Whereas the genomic landscape of endocrine‐resistant breast cancer has been intensely characterized in previously treated cases with local or distant recurrence, comparably little is known about genomic alterations conveying primary non‐responsiveness to endocrine treatment in luminal early breast cancer.ARID1A, BRAF, ERBB2, ESR1, GATA3, HRAS, KRAS, NRAS, PIK3CA, and TP53) by next‐generation sequencing. Amplifications of CCND1, FGFR1, ERBB2, and PAK1 were determined by digital PCR or fluorescence in situ hybridization.In this study, 622 estrogen receptor‐expressing breast cancer cases treated with short‐term preoperative endocrine therapy (pET) from the WSG‐ADAPT trial (NCT01779206) were analyzed for genetic alterations associated with impaired endocrine proliferative response (EPR) to 3‐week pET with tamoxifen or aromatase inhibitors. EPR was categorized as optimal (post‐pET Ki67 <10%) versus slightly, moderately, and severely impaired . Recently described gene mutations frequently found in previously treated advanced breast cancer were analyzed (ERBB2 amplification (p = 0.0015) and mutations of TP53 (p < 0.0001) were significantly associated with impaired EPR. Impaired EPR in TP53‐mutated breast cancer cases was independent from the Oncotype DX Recurrence Score group and was seen both with tamoxifen‐ and aromatase inhibitor‐based pET (p = 0.0005 each).TP53‐mutated luminal cancers might not be sufficiently treated by endocrine therapy alone.We conclude that impaired EPR to pET is suitable to identify cases with primary endocrine resistance in early luminal breast cancer and that  TP53 mutations.Early recognition of endocrine resistance could be enabled by impaired suppression of proliferation after short‐term preoperative endocrine therapy (pET). Impaired endocrine proliferative response  sorts out cancers carrying  Hormonal blockade with tamoxifen or aromatase inhibitor (AI) therefore provides an effective growth suppressive therapy for the majority of BCs. However, in a considerable proportion of luminal BC, that is, about 20%–30%, local or distant recurrence during or after endocrine treatment indicate resistance to endocrine therapy.ESR1).ESR1 mutations. ESR1 mutations are enriched in metastatic BC and with significant differences in metastatic site.ESR1‐mutated.Two general patterns of endocrine therapy resistance are recognized clinically: primary, intrinsic resistance, whereby estrogen receptor‐expressing (ER‐positive) cancers never adequately respond to endocrine treatment, and secondary, acquired resistance, which develops following an initial response.ARID1A, ESR1, ERBB2, and TP53.Efforts have been undertaken to identify the genetic characteristics of endocrine‐resistant BC.TP53 gene mutation is frequent in the triple‐negative BC with up to 80% of cases showing this mutation.TP53 mutation is encountered in about 12%–29% of cases.TP53 mutations are somatic because selection for familial BC cases yielded lower proportions of TP53‐mutated cases indicating that germ‐line mutations (Li‐Fraumeni syndrome) are considerably rarer than somatic changes.TP53 alterations were most frequent in metastatic luminal BC (29%).TP53 mutations evolve in the context of clonal evolution in recurring BC or are acquired during progression is not clear.TP53 mutation was found in primary tumors as well as in metastatic deposits.TP53 in BC treatment response and survival have been summarized as contradictory and inconclusive.Neoadjuvant therapy of BC has generated new endpoints to evaluate therapy efficacy. In triple‐negative and HER2‐positive early BC, complete pathological remission after neoadjuvant therapy is commonly used as a surrogate marker for therapy responsiveness and favorable prognosis. In 70%–80% of BCs, this surrogate marker is not readily available because ER‐positive, luminal BCs will usually not regress completely when exposed even to long‐term preoperative endocrine therapy. Endocrine responsiveness, however, is indicated very precisely by a decrease in tumor cell proliferation, even after a short‐term exposure of only 2–3 weeks of endocrine therapy.ARID1A, CCND1, ERBB2, ESR1, FGFR1, PAK1, PIK3CA, and TP53.In this study, luminal early BCs from the prospective WSG‐ADAPT trial which did not respond to short‐term preoperative endocrine therapy (pET) were analyzed for alterations of genes which had recently been implicated in endocrine resistance in metastatic BC including 22.1Pre‐ and postmenopausal patients with ER‐ and/or PR‐positive, HER2‐negative early BC as determined by local pathologic assessment, were treated with short‐term pET in the West German Study Group (WSG) ADAPT trial NCT01779206). The details on study design were published previously.779206. Tn = 301 unselected consecutive BC cases from the run‐in phase of the WSG‐ADAPT trial.n = 400 consecutive cases from the main phase of the WSG‐ADAPT trial. A total of n = 79 cases were excluded due to (i) missing Ki67 at baseline or post‐pET, (ii) unavailable tissue blocks , (iii) divergent histological subtype at baseline and post‐pET, (iv) triple‐negative hormone receptor status, and (v) insufficient DNA amount and/or quality. The total number of specimens available for statistical analysis was n = 622 (Table Tumor specimens correspond to 2.2Immunohistochemistry (IHC) for estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 was performed in the central reference pathology unit of the ADAPT trial  using a Benchmark Ultra automated stainer (Ventana). Immunological reagents and central IHC scoring methods are summarized in the Table .Ki67 index assessment was supported by digital quantification as follows: First two experienced pathologists independently scored the Ki67 index by eyeballing in a minimum of 500 tumor cells . Next, Ki67‐positive tumor cell nuclei were quantified using the digital pathology platform iScan Coreo (Ventana) and Virtuoso quantification software  as described previously.Endocrine proliferative response (EPR) was determined by the post‐pET consensus Ki67 index. The EPR was categorized in four categories corresponding to optimal (post‐pET Ki67 <10%) versus slightly, moderately, and severely impaired proliferative response  Figure . These pHER2 was scored as 0, 1+, 2+, or 3+, in accordance with the Dako HercepTest. BCs with a HER2 score of 2+ or 3+, were subjected to HER2 fluorescence in situ hybridization (FISH). FISH categorization was in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guidelines.2.3® RSC DNA FFPE Kit on a Maxwell® RSC instrument (Promega) according to the manufacturer's recommendations. DNA concentration was quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and the dsDNA high sensitivity kit (Thermo Fisher Scientific).Depending on tumor size 6–8 sections (8 µm) were taken. Genomic DNA was extracted from FFPE specimens with the Maxwell2.4ARID1A, BRAF, ERBB2, ESR1, HRAS, KRAS, NRAS, and TP53) that were identified by Razavi et al. by comparing alterations in ER‐positive metastatic BCs with primary tumors.PIK3CA mutation status was determined because of its potential as a target for selective inhibition.GATA3 mutation status, a potential marker for sensitivity toward aromatase inhibitors, was also included.Within this study, we analyzed candidate endocrine resistance genes  and mean depths per base was 9460 . The second panel covered the complete protein‐coding sequence of ARID1A, BRAF, GATA3, HRAS, KRAS, and NRAS. Mean mapped reads per case was 282,100  and mean depths per base was 1,213   from all cases was carried out retrospectively by next‐generation sequencing (NGS) in the years 2018–2020. Targeted sequencing was performed using two customized amplicon‐based panels. Library preparation was performed with Ion AmpliSeq™ Library Kit 2.0 (Thermo Fisher Scientific). For quantification of prepared libraries, the Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific) was used. Sequencing was performed on an Ion S5 instrument (Thermo Fisher Scientific). The first panel covered