The use of molecular markers in plant breeding has become a routine practice, but the cost per accession can be a hindrance to the routine use of Quantitative Trait Loci (QTL) identification in breeding programs. In this study, we demonstrate the use of targeted re-sequencing as a proof of concept of a cost-effective approach to retrieve highly informative allele information, as well as develop a bioinformatics strategy to capture the genome-specific information of a polyploid species. SNPs were identified from alignment of raw transcriptome reads (2 × 50 bp) to a synthetic tetraploid genome using BWA followed by a GATK pipeline. Regions containing high polymorphic SNPs in both A genome and B genomes were selected as targets for the resequencing study. Targets were amplified using multiplex PCR followed by sequencing on an Illumina HiSeq. Eighty-one percent of the SNP calls in diploids and 68% of the SNP calls in tetraploids were confirmed. These results were also confirmed by KASP validation. Based on this study, we find that targeted resequencing technologies have potential for obtaining maximum allele information in allopolyploids at reduced cost. Marker-assisted breeding often requires a large number of samples to be genotyped, especially in early generations, for effective selection ,2. MolecArachis hypogaea L., along with the closely-related wild tetraploid species A. monticola, is allotetraploid (2n = 4x = 40), wherein two related genomes are present. A. hypogaea is widely considered to be derived from a single recent hybridization event between the two diploid species A. duranensis and A. ipaënsis, followed by chromosome doubling to form a polyploid [Cultivated peanut makes an ideal system for testing resequencing in a complex polyploid. olyploid ,8,9,10. olyploid . A and Bolyploid . The higolyploid ,14. Despolyploid . The sofolyploid . SWEEP uSNP genotyping methods mentioned above have been useful at identifying or measuring of variation in peanut. The KASP  assay that is based on single allele discrimination is used for MAS (Marker Assisted Selection) where there are many individuals x a few numbers of SNPs to identify. Genotype by sequencing and SNP chip are cost-effective when there are few individuals ×x thousands of SNPs to screen ,18. Howehttp://www.fluidigm.com). High read depth of targeted resequencing coupled with multiplexing could potentially be used for cost-effective QTL analysis in peanut and other allopolyploids.Targeted resequencing is a method of sequencing regions of interest from the genome. Sequencing a subset of the genome is performed to lower the costs relative to whole genome sequencing and can be effective of detecting low levels of variation due to higher read depth. Several studies for targeted resequencing have been reported in plants, especially polyploids ,20,21,22In the current work, we tested targeted resequencing as a potential cost-effective method for QTL analysis in peanut. Forty-eight SNP targets were used, and 48 accessions were evaluated by multiplexed PCR amplification of targets, followed by mass pooled sequencing. We demonstrate that bioinformatic analysis of sequence products was able to validate target SNPs.A. duranensis V14167 and A. ipaënsis GKPSSc 30076 diploid genome sequences (pre-release sequences builds) from PeanutBase [http://broadinstitute.github.io/picard). BAM files were ordered based on the position coordinates using SortSam from Samtools [Four approaches were used to select SNP datasets, with different potentials for understanding the peanut tetraploid genome . The firanutBase ,25. The anutBase  with staSamtools . PCR dupSamtools  with staThe second set of SNPs was obtained using the SWEEP filter version 1  to filtehttps://peanutbase.org). Primer sequences are given in The SNPs obtained from the four approaches mentioned above were filtered for Polymorphic Information Content (PIC) greater than 30% and a read depth of greater than 100 when summed across accessions using a custom python script . InDels DNA was extracted from seed samples of 48 accessions, which included 12 diploids and 36 tetraploids . Small sThe Fluidigm Access Array protocol was used, and samples were preamplified prior to the amplification step . AmplifiPooled amplicons from the Fluidigm Access Array were sequenced on an Illumina MiSeq at the College of Veterinary Medicine, Texas A&M University, College Station, TX using paired-end (2 × 250 bp) sequencing. PhiX174 DNA was added to 5% final concentration relative to the other DNA samples to make up for low diversity libraries. Samples were demultiplexed by identification of barcodes.Bioinformatic analyses were performed using the Texas Tech University Centos based Hrothgar High Performance Computing Cloud (HPCC) or on laboratory servers running Red Hat Enterprise Linux or CentOS 6.7. Sequencing results from the MiSeq consisted of 48 files, which were produced by demultiplexing by barcode sequence, each plant having a unique barcode. Each file included sequences from the 48 targets for that plant sample. Forward and reverse reads from each file were combined using FLASH . The filA subset of eight targets representing all four approaches was selected from the original 48 targets for validation using Kompetitive Allele-Specific PCR (KASP) assays. For this, a subset of 24 genotypes representing all four market types plus selected wild species was selected from the original set of 48 genotypes. The KASP assay was performed on a Lightcycler 480  using filters for dyes FAM and HEX dyes in the master mix. The PCR conditions and primer sequences are given in Sequencing cost was estimated in three ways, two using the Fluidigm Access Array. The first used the Fluidigm Access Array instructions  and prices of 2012; in this, primers were designed and ordered from IDT , and reagents except the barcode set were ordered from third parties. The chemistry was limited in ability to multiplex samples up to 10-fold, as per the manufacturer’s instructions. In the second, the updated Fluidigm kit was used, with newer chemistry and all reagents purchased from Fluidigm; likewise, primers were designed and synthesized from Fluidigm ; current prices were used. Multiplexing was allowed up to 50-plex. Results from this method are presented below. In the third, samples were all custom designed as per Shirasawa et al.  and amplFrom the GATK pipeline, approximately 1.6 million raw SNPs were identified from the SNP identification pipeline . These SThe Illumina MiSeq run for 48 samples × 48 targets generated 30,153,796 reads, of which 24,430,536 were identified with barcodes . AmplifiAmong the four methods of SNP identification and selection compared, the three methods based on use of a genomic reference gave the highest percentage of validation. GATK had 52.6% of the total polymorphic targets, and 71.4% of SNPs in targets that were successfully identified by sequence analysis validated in the MiSeq run; targets obtained from the SNP chip had 66.7% polymorphism and 66.7% validation, and SNPs filtered through SWEEP had a polymorphism rate of 55.5% and a validation ratio of 62.5%. Targets based on the OLin transcriptome reference  had the A. duranensis KSSc38901, A. duranensis K7988, A. cardenasii GKP10017 and A. diogoi GK10602). Among the four B genome diploid accessions , 313 SNPs representing 39 scaffolds were highly polymorphic.In the first approach for compiling reads, reads were mapped to the pooled target reference of 48 target regions, and 111 SNPs were identified among the 35 tetraploid accessions. These mapped reads represented 25 genome scaffolds (target regions), as there were many cases where two targets were on a single scaffold. Among these 111 SNPs, 81 SNPs were highly polymorphic with PIC values higher than 0.3 . In contA. duranensis KSSc38901, A. duranensis K7988, A. cardenasii GKP10017 and A. diogoi GK10602), 207 SNPs were highly polymorphic. Among the four B genome diploid accessions , 262 SNPs were highly polymorphic.In the second approach, reads were mapped to the whole genome reference, from which 73 SNPs were identified among the 35 tetraploid accessions, representing 22 contigs. Among these 73 SNPs, 56 SNPs were highly polymorphic with PIC values higher than 0.3. Among the four A genome diploid accessions  to 24.4% (TxAG-6). After correction for a 20% heterozygote threshold, the percentage of heterozygotes ranged from 0.1% to 0.3%  for the Access Array and $14.08 using all generic components .Comparing to other means of analysis, prices per sample for resequencing ($28.75) were lower using the 2012 instructions than the prices for RADSeq and the SNP chip in the 2016–2018 time frame. Since then, the prices for the latter two have dropped to $28/sample for RADSeq and the Axiom_Array2 chip. The Fluidigm prices chemistry changed in 2016 to allow higher multiplexing, but the newer protocol dropped instructions on primer design from third parties; the revised prices at $66/sample are higher than other means of analysis. However, we have estimated that running the analysis using generic components in 384-well plates can be as low as $14/sample, half the cost of other analyses, but less convenient than using the Access Array.The current experiment was designed as a proof of concept, to test resequencing in peanut, and also to determine whether it could provide a lower cost method of genotyping, at a marker density lower than high throughput methods, but sufficient for QTL analysis and marker-assisted selection. Ultimately, a price point of $5/sample would be ideal for breeding programs. The current work suggests that we do not have the ability to perform this at that price; however, it may be possible to drop the price by 50% compared to other methods. This would still be useful. The major costs of such an approach are primer synthesis and sequencing. Lower primer costs are possible; they have been estimated at the 500pmol scale and $0.10/base. Smaller-scale syntheses could reduce costs by 75% at 10 pmol to 50 pmol scales, but the volumes needed for PCR reactions in 384-well plates would not make it possible to synthesize sufficient primer amounts without miniaturization to use the IFCs; however, the costs of the IFCs is greater than the cost savings that could be realized. Throughput of newer sequencers is higher, but the higher cost per run offsets much of the advantage.The re-sequencing effort with three approaches, GATK, SWEEP and SNP chip, was successful, with 81% of the targets for the diploids and 68% of the targets for tetraploids being found polymorphic by resequencing . HoweverThree potential difficulties could explain the low 68%) agreement between resequencing and the initial transcriptome sequence results. The first is that SNPs from the GATK, SWEEP and OLin methods were developed from a transcriptome sequence. Some of the SNP targets may have been spurious, a result of lack of expression of some genes; when resequencing or KASP assays were performed, the genotyping was based on genomic DNA and not RNA expression. However, SNP targets selected from the SNP chip, identified by whole genome sequencing, were not more accurate. A second possible explanation is polyploidy; indeed, difficulty in tetraploid SNP validation has been reported before 8% agreem,33,34. HVariant call files (vcf) of sequences from analysis of MiSeq data had heterozygous SNP calls with means of 11.1% for diploids and 7.5% for tetraploids. These were too low to be likely for any given locus, as 50%, called heterozygosity, is expected for a true diploid heterozygote, or for a pair of tetraploid homoeologs wherein the allele calls for A and B genomes were different but each was homozygous. The observed mean values are too high for the set of targets overall as the peanut accessions are considered to be self-pollinated lines. Diploid accessions not only lack homoeologs, but many are incompatible with each other, and thus, could not be the products of recent hybridization.Three reasons can be hypothesized for the apparent unexpectedly high proportions of heterozygous allele calls: (1) sequencing error, (2) inaccurate mapping to the A- or B-genome in tetraploids, resulting in overlap of homoeologs or (3) co-mapping of paralogs. Each of these possibilities was considered.