimated that about 1.5 million people suffer from IBD, causing considerable suffering, with a prevalence of Crohn’s disease (CD) of 201 per 100,000 population, UC is being equally prevalent . p < 0.05%) suggesting that the specific PDE4 inhibitor may be effective in Crohn’s disease [Because Crohn’s disease is mainly a chronic inflammation, the inflammatory cytokine productions in intestinal biopsies have been studied in relation to the pathological grade. This study originally shows that TNF-α and IL-1β are significantly increased in endoscopic biopsies, as well as for TNF-α, IL-1β, and IL-6 in the individual culture supernatant of intestinal biopsies, opening a new way of investigation . Thus, t disease . In that disease . As a re disease . Further disease . Interes disease .Overall, today, no PDE4 inhibitor has been developed for IBDs, although it appears to be more effective than methylprednisolone . This maOsteoporosis is a common disease characterized by reduced bone mineral density (BMD) and increased risk of fragility fractures, which is usually caused by osteoclasts, whereas osteopenia corresponds to a lower bone density than normal and represents the stage before osteoporosis. By 1970, no medications against osteoporosis were being investigated. At the time, fractures were not even recognized as an illness but were considered part of normal aging . NonetheIn that way, a study showed that denbufylline, an inhibitor of PDE4 ,113, wasIn addition, pentoxifylline and rolipram have been shown to increase bone mass in normal mice mainly primarily by accelerating bone formation ,117. ThiThis human cell study, combined with previous studies ,106,107,In total, these data point to the participation of PDE4 in osteoporosis . FurtherAlthough it was shown that PDE4-Is are effective in decreasing osteoporosis, this potential therapeutic approach always seems to not be recognized for osteoporosis . Hopefulhttps://medlineplus.gov/enc/article/002253.htm, accessed on 13 November 2020). The global cancer incidence and mortality in 2020 were estimated at 19.3 million, almost a million new cases of cancer and 10 million deaths from cancer. The global cancer burden is expected to be 28.4 million cases in 2040 [According to MedlinePlus Medical Encyclopedia, “the malignancy” refers to the presence of cancerous cells that have the ability to spread to other sites in the body  genetically engineered mouse (GEM) model is sufficient to induce gliomagenesis in a mouse model of NF1, and conversely, rolipram dramatically inhibits in vivo optic glioma growth and tumor size in the model . In ovarSome PDE4-Is were recently considered cancer therapeutics, including apremilast . In thisFatty liver disease, currently named nonalcoholic fatty liver disease (NAFLD) according to the practice guidance commissioned by the American Association for the Study of Liver Diseases (AASLD), is defined by: (1) evidence of hepatic steatosis (HS), either by imaging or histology, and (2) lack of secondary causes of hepatic fat accumulation such as significant alcohol consumption, long-term use of a steatogenic medication, or monogenic hereditary disorders . The oveOverall, these data suggest that today PDE4-Is have failed in the treatment of NAFLD but have induced interesting positive data regarding ALD treatment. Thus, it has been shown that altered PDE4B plays a critical role in alcohol-induced steatosis . A recenMajor depression is a common illness that severely limits psychosocial functioning and diminishes quality of life. In 2008, the WHO ranked major depression as the third cause of burden of disease worldwide and projected that the disease will rank first by 2030, the 12-month prevalence of major depressive disorder (MDD) being approximately 6.6%, and the lifetime risk being 15–18% . −/−) mice, the loss of PDE4D in the cerebral cortex and hippocampus, interestingly, induces an antidepressant-like effect on behavior and reduces sensitivity to rolipram. Furthermore, rolipram potentiated isoproterenol-induced cyclic AMP formation only in the PDE4D+/+ mice. Interestingly, the PDE4D-regulated cyclic AMP signaling may play a role in the pathophysiology and pharmacotherapy of depression [The potential antidepressant activity of rolipram, as a cAMP phosphodiesterase inhibitor, was canonically demonstrated in mice by Wachtel H, 1983 . In thatpression .