the complete protein‐coding sequence (as well as 10 base pairs of flanking intron sequence to cover the splice sites) of 6) Table .http://annovar.openbioinformatics.org/en/latest/).Evaluation of sequencing data and variant annotation was performed with the ANNOVAR software and database tools  and the classification according to Molina‐Vila et al. (classification as disruptive vs. non‐disruptive).Alterations in the 2.5CCND1, FGFR1, and PAK1 was performed using digital PCR (dPCR) in all tissue specimens with a tumor cell amount >50% (n = 199).DNA copy number analysis of CCND1 assay ID: Hs02559587_cn; FGFR1 assay ID: Hs05052584_cn and PAK1 assay ID: Hs01931361_cn; Thermo Fisher Scientific) were used with 15.0 ng of FFPE DNA input. If less than 15.0 ng of DNA was present, the maximum input volume of 7.13 µl was used. The range of DNA input was 2.21–15.0 ng. Two reference gene assays were used for each target assay to determine the found amplifications. For detection of CCND1 and PAK1 amplifications RPPH1  and PMP22  were used as reference assays. The reference assays for FGFR1 amplifications were TERT  and RPPH1.TaqMan™ Copy Number Assays , 3D PCR Master Mix v2 (Thermo Fisher Scientific), and 3D PCR 20K Chip Kit v2 (Thermo Fisher Scientific). PCRs were performed on a flat block thermocycler. The reaction conditions were as follows: hot start at 96°C for 10 min, annealing at 56°C for 2 min, and denaturation at 98°C for 30 s for a total of 39 cycles, followed by a final extension step at 60°C for 2 min. Data were analyzed with QuantStudio™ 3D AnalysisSuite™ Software . The threshold for amplification was a ratio of target‐gene calls to reference‐gene calls of 2.2. Within this study, only those cases that showed amplification with both reference assays are considered to be amplified Table .2.6p ≤ 0.05.For statistical evaluation of the association between genetic alterations and pathologic parameters, we focused on candidate genes with a mutation frequency of ≥2.5%. The two‐sided Fisher's exact test and the Chi‐squared test for trends were used for contingency analysis. The results were considered to be statistically significant if TP53 status , pET (AI vs. tamoxifen), pT stage (pT2 vs. pT1 and pT3/4 vs. pT1), pN stage (pN1+ vs. pN0), histological grade (G3 vs. G1/2), Oncotype DX RS (26–100 vs. 0–25), baseline Ki67 , baseline ER status , and baseline PR status  to predict impaired EPR (post‐pET Ki67 ≥10%). Statistical analysis was performed with GraphPad Prism software Version 5.00 (GraphPad Software) and Stata/IC Volume 16.1. (Stata Corp).Multivariate logistic regression was used to analyze the independent ability of 33.1We performed targeted sequencing of 622 HR+/HER2‐early BCs from patients, enrolled in the WSG‐ADAPT trial NCT01779206). Tumor specimens corresponded to unselected consecutive cases from the run‐in phase of the WSG‐ADAPT trial.779206. T3.2ARID1A, BRAF, ERBB2, ESR1, HRAS, KRAS, NRAS, and TP53) were determined by next‐generation sequencing in resection specimens following short‐term pET.PIK3CA was included because the therapeutic potential of PI3K inhibitors in PIK3CA‐mutated BCs.GATA3 mutation status, a potential marker for sensitivity toward AIs, was also included.CCND1, FGFR1, and PAK1 in a subgroup of cases (n = 199).ERBB2 were determined conventionally by immunohistochemistry and FISH in the context of the central pathology review.Genetic alterations in candidate endocrine resistance genes (n = 412). A subset of these cases revealed more than one genetic alteration under study . Figure ARID1A were found in 5.3% of cases. Other somatic mutations with rates >5% of cases were GATA3 (16.6%), PIK3CA (43.3%), and TP53 (9.0%). Mutations of BRAF, ERBB2, ESR1, HRAS, KRAS, and NRAS occurred less frequently. Some genes like PIK3CA were mainly affected by missense mutations, whereas in other genes like ARID1A or GATA3 truncating mutations were more frequent. In a total of 36 tumors (18.1%) CCND1, FGFR1, and/or PAK1 amplifications were observed , FGFR1 (n = 6), and PAK1 (n = 5) were detected by dPCR in 16.1%, 3.0%, and 2.5% of cases under study, respectively  in a small subset of tumors (n = 8), which had been classified as HER2‐negative by local assessment (data not shown). These cases are termed ERBB2‐amplified herein. For subsequent statistical analysis, we focused on genes, which were altered in ≥2.5% of all cases and also included the ERBB2‐amplified tumors.Somatic mutations or gene amplification were present in 66.2% n = 42. A subs3.3n = 142) were in the group with low RS (RS 0–11). The majority  had intermediate RS (RS 12–25) and 16.2% (n = 101) were categorized as high‐risk RS (RS ≥26). Figure TP53 mutations were significantly more frequent in cases with high‐risk RS , than in cases with low RS 0–11  (p = 0.0083). Nevertheless, in absolute numbers, the majority of TP53 mutations were in the low and intermediate RS groups  and were rarer in cases with high RS  (p = 0.0027). Mutations of ARID1A, ERBB2, and GATA3 were equally distributed among RS groups  and ERBB2 (0 to 4.0%) was significantly more frequent in cases with high RS   Figure . CCND1 ad Figure .3.4n = 327) showed an optimal EPR . In total, 29.9% of cases (n = 186) displayed a slightly impaired EPR . In total, 14.0% and 3.5% of BC cases (n = 87 and n = 22) showed moderately and severely impaired EPR   to short‐term pET was determined by the post‐pET Ki67 index. In the present molecular analysis, EPR was categorized in four categories corresponding to optimal response (post‐pET Ki67 <10%) versus slightly, moderately, and severely impaired proliferative response  Figure . In tota) Figure .TP53 mutations increased significantly with increasing post‐pET Ki67 category. In detail, the frequency of TP53 mutations increased from 4.9% in BC cases with optimal EPR (group I) to 22.7% in BCs with severely impaired EPR   Figure . This inTP53 mutations can be associated with variable loss of p53 transcriptional activity. According to IARC, the functional impact of TP53 mutations is categorized as  functional or non‐functional.TP53 mutations.TP53 mutations in 56 patients. All detected TP53 mutations were located in the p53 DNA‐binding domain  were missense mutations. Truncating mutations were present in 18% (n = 10) of the patients and 5% (n = 3) harbored splicing mutations or in‐frame deletions. The majority of these TP53 mutations  encoded for p53 mutants that are classified as non‐functional, according to IARC , or  functional , according to IARC  compared to TP53‐mutated BCs with severely impaired EPR  to 18.2% in BCs with severely impaired EPR  were also associated with EPR. Despite the small number of ERBB2‐amplified cases (n = 8) in this study, we observed a statistically significant correlation between ERBB2 amplification status and impaired EPR  and was not associated with impaired EPR , pT stage, pN stage, baseline histological grade, Oncotype DX RS group, baseline Ki67, baseline ER status , and baseline PR status . Independent predictive parameters for impaired EPR included TP53 mutation status, type of pET, RS group, baseline Ki67 index, baseline ER status, and baseline PR status  had higher odds of impaired EPR  compared to TP53 wild‐type tumors. Risk of impaired EPR was 3.5 times higher in cases with RS 26–100, than in cases with RS 0–25. Cases with high baseline Ki67 also had higher odds for an impaired EPR. pT stage, pN stage, and histological grade did not independently predict impaired EPR , than in tumors without TP53 mutations. Hence, TP53 mutation predicts impaired EPR independently from Oncotype DX RS and other clinicopathological parameters.For completeness, we used a refined multivariate logistic regression considering the IARC classification to compare wild‐type s Figure . In this4The greatest part of our knowledge regarding endocrine resistance in BC stems from experimental in vitro studies using cell lines and animal models. Over the last decades, tamoxifen sensitivity has been the subject of many, primarily