(1)Sequence data used a minimum quality (Q) score of 30, thus, an error rate of less than 0.1% might be expected, far less than the heterozygote percentage in the range of 7 to 11% that was observed. A study on MiSeq error profiling suggests that errors from MiSeq sequencing are not random and are more prone to G/A and A/C base substitutions, and there is an increase in sequencing error towards the end of the sequence . To esti(2)A. ipaënsis GKPSSc30076, A. duranensis GKP10017 and A. batizicoi K9484, respectively.Even though we expect all A genome reads to map against the A genome part of the synthetic tetraploid reference, and all B genome reads to map against B genome part, there is little control over the alignment due to the fact of high similarity between the two genomes. Additionally, the BWA aligner used in this analysis is not a specialized tetraploid aligner and the power for BWA aligner to discern the few differences between the sub-genomes is unclear. However, even though homoeologous sequences were not completely resolved, they were distinguished to some extent by aligning these sequences against corresponding A and B genome diploid sequences A. Arguin(3)Due to the highly-repetitive nature of the peanut genome, reads co-mapping to paralogous sequences could contribute towards apparent heterozygosity. To check the presence of paralogs, target primer sequences were compared against the A diploid genome using BLAST . There wA. cardenasii GKP10017, A. diogoi GK10602 and A. batizicoi K9484) was 16.0%, while the observed rate for TxAG-6 was 24.4%.If paralogs were a major contributing factor to apparent heterozygosity, the rate of heterozygous SNPs in the tetraploids would be expected to be similar to the average of the two diploid parents. An expected frequency of SNP heterozygous calls for UF439-16-10-3-2 could be estimated as 6.2%, and was observed to be 5.7%. Similarly, estimation of heterozygous calls of TxAG-6  or between 70% and 80% heterozygosity . Peaks in these three ranges also occurred in the tetraploids TxAG-6 and UF439-16-10-3-2. These were explained as follows: In the case of diploids, a peak of 50% would indicate a true SNP in one gene. The expected percentage heterozygosity would be 50%. In cases of tetraploids, this range can be considered to correspond to cases of a homozygous SNP distinguishing A- and B-genome homoeologs of that accession. In the case of a heterozygous SNP in one genome while the homoeologous allele was homozygous, 25% or 75% would be called heterozygous. In the case of two paralogous genes present, an SNP in one would result in a peak at 25% or 75% assuming equal representations of both genome sequences. This would correspond to the peaks observed in the 20% to 30% range and in the 70% to 80% range for the tetraploids. For diploids, this could represent presence of two paralogs, one homozygous and the other heterozygous and sharing one target nucleotide identical with the homozygous paralog.Based on observation of the histograms , it was The original SNP call dataset was divided into three sub-datasets using cutoff values of 25%, 20% and 15% correction for the minor allele using python scripts. If the percentage of heterozygotes in the minor allele was less than the cutoff value, the minor allele was rescored as a homozygote instead of the heterozygote. We observed validation values of 85.4%, 85.7% and 80.5%, respectively, for 25%, 20% and 15% cutoff-corrected SNP calls. With less correction (15% cutoff), there was a lower validation rate, but the 20% cutoff would allow for identification of true heterozygotes in tetraploids while not reducing the validation rate. This correction reduced an average of heterozygous SNP calls to 0.18% from the initial 8.5% . Minor aA. hypogaea, these were approximately double the number of the targeted SNPs validated; for the diploids, the numbers were much higher. Such SNPs may be in linkage disequilibrium [In addition to scoring targeted SNPs, sequencing also identified some non-targeted SNPs present in the surrounding regions. In the case of accessions of ilibrium  and may Given that paralogs were found for 37 of 48 targets , it is pThe current experiment was designed as a proof of concept, using the Fluidigm Access Array for multiplexing, to demonstrate the possibility of resequencing in peanut, and also to determine whether it could provide a lower cost method of genotyping, at a marker density sufficient for initial QTL analysis and marker-assisted selection. Validation of results obtained from targeted resequencing suggests that targeted resequencing could be used for QTL mapping studies. For QTL identification, a set of 500 to 2000 highly-polymorphic SNPs can be selected that could potentially be used for any cultivated x cultivated cross. Amplification of targets from both sub-genomes simultaneously can effectively double the number of target loci, increasing the genomic information that can be extracted. Although a price point of $5–10/sample would be ideal for breeding programs, the current work suggests that it may be possible to drop the price by 50% compared to other methods . In addition, as much of the price of resequencing is the primer cost, once this is paid upfront, genotyping cost per sample drops by 50% to $7/sample thereafter. We believe such an approach can be readily adopted in many allopolyploid species. GBS or the SNP chip could be used subsequently for high-resolution mapping of the target region for genetic studies, if desired.


Eph receptors and the corresponding Eph receptor-interacting (ephrin) ligands jointly constitute a critical cell signaling network that has multiple functions. The tyrosine kinase EphA2, which belongs to the family of Eph receptors, is highly produced in tumor tissues, while found at relatively low levels in most normal adult tissues, indicating its potential application in cancer treatment. After 30 years of investigation, a large amount of data regarding EphA2 functions have been compiled. Meanwhile, several compounds targeting EphA2 have been evaluated and tested in clinical studies, albeit with limited clinical success. The present review briefly describes the contribution of EphA2-ephrin A1 signaling axis to carcinogenesis. In addition, the roles of EphA2 in resistance to molecular-targeted agents were examined. In particular, we focused on EphA2’s potential as a target for cancer treatment to provide insights into the application of EphA2 targeting in anticancer strategies. Overall, EphA2 represents a potential target for treating malignant tumors. Ephrin receptors (Eph) represent the most important class of receptor tyrosine kinases (RTKs) . EphA1, Eph receptors are single transmembrane proteins with extra-  and intracellular domains with ligand-binding and intrinsic enzymatic activities, respectively , 8. Eph Some Eph receptors, especially EphA2, attract increasing attention because of demonstrated or hypothesized contributions to modulatory processes controlling carcinogenesis and tumor progression Fig. . The preThe EphA2 receptor is a 130-kDa transmembrane glycoprotein with 976 amino acids . The EphEphA2 interacts with any of the eight different ephrin A-family ligands, with overt preference to ephrin A1 , 15. EphUnder normal conditions, EphA2 interacts with ephrin A1 on the neighboring cell and induce diverse signaling networks following cell-to-cell contact. As membrane proteins, ephrins are engaged in both forward (termed ephrin:EphA2 forward) and reverse  signaling from ephrin ligands to EphA2 and vice versa; this is also known as ephrin-EphA2 bidirectional signaling , 20. ForDifferent from the majority of Eph kinases that are mostly synthesized during the developmental process, EphA2 is mainly restricted to proliferating epithelial cells in adults . EphA2 eThere is considerable interest in the mechanisms that govern EphA2 expression and in understanding how these mechanisms are subverted in cancer. Emerging evidence links high EphA2 protein amounts with EphA2 regulation at the mRNA level as well as protein stability, although the precise mechanisms governing EphA2 upregulation in cancer remain largely undefined , 34.EphA2 mRNA is tightly regulated. To date, a few somatic mutations of EphA2 have been reported –37. In aDecreased ligand-mediated receptor internalization and degradation, consequently enhancing protein stability, might help increase EphA2 amounts in malignant cells. An interesting consequence of EphA2 stimulation (by ligand or antibody) is EphA2 phosphorylation, internalization, and degradation –45. AfteOverexpression and aggressive features of EphA2 in tumor cells and relatively low expression in most normal adult tissues make this protein a potential therapeutic target in cancer. The EphA2/ephrin A1 system could be targeted for cancer treatment at least via two mechanisms. First, EphA2’s oncogenic features could be inhibited, e.g., decreasing EphA2 expression, promoting EphA2 degradation, and blocking endogenous EphA2 activation. Alternatively, the EphA2 receptor could be employed to deliver therapeutics (exogenous drugs or endogenous immune cells) to cancer cells and associated vessels. Therapies targeting EphA2 in cancer are shown in Table Given the positive association of EphA2 overexpression with aggressive clinical and pathological features in human cancers, investigators have examined the potential of downregulating EphA2 in preclinical models. Short interfering RNAs (siRNAs) for gene knockdown constitute a great tool for protein function