−/−) displayed long-term depression as compared to wild-type littermates [According to a review on CNS and PDEs, it is pointed out that PDE4A, 4B and 4D may participate in depression . Mice determates . In thistermates . Howevertermates . The devtermates . In thattermates .Therefore, strategies to develop antidepressant PDE4 inhibitors is to develop a new generation of PDE4 inhibitors which act specifically on long PDE4 isoforms. They might also be capable of targeting the interactions with their signaling “partner” proteins, such as RACK1, β-arrestin, and DISC1 in order to affect cAMP levels in specific cellular compartments . In addiAs previously  stated, 1R agonist, induces PDE4 up-regulation, with a 44% increase in the PDE4A protein, mediating cascade inflammation and oxidative stress. Altogether, this opens a new way of PDE interaction with the renin–angiotensin system [Interestingly, a paper published in Nature Medicine (2005), demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a strategic role in SARS-CoV-2-induced lung injury. As expected, the injection of SARS-CoV-2 spikes into mice worsens acute lung failure in vivo which can be alleviated by blocking the renin–angiotensin pathway governed by ACE2 . Furthern system . More ren system ,179,180.n system .Altogether, these data reported for COVID-19 suggest the hypothesis that a PDE4 inhibitor might alleviate both viral infection and tissue inflammation induced by SARS-CoV-2 . Altogether, PDE4s are implicated in various human pathologies, such as cardiovascular diseases, obesity, diabetes, ulcerative colitis and Crohn’s, osteoporosis, malignancies, fatty liver disease, and depression, as well as COVID-19 . Given tTo further explore the therapeutic potential of PDE4-Is, different strategic approaches could be used to reduce adverse effects. Since PDE4 could act in interactome signaling, it should be possible to act on the protein–protein interactions governing PDE activity in various cell compartments ,185. ThiThe present paper reports the implication of dysregulation of PDE4 activity in various pathologic fields pointing out the effectiveness of PDE4 inhibitors as well as the increases in the knowledge of PDE4 functions related to their multiplicity at cellular, subcellular, and molecular levels. This introduces new approaches to powerful, selective, and effective treatment, with lesser adverse effects, opening up a new generation of more suitable PDE4 inhibitors. This was just newly discussed and reviewed for clinical implications of PDE4-Is .


In this study, using a bioinformatics approach and Microarray data were obtained from GSE67916 and GSE85871 datasets to identify differentially expressed genes (DEGs). DEGs common between HON-treated and TAM-resistant cells were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and protein-protein interaction (PPI) networks were constructed. Selected genes were analyzed for genetic alterations, expression, prognostic value, and receiver operating characteristics (ROC). TAM-resistant MCF-7 (MCF-7 TAM-R) cells were generated and characterized for their resistance toward TAM. A combination of HON and TAM was used for cytotoxicity and gene expression analyses. Molecular docking was performed using the Molecular Operating Environment software.FN1, FGFR2, and RET were the top three genes with the highest scores. A genetic alteration study of potential target genes revealed MMP16 and ERBB4 as the genes with the highest alterations among the breast cancer samples. Pathway enrichment analysis of FGFR2, RET, ERBB4, SOX2, FN1, and MMP16 showed that the genetic alterations herein were likely to impact the RTK-Ras pathway. The expression levels of RET, MMP16, and SOX2 were strongly correlated with prognostic power, with areas under the ROC curves (AUC) of 1, 0.8, and 0.8, respectively. The HON and TAM