cell biological studies, but none of the many potential markers had found its way into the clinic. The major reason for this deficit in translation is the lack of clinical studies suitable to validate the in vitro findings.ESR1 mutation which is far more frequent in previously AI‐treated luminal cancers than in treatment‐naïve cases.GATA3, PIK3CA, and TP53.TP53 mutation was found in 37% of BC, with a frequency of 12% in luminal A, and 29% in luminal B type of BC, respectively.A novel approach to understand endocrine resistance was enabled by large‐scale mutational analysis of relapsing and metastatic BC which had been treated with adjuvant endocrine therapy after surgery.Despite the huge number of BC cases which have undergone next‐generation sequencing, there are no prospective clinical trials in primary, non‐metastatic BC which exploit the available bulk of sequence data in order to stratify cases according to their endocrine responsiveness. Currently, gene expression profiles like Oncotype DX RS are utilized to achieve this goal.TP53 mutation has not been clearly identified as a potential source of endocrine resistance.TP53 mutations compared with good responders.n = 66, their p = 0.8).TP53 mutations and higher Ki67 levels at baseline and at surgery, as well as a correlation with the luminal B subtype.TP53, ATR, APAF1, and THBS1 combined), but predictive relevance of TP53 mutations per se was not evaluated.TP53 mutation and impaired EPR in a limited tumor collection from a clinical trial at the Vanderbilt University (n = 140).TP53 dysfunction signature was associated with impaired EPR after 2 weeks of pET with AI, but TP53 gene mutation status was unknown.TP53 mutation is currently not considered as a determinant of endocrine resistance.TP53 mutation, in AI‐ as well as tamoxifen‐treated BC. Different reasons may account for the fact that TP53 mutation was associated with EPR in our study, but not in previously reported studies. One possible reason is the considerably larger sample sizes in the present study.TP53 analysis was based on slightly different methods.TP53 mutation status was assumed based on a gene expression signature.TP53 gene was analyzed for mutations by NGS.With regard to luminal BC, identification of the resistant sub‐cohort by impaired EPR after pET could enable recognition of primary aberrations associated with endocrine resistance. In previous smaller studies which have used this approach, TP53 mutations in the BC collection analyzed in this study was comparatively low (9%).ESR1 mutations than expected.The overall frequency of TP53 mutation as a predictive marker in BC, indicating either increased sensitivity to cytotoxic drugs or on the contrary potential resistance.TP53 mutation on survival of BC cases was studied. Thereby, two effects of the mutation could be observed. First, TP53 mutant cancers displayed a superior overall survival when treated by chemotherapy and irradiation. Second, TP53 wild‐type cases revealed substantial benefit when ER‐expressing cancers were treated with endocrine therapy. Overall survival in this study was worse in endocrine‐treated cases with TP53 mutation than in TP53 wild‐type cancers, corresponding to our findings that TP53 might be an effector of endocrine resistance.TP53 and endocrine responsiveness in more detail. In vitro cell models of HR+BC cell lines harboring TP53 mutations introduced by the CRISPR‐Cas9 method might help to clarify whether or not sensitivity to estrogen deprivation is directly dependent on the TP53 status. Furthermore, large‐scale sequencing studies in clinical cohorts with long‐term follow‐up may document the relevance of TP53 mutation for primary endocrine resistance.There have been conflicting data regarding TP53 mutation, only RS group, type of pET (associated with age), and baseline Ki67 were associated with impaired EPR. For recurrence score, Paik et al. have similar findings in cases treated with tamoxifen.FGFR1 amplifications and PIK3CA mutations.Besides of TP53 mutations indicates primary endocrine resistance in about 10% of luminal early BC cases. As TP53 mutations have also been implicated with conveying sensitivity toward conventional chemotherapy, further studies are needed in order to clarify the clinical consequences of our findings.In conclusion, the current WSG‐ADAPT translational study demonstrates that impaired EPR is suitable to identify genetic mechanisms of primary endocrine resistance already early during the course of early luminal BC. The presence of OG has minority ownership interest in WSG GmbH, received honoraria from Genomic Health/Exact Sciences, Roche, Celgene, Pfizer, Novartis, NanoString Technologies, AstraZeneca, served in consulting/advisory role for Celgene, Genomic Health/Exact Sciences, Lilly, MSD, Novartis, Pfizer, Roche, and received travel support from Roche.UN has minority ownership interest in WSG GmbH, received honoraria from Agendia, Amgen, Celgene, Genomic Health, NanoString Technologies, Novartis pharma, Pfizer Pharmaceuticals, Roche/Genentech, Teva, served in consulting/advisory role for Genomic Health, Roche, provided expert testimony for Genomic Health, received travel support from Genomic Health, Pfizer Pharmaceuticals, Roche, and her institution received research funding from Agendia, Amgen, Celgene, Genomic Health, NanoString Technologies, Roche, Sanofi.SK has minority ownership interest in WSG GmbH, received personal fees from Lilly, Roche, Genomic Health, Novartis, Amgen, Celgene, Daiichi Sankyo, AstraZeneca, SOMATEX Medical Technologies, MSD, Pfizer, Puma Biotechnology, PFM medical, and non‐financial support from Roche, Daiichi Sankyo, Sonoscope.MB received honoraria from AstraZeneca, Exact Sciences, Novartis, Pfizer, Roche, Teva, travel support from AstraZeneca, Celgene, Medac, Novartis, Roche and served in consulting/advisory role for AstraZeneca, Exact Sciences, Novartis, Puma, Roche.BA reports a potential financial conflict of interest as follows: Pfizer Pharma GmbH, Roche Pharma AG, Novartis Pharma GmbH, AstraZeneca GmbH, PharmaMar GmbH, MSD Merck Sharp & Dohme GmbH, Onkowissen.de GmbH, Lilly Deutschland GmbH, ProMedicis GmbH.RK served in consulting/advisory role for West German Study Group, and reports for an immediate family member: ownership interest in WSG GmbH, honoraria from Amgen, AstraZeneca, Genomic Health, Novartis, Pfizer, Pierre Fabre, Roche, Zodiac Pharma, consulting/advisory role for Agendia, AstraZeneca, Celgene, Daiichi Sankyo, Lilly, Merck Sharp & Dohme, Novartis, Odonate Therapeutics, Pfizer, Pierre Fabre, Roche/Genentech, Sandoz, Seattle Genetics, and research funding from Lilly, Merck Sharp & Dohme, Novartis, Pfizer, Roche/Genentech.RW served in consulting/advisory role as well as on speakers’ bureau for and received travel support from Agendia, Amgen, Aristo, AstraZeneca, Boehringer Ingelheim, Carl Zeiss, Celgene, Clinsol, Daiichi‐Sankyo, Eisai, Genomic Health, Glaxo Smith Kline, Hexal, Lilly, Medstrom Medical, MSD, Mundipharma, NanoString, Novartis, Odonate, Onkowissen, Paxman, Palleos, Pfizer, Pierre Fabre, Puma Biotechnology, Riemser, Roche, Sandoz/Hexal, Seattle Genetics, Tesaro Bio, Teva, Viatris.NH has minority ownership interest in WSG GmbH, received honoraria from Amgen, AstraZeneca, Genomic Health, Novartis, Pfizer, Pierre Fabre, Roche, Zodiac Pharma, served in consulting/advisory role for Agendia, AstraZeneca, Celgene, Daiichi Sankyo, Lilly, Merck Sharp & Dohme, Novartis, Odonate Therapeutics, Pfizer, Pierre Fabre, Roche/Genentech, Sandoz, Seattle Genetics, an immediate family member served in consulting/advisory role for West German Study Group, and her institution received research funding from Lilly, Merck Sharp & Dohme, Novartis, Pfizer, Roche/Genentech.IG, SB, LK, LB, HC, MGro, MR, UL, CzE, EMG, CS, KLH, MGra, MC, and HK, declare no potential conflict of interest.The study design is following the guidelines of the local ethics committee .Figure S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.