assessment, gene discovery, and drug development , 84, andArtificial ligands or antibodies interacting with EphA2 could suppress signaling by promoting internalization and degradation.Plasma membrane–bound ephrins and soluble ephrins with artificial clustering/dimerization associated with antibodies targeting COOH-terminal epitope tags or fusion to immunoglobulin g (IgG) Fc potently promote EphA2 phosphorylation and degradation .Ephrin A1 has been demonstrated to be present at low levels and to possess tumor suppressing properties dependent on cell-to-cell contact in a variety of tumors , 30, 91.In addition to soluble ephrin A1, ephrin A1-Fc, obtained by fusing recombinant Ephrin A1 to human IgG Fc for dimerization, shows ephrin-like features and induces EphA2 phosphorylation . TreatmeThe large extracellular domain of EphA2 provides an antigen that is frequently upregulated on tumor cells , 96. In Studies have shown that several agonist monoclonal antibodies raised against EphA2 induce its internalization and degradation, suppressing its malignant features. For example, Kinch et al.  isolatedAntibody-dependent cellular cytotoxicity (ADCC) kills cells via perforin/granzyme, TRAIL, and FasL . ADCC alCompounds binding to EphA2 or ephrin A1 could suppress signaling by direct antagonist effects.Small molecules that block EphA2 could represent efficient alternatives to peptides and antibodies. Recently, small molecules disrupting the Eph-ephrin complex have been described, with most exerting pharmacological activities through targeting of the ligand-binding domain of EphA2, thereby acting as common protein-protein interaction (PPI) inhibitors. The ephrin binding site in Eph receptors allow high-affinity binding of small molecules , 100.It was hypothesized that soluble receptors repress EphA signaling by suppressing the interactions of endogenous ephrins with EphA receptors. EphA2-Fc represents a soluble protein chimera involving the fusion of EphA2’s extracellular domain with human IgG1 Fc, preventing interactions of several ephrin A ligands with endogenous receptors and potently inhibiting EphA receptor activation in cultured cells. By interacting with ephrin A1, EphA2-Fc could induce ephrin-initiated reverse signaling. Treatment with EphA2-Fc was shown to dose-dependently inhibit EphA2 receptor phosphorylation and activity. In addition, EphA2-Fc strongly inhibited angiogenesis and microvessel growth in vitro as well as growth in pancreatic tumor xenografts . FurtherLithocholic acid -3-hydroxycholan-24-oic acid), a secondary bile acid produced by prokaryotic transformation of chenodeoxycholic acid, is considered an EphA2 antagonist. LCA interacts with the nuclear receptor farnesoid X receptor (FXR) and the G-protein-coupled receptor G-protein-coupled bile acid receptor 1  under physiological conditions , 102. MoThe specificity of LAC in antagonizing Eph receptor has been demonstrated, with no detected effects on other RTKs, including EGFR, vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor 1 receptor (IGF-1R), and the insulin receptor. However, LCA is also considered to interact with EphA and EphB receptors, indicating an interaction with the highly conserved region of Eph receptor family members . Thus, LBesides LCA and its analogues, small molecules that interfere with the EphA2-ephrin A1 system comprise the following: (i) the FXR agonist GW4064 , a stilbThe successful development of specific RTK inhibitors has prompted subsequent efforts for identifying comparable targets. Unlike other anticancer approaches, targeted therapies are relatively less toxic. Multiple small molecule EphA2 inhibitors interacting with the intracellular kinase domain have been described.Dasatinib (BMS-354825) represents an oral kinase inhibitor simultaneously targeting breakpoint cluster region-Abelson (BCR-ABL), c-KIT, platelet-derived growth factor receptor (PDGFR), and SFKs , 106. ItALW-II-41-27, a type II small molecule inhibitor targeting the ATP-binding region of the kinase domain as well as an allosteric site following the “DFG” motif in EphA2, has been shown to bind to and potently inhibit EphA2 kinase activity , 108. TrPeptides and antibodies selectively binding cancer cells followed by internalization provide a powerful vehicle to guide therapeutic delivery to specific cell types and to determine the format of peptide/antibody-drug conjugates or antibody-directed nanotherapeutics. These conjugates or targeted systems could deliver toxic compounds selectively to the tumors while sparing noncancerous tissues. For example, EphA2 could selectively deliver therapeutics to EphA2-overexpressing cancers while simultaneously regulating EphA2-signaling related events.Ephrin A1-Fc has been utilized in EphA2 targeted therapy upon conjugation with PE38QQR, a derivative of Pseudomonas aeruginosa endotoxin A, to generate ephrin A1–PE38QQR conjugated cytotoxin . Pseudom1C1 is a fully human monoclonal antibody with selective binding to EphA2, but no other Eph receptor family member . After cClinicalTrials.gov: NCT03076372).Off-target drug toxicity frequently causes treatment discontinuation, restricted dose escalation, and worse outcomes. Antibody-mediated tumor targeting and nanoparticle encapsulation decrease the toxic effects of anticancer agents, improving treatment efficacy. Trametinib (TMB) is a MAP/ERK kinase (MEK) inhibitor, but its off-target toxicities frequently prompt dose interruption as well as treatment discontinuation . Thus, YImmunotherapy, which relies on enhancing the patient’s immune defenses to combat tumor cells, has become a game changer in cancer treatment. EphA2 overexpression on tumor cells could constitute a new antigen for tumor immunotherapy.Dendritic cell (DC)-based vaccines represent attractive anticancer tools, since DCs induce both tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T cells . Tumor aA strategy to treat cancer is chimeric antigen receptors (CARs) modified T (CAR-T) cell therapy, a cell-based tumor immunotherapeutic approach , 116. CASeveral EphA2-specific T cells have been developed and evaluated in preclinical studies, recognizing EphA2-expressing tumors as assessed by interferon-γ (IFN-γ) and IL-2 synthesis and conferring cancer cell cytotoxicity. A second-generation EphA2-specific CAR was engineered on the basis of the humanized EphA2 monoclonal antibody 4H5, a CD28.ζ signaling domain, a CD28 transmembrane domain, and a CH2CH3 spacer. The final T cell populations comprised both CD4+ and CD8+ cells, all expressing EphA2-specific CARs . These EVarious modalities of combination therapy based on EphA2 targeting have been evaluated in preclinical studies . The NCT02004717 trial was a two-step, study with step 1 evaluating a dose escalation cohort (six dose levels from 0.1 to 20 mg/kg) in patients with advanced solid tumors and step 2 assessing dose expansion in individuals with EphA2-positive esophageal and gastric cancers. The maximum tolerated dose was not reached in step 1 and the planned highest dose (20 mg/kg) was used in step 2. A total of 37 cases  were included, but all discontinued the study for overt disease progression  or adverse events  . Similarn = 3) and coagulation (n = 2) events, and the planned dose escalation was not pursued. Clinical responses comprised disease progression  and stable disease . The safety profile of MEDI-547 prevents its further clinical development for advanced-stage solid tumors. However, the causes of the detected AEs remain unclear.The safety, pharmacokinetic features, and anticancer effects of MEDI-547 were evaluated in a phase 1, open-label trial with included dose-escalation and dose-expansion cohorts . Cases uThe crucial roles in tumor biology have defined EphA2 as a promising therapeutic target. Due to intensive investigation and remarkable advances in understanding some of the mechanisms associated with EphA2 effects, multiple potential targets have been described. The advantages of the EphA2 as the target of tumor therapy include (i) possibility of targetable by cellular, molecular, and pharmaceutical approaches; (ii) possibility of developing anticancer immune-therapy; and (iii) possibility of combination with conventional therapeutics to improve efficacy.However, to date, the precise mechanisms of EphA2, especially the effects of EphA2:ephrin reverse signaling, are largely unknown, and the outcomes of its regulation cannot be predicted with confidence. Indeed, EphA2-ephrin A1 signaling is extremely complex, with both interacting cells receiving interdependent signals from the identical signaling complex that is frequently associated with other Eph receptors and receptor tyrosine kinases, as well as additional signaling pathways. Meanwhile, EphA2 responses following stimulation could result from cell/tissue-specific kinase-dependent or kinase-independent signaling pathways. However, most of the preclinical work presented in this review tend to simplify the EphA2-ephrin A1 system. EphA2 inhibition or targeting confers marked benefits in experimental studies. However, these findings could not be translated into clinical use. For the currently developed EphA2-based targets, existing challenges include the following: (i) downregulation of EphA2 by siRNA affects both the forward and reverse signaling, together with compensatory stimulation of other Eph receptors and oncogenic signalings, which potentially regulate the biological behavior of a cell; (ii) compound that act on the extracellular ligand-binding domain of EphA2 block both reverse and forward signaling, and show poor physicochemical characteristics; (iii) the highly conserved cytoplasmic domain, particularly the kinase domain, among different Eph kinases, could lead to non-specific inhibition of other Eph family members and unwanted toxicities by the use of small molecule inhibitors to antagonize EphA2’s enzymatic activity; (iv) although antibody-drug conjugates are highly specific and stable, and possess antibody-like pharmacokinetic features, the non-negligible technological limitations impact drug activity and/or safety; (v) the significant proportion of non-responsive cases and treatment-related toxicities remain obstacles to successful immunotherapy treatment.Sustained and systematic efforts in the future may involve an in-depth understanding of EphA2-ephrin A1 signaling and a precise elucidation of the crosstalk with other oncogenic pathways. The performed clinical studies and novel biological findings provide clues for developing next-generation EphA2-targeting therapies: (i) focus should be placed on enhancing efficacy and selectivity while preventing off-target secondary effects; (ii) combination with other therapies may be helpful. Despite multiple challenges due to the complex biological properties of the EphA2-ephrin A1 system, exciting possibilities still exist for novel treatment approaches based on these molecules.