combination increased TAM cytotoxicity in MCF-7 TAM-R cells by regulating the expression of potential target genes ret, ERBB4, SOX2, and FN1, as well as the TAM resistance regulatory genes including HES1, VIM, PCNA, TP53, and CASP7. Molecular docking results indicated that HON tended to bind RET, ErbB4, and the receptor protein Notch1 ankyrin domain more robustly than its native ligand.PPI network analysis revealed that FGFR2, RET, ERBB4, MMP16, FN1, and SOX2. However, further studies are required to validate these results.HON could overcome breast cancer resistance to TAM, potentially by targeting  Tamoxifen (TAM), a selective estrogen receptor modulator, is the most commonly used drug in the estrogen receptor (ER)-positive breast cancer treatment . The effin vitro and in vivo showed that HON inhibits the proliferation, invasion, and migration of cancer cells. Moreover, the molecular targets of HON in cancer cells include NF-κB, mTOR, EGFR, BMP7, STAT3, and hedgehog  . HON hashedgehog .HON has also been used in combination with other anticancer agents. HON increased the cytotoxicity of bleomycin in MCF-7, PANC1, and UACC903 cells . The comThis study aimed to explore the potential downstream targets and mechanisms of HON’s action in circumventing breast cancer resistance to TAM using a bioinformatics approach.https://www.ncbi.nlm.nih.gov/geo/geo2r/). DEGs between TAM-sensitive and TAM-resistant cells, and HON-treated and control were screened. The adjusted p-value < 0.05 and log |Fold Change| > 2 were used to select significant DEGs.Microarray data of MCF-7 TAM-R cells were obtained from GSE67916 . It contp < 0.05 was considered the cutoff value.GO and KEGG pathway enrichment analyses were conducted using DAVID v6.7 . A p < 0https://genemania.org/). Genes having the highest degree score of 5, analyzed using the cytoHubba plugin, were selected as hub genes   , 18. In FGFR2, ERBB4, RET, FN1, MMP16, and SOX2 in breast cancer patients from the TCGA study was performed using Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia2.cancer-pku.cn)  . BrieflyFGFR2, ERBB4, RET, FN1, MMP16, and SOX2 were analyzed using the KMPlotter of breast cancer patients (https://kmplot.com)  . Brieflyhttp://www.rocplot.org)  . Briefly2 incubator. MCF-7 TAM-R cells were prepared as previously described (MCF-7 cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium-high glucose (Gibco) containing 20% of fetal bovine serum , L-glutamine (Gibco), and penicillin-streptomycin, and kept at 37 °C in a 5% COescribed . Brieflyp < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001.Cells were seeded  in a 96-well plate and incubated until they reached 80% confluency. TAM (purchased from Sigma), HON , or a combination of both was added to the wells and incubated for 72 h. After incubation, MTT solution was added and cells were reincubated for 3 h. The resulting formazan crystals were dissolved in DMSO. The cell viability was calculated to be 5 for the control. We calculated the IC50 value with GraphPad Prism 5.0 using non-linear regression (curve fit) log (agonist) vs. normalized response-variable slope. Moreover, statistical analyses for the cytotoxicity data of TAM alone in MCF-7 and MCF-7 TAM-R, and HON alone and in combination in MCF-7 TAM-R, were conducted using two-way ANOVA with Sidak’s multiple comparison test. *6 cells in a 10-cm plate), incubated until 80% confluency, and treated with 10 μM of TAM, 10 μM of HON, a combination of TAM and HON, or DMSO for 72 h. Next, the cells were lysed and RNA was extracted using a QIAGEN RNA isolation kit. RNA was then transcribed into cDNA using a SensiFAST cDNA synthesis kit (Meridian Bioscience). Gene expression was measured in Bio-Rad CFX using the SensiFAST SYBR® No-ROX Kit (Meridian Biosciences) with the selected primers  was used for docking simulation, RMSD-docking score calculation, and visualization interaction. The PDB ID of the RET (2IVU), ErbB4 (3BBT), FGFR2 (2PVF), and the ankyrin domain of Notch1 (2HE0) was used for the search on rcsb.org. The chemical structure of HON and the natural ligand of the protein, were constructed