Compound 3c inhibited leukemia cancer panel better than other cancer panels with growth inhibition of 50% (GI50) values ranging from 1.48 to 2.73 µM, and the most promising inhibition with GI50 of 1.25 µM was observed against leukemia cell line SR, while the least inhibition was found against non-small lung cancer cell line NCI-H226 with GI50 value of 7.29 µM. Compounds 3b,c demonstrated superior antiproliferative activity than curcumin and gefitinib. In molecular docking, compound 3c had the most significant interaction with four H-bonds and three π–π stacking, and compound 3c was found to moderately inhibit EGFR. The curcumin analogs discovered in this study have the potential to accelerate the anticancer drug discovery program.Cancer is the world’s second leading cause of death, accounting for nearly 10 million deaths and 19.3 million new cases in 2020. Curcumin analogs are gaining popularity as anticancer agents currently. We reported herein the isolation, molecular engineering, molecular docking, antiproliferative, and anti-epidermal growth factor receptor (anti-EGFR) activities of curcumin analogs. Three curcumin analogs were prepared and docked against the epidermal growth factor receptor (EGFR), revealing efficient binding. Antiproliferative activity against 60 NCI cancer cell lines was assessed using National Cancer Institute (NCI US) protocols. The compound  Sorangiumcellulosum, has been chemically modified to Ixabepilone [Camptotheca acuminate, has been modified into various anticancer agents including Karenitecin (BNP-1350), Diflomotecan (BN80915), and Gimatecanetc [Podophyllum peltatum [Acronychiabaueri that has been modified to S23906-1 [Combretum caffrum thathas been converted into anticancer agents such as Combretastatin A-4 phosphate, Combretastatin A-4 diphosphate and Ombrabulin (AVE8062) [O-methyl-nordihydroguaiaretic acid, a lignin isolated from Larreadivaricatta [Pueraria Mirifica, soybeans, and soy products, has been converted to Triphendiol and Genistein [Natural products (NPs) have been used to treat human ailments since the dawn of medicine. NPs have historically been a highly prolific source of new medicines since the time immemorial in most of the civilizations. NPs continue to offer a diverse set of structural templates for drug discovery and development, which was heightened in the 1970s and 1980s . Many ofbepilone . Camptottecanetc ,7,8. TafS23906-1 . CombretAVE8062) ,12,13. Taricatta . Daidzeican BN809, and Gimenistein .Curcuma longa L. of the family (Zingiberaceae), has had its structure modified in order to produce biologically active compounds. The chemical modification improved not only the biological activities but also the stability, reducing rotational freedom and minimizing metal-chelation properties [Curcumin, one of the active constituents of operties . Curcumioperties ,26,27,283a,b). In silico molecular docking analysis was carried out against EGFR through the Schrodinger’s software. As molecular docking is now an integral part of computer-aided drug design (CADD), in which ligands with computed generated 3D structures are positioned into a receptor or biological target in a variety of orientations, conformations, and positions [Cancer is one of the most dreadful diseases and the leading cause of death today, with nearly 10 million deaths and 19.3 million new cases expected in 2020 . Chemothositions , it has ositions . Indeed,ositions .All of the chemicals were obtained from a local vendor. The standard curcumin was obtained from Konark Herbals in India, and the remaining chemicals and reagents were obtained from Merck Chemicals, SD Fine, and CDH. All of the chemicals were either analytical or synthetic grades in nature. The dry turmeric was purchased from a local market and ground into powder.Eighty grams of ground turmeric was refluxed in 200 mL of dichloromethane for one hour with continuous stirring, followed by filtration under vacuum. The filtrate was concentrated under vacuum while the temperature was kept at 50 °C. The reddish yellow oily residue was triturated further with 80 mL of hexane to yield 2.47 g of solid residue. The resulting solid residue was dissolved in a small amount of dichloromethane/ methanol (99:1) and loaded into a column packed with silica gel. The column was eluted with the same mobile phase, and curucmin was isolated, collected, and analyzed using thin layer chromatography (TLC) with standard curcumin as a reference. For the isolation of curcumin the conventional method was used and with 80 g of curcumin powder nearly 244 mg of curcumin was isolated .1)  and 3-chlorophenyl thiourea (2a)  in glacial acetic acid in 50 mL round-bottom flask placed in sand bath was heated at 80 °C with continuous stirring on magnetic stirrer for 10 h to obtain the compound 3a, while an equimolar amount of curcumin (1)  and N-(4-substitutedphenyl)hydrazine carboxamides  (0.25 mmol) in glacial acetic acid in 50 mL round-bottom flask placed in sand bath was heated at 80 °C with continuous stirring for 8–10 h on magnetic stirrer to obtain the compound 3b,c. The completion of reaction was monitored throughout by thin layer chromatography using mobile phase, n-hexane: ethylacetate (6:4). The excess of solvent was removed under vacuum distillation. Cooled the reaction mixture to room temperature and poured into the crushed ice to obtain the crude product, filtered, and dried. The crude products were further re-crystallization with ethanol to obtain the compounds, 3a–c. The synthetic route followed is shown in The isolated curcumin was used as a starting compound for the preparation of curcumin analogues via chemical modification of curcumin according to the methods described ,19. Figu3a–c were saved as mol files, and docking was performed according to the protocol described by Sogabe et al. (2013) [The docking was done against epidermal growth factor receptor (EGFR), which is one of the most appealing targets for many anticancer drugs. The protein data bank provided the EGFR (PDB:3W2R) X-ray crystal structure with a resolution of 2.05 Å; R-value 0.220 (observed) . The lig. (2013) .50), 50% lethal concentration (LC50), and total growth inhibition TGI [The antiproliferative activity of tested compounds was done against five dozen (60) cancer cell lines derived from nine different panels. All of the compounds were evaluated for antiproliferative activity in a single dose at 10 µM. Growth percent (GP) and percent growth inhibition (% GI) were used to calculate antiproliferative activity. The tested compounds that demonstrated significant activity were subjected to a five-dose assay (0.01 to 100 µM). The antiproliferative results of five-dose assay were calculated as three dose related parameters like, 50% growth inhibition  after 24 h to represent a cell population measurement for each cell line at the time of drug addition (Ti). The experimental drugs were frozen after being solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration. An aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with medium containing 50 µg/mL gentamicin at the time of drug addition. Additional four, 10-fold, or ½ log serial dilutions serial dilutions were performed to provide a total of five drug concentrations plus the control. Aliquots of 100 µL of these different drug dilutions were added to the appropriate microtiter wells already containing 100 µL of medium, resulting in the required final drug concentrations. Following drug addition, the plates are incubated for an additional 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µL of cold 50% (w/v) TCA  and incubated for 60 min at 4 °C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 min at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 µL of 80% TCA .Human tumor cell lines were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM max 540 nm) was performed in human A431 cells, which express higher levels of EGFR protein [According to the reported method, a spectrophotometric assay for EGFR tyrosine kinase inhibition (absorbance at λ protein .3a–c) are given in 3a displayed only π–π stacking interaction with the amino acids Asp855, Asp800, and Leu718, while ligand 3b displayed two H-bond with the residues Leu718 and Asp800 and two π–π stacking with the residues Leu718 and Asp800 as showed in 3c displayed significant interaction within the active site of EGFR having three H-bonds with the residues Met793, Gln791, and Ser720, while three π–π stacking with the residues Asp855, Asp800, and Leu718 as shown in EGFR was chosen for docking studies because it is a potential target for many anticancer drugs, including gefitinib, erlotinib, cetuximab, and panitumumab . Curcumi3a–c demonstrated significant interactions  in the molecular docking studies and were further tested for antiproliferative activity against five dozen (60) human cancer cell lines derived from nine different panels, including breast, colon, CNS, leukemia, melanoma, non-small cell lung, prostate, and renal cancer cell lines, according to National Cancer Institute (NCI US) protocols [3a inhibited growth by more than 50% in three cancer cell lines—MOLT-4 (% GI = 53.78), HT29 (% GI = 53.43), and SR (% GI = 51.28), while the remaining cell lines (56 cell lines) inhibited growth by less than 50%. Compound 3b was found to be lethal against two cell lines—NCI-H522 and MDA-MB-435, with percent GIs of 113.52 and 136.25, respectively, whereas compound 3c had lethal effect against RPMI-8226 and HCT-116, with percent GIs of 100.21 and 153.67, respectively  against 13 cell lines, while compound 3c demonstrated >50% GIs against 12 cell lines .The ligands rotocols ,45,46,47R % GI = .28, whilat 10 µM . The ord3b,c, which demonstrated promising antiproliferative activity against some of the cancer cell lines in a single dose assay with GIs of >68 (GP 32 percent), were chosen for further evaluation in a five-dose assay [3b,c) was represented as a three-dose-related parameter: GI50, TGI, and LC50. The compound 3b had GI50 values ranging from 1.25 to 18.8 µM, TGI values ranging between 6.69 and >100 µM and LC50 > 100 µM, whereas the compound 3c had GI50 values ranging from 1.48 to 7.29 µM, TGI values ranging between 2.06 and >100 µM and LC50 > 100 µM. Compounds 3b,c