Acute myocardial injury (AMI), which is induced by renal ischemia-reperfusion (IR), is a significant cause of acute kidney injury (AKI)-related associated death. Obesity increases the severity and frequency of AMI and AKI. Tanshinone IIA (TIIA) combined with cyclosporine A (CsA) pretreatment was used to alleviate myocardial cell apoptosis induced by renal IR, and to determine whether TIIA combined with CsA would attenuate myocardial cell apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats.Male rates were fed a high fat diet for 8 weeks to generate obesity. AKI was induced by 30 min of kidney ischemia followed 24 h of reperfusion. Obese rats were given TIIA (10 mg/kg·d) for 2 weeks and CsA (5 mg/kg) 30 min before renal IR. After 24 h of reperfusion, the rats were anaesthetized, the blood were fetched from the abdominal aorta and kidney were fetched from abdominal cavity, then related indicators were examined.TIIA combined with CsA can alleviate the pathohistological injury and apoptosis induced by renal IR in myocardial cells. TIIA combined with CsA improved cardiac function after renal ischemia (30 min)-reperfusion (24 h) in obese rats. At the same time, TIIA combined with CsA improved mitochondrial function. Abnormal function of mitochondria was supported by decreases in respiration controlling rate (RCR), intracellular adenosine triphosphate (ATP), oxygen consumption rate, and mitochondrial membrane potential (MMP), and increases in mitochondrial reactive oxygen species (ROS), opening of the mitochondrial permeability transition pore (mPTP), mitochondrial DNA damage, and mitochondrial respiratory chain complex enzymes. The injury of mitochondrial dynamic function was assessed by decrease in dynamin-related protein 1 (Drp1), and increases in mitofusin1/2 (Mfn1/2), and mitochondrial biogenesis injury was assessed by decreases in PPARγ coactivator-1-α (PGC-1), nucleo respiratory factor1 (Nrf1), and transcription factor A of mitochondrial (TFam).We used isolated mitochondria from rat myocardial tissues to demonstrate that myocardial mitochondrial dysfunction occurred along with renal IR to induce myocardial cell apoptosis; obesity aggravated apoptosis. TIIA combined with CsA attenuated myocardial cell apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats. Acute kidney injury (AKI) is a very severe, even life-threatening complication of critically ill patients . Renal iSalvia miltiorrhiza Bge [Reducing the inflammatory response and resisting oxidant stress are the main biological activities of tanshinone IIA (TIIA), the main active ingredient in hiza Bge . In addihiza Bge . The stuhiza Bge . Cyclosphiza Bge . Injectihiza Bge . HoweverCell survival is associated with the phosphoinositide-3 kinase (PI3K) pathway, which relies on Akt kinase phosphorylation and activation followed by the proapoptotic Blc-2 family protein Bad phosphorylation and inhibition. PI3K plays an important role in growth factor signal transduction. Under various cytokines and the activation of physiochemical factors, PI3K generates myoinositol as a second messenger. Akt also plays crucial roles in many biological processes, such as the cell cycle, cell metabolism, apoptosis, and cell growth . MitochoThe current research focused on assessing an AKI method (induced by IR) leading to dysfunction of myocardial mitochondria, using a combination of TIIA and CsA to identify new therapeutic targets.We used male Sprague–Dawley rats (weight 180–220 g and 8 weeks old) in our experiments. The animals were acquired from Liaoning Changsheng Biotechnology Co., Ltd. (Production License: SCXK (Liao) 2015–0001). The rats were kept in cages under controlled conditions of 20 ± 3 °C and 45–65% humidity, and with a 12 h light/dark cycle (lights on at 06:00 h), and fed water and a pelleted diet ad libitum. Animal experimental design and husbandry procedures were approved by the Ethical Committee of Animal Handling (2019019) at Liaoning University of Traditional Chinese Medicine and complied with the guidelines of the Use and Care of laboratory animals published by US National Institutes of Health. We did our best to decrease the number of rats and suffering during the study.TIIA  was produced at the Shanghai NO. 1 Biochemical Pharmaceutical Co., Ltd. . TIIA was dissolved in deionized water to obtain a stock solution (5 mg/ml). CsA (20 mg each) was produced at Beijing Solarbio Science & Technology Co., Ltd.  and was dissolved in 0.1% dimethylsulfoxide to a stock solution (2.5 mg/ml), which was further diluted to achieve the ideal concentration. All of the above stock solutions were used in a timely manner.A total of 120 rats were randomly (random number table method) divided into six groups, including the sham operation group, the IR group, the IR (obese) group, the TIIA group, the CsA group, and the TIIA+ CsA group. Each group included 20 rats. All rats were fed a general maintenance feed for 2 weeks to adapt to the environment. Then, the sham and IR groups continued to eat the general maintenance feed for 8 weeks, whereas the other four groups were fed a high-fat diet (HFD) for 8 weeks. Rats in the sham group, the IR, and IR (obese) groups were given deionized water. The TIIA group was given an intraperitoneal injection of TIIA (10 mg/kg·d) for 2 weeks before renal IR; the CsA group was given an intraperitoneal injection of CsA (5 mg/kg) 30 min before renal IR; and the TIIA + CsA group was given an intraperitoneal injection of TIIA (10 mg/kg·d) for 2 weeks before renal IR+ intraperitoneal injection CsA (5 mg/kg) 30 min before renal IR. The components of the HFD included 25% total fat, 18% protein, 44% carbohydrate, 13% fiber, and 11% unsaturated fat, ash, and other ingredients . Rats thThe rats were anaesthetized with thiopental sodium (120 mg/kg) by intraperitoneal injection, and pinching of the tail and paw was used to evaluate the anesthetic effect. The abdomen was opened to expose the right kidney, and the renal pedicle was separated to expose the renal vessels. A 3–0 silk suture was used to ligate the renal vessel, and the right kidney was removed. The left kidney was exposed and the renal vessels were separated. An arterial clamp was used to clamp off the left renal artery for 30 min to build ischemia. After 30 min of ischemia, the arterial clamp was removed for 24 h of reperfusion. The left kidney was observed for 15 min to ensure normal blood reperfusion, which was shown by a red color . The incArterial blood samples were used for the myocardial enzyme spectrum analysis. A 0.5 mL aliquot of arterial blood was drawn from the abdominal aorta (at the end of the reperfusion period), and creatine kinase isoenzymes-muscle/brain (CK-MB), cardiac troponin I (cTNI), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were measured with kits .Noninvasive transthoracic echocardiography  was used to evaluate the function and morphology of the left ventricle in anesthetized animals (Matrx VIP 3000) . The specimens were fixed in glutaraldehyde (2%) at 4 °C, washed with pH 7.4 PBS (0.1 mol/L), fixed with osmium tetroxide (1%), and stained with aqueous uranyl acetate (1%). Place the pecimens at 70 °C for about 48 h with capsules embedding medium. The sections were stained with uranyl acetate and alkaline lead citrate for observation with an electron microscope .Myocardial tissues were obtained immediately after anesthesia and cut into small pieces  assay was used to detect apoptosis with an In Situ Cell Death Detection Kit, POD . We used 5-μm thick sections for TUNEL staining . The secCaspase-3/9 activity were detected in myocardial tissues by the fluorescent caspase-specific substrate AcDEVD-7-pNA (Solarbio). A 10 mg portion of myocardial proteins was added to reaction buffer and incubated at 37 °C for 2 h. Enzyme-catalyzed release was quantified by a fluorimeter at 405 nm.2PO4, 2 mM MgCl2, 100 mM KCl, 5 mM HEPES, and 1 mM EDTA) to generate a 5 mg/mL mitochondrial protein suspension, which was placed on ice for immediate use. The bicinchoninic acid (BCA) reagent box (Beyotime) was used to measure the protein concentration of the mitochondrial suspension (100–1000 μg/ml). The mitochondrial suspension was used to measure the mitochondrial membrane potential (MMP), ATP synthesis, opening of the mitochondrial permeability transition pore (mPTP), reactive oxygen species (ROS), and the respiratory control rate (RCR).As previously reported in liver , the aniA mitochondrial membrane potential assay kit (Beyotime) was used to detect the MMP. The prepared JC-1 working solution was diluted five times with JC-1 Dyeing Buffer (1X); a 0.9 ml aliquot of the diluted JC-1 working solution was added to 0.1 ml of the purified mitochondria (protein content 100–1000 μg/ml). We used a fluoroenzyme label to detect MMP; 1 ml of the mixture was added to the fluoroenzyme-labeled assay with the emission wavelength set to 530 nm and excitation wavelength set to 490 nm, and the red/green value was calculated.2; “-”: treatment without Ca+; “+”: treatment with Ca+. The Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit  was used according to the manufacturer’s protocol. CaCl2 (200 μmol/L) was used to induce opening of the mPTP. The optical density (OD) value from 0 to 10 min at 520 nm was read from an ultra-micro microporous plate spectrophotometer . A decrease in the OD value reflects opening of the mPTP. The OD value recorded at the onset of the experiment (0 min) represents minimum optical density (min OD); the OD value recorded at the end of the experiment (10 min) represents maximum optical density (min OD). Min/max OD is negatively associated with the extent of MPTP opening [Opening of the mPTP was measured by detecting absorbance at 540 nm of mitochondria exposed to 250 μM CaCl opening , 25.ROS were detected using the multi-mode microplate reader and the 2′,7′-dichlorodihydrofluorescein diacetate fluorescent probe (DCFH-DA).Mitochondrial oxygen consumption rate was detected using a Clark-type electrode connected to a thermostatic water bath and calibrated with using air-saturated medium (contain 432 nmol O/ml at 32 °C).2PO4, and 2 mM MgCl2, pH 7.2), and stirred at 37 °C. The slope of the response of mitochondria to consecutive administrations of respiration substrates was defined as the rate of oxygen consumption for each respiratory state, as reported previously [RCR (ratio of state III to state IV) is an index used to assess oxidative phosphorylation, respiratory chain function, and integrity of isolated mitochondria. The RCR was evaluated using an Oxytherm Clark-type oxygen electrode . Mitochondria (60 μg isolated from myocardiu) were placed in the oxytherm chamber containing respiration buffer  was assessed through the ratio of long and short fragments using the real-time quantitative polymerase chain reaction (RT-qPCR). DNA was isolated by extracting total DNA using the Genomic-tip 20/G kit  according to the manufacturer’s protocol. The PCR products and purified DNA were quantified fluorometrically using the Picogreen ds DNA reagent . RT-qPCR was performed on the DNA extracts as reported previously using the following modifications . PCR ampThe myocardial tissues (100 mg) were homogenized in an isolation buffer. The entire isolation process was performed at 4 °C. Mitochondrial respiratory chain complex enzymes I, II, III, IV, and V were independently assessed using the independent Assay Kit (Solarbio). Enzyme-catalyzed release were quantified by a