using MarvinSketch. These structures were then subjected to conformational search and minimized in MOE using the energy-minimizing menu. For the docking simulation setting, London dG was used for both Rescoring 1 and Rescoring 2, Triangle Matcher was used for score function and placement setting, and Forcefield was used to refine the docking results from 30 retained settings. The results of this method helped determine which conformation has the lowest binding energy for interaction between the ligand and its receptor.To predict the binding characteristics between HON and RET, ErbB4, FGFR2, and the ankyrin domain of Notch1, molecular docking was conducted. The computational prediction was simulated on a Windows 10 operating system, Intel Core(TM) i5-10http://www.interactivenn.net) (To explore the target genes of HON in circumventing breast cancer resistance to tamoxifen (TAM), we first used GSE67916, which contained microarray data from MCF-7 TAM-R cells and MCF-7 parental cells. We obtained DEGs and considered them to be regulatory genes of TAM-R in MCF-7 cells. We also used GSE85871, which contained microarray data from HON-treated and DMSO-treated MCF-7 cells to retrieve the DEGs of HON-treated MCF-7 cells to obtain the DEGs of HON-treated cells. A total of 1,030 and 1,111 genes were extracted from the GSE67916 (enn.net) , resultiThe GO analysis was divided into three categories: biological processes, cellular components, and molecular functions. Several DEGs Table 1 p-value of 0.00288  were analyzed across breast cancer studies using the cBioPortal to assess genomic alterations. FGFR2, RET, and ERBB4 were selected based on the KEGG pathway enrichment analysis results, which suggested the regulatory function of these genes in the endocytosis process. The genes, FN1, SOX2, and MMP16, were selected given their highest scores. ERBB4 encodes HER4, a member of the tyrosine kinase receptor family, and a large membrane glycoprotein, playing a critical role in response to external stimuli (FGFR2 encodes fibroblast growth factor receptor 2 (FGFR2), a member of the tyrosine kinase receptor family. MMP16 encodes matrix metalloproteinase 16, a proteolytic enzyme that plays a role in extracellular matrix degradation during cancer metastasis , 2.8% (FN1), 3% (FGFR2), 3% (SOX2), 9% (ERBB4), and 13% (MMP16) (ERBB4–FN1) exhibited significant co-occurrence (p < 0.05) in a breast cancer study of the INSERM project  , whereas patients with high levels of ERBB4 had a better RFS than the opposite group (p = 9.5 × 10-5) .The prognostic value of mRNA expressions of FGFR2, ERBB4, and MMP16 were significantly moderately correlated with AUC values of 0.568, 0.547, and 0.549, respectively  based on transcriptome data from patients with breast cancer was analyzed. The expression levels of MDR1, BCRP1, and MRP1  alone and in combination with 10 μM of TAM in both parental and MCF-7 TAM-R cells. The results revealed that the combination of TAM with HON at concentrations of 10 μM significantly increased the cytotoxicity compared to HON treatment alone in MCF-7 parental cells  docked with HON and its native ligand, vandetanib. ErbB4 (PDB ID: 3BBT) docked with HON and its native ligand, lapatinib. FGFR2 (PDB ID: 2PVF) docked with HON and its native ligand, phosphomethylphosphonic acid guanylate ester. The ankyrin domain of Notch1 (PDB ID: 2HE0) docked with HON and its native ligand, 1,2-ethanediol. An RMSD < 2 indicated the validity of the docking method , a tyrosine kinase receptor that regulates signaling pathways through the proteolytic release of intracellular and extracellular receptor fragments  which showed the downregulation of PCNA and genes involved in the cell cycle , in which the downregulation of RET increases the sensitivity of MCF-7 cells toward TAM  can be found in the article/AH conceptualized and designed the study; acquired, analyzed, and interpreted the data; and drafted and revised the article. HP and NH analyzed, and interpreted the data, and drafted the article, NF and HP acquired the data. All authors contributed to the article and approved the submitted version.