showed promising inhibitions against SR in leukemia cell lines’ panel, with GI50 values of 1.52 and 1.48, respectively, whereas the compounds 3b,c showed promising inhibitions against NCI-H522 in non-small lung cancer cell lines‘ panel with GI50 values of 1.76 and 1.78, respectively. Likewise, compounds 3b,c showed promising inhibitions against SF-539 in CNS cell lines’ panel, with GI50 values of 2.35 and 2.37 respectively, whereas the compounds 3b,c showed promising inhibitions against UO-31 in renal cancer cell lines’ panel with GI50 values of 1.52 and 1.94, respectively. Compound 3b,c showed promising inhibitions against BT-549 in breast cell lines’ panel with GI50 values of 2.49 and 1.96, respectively. Compounds 3b,c showed promising inhibitions, respectively, against HCT-15 and HCT-116 in colon cancer cell lines’ panel with GI50 values of 2.98 and 1.51 respectively, whereas compounds 3b,c showed promising inhibitions, respectively, against MDA-MB-435 and LOXIMVI in melanoma cell lines panel with GI50 values of 1.25 and 1.77 respectively. Compounds 3b,c showed promising inhibitions respectively against OVCAR-3 and OVCAR-5 in ovarian cancer cell lines’ panel with GI50 values of 2.98 and 1.16, respectively, whereas compounds 3b,c showed promising inhibitions, respectively, against DU-145 and PC-3 in prostate cancer cell lines’ panel with GI50 values of 3.99 and 3.21, respectively. Furthermore, the selectivity ration (SR) of compound 3b ranging between 0.76 and 1.89, whereas the selectivity ration of compound 3b ranging between0.82 and 1.31 indicated their non-selectivity towards the cancer cell line panels. 50 of each panel was calculated and compared to that of curcumin. Except for the panel colon cancer by compound 3b, both the curcumin analogs  demonstrated more promising antiproliferative activity with better inhibition than curcumin as shown in 3c is shown in 3b against nine panels of 60 NCI cancer cell lines in terms of GP and Log10 molar concentration are shown in 3c > 3b > curcumin. Curcumin molecular engineering into semi-synthetic curcumin analogues  resulted in increased antiproliferative activity.Compounds se assay ,51. The 3b,c) with promising antiproliferative activity and displayed significant interaction within the active site of EGFR in molecular dockingwere then tested for anti-EGFR activity in human A431 cells, which express high levels of EGFR protein, using a spectrophotometric assay (absorbance at λmax 540 nm). Both the compounds  moderately inhibited the EGFR with IC50 values of 3.89 and 4.18 µM, respectively, while the standard drug, gefitinib inhibited EGFR with IC50 value of 0.017 µM.The ligands , SR (% GI = 51.28), and HT29 (% GI = 53.43). The compound 3b displayed two H-bonds with the phenolic functions with the residues Asp800 and Leu718, as well as two π–π stacking one between the methoxyphenyl moiety and the residue Asp855 and another with one of the hydrogen of doubly bonded carbon (C=C). Compound 3b exhibited significant interaction within the active site of EGFR in molecular docking studies, and demonstrated better inhibition than curcumin against 35 of the 56 cell lines tested in common in single dose assay at 10 µM. Compound 3b inhibited leukemia cancer panel better with GI50 values ranging from 1.52 to 3.38 µM, but the most promising inhibition with GI50 of 1.25 µM was observed against MDA-MB-435, while the least sensitivity was found against OVCAR-5 with GI50 value of 18.8 µM. Compound 3c displayed three H-bonds with the phenolic functions with the residues Met793, Ser720, and Gln791, as well as four π–π stacking three between the p-trifluoromethylphenyl moiety and the residues Leu718 and Asp800 and another one with the pyrazoline CH and residue Asp855. The p-trifluoromethyl phenyl function lies within the hydrophobic cavity containing important residues Leu844, Cys797, and Met793. Compound 3c inhibited leukemia cancer panel better with GI50 values ranging from 1.48 to 2.73 µM, and the most promising inhibition with GI50 of 1.25 µM was observed against SR, while the least inhibition was found against NCI-H226 with GI50 value of 7.29 µM. The SI values indicated that both the curcumin analogues  were found to be non-selective against all the nine panels tested.The molecular docking revealed that compound 3a–c) demonstrated promising antiproliferative activity and significant interaction within the active site of EGFR in molecular docking. Previous research has shown that curcumin has anti-EGFR activity [3a–c) were found to be moderately inhibited EGFR. Compound 3a had a higher docking score than compounds 3b,c, but it showed moderate antiproliferative and anti-EGFR activities. Compounds 3b,c, on the other hand, demonstrated significant antiproliferative and anti-EGFR activity, as we observed better interaction within the EGFR active site. Only π–π stacking interaction was observed with compound 3a, while H-bond and π–π stacking interactions were observed with compounds 3b,c.The curcumin analogs  were found to be much more potent than the reported curcumin analogues [In most of the cases, the chemical modifications of diketonic function of curcumin into pyrazole and primidinone analogues were found to be promising, whereas chemical modification of diketonic function that resulted in bigenelli-type curcumin compounds were found to be least significant ,19,20,54nalogues .3a–c). Compounds (3a–c) had docking scores >−6.337 kcal/mol, indicating efficient binding to the active site of EGFR. Compounds 3b,c demonstrated promising antiproliferative activity in both one and five-dose assay. In a one-dose assay, compound 3b demonstrated the most promising antiproliferative activity with mean % GI of 66.46, while compound 3c demonstrated the most promising antiproliferative activity in five-dose assay with mean GI50 value of 2.84 µM, but none of the compounds were found to be selective against any panel in five-dose assay, as indicated by their selectivity ratio values. Except for compound 3a, both compounds  effectively inhibited the tested cancer cell lines in single and five dose assays. The curcumin analogues moderately inhibited the EGFR, a potential target for anticancer agents. Because the compounds demonstrated promising antiproliferative activity, the current report may lead to an expansion of the anticancer research development program in the future.Three curcumin analogs were prepared and tested for antiproliferative activity (


Moreover, 2ThioUTP treatment was able to significantly reduce pro-fibrotic gene expression (p < 0.05), as well as that of protein α-smooth muscle actin (p = 0.004). Conclusions—Our data suggest that P2RY2 activation should be further investigated as a pharmacological target for the prevention of CAVS progression, acting on both calcification and myofibroblastic activation.Rationale—Calcific aortic valve stenosis (CAVS) is a pathological condition of the aortic valve with a prevalence of 3% in the general population. It is characterized by massive rearrangement of the extracellular matrix, mostly due to the accumulation of fibro-calcific deposits driven by valve interstitial cells (VIC), and no pharmacological treatment is currently available. The aim of this study was to evaluate the effects of P2Y2 receptor (P2RY2) activation on fibro-calcific remodeling of CAVS. Methods—We employed human primary VICs isolated from CAVS leaflets treated with 2-thiouridine-5′-triphosphate , an agonist of P2RY2. The calcification was induced by inorganic phosphate (2 mM) and ascorbic acid (50 µg/mL) for 7 or 14 days, while the 2ThioUTP was administered starting from the seventh day. 2ThioUTP was chronically administered for 5 days to evaluate myofibroblastic activation. Results—P2RY2 activation, under continuous or interrupted pro-calcific stimuli, led to a significant inhibition of VIC calcification potential ( Calcific aortic valve stenosis (CAVS) is a slow and progressive pathological disorder of the aortic valve with a prevalence of 3% in the general population over 65 years of age . To dateThirty-three patients who underwent surgical aortic valve replacement (AVR) were enrolled in the study at Centro Cardiologico Monzino IRCCS. Patients’ characteristics are summarized in l-glutamine ). After the endothelial cell removal, the leaflets were moved away in new fresh medium and mechanically disrupted using a scalpel, before incubating for at least 4 h in 2 mg/mL type II collagenase in cell culture medium at 37 °C to process the extracellular matrix proteins.The collected aortic valve leaflets were employed to isolate valve interstitial cells (VICs) by using a modified method previously described ,23. Brie® 200pro (TECAN) spectrophotometer. Each datum was normalized on the total protein quantification.Cells were seeded and treated every 2 days, changing medium every time, for 7 or 14 days with ascorbic acid  and inorganic phosphate , with or without 2ThioUTP (10 uM). At the end of the assay, after medium removal, we employed 0.6 M HCl to dissolve extracellular calcium. After 5 h of HCl treatment in gentle agitation, samples were collected and stored at +4 °C. Cells on the bottom of the plate were incubated a +4 °C in 0.1 M NaOH with 0.1% sodium dodecyl sulfate (SDS) for total protein quantification using bicinchoninic acid (BCA) Protein Assay . To quantify extracellular calcium, we used a calcium colorimetric assay kit , following the manufacturer’s protocol. The absorbance was detected using the InfiniteFor the fibrosis assays, cells were seeded and treated with 2ThioUTP (10 µM) every day without changing media for 5 days. At the end of the assay, total mRNA and protein were extracted and preserved at −20 °C until analyzed.Western blot analysis was carried out to assess protein expression using specific antibodies against α-smooth muscle actin  and α-tubulin  as an endogenous control. Briefly, we collected and lysed untreated or 2ThioUTP-treated cells in RIPA buffer with a protease and phosphatase inhibitor cocktail . Total protein concentration was assessed through the use of a BCA kit . Afterward, proteins separation was performed using Bolt™4–12% Bis-Tris Plus Gels ; after the run, we transferred proteins onto a nitrocellulose membrane and blocked