fluorimeter at 340, 605, 550, 550, and 660 nm separately.The concentration of ATP in isolated mitochondria was detected using a luciferase-based luminescence enhanced ATP assay kit (Beyotime). Isolated mitochondria (1 mg/mL protein) were incubated in respiration buffer (0.5 mL) with 2.5 mM malic acid, 2.5 mM succinate, and 2.5 mM ADP for 10 min. The ATP concentrations in the mitochondrial suspension and cell lysates were determined using a SpectraMax Paradigm Multi-Mode Microplate Reader .Trizol reagent  was used to isolate total genomic RNA, and the quality of isolated RNA was determined by spectrophotometry (260 nm). Reverse transcription was performed with total RNA (1 μg) and the M-MLV Reverse Transcriptase Kit . Briefly, the total reaction volume (40 μL) was used in a Veriti 96 Well Thermal Cycler long PCR system  with the following reaction conditions: 72 °C for 3 min, 42 °C for 90 min, 70 °C for 15 min, and hold at 4 °C.RT-qPCR was used to detect the copy number of specific genes at the transcriptional level using a cDNA template. PCR was performed on a Rotor-Gene Q Sequence Detection System  using SYBR Premix Ex TaqII  . PCR wasTotal proteins were extracted from tissues using RIPA Lysis Buffer. Protein concentration was measured using the BCA Protein Assay Kit. An equal amount of total protein was subjected to 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to a PVDF membrane. After blocking in skim milk solution, the membrane was incubated overnight separately with anti-GAPDH, anti-PI3K, anti-p-Akt, anti-Akt, anti-p-Bad, anti-Bad, anti-Bcl-2, anti-Bax, anti-Cyt-c, anti-caspase-9, anti-cleaved caspase-9, anti-caspase-3, anti-cleaved caspase-3, anti-PARP, anti-Drp1, anti-Mfn1, anti-Mfn2, anti-PGC-1, anti-NRF1, and anti-TFam antibodies  was used for the statistical analysis. Data are expressed as mean ± standard error. One-way analysis of variance (ANOVA) was used to compare the six independent groups and the 2 × 2 comparisons between the groups. The Tukey-Eventually, 66 rats (13 (65.00%) in the sham group, 13 (65.00%) in the IR group, 10 (50.00%) in the IR (obese) group, 10 (50.00%) in the TIIA group, 11 (55.00%) in the CsA group, and 9 (45.00%) in the TIIA+CsA group) completed this study; 54 rats died and the reasons are described in Table We assessed cardiac function Fig. b, myocarp < 0.05), which was increased by pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA was higher than TIIA and CsA (p < 0.05). LVIDs and LVIDd increased in the IR and IR (obese) groups, particularly in the IR (obese) group, compared with the sham group (p < 0.05), which was decreased by pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05) , which was decreased by pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05) , which decreased after the pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05)  groups, particularly in the IR (obese) group, compared with the sham group (05) Fig. b. The CK05) Fig. c. Serum 05) Fig. d.p < 0.05), all injury scores decreased in the TIIA, CsA, and TIIA+CsA groups, but only the TIIA+CsA group have the statistical difference (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA but the difference is insignificant (p > 0.05)  Fig. a.Fig. 2We evaluated mitochondrial function to assess myocardial injury and the protective effect of TIIA+CsA. We used an electron microscope to observe the changes in mitochondrial morphology Fig. b.p < 0.05). However, pretreatment with TIIA, CsA, or TIIA+CsA decreased percentage of damaged mitochondria (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05)  of the myocardial tissues revealed that myocardial cells in the IR and IR (obese) groups exhibited abnormal mitochondrial morphology with swelling or membrane rupture (paired white arrows) following renal IR. The sham group exhibited normal mitochondrial morphology (single white arrow). The number of abnormal mitochondria increased in the IR and IR (obese) groups, particularly in the IR (obese) group, compared with the sham group. Pretreatment with TIIA, CsA, or TIIA+CsA improved the changes in mitochondrial morphology Fig. b. Percen05) Fig. b.p < 0.05). However, pretreatment with TIIA, CsA, or TIIA+CsA alleviated myocardial cell apoptosis (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05) . However, pretreatment with TIIA, CsA, or TIIA+CsA decreased caspase-9/3 activity in myocardial cells (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05) . However, pretreatment with TIIA, CsA, or TIIA+CsA can decrease the cleaved caspase-9/3 (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05)  Fig. a. Caspasp < 0.05). The ROS level and opening of the mPTP (%) were decreased by pretreatment with TIIA, CsA, or TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA were lower than TIIA and CsA (p < 0.05). Renal IR decreased the mitochondrial oxygen consumption rate, RCR, and the MMP (ratio of red/green), particularly in the IR (obese) group, compared with the sham group (p < 0.05). Pretreatment with TIIA, CsA, and TIIA+CsA significantly increased these factors (p < 0.05), pretreatment with TIIA+CsA were higher than TIIA and CsA (p < 0.05). Real-time qPCR was used to measure the levels of mtDNA oxidative damage. The ratio of long/short fragments decreased in the IR and IR (obese) groups, particularly in the IR (obese) group, compared with the sham group (p < 0.05). Pretreatment with TIIA, CsA, and TIIA+CsA increased the ratio of long/short mtDNA fragments (p < 0.05), pretreatment with TIIA+CsA were higher than TIIA and CsA (p < 0.05) . Pretreatment with TIIA, CsA, and TIIA+CsA significantly increased these enzymes (p < 0.05), pretreatment with TIIA+CsA were higher than TIIA and CsA (p < 0.05) , opening of the mPTP (%), ROS, mtDNA, oxygen consumption rate, RCR, ATP, and the activity of mitochondrial respiratory chain complex enzymes  to assess myocardial mitochondrial function after renal IR and the protective effect of TIIA+CsA. Mitochondria were isolated from rat myocardial tissues. Renal IR increased the ROS level and opening of the mPTP (%), particularly in the IR (obese) group, compared with the sham group (05) Fig. a. Renal 05) Fig. b.Fig. 4p < 0.05). Pretreatment with TIIA, CsA, and TIIA+CsA increased these mRNA levels (p < 0.05), pretreatment with TIIA+CsA were higher than TIIA and CsA (p < 0.05). Mfn1 and Mfn2 mRNA expression increased in the IR and IR (obese) groups, particularly in the IR (obese) group, compared with the sham group (p < 0.05). The mRNA levels of these factors decreased after pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA were lower than TIIA and CsA (p < 0.05) , nucleo respiratory factor1 (Nrf1), and transcription factor A of mitochondrial (Tfam) to represent mitochondrial biogenesis, and dynamin-related protein 1  and mitofusins , and Drp1 to represent mitochondrial dynamics (fusion and fission processes). Our results show that PGC-1α, Nrf1, Tfam, and Drp1 mRNA levels decreased in the IR and IR (obese) groups, particularly in the IR (obese) group, compared with the sham group p < 0.0. Pretreap < 0.05). The mRNA level expression of these factors decreased after pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA were lower than TIIA and CsA (p < 0.05). Renal IR decreased the mRNA level expression of PI3K, Bad, Akt, and Bcl-2 significantly, particularly in the IR (obese) group, compared with the sham group (p < 0.05). The mRNA level expression of these factors increased after pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA were lower than TIIA and CsA (p < 0.05)  group, compared with the sham group (05) Fig. a. The we05) Fig. b. TIIA, 05) Fig. b.Fig. 6The main cause of AKI in critically patients is renal ischemia-reperfusion injury (IRI). Continuous renal replacement therapy is the main AKI treatment for critically ill patients, however, it is not sufficient to reduce mortality, multiple organ dysfunction syndrome, and interorgan crosstalk induced by extrarenal complications, which are causes of death from AKI . Renal Ip < 0.05), and that TIIA, CsA, and TIIA+CsA improved cardiac function (p < 0.05), pretreatment with TIIA+CsA was better than TIIA and CsA (p < 0.05) . In contrast, TIIA, CsA, and TIIA+CsA decreased myocardial enzyme (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05)  is still very high , so a ne05) Fig. b. Additi05) Fig. c. Taken Renal IRI is involved in the adaptive and innate immune responses and releases cytokines in the kidney . Once thp < 0.05), which decreased after the pretreatment with TIIA, CsA, and TIIA+CsA (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05)  Fig. d. Systemp < 0.05). The score in the IR (obese) group was higher than that in the IR group (p < 0.05). All injury scores decreased in the TIIA, CsA, and TIIA+CsA groups, but only the TIIA+CsA group have the statistical difference (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA but the difference is insignificant (p > 0.05) . However, TIIA, CsA, or TIIA+CsA decreased caspase-9/3 activity in myocardial cells (p < 0.05), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05)  (p < 0.05)  group, combined with interstitial edema. However, pretreatment with TIIA, CsA, and TIIA+CsA alleviated the myocardial injury, particularly pretreatment with TIIA+CsA, as shown by improvements in interstitial edema and decreases in the number of disordered myocardial cells Fig. a. Struct05) Fig. a. At the05) Fig. a, wherea05) Fig. a. CaspasAccording to previous studies, some anti-inflammatory agents have been discovered, including α-melanocyte stimulating hormone, anti-apoptotic agents, and IL-6 inhibitors , 38. A pmiltiorrhiza Bge eliminates toxic substances from the blood, accelerates fibrinolytic enzyme activity and thrombolysis, reduced blood viscosity, and protects the cardiovascular system and blood vessels [S. miltiorrhiza Bge. Pretreatment with TIIA relieves renal injury induced by IR by downregulating the expression of myeloperoxidase (MPO), caspase-3, and inflammation [As a universal medicinal herb, it has been demonstrated that S.  vessels . TIIA isammation . TIIA deammation . Becauseammation . It is nammation . Openingammation . Howeverp < 0.05). Pretreatment with TIIA, CsA, and TIIA+CsA alleviated the decrease in the oxygen consumption rate and RCR, pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05) . TIIA, CsA, and TIIA+CsA significantly decreased the ROS level and opening of the mPAP (%), pretreatment with TIIA+CsA was lower than TIIA and CsA (p < 0.05) , whereas TIIA, CsA, and TIIA+CsA increased the mitochondrial MMP (ratio of red/green) significantly, pretreatment with TIIA+CsA was higher than TIIA and CsA (p < 0.05)  Fig. a. Renal 05) Fig. a. Renal 05) Fig. a. These p < 0.05), which increased after pretreatment with TIIA, CsA, and TIIA+CsA, pretreatment with TIIA+CsA was higher than TIIA and CsA (p < 0.05) (p < 0.05)  Fig. a. As mtDp < 0.05).Respiration is one of the most vital and basic features of living organisms. In mammals, respiration is accomplished by respiratory chain complexes located on the mitochondrial inner membrane to transfer electrons and establish the proton gradient for complex V to synthesize ATP . As showAs an important adaptation to exposure to chronic energy deprivation, mitochondrial biogenesis is modulated by many complex elements, such as Tfam and Nrf1. Nrf1 fosters transcription of nuclei-encoded mitochondrial protein expression, including factors involved in the respiratory