them with 5% skim milk . To detect target proteins, we incubated membrane overnight at 4 °C with specific primary antibodies. Then, the membranes were washed with TBS plus 0.1% Tween-20 and incubated with IRDye-conjugated secondary antibody (LI-COR Biosciences) for 20 min. Finally, the membranes were washed to remove any excess secondary antibody and acquired using an Odyssey Infrared Imaging System (LI-COR Biosciences). Densitometric analysis was performed using the software ImageJ .RNA extraction was performed from VICs using the Total RNA Purification Plus Kit , according to the manufacturer’s instructions. Briefly, cells were lysed in RL buffer, and gDNA was removed by a “gDNA removal column”; then, RNA was purified by an RNA extraction column, washed with ethanol 100%, and diluted in “elution solution A”. Next, 10 µg of total RNA underwent DNase treatment using a TURBO DNA-free™ Kit , following the manufacturer’s instructions. RNA obtained was then precipitated in pure ethanol and ammonium acetate (125 mM), washed with ethanol 70%, and resuspended in deionized water, to ensure purity before being quantified by Nanodrop One. Total RNA (1 μg) was converted into cDNA, using Luna RT supermix . Real-time PCR (qPCR) was carried out on an AB Prism 7900 HT (Applied Biosystems), according to the manufacturer’s instructions; analysis was performed using software SDS2.4 .4Cl 50 mM to break down cell auto-fluorescence, permeabilized in saponin 0.2%, and resuspended in 100 μL of PBS with 5 mM ethylenediaminetetraacetic acid (EDTA), 1% bovine serum albumin (BSA), and 0.2% saponin. Then, cell samples were incubated for 30 min at room temperature using specific conjugated-antibodies against CD90–phicoeritrin , vimentin–AlexaFluor 488 , and DRAQ5 , a nuclear stain. For the P2RY2 staining, cells were detached and incubated in the same way, then stained with anti-P2RY2 (Santa Cruz Biotechnology) for 30 min, and conjugated with an anti-rabbit–AlexaFluor 647 for 30 min. Samples were then washed with FACS buffer (PBS containing 5 mM EDTA and 1% BSA) by centrifugation for 5 min at 600× g. Finally, cells were resuspended in FACS buffer and analyzed. A total of 30,000 events were acquired in the single cells and focus gated area. Image analysis was performed using the IDEAS 6.2 software.VIC characterization was performed by an ImageStream X flow cytometer, which combines flow cytometry with microscopy technology . VICs were detached through TripLE Select , an enzymatic method useful to preserve the extracellular epitopes of proteins on the cell surface. Then, we fixed cells in paraformaldehyde, incubated in NHThe capillary Western blot was performed using WES for Simple Western . Briefly, a total of 2 μg of protein was diluted in fluorescent master mix 5×, sample buffer 10×, and DTT, following the manufacturer’s instructions. Then, samples were thermically reduced at 95 °C for 5 min and loaded in a separation module 12–230 kDa . The running protocol was the standard one. To detect P2RY2 expression, we used anti-P2RY2  and anti-rabbit HRP-conjugated antibody . Image analysis was performed using Compass for Simple Western software.t-test or one-way ANOVA. A p-value < 0.05 was deemed statistically significant.All continuous variables are represented as the mean ± standard error of mean (SEM), while categorical are represented as percentages (%). Statistical significance was evaluated with GraphPad Prism . Variables were analyzed by Student’s n = 15), and protein, analyzed by capillary Western blot (WES). WES evaluation revealed three different bands at 50, 58, and 65 kDa, probably due to post-translational modifications of P2RY2 [p < 0.0001; n = 12; n = 6) and AS patients , with no statistical differences between the two groups . Under continuous pro-calcific stimulus, the activation of P2RY2 led to a mild reduction in VIC extracellular calcification in AVSc patients  and AS patients .To verify the extent of extracellular calcification, we employed an in vitro assay with ascorbic acid  and inorganic phosphate , widely recognized pro-calcific stimuli. The calcification was induced for 7 days, shown in Taken together, these data suggested that the activation of P2RY2 by 2ThioUTP prevents the extracellular calcium deposition in pathological VICs even under continuous stressed conditions.n = 3; 2(FC) vs. untreated; p = 0.07). All other screened genes were downregulated by 2ThioUTP: collagen 3A1 (COL3A1 −0.42 ± 0.2 log2(FC); p = 0.03), transforming growth factor β (TGFβ1 −0.53 ± 0.1 log2(FC); p = 0.0006), TGFβ2 (−0.48 ± 0.2 log2(FC); p = 0.02), and actin α2 (ACTA2 −1.28 ± 0.3 log2(FC); p = 0.002). Since the upregulation of ACTA2 mRNA and, thus, αSMA is a widely recognized marker of VIC activation (myofibroblast-like phenotype), we evaluated the protein expression. We found that αSMA expression was significantly downregulated by 2ThioUTP treatment  for 5 days in normal medium. We evaluated the expression of several pro-fibrotic genes by qPCR, and we found that 2ThioUTP treatment was able to significantly reduce their expression n = 3; a. In parAll these data corroborate the hypothesis that P2RY2 stimulation inhibits myofibroblastic gene and protein expression in AS patients.Aortic valve replacement is one of the most common procedures performed in cardiac surgery, and there is no available pharmacological approach for CAVS treatment able to revert the accumulation of ectopic calcific nodules. For this reason, it is imperative to investigate new pharmacological targets capable of blocking CAVS progression and possibly reverting the pathological phenotype. The activation of P2RY2 by 2ThioUTP has been reported to prevent extracellular calcification, and its administration has already been tested in a mouse model, proving its safety  . The samFurther studies are needed to better investigate the molecular pathway activated by 2ThioUTP and to prove its antifibrotic effects in vivo. In this regard, the multi-omics approaches implemented in the last few years to study the CAVS progression ,17,29 coIn conclusion, our findings reveal the potential therapeutic effects of 2ThioUTP in CAVS patients, even at the early stage of the pathology. Its ability to prevent calcium deposition could be of interest to fill the absence of pharmacological treatments for patients with AVSc and avoid the CAVS onset, thus reducing the need for more invasive approaches such as surgery or transcatheter aortic valve replacement. Moreover, these data could also be of high importance for the future development of tissue engineering, considering its capacity to maintain the inactive form of CAVS VICs.


Skeletal muscle atrophy, resulting from states of hypokinesis or immobilization, leads to morphological, metabolic, and functional changes within the muscle tissue, a large variety of which are supported by the stromal cells populating the interstitium. Telocytes represent a recently discovered population of stromal cells, which has been increasingly identified in several human organs and appears to participate in sustaining cross-talk, promoting regenerative mechanisms and supporting differentiation of local stem cell niche. The aim of this morphologic study was to investigate the presence of Telocytes in the tibialis anterior muscle of healthy rats undergoing an endurance training protocol for either 4 weeks or 16 weeks compared to sedentary rats. Histomorphometric analysis of muscle fibers diameter revealed muscle atrophy in sedentary rats. Telocytes were identified by double-positive immunofluorescence staining for CD34/CD117 and CD34/vimentin. The results showed that Telocytes were significantly reduced in sedentary rats at 16 weeks, while rats subjected to regular exercise maintained a stable Telocytes population after 16 weeks. Understanding of the relationship between Telocytes and exercise offers new chances in the field of regenerative medicine, suggesting possible triggers for Telocytes in sarcopenia and other musculoskeletal disorders, promoting adapted physical activity and rehabilitation programmes in clinical practice. The skeletal muscle is a highly dynamic and plastic tissue that promptly responds to physical activity and sedentary behavior . SkeletaAlexander Mauro described, for the first time, in 1961, a population of mononucleated cells which had been called “satellite cells” (SCs) by virtue of their localization: underneath the surrounding basal lamina and outside the plasma membrane of the muscle fiber they are associated with . These cLastly, another type of cell, recently identified in the muscle interstitium, is represented by the so-called “Telocytes” (TCs), which appear to physically reside near satellite cells, nerve, and microvascular network . The disIt is noteworthy to mention that this discovery raised skepticism and controversy, since ICLCs were also described merely as CD34-positive stromal cells acting as stem cells during regeneration processes ,40, or tAlthough it is still unclear, the role of TCs seems to participate in sustaining cross-talk communication between stromal cells through signaling transmission via exosomes ,45, secrFinally, the aim of this morphologic study was to investigate the presence/absence of TCs in tibialis anterior muscle of healthy rats who underwent a protocol of endurance training for either 4 weeks or 16 weeks in comparison to sedentary rats who were inactive, i.e., not engaging in any physical exercise, throughout the duration of the experiment.All the procedures involving alive animals were performed at the Center for Advanced Preclinical In Vivo Research (CAPIR), University of Catania. The guidelines of the Institutional Animal Care and Use Committee (I.A.C.U.C.) of the University of Catania  have been complied with. Animal care and handling were carried out in accordance with the EU Directive 2010/63/EU, as well as the Italian law (D.Lgs. 26/2014).Two-month-old healthy female Wistar outbred rats, with a bodyweight of 200 ± 20 g, were purchased from Charles River Laboratories, Milan, Italy and bred in the animal facilities at the University of Catania. Rats were maintained and kept in polycarbonate cages (10.25″ W × 18.75″ D × 8″ H) in stable hygrometric and thermic conditions (20–23 °C) on 12 h light/dark cycle with ad libitum access to water and food, throughout the whole period of the experiment. It was used a standard rat chow: carbohydrates (40%), proteins containing all essential amino acids (30%), and lipids (30%). Lipids were a mixture of neutral fatty acids, saturated fatty acids, and unsaturated fatty acids. Diets were provided by Laboratorio Dottori Piccioni, Gessate (Milan), Italy. Twenty rats were used in this study equally divided into two groups, sedentary and undergoing physical exercise, respectively CTRL and PA, sacrificed at two different time points, 4 and 16 weeks: CTRL4W, control sedentary rats sacrificed at 4 weeks; PA4W, rats performing physical exercise sacrificed at 4 weeks; CTRL16W, control sedentary rats sacrificed at 16 weeks; PA16W, rats performing physical exercise sacrificed at 4 weeks. All animals were randomly distributed to groups. All efforts were made to minimize the number of mice, according to the principles of the 3Rs and the resource equation approach, and reduce their suffering . ThroughTwo groups of rats (PA4W and PA16W) performed physical activity in the form of running on a treadmill  . Rats weTibialis anterior muscle samples were washed in phosphate-buffered saline , fixed in 10% buffered-formalin  for 24 h at room temperature. Afterwards, the samples were dehydrated in graded ethanol , cleaned in xylene  and paraffin-embedded , being careful to preserve the desired anatomical orientation. Slides of 5 μm thickness were cut from the obtained paraffin blocks and hematoxylin and eosin-stained  following a protocol described elsewhere . The sam2, using a software for image acquisition  [Each H&E stained muscle cross section was subjected in triplicate to morphometric analysis by calculating the area of twenty muscle fibers of five randomly selected fields with a total area of about 35.000 μmGermany) . Data we2O2/PBS, for 30 min, to block endogenous peroxidase activity. Non-specific antibody binding sites were blocked by applying a solution of 1% bovine serum albumin  in PBS 1X for 1 h at room temperature. Tissue slides were washed in PBS and, then, incubated overnight at +4 °C with a mixture of mouse and rabbit primary antibodies at appropriate dilution in antibody dilution buffer: mouse monoclonal anti-CD34 , rabbit monoclonal anti-CD34 , rabbit polyclonal anti-CD117 , mouse monoclonal anti-Vimentin . Primary antibodies were revealed using specific fluorescent-dye Goat anti-Mouse Alexa Fluor 488-conjugated IgG  and Goat anti-Rabbit 594-conjugated IgG  for 1 h at room temperature. Details on primary and secondary antibody sources and dilutions are shown in Paraffin-embedded muscle tissue sections of 5 μm thickness were subjected in triplicate to double immunofluorescence (IF) combining anti-mouse and anti-rabbit goat secondary antibodies with either mouse or rabbit primary antibodies. Muscle sections were deparaffinized with xylene and rehydrated in graded ethanol scale. Afterwards, the slides were cleaned for 20 min with phosphate-buffered saline  and unmasked in citrate buffer , or in ethylenediaminetetraacetic acid-Tris buffer  for the antigenic retrieval and incubated in 0.3% H2)/(pixel2) of double immunostaining on muscle tissue. Three blinded investigators  made the evaluations that were assumed to be correct if values have not statistically significant difference. If disputes concerning interpretation occurred, unanimous agreement was reached after sample re-evaluation.Digital micrographs of double immunofluorescence sections were taken using a confocal laser scanning microscopy  with ZEN-2010 software. In order to detect the fluorophore signal, three lasers with 405, 488, and 555 nm wavelengths were used for the analysis of blue, green, and red signals, respectively. Image analysis software , which quantifies the level of double positive staining of anti-CD34/anti-CD117 and anti-CD34/anti-Vimentin immunolabeling, was used to calculate the densitometric count in five fields, 10× magnification, randomly selected from each section. Statistical results are expressed as densitometric count . After blocking, the sections were incubated overnight at 4 °C with rabbit monoclonal anti-CD34 . Immune complexes were then treated with biotinylated link antibodies (horseradish peroxidase polymer (HRP)-conjugated anti-rabbit and anti-mouse were used as secondary antibodies) and then detected with peroxidase-labelled streptavidin, both incubated for 10 min at room temperature . Immunoreactivity was visualized by incubating the sections for 2 min in 0.1% 3,3′-diaminobenzidine (DAB) . The sections were lightly counterstained with Mayer’s hematoxylin , mounted in Glycerol Vinyl Alcohol (GVA) , observed with an Axioplan Zeiss light microscope , and photographed with a digital camera .Skeletal muscle samples, 5 μm-thick, were processed for immunohistochemical analysis. Briefly, the slides were dewaxed in xylene, hydrated using graded ethanols, and therefore heated (5 min × 3) in capped polypropylene slide-holders with citrate buffer—pH 6 , using a microwave oven  to unmask antigenic sites. The slides were incubated for 30 min in 0.3% H® Biostatistics version 8.0 software . The sample size calculation for this study was established using the resource equation approach, including minimum and maximum sample sizes, because it was not possible to assume standard deviation and effect size [p-values of less than 0.05 (p < 0.05) were considered statistically significant; p values of less than 0.01 (p < 0.01) were considered to be highly statistically significant. The data are presented as the mean value ± SD. Cohen’s κ was applied to measure the agreement between the three blinded observers and averaged to evaluate overall agreement.Statistical analysis was performed using GraphPad Instatect size . Data wep > 0.05), as expected  and PA4W (27.47 ± 1.61 μm) (* p < 0.001) vs. CTRL16W (21.39 ± 2.19 μm). PA16W (27.47 ± 1.61 μm) also shows a predictable hypertrophy when compared to CTRL4W (24.01 ± 1.57 μm), (* p < 0.001). On the contrary, PA16W (30.46 ± 1.43 μm) does not show a statistically significant hypertrophy when compared with group PA4W (27.47 ± 1.61 μm), (ns)  (mean ± SD) of the muscle fibers highlights a significant hypertrophy of the groups PA16W (30.46 ± 1.43 μm) (** m), (ns) . No sign2)/(pixel2) of double immunostaining on muscle tissue. CD34 and CD117 double labeling analysis indicates a statistically significant increase in the expression of TCs in PA16W (7.7 × 10−4 ± 1.3 × 10−4 (pixel2)/(pixel2)) vs. CTRL16W (3.9 × 10−4 ± 2 × 10−4 (pixel2)/(pixel2)) (* p < 0.05) (−4 ± 1.4 × 10−4 (pixel2)/(pixel2) and 6.1 × 10−4 ± 2.5 × 10−4 (pixel2)/(pixel2). Similarly, the number of interstitial TCs is highly significant higher in PA16W (1.3 × 10−3 ± 3.1 × 10−4 (pixel2)/(pixel2)), vs. CTRL16W (6.9 × 10−4 ± 1 × 10−4 (pixel2)/(pixel2)) (** p < 0.01) as determined by CD34/VIM double-immunostaining (−3 ± 2.6 × 10−4 (pixel2)/(pixel2) and 9.3 × 10−4 ± 2.5 × 10−4 (pixel2)/(pixel2). No other statistically significant differences are highlighted between groups, in both experiments.Although transmission electron microscopy (TEM) examination is the golden standard for TCs identification ,61, doub < 0.05) B. Densitstaining B. DensitSkeletal muscle sections showed CD34+ cells at the periphery of the fibers, within the interstitium between muscle fibers . These cTCs have been largely identified, over the last ten years, as populating the stromal compartments of a variety of organs, belonging to the tissue stem cell niche ,49,65. WIn the present morphological study, the presence of TCs in tibialis anterior muscle of healthy rats who underwent a protocol of endurance training for either 4 weeks or 16 weeks was investigate in relation to sedentary rats who were inactive, i.e., not engaging in any physical exercise, throughout the duration of the experiment.p < 0.0001) and 4 weeks (* p < 0.001) (p > 0.05) in body weights in all groups, indicating a physiological growth of the rats during the weeks, as expected  when compared to physically active rats undergoing treadmill training for both 16 weeks (** < 0.001) . Muscle expected . Whether atrophy may affect muscle stem cells numbers or behavior is still controversial and should be further elucidated to assess the role of daily mechanical stress administered through exercise . MitchelAlthough SCs are the main characters of renewal programme in skeletal muscle, other tissue residents and recruited stromal cells, e.g., fibroblasts, fibro-adipogenic progenitors, endothelial cells, perycites, and macrophages, are paramount supporting players ,22,69. AThis study provides a novel finding that interstitial TCs are decreased in the muscles of sedentary rats compared to exercised rats, supporting the recent reports that TCs are, otherwise, increased in exercise-induced cardiac growth , particip < 0.05) (p < 0.01)  surface but it has also been found in other tissue-specific stem cells . Vimenti < 0.05) . This re < 0.01) . These rFinally, immunohistochemistry showed CD34+ cells, within the interstitium between muscle fibers , with chThese findings herein are intended to encourage knowledge about TCs population and their role in the stem cells niche of skeletal muscles. Further studies investigating TCs in response to different types of exercise , sedentary behavior, ageing, and pathophysiological conditions, as well as studies on the possibility of triggering TCs through exercise to reverse atrophic conditions, would be scientifically valuable. This study may be framed in a field still in its infancy, although it is rapidly attracting the attention of the scientific community. The understanding of the above-mentioned mechanisms between the cells involved in the tissue remodeling process could offer new chances in regenerative tissue strategies and insights about finding possible triggers for TCs in sarcopenia and other musculoskeletal disorders, in clinical medicine.Finally, since exercise training has been shown to exert protective effects against sedentary-induced atrophy, rather by sustaining muscle remodeling and maintenance of TCs, this finding might promote further skeletal muscle adapted physical activity and rehabilitation programmes for humans.