complexes and oxidative phosphorylation. Tfam increases gene transcription and DNA replication in mitochondria by directly binding to the mitochondrial genome. PGC-1α is an important transcriptional co-activator that modulates key factors, including Nrf1 and Tfam, which increase mitochondrial biogenesis . MitochoMitochondria normally undergo a dynamic process of fusion and fission. This dynamic process is important to maintain constant changes in size, shape, and network of mitochondria. Mitochondria are under the control of regulatory proteins, including Drp1 and Mfns . Our resThe PI3K/Akt/Bad signaling pathway plays an important role inhibiting apoptosis mediated by mitochondria . This paIn our study, we used isolated mitochondria to show that renal IR promotes the production of ROS, which damages mtDNA, mitochondrial respiratory function, biogenesis, and dynamic function. Swollen mitochondria were induced by opening of the mPTP after renal IR. Opening of the mPTP induced back flow of protons from the mitochondrial membrane space into the matrix, thereby reducing ATP synthesis and MMP, leading to metabolic abnormalities. A reduction of MMP and ATP synthesis and an increase of Cyt-c were induced by opening of the mPTP, which led to back flow of protons from the mitochondrial membrane space to the matrix to induce metabolic abnormalities and eventually leading to myocardial cell apoptosis. The TIIA+CsA pre-treatment inhibited apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats. But in our study, considering of the combination therapy, we did not use the PI3K/Akt/Bad pathway inhibitor, so the mechanism of drug intervention can not be fully revealed,in the future, we will use the LY294002 (PI3K inhibitor) in the in vitro cell experiment in order to fully reveal the mechanism of its intervention in the future.Renal IR can induce the mitochondrial dysfunction and apoptosis (obesity increases the severity) in myocardial cell, TIIA combined with CsA can attenuated myocardial cell apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats.


Genetic diversity of viruses is driven by genomic mutations and selection through its host, resulting in differences in virulence as well as host responses. For baculoviruses, which are naturally occurring pathogens of insects and which are frequently sprayed on hundred thousands to millions of hectares as biocontrol agents of insect pests, the phenomenon of virus–host co-evolution is of particular scientific interest and economic importance because high virulence of baculovirus products is essential and emergence of host resistance needs to be avoided as much as possible. In the present study, the population structure of twenty isolates of the Cydia pomonella granulovirus (CpGV), including twelve isolates from different geographic origins and eight commercial formulations, were studied on the basis of next-generation sequencing data and by analyzing the distribution of single nucleotide polymorphisms (SNPs). An entirely consensus sequence-free quantitative SNP analysis was applied for the identification of 753 variant SNP sites being specific for single as well as groups of CpGV isolates. Based on the quantitative SNP analysis, homogenous, heterogenous as well as mixed isolates were identified and their proportions of genotypes were deciphered, revealing a high genetic diversity of CpGV isolates from around the world. Based on hierarchical clustering on principal components (HCPC), six distinct isolate/group clusters were identified, representing the proposed main phylogenetic lineages of CpGV but comprising full genome information from virus mixtures. The relative location of different isolates in HCPC reflected the proportion of variable compositions of different genotypes. The established methods provide novel analysis tools to decipher the molecular complexity of genotype mixtures in baculovirus isolates, thus depicting the population structure of baculovirus isolates in a more adequate form than consensus based analyses. After filtering, the reference nucleotide and first alternative nucleotide counts explained approximately 100 per cent of the data, a finding that was described previously , and thet test. This data matrix file was loaded into R software package.SNP density in every ORF was defined by the average SNP number in each ORF per kbp. On the basis of their annotation, ORFs were classified into biological regulation, DNA replication, metabolic process, structure protein, virus transcription and unknown function . Prior tThe variability of CpGV isolates represented by SNP positions and frequencies can be assessed by the distances between individuals using factorial analysis (PCA), HC and k-means clustering, of which three methods constitute HCPC. For the clustering of CpGV isolates, the filtered SNP frequency table with the entity of 753 SNP positions were used by applying the HCPC method as implemented in the FactoMineR v1.41 package  for R R Table 2Table 2. pe38 in breaking type I resistance (pe38 repeat region were counted when they fulfilled the following conditions: (1) they were defined by nonrepetitive flanking sequences of 12 bp upstream and 10 bp downstream of the 12 bp repeat motif and (2) both non-repetitive flanking sequences were present on a single read within the data set. From this entire set of reads covering the entire pe38 repeat region the distance between the nonrepetitive flanking sequences was calculated for each read and isolate by the help of the ShortRead v1.40.0  (P < 0.05)  was detected in all aligned reads. The SNPs included 544 transitions and 209 transversions. Six hundred sixty-five SNPs (88.3%) were distributed over 124 ORFs, whereas 88 SNPs (11.7%) were found in noncoding regions . Only 18 = 0.07) . A signi < 0.05) , indicatThe SNP frequencies at all variable SNP positions of each CpGV isolate were plotted against the CpGV-M consensus sequence as reference . The lowa), frequency for genotype b (ƒb), and isolate frequency ƒa + ƒb that was often ƒa+b = 1 when genotype a and b shared the same SNP position. Only CpGV-M, -S, -WW, and -V003 appeared to be highly homogenous showing few genomic variations , ORF2, ORF6, ORF7 (ie1), ORF10 (chitinase), ORF139, ORF140 (fgf-3), and ORF141 (egt). An obvious SNP island I/II with a SNP frequency <5 per cent, covering 8.6 kbp from genome map position 119,522 to 6,149 was noted in the circular genome of CpGV-M (granulin (1 SNP), ORF2 (2), pk-1 (1), ORF6 (2), ie1 (1), ORF8 (1), chitinase (1), ORF139 (1), fgf-3 (4) and egt (3).The assembly of the re-sequenced isolate CpGV-M against its own reference revealed a low number of variable sites. Only 51 SNPs were identified, of which only five exceeded an alternative frequency of 20 per cent. In particular, only one SNP had a frequency above 95 per cent . In the remaining 19 CpGV isolates, the alternative frequencies of this position ranged from 92 per cent to 100 per cent, strongly proposing that this position is most likely an annotation error in the consensus of the CpGV-M reference genome. On the other hand, the high accuracy of the reference sequence was confirmed by the absence of highly variable sites in this analysis . It is if CpGV-M . These SCpGV-V003, a resistance-breaking isolate selected from CpGV-M, also contained 51 SNPs. But only ten SNPs, at position 1,649, 11,147, 18,799, 20,332, 20,360, 2,859, 41,473, 76,292, 85,126 and 105,178, were identical to those positions found in CpGV-M . The oveThe overall SNP patterns of CpGV-WW and -S were highly identical in their location and frequency . The majThe three isolates CpGV-KS1, -KS2, and -ZY exhibited a similar SNP pattern in their position and frequency . No SNP The two Iranian isolates CpGV-I12 and CpGV-I0X differed in their SNP abundance and density. CpGV-I12 was identified as a mixture of CpGV-M (56%) and CpGV-S (42–46%) according to CpGV-M and -S specific group SNPs, though frequencies of CpGV-M specific SNP showed a highly uneven distribution. The CpGV-WW fingerprint regions a and b were lacking. CpGV-I0X was measured to only consist of mainly 93 per cent CpGV-M . Except A total number of 320 variant SNP positions were detected for CpGV-ALE, the reference isolate of genome group G . Ninety-CpGV-JQ and -ZY2 shared 239 SNPs with -WW, echoing that genome group E was within these isolates. A total number of 89 SNP positions were identified to be specific for CpGV-JQ and CpGV-ZY2 only, representing their own genetic marker for genome group F . AccordiCpGV-E2 and -0015 were unique as they showed a highly uneven SNP frequency distribution, resembling a ‘wave’-like pattern, clearly visible for the alternative SNP frequencies between genome positions from 10,000 to 113,000. Another characteristic was the presence of two cluster regions with SNP frequencies of 100 per cent at genome positions (1) 10,000 to 13,000 and (2) 39,000 to 43,000. Despite the heterogenous SNP pattern, a total number of 68 for only CpGV-E2 specific SNP positions was detected . These sin vivo propagation of CpGV-E2. SNP positions and frequency of both isolates of CpGV-E2 and -0015 were identical  and 3,752 (CpGV-0015) reads were used for this analysis. Between one to five copies of the 12 bp repeat motifs (1–5 × 12 bp) were identified in the pe38 of the twenty CpGV isolates  of the 1 × 12 bp repeat motif. Its abundance increased to 95 per cent  in CpGV-V003, a selection from CpGV-M, whereas the rest of the genome sequences of CpGV-M and -V003 showed highly similar SNP patterns  covered 95.2 per cent information (variances) presenting in the SNP data set and excluded the noise from data , an indiHigh throughput sequencing methods with high genome coverage and read depth provide new tools to study the population structure of virus isolates. As a consequence, more comprehensive genetic information can be unveiled than in previous studies. The data sets of re-sequenced genomes of CpGV-M, -S, -E2, and -I12, with mean read depth from 3,320 to 4,045 was between 100 and 1,000-fold higher than previous data sets of these viruses, ranging from 3.9- to 243-fold coverage . This hipe38, corresponding to the ability to overcome type I resistance in CpRR1, was quantified. When correlating bioassay data (× 12 bp repeats in different CpGV isolates (× 12 bp repeats larger than 46%) and a non-resistance-breaking activity was underlined , pep (ORF23), and pe38 (ORF24). As most nucleotide frequencies of CpGV-V003 were close to 100 per cent, it can be concluded that the virus selection successfully resulted in a notably pure genotype that was even more homogeneous than the original CpGV-M.On the other hand, only ten SNP positions were shared between CpGV-M and CpGV-V003, also excluding those from SNP island I/II, proposing that they were lost during the selection process, while nucleotide frequency at other SNP positions were enriched or new SNPs were selected, for example in the CpGV-V003 specific SNP hotspot covering p45, dnapol, desmoplakin, lef3, iap5, lef9, dna-ligase, which are supposed to be involved in viral DNA replication and BV production  or with other ArGV strains, revealed in most cases the presence of ArGV1 or recombinants between inoculums and ArGV1, suggesting ArGV1 is present as latent genotype in the host population .The raw SNP counts and frequencies that were used in this study have been provided in the Conflict of interest: None declared.veaa073_Supplementary_DataClick here for additional data file.