The proportion of patients with high on-treatment platelet reactivity  dropped from 23.5 to 10% (p < 0.001). Female sex was associated with a greater PRU reduction with prasugrel, whereas HPR at baseline, age ≥ 65 years, and body mass index ≥ 25 best predicted HPR at Week 4. Throughout the 48-week treatment with prasugrel, the incidences of MACE (1.0%) and TIMI major bleeding (2.0%) were rather low, accompanying an acceptable safety profile of TIMI minor (6.4%) and non-major, non-minor clinically relevant bleeding (3.0%). Overall, switching to the maintenance dose of prasugrel (3.75 mg) was observed to be effective and well tolerated among post-PCI ACS patients in Taiwan. Clinical Trial Registration Number: NCT03672097.The recommended maintenance dose of prasugrel for East Asian populations  is 3.75 mg as part of dual antiplatelet therapy (DAPT) for the prevention of recurrent ischemia and stent thrombosis in acute coronary syndrome (ACS). This modified dosage regimen has been established in studies conducted in Japan; however, the efficacy and safety of switching from clopidogrel to prasugrel DAPT among Taiwanese patients remain to be explored. In this phase IV, multicenter, single-arm, open-label study, we evaluated the 4-week pharmacodynamic response, and the 48-week safety outcomes of prasugrel 3.75 mg after a switch from clopidogrel in Taiwanese ACS patients. A total of 203 prasugrel-naïve ACS patients  who had received post-PCI clopidogrel DAPT for at least 2 weeks were enrolled from ten medical centers in Taiwan and subsequently switched to prasugrel 3.75 mg DAPT. Four weeks after the switch, P2Y12 reaction unit (PRU) values were significantly decreased in the total cohort (mean − 18.2 ± 48.1; 95% confidence interval − 24.9 to − 11.5, The online version contains supplementary material available at 10.1007/s12928-021-00771-w. CYP2C19 loss-of-function mutations, compliance issues, adverse events, cost, and drug availability)  criteria to extrapolate the study results to data from other investigators. The ARC-HBR criteria, an identifier of high bleeding risks based on characteristics of ACS patients at the time of PCI, was adopted for exploratory analyses in patient characteristics and safety outcomes –14.® system , the method of which has been published previously [PRU was assessed with the VerifyNoweviously . Based oeviously –18.α) and an 80% power.Based on the PRU results from a prior study , we postDescriptive analyses were applied for baseline characteristics using means and SD for continuous variables, and frequencies and percentages for categorical variables.t tests and Wilcoxon signed-rank tests were used where appropriate. For secondary endpoints, McNemar’s test was applied for comparing PRU values.The primary analyses were the PRU values at and the PRU differences between baseline and Week 4. These were described by the number of analyzed cases, means, SD, and the 95% confidence intervals (CIs) in both total and prespecified subgroups. The secondary analysis was HPR at baseline and Week 4, which was expressed as frequencies/percentages. Both primary and secondary analyses were tested for statistical significance. For primary endpoint, p values. Statistical analyses were performed using SAS® version 9.4.We adopted an analysis of covariance (ANCOVA) model and a multivariate logistic regression model to explore factors associated with change from baseline PRU values and with HPR at Week 4 (PRU > 208 as the exploratory cutoff), respectively. In both models, factors were step-wisely selected from the following variables: age, sex, body mass index, compliance, diabetic status, lipid disorder status, and decreased GFR status (eGFR < 60). A probability of < 0.2 during the stepwise ANCOVA further determined the entering and staying of a variable in the model. For factors selected into the final models, adjusted least squares (LS) means with 95% CI, or adjusted odds ratios (ORs) with 95% CI were shown along with the respective The primary safety endpoint, non-CABG-related TIMI major bleeding for 28 weeks  was evaluated using Kaplan–Meier estimates. Secondary safety endpoints were the incidences of events  during the 4-week maintenance dose of prasugrel (3.75 mg) treatment (Period 1), and the incidences of events  during the 28-week  maintenance dose of prasugrel (3.75 mg) (Period 2). Additional safety endpoints included the incidences of AEs and deaths were also recorded. Bleeding events by Bleeding Academic Research Consortium (BARC) classification were also included in the exploratory analysis. Major and minor bleeding events according to BARC criteria were analyzed and summarized as pre-treatment AEs and treatment-emergent AEs (TEAEs) for Study Period 1 and Period 2. The incidence of BARC type 2, 3 or 5 bleeding was analyzed using Kaplan–Meier estimates, and the association between these events and prognostic factors was analyzed using a multivariate logistic model. The association between the presence of at least one net adverse clinical event  and prognostic factors was evaluated using a multivariate logistic model. Additional safety analyses by prespecified subgroups included the incidences of TIMI major bleeding, BARC type 2, 3 or 5 bleeding and MACEs using Kaplan–Meier estimates, by HPR (PRU ≥ 208 versus PRU < 208) at Week 4, and their Academic Research Consortium-High Bleeding Risk (ARC-HBR) (HBR versus non-HBR) status.Among the 203 patients who were enrolled and received treatment, 200 completed the 4-week initial trial Period 1). Three withdrew informed consent and one withdrew because of a treatment-emergent AE , and all Asian in ethnicity  and 85.2% as non-HBR.p < 0.001; Fig. p = 0.002) and those with NSTEMI  rather than in those with UA . The reported mean baseline PRU levels were 153.8 ± 53.8 in STEMI, 168.6 ± 67.6 in NSTEMI and 149.3 ± 56.6 in UA patients. PRU decreases were significant in both male and female patients  of 88. After 4 weeks of treatment, the graph was shown to be narrower, with a median PRU of 142 and an IQR of 67, indicating the pharmacodynamic effects of the shift from clopidogrel to prasugrel among treated patients . To facilitate the exploratory risk factor analysis, a more stringent PRU cutoff (PRU > 208) was utilized. The result remained consistent at a cutoff PRU of 208—where the percentage of patients with HPR dropped from 23.5% at baseline to 10.0% by the end of Period 1 (p < 0.001).Accordingly, the percentage of patients with HPR, as defined by PRU > 235 per protocol, significantly decreased from 11.3% at baseline to 3.0% by the end of Period 1 , diabetic status (p = 0.091), and BMI < 25 (p = 0.136). With the multivariate logistic regression model, we identified that HPR (PRU > 208) at baseline best predicted HPR (PRU > 208) at Week 4 . In addition, BMI ≥ 25  and age ≥ 65  were also significant predictors of HPR at Week 4  had at least one TIMI major bleeding event during Period 2 with an increase in cumulative incidences from 0.5% at Week 28 to 2.5% at Week 48. Thirteen patients (6.4%) reported at least one treatment-emergent TIMI minor bleeding event; six (3.0%) had at least one treatment-emergent, non-major, non-minor, clinically relevant bleeding event, and two (1.0%) reporting treatment-emergent MACEs . The cumulative incidences at Week 48 were 7% for TIMI minor bleeding; 3.1% for non-major, non-minor clinically relevant bleeding; and 1.1% for MACE.p = 0.1846, log-rank test). There were similar rates of BARC type 2, 3, and 5 bleeding in the high bleeding risk (HBR) subgroup (9.1%) versus non-HBR (7.7%) patients and no difference in cumulative incidences between HBR subgroups . All four cases with TIMI major bleeding events were found in non-HBR patients.Among all patients, 2.5% of patients reported BARC type 3 and 5 bleeding; 5.9% reported any BARC type 2 bleeding; and 7.9% reported any BARC type 2, 3, and 5 bleeding. There were numerically more BARC type 2, 3, and 5 bleeding events in non-HPR patients at Week 48 (8.9%) versus HPR patients (0%), but the difference in cumulative incidences between the HPR subgroups