Targeted and untargeted selections including domestication and breeding efforts can reduce genetic diversity in breeding germplasm and create selective sweeps in crop genomes. The genomic regions at which selective sweeps are detected can reveal important information about signatures of selection. We have analyzed the genetic diversity within a soybean germplasm collection relevant for breeding in Europe (the EUCLEG collection), and have identified selective sweeps through a genome-wide scan comparing that collection to Chinese soybean collections. This work involved genotyping of 480 EUCLEG soybean accessions, including 210 improved varieties, 216 breeding lines and 54 landraces using the 355K SoySNP microarray. SNP calling of 477 EUCLEG accessions together with 328 Chinese soybean accessions identified 224,993 high-quality SNP markers. Population structure analysis revealed a clear differentiation between the EUCLEG collection and the Chinese materials. Further, the EUCLEG collection was sub-structured into five subgroups that were differentiated by geographical origin. No clear association between subgroups and maturity group was detected. The genetic diversity was lower in the EUCLEG collection compared to the Chinese collections. Selective sweep analysis revealed 23 selective sweep regions distributed over 12 chromosomes. Co-localization of these selective sweep regions with previously reported QTLs and genes revealed that various signatures of selection in the EUCLEG collection may be related to domestication and improvement traits including seed protein and oil content, phenology, nitrogen fixation, yield components, diseases resistance and quality. No signatures of selection related to stem determinacy were detected. In addition, absence of signatures of selection for a substantial number of QTLs related to yield, protein content, oil content and phenological traits suggests the presence of substantial genetic diversity in the EUCLEG collection. Taken together, the results obtained demonstrate that the available genetic diversity in the EUCLEG collection can be further exploited for research and breeding purposes. However, incorporation of exotic material can be considered to broaden its genetic base. Crop improvement relies on genetic diversity of plant genetic resources. A high genetic diversity provides an opportunity for plant breeders to develop cultivars with desirable characteristics . Today’sSelective sweeps have been analyzed in several crops to reconstruct their history of domestication and selection and to identify the genetic loci and their underlying genome sequence that were mainly affected by these processes. For example, Modern cultivated soybean was domesticated approximately 5000 years ago from its wild progenitor Glycine soja, which is distributed throughout Eastern Asia, including most of China, South Korea, and Japan . SoybeanBased on the responsiveness of soybean flowering and maturity to photoperiod and temperature, a total of 13 distinct maturity groups have been defined, of which only early maturing types (maturity groups MG000 to MGII) are suitable for cultivation in Europe . Previou1. In this context a unique collection of 480 soybean accessions considered relevant for European breeders, originating from 25 countries and covering a broad range of genetic diversity was assembled (named the EUCLEG collection in what follows). This offers a unique opportunity to compare the genetic diversity of the EUCLEG collection to that contained in reference materials from China, helping us to understand the main forces that have shaped the soybean genepool currently being used in breeding programs outside China.Our current knowledge on the origin and genetic relationships within European soybean germplasm is still fragmented. Main reasons are the limited number of accessions  that were included in previous studies  000, 00, 0 and I/II, and includes 210 improved varieties, 216 breeding lines and 54 landraces. A detailed description of this collection is provided in ® Plant Mini Kit2. Pure and good quality DNA samples with an average concentration of 106 ng/μl (range 24–731 ng/μl) were used for genotyping using the 355K SoySNP Axiom microarray , we first checked the performance of the 355K SoySNP microarray on the 480 EUCLEG samples separately. This step was considered necessary, as the 355K SoySNP microarray was developed using the NJAU collection and might perform sub-optimally with plant materials of a different origin. In brief, the Affymetrix® Power Tools (APT) software package, version 1.15.0 implemented in AAS performed sample quality control based on 20,000 non-polymorphic probe sets and considering the parameters Dish Quality Control  and Sample Call Rate . Based on criteria DQC > 0.82 and QC-CR ≥ 97, AAS filtered out four poor-quality samples. The R package SNPolisher version 1.3.6.7 implemented in AAS was used for SNP calling using 609,883 probe sets targeting 355,595 SNPs. Its Ps_Classification function classified the SNPs/probe sets into six categories based on the following SNP QC metrics: call rate (CR) ≥97%, Fisher’s linear discriminant (FLD) ≥ 3.6, heterozygous strength offset (HetSO) ≥−0.1, and homozygote ratio offset (HomRO) ≥0.3 for one-cluster or two-cluster SNPs or ≥−0.9 for three-cluster SNPs. A summary of the SNP classification was obtained for the 476 good quality samples of the EUCLEG collection. We compared this summary with the SNP classification summary obtained from the NJAU collection by SNP calling was performed using the software Axiom Analysis Suite (AAS) from AffymetrixIn a second step, we genotyped the combined dataset (EUCLEG and NJAU), starting from the raw fluorescence data following the procedure described above. In the quality control step, 69 poor quality samples were excluded. SNP calling was performed on the remaining 805 good quality samples. After genotyping, low quality SNPs based on SNP QC metrics were excluded and a final genotyping dataset containing 229,557 SNPs was generated. This dataset was divided in three subsets for further processing: EUCLEG, NJAU-Wild and NJAU-Cultivated, comprising 477, 82, and 246 accessions, respectively. For the divisions NJAU-Wild and NJAU-Cultivated, we refer to For some of the downstream analyses, the genomic coordinates of the SNPs were required. Because during the development of the 355K SoySNP microarray SNP coordinates were assigned using an older version of the soybean reference genome sequence (Glyma.Wm82.a1), we positioned SNPs onto the novel reference genome sequence Glyma.Wm82.a2 . Finally, the 224,993 SNPs corresponding to probes that could be positioned onto the 20 soybean chromosomes using a blast query were considered for further analyses.The results of population structure of the NJAU collection are available in ST) value per SNP site in VCFtools v. 0.1.15  was determined considering the delta log-likelihood criterion. For the interpretation of the results of fastSTRUCTURE, once an optimal value of K was identified, each accession was assigned to a subgroup “n” for which the ancestral coefficient reached a value Qn ≥ 0.4; where n is the number of subgroup (1 to K). Accessions for which the two highest Q values differed by less than 0.2 were considered “Admixed”. Finally, the degree of divergence between the EUCLEG and the NJAU collections was estimated by calculating a fixation index  analysis was performed in VCFtools v. 0.1.15 considering the filtered genotyping data including 139,986 SNPs, 162,098 SNPs and 185,194 SNPs with MAF of at least 5% in the sample sets EUCLEG, NJAU-Cultivated, and NJAU-Wild, respectively. LD was estimated for each chromosome by computing the Diversity estimates were determined for EUCLEG, NJAU-Cultivated, and NJAU-Wild separately, including all 224,993 SNPs. The EUCLEG collection contained 21 accessions from Chinese origin, but to avoid any possible confounding effect of those accessions while comparing genetic diversity between EUCLEG and NJAU, these 21 accessions were not considered. The average pairwise divergence among genotypes within each collection was then determined by computing the nucleotide diversity index (π) per SNP site in VCFtools v. 0.1.15.To detect signals of selection in the EUCLEG collection, the cross-population composite likelihood ratio test (XP-CLR) implemented in XP-CLR v. 1.0  was usedr2 values can provide redundant information, corrLevel was set to 0.95 to weight the XP-CLR value of a window containing highly correlated SNPs. Windows with weighted XP-CLR scores in the top 1% of the empirical distribution of the genome-wide XP-CLR values were used to delineate regions of interest. To define the regions of interest , we combined neighboring windows when the gap was less than 1 Mbp.The whole genome was scanned in XP-CLR choosing a sliding window of 1 Mbp at steps of 5 kbp. XP-CLR options were as follows: XPCLR -xpclr genofile1.txt genofile2.txt mapfile outputfile -w1 snpWin 0.01 gridSize 5000 chrN -p0 corrLevel 0.95; where genofile1.txt and genofile2.txt correspond to the object (EUCLEG) and reference (NJAU-Wild) collections, respectively. Because two SNP loci with high pairwise 4  belonging to the recommended categories including PolyHighResolution , MonoHighResolution  and NoMinorHom . These categories refer to SNPs exhibiting all three genotypic classes with a good cluster resolution (PHR), SNPs with good cluster resolution but displaying only one of the homozygous clusters (MHR) and SNPs with good cluster resolution but for which one of the two homozygous clusters is missing (NMH). These proportions correspond quite well with those previously reported for the NJAU collection by Genotyping of the EUCLEG and NJAU combined collection rendered 229,557 SNPs  belonging to the recommended categories . For the remaining 126,038 SNPs, at least one of the QC metrics were below the threshold and hence they were assigned to non-recommended categories. There were 16%  more SNPs of non-recommended categories in the combined analysis as compared to the separate analysis of the EUCLEG collection. This was because a number of SNPs  of recommended categories  in the EUCLEG separate analysis were assigned to the non-recommended categories in the combined analysis. Taken together, these results indicate that SNP calling on EUCLEG and NJAU combined is essential to get a more precise classification of SNP markers as compared to a separate analysis for each collection. In addition, these results indicate that the SNP dataset of EUCLEG and NJAU combined contains a high number of SNPs from the recommended categories.For 224,993 SNPs of the total 229,557 (98%), new coordinates could be positioned onto 20 chromosomes in the Glyma.Wm82.a2 soybean reference genome sequence using a BLAST query. The probes targeting the remaining SNPs  were either missing in the novel soybean genome assembly or were assigned to the non-anchored scaffolds and not to chromosomes, and were excluded from subsequent analyses. The genome-wide distribution of the final set of 224,993 SNPs used for downstream data analyses is given in ST values were 0.14, 0.34 and 0.22 for EUCLEG vs. NJAU-Cultivated, EUCLEG vs. NJAU-Wild and NJAU-Cultivated vs. NJAU-Wild comparisons, respectively. This indicates that the set of 805 soybean accessions considered in this study consists of three major groups including EUCLEG, NJAU-Cultivated and NJAU-Wild. The relatively low FST between EUCLEG and NJAU-Cultivated in relation to comparisons with NJAU-Wild supports a strong differentiation between cultivated and wild soybean accessions.We analyzed the presence of population structure using two approaches, fastSTRUCTURE and PCA. In the EUCLEG and NJAU combined analysis comprising 805 accessions, the marginal likelihood of the fastSTRUCTURE-output from K = 2 to K = 10 indicated the optimum K between 2 and 4 . The EUCIn the separate analysis of the EUCLEG collection the marginal likelihood of the fastSTRUCTURE-output from K = 2 to K = 10 indicated the optimum at K = 5 . AlthougThe results of the cluster analysis were similar with those of the fastSTRUCTURE analysis of EUCLEG and NJAU combined (see above). The accessions of the two collections were assigned to different clusters. NJAU-cultivated and NJAU-wild grouped into two separate clusters as in A higher number of fixed SNP sites (MAF = 0) was observed in the EUCLEG collection (20%) compared to the NJAU collections  . MoreoveLinkage disequilibrium (LD) dropped to half of its maximum at 175, 100, and 50 kbp in EUCLEG, NJAU-Cultivated and NJAU-Wild, respectively , suggestAverage pairwise divergence among genotypes per site (π) decreased from 0.31 in NJAU-Wild to 0.26 in NJAU-Cultivated and to 0.23 in EUCLEG . This is7 delivered 248 of the total 2,880 previously published QTLs coinciding with the selective sweep regions  , account regions . A total regions .A nearly equal and relatively low nucleotide diversity (π) was observed in selective sweep regions 6.2, 12.2, and 15.1 in both EUCLEG  and NJAU-Cultivated  when compared to NJAU-Wild  . This inGlyma.08G190300, Glyma.08G190500).Interestingly, π was extremely low (0.04 − 0.13) in selective sweep regions 1.1, 1.2, 7.4, 7.5, 8.2, 18.2 and 19.1 in EUCLEG as compared to NJAU-Cultivated (0.16 − 0.33) and NJAU-Wild (0.25 − 0.35), indicating a greater strength of selection on these regions in EUCLEG . Such a MtN3, nodulin MtN21; E2 (Glyma.10G221500) and E4 , and genes related to hormonal control of plant growth including auxin response factor, gibberellin-regulated protein, brassinosteroid signaling, jasmonic acid biosynthesis and strigolactone biosynthesis are located in these selective sweep regions  have low to medium π (0.11 − 0.25) in the EUCLEG collection compared to that in NJAU-Cultivated (0.21 − 0.35) and NJAU-Wild (0.27 − 0.34) . Of the  regions .We also observed known QTLs related to hilum color and seed coat color that coincide with selective sweep region 8.1 . This reIn soybean, different genotyping microarrays are available including the SoySNP50K iSelect Bead chip from Illumina  and the Soybean is native to China, Japan and Korea, and has been introduced to Europe and other parts of the world . EvidencThe level of genetic diversity (π) in this study was the lowest for the EUCLEG collection, followed by NJAU-Cultivated, and with NJAU-Wild containing the highest level of genetic diversity. This agrees with the model for soybean breeding history presented by Nevertheless, the EUCLEG collection is strongly structured, with a distribution of genetic diversity over five subgroups. In agreement with previous reports by We have applied the XP-CLR methodology to determine selective sweeps because compared to other approaches, XP-CLR is robust to determine selective sweeps even in structured populations and has a higher power to detect signals of selection. Moreover it can be used with un-phased genotyping data . We haveE1 to E10) , suggesting strong signals of selection at these loci in the EUCLEG collection. These results are consistent with previous reports. According to E1 to E4) with the recessive e1 and e2 alleles being more frequently found in Central Europe and the dominant E3 and E4 alleles being more frequent in Southern European accessions. The genetic diversity of region 10.2 (π = 0.11) and 20.1 (π = 0.25) indicate that the E2 locus is more fixed than the E4 locus in the EUCLEG collection. Furthermore, the absence of any previously reported QTL coinciding with the selective sweep region 12.2 containing genes related to time to flowering and hormonal signaling  . Except ncreases . In thisignaling  providesSurprisingly, no significant signatures of selection were detected for stem determinacy, which is an important adaptive trait affecting grain yield in soybean . DetermiIn Europe, soybean is considered a protein crop and, together with yield, seed protein content is one of the main breeding goals . We havePreviously known QTLs related to high seed oil content also coincided with the selective sweep regions in the EUCLEG collection compared to NJAU-Wild. This is in line with the observation that wild soybean seeds have low oil content . AlthougOur analysis has also revealed some signatures of selection related to seed fatty acids including the polyunsaturated fatty acids (linolenic acid and linoleic acid) and monounsaturated fatty acids (oleic acid). Improving the quality of seed oil by minimizing the level of polyunsaturated fatty acids is an important consideration for improved stability of soybean oil . MoreoveSoybean, a member of the Fabaceae family, has the ability to fix atmospheric nitrogen through symbiosis with rhizobium bacteria. It has been reported that high yielding soybean varieties have a better ability to fix nitrogen . While dSclerotinia sclerotiorum, is considered important in European soybean breeding programs because SCL is widely spread throughout Europe  caused by t Europe . Pannecot Europe . In our t Europe . Of the To the best of our knowledge this is one of the first studies that explores the genetic diversity of a large soybean collection relevant for breeding in Europe, in comparison to Chinese germplasm. Although we have found several selective sweeps that could be linked to useful traits in soybean through XP-CLR analysis, some methodological aspects require attention. First, genotyping using whole genome sequencing rather than microarray data could provide more variants and thus a more detailed description of the genomic regions that have experienced selective sweeps . For exaThe present study focused on the exploration of a representative sample of soybean accessions relevant for breeding in Europe, the EUCLEG collection. This is one of the first studies in which the patterns of genetic diversity in a large soybean germplasm set relevant for breeding in Europe has been compared to the genetic diversity contained in Chinese cultivated and wild soybean germplasm. Our study has demonstrated a relatively lower genetic diversity in the EUCLEG collection compared to Chinese collections of cultivated and wild accessions, which indicates a narrow genetic base of the EUCLEG collection. However, a more detailed analysis of the patterns of genetic diversity in the EUCLEG collection has revealed substantial sub-structuration in five subgroups associated with geographical origins, and without a clear association with maturity classes. A selective sweep analysis has revealed the presence of multiple signatures of selection in the EUCLEG collection, compared to Chinese wild germplasm. In particular, genomic regions previously reported to influence grain protein, yield and disease resistance have been identified, whose exploration in future work might facilitate further selection efforts. No signals of selection have been detected for loci involved in stem determinacy, probably because no directed selection has been performed for this trait among the germplasm represented in the EUCLEG collection. On the other hand, clear signatures of selection were detected for at least two loci involved in photoperiod sensitivity and time to flowering, which are main traits considered by breeders in order to adapt soybean for cultivation in Europe (only maturity classes 000 to I/II). Taken together, our results have identified relevant genomic regions that can be further exploited to improve soybean for the European agricultural sector, either through further improvement of genetic resources that are available in Europe, or through incorporation of exotic soybean material in European breeding programs.The original contributions presented in the study are included in the article/AS, HM, JA, and IR-R conceived the study. AS and HM performed the research and analyzed the data. AS and IR-R drafted the manuscript. AS, HM, JA, TR, JW, DY, and IR-R interpreted the results and elaborated the manuscript. All authors read and approved the final version of the manuscript.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. The reviewer, MS, declared a past co-authorship with one of the authors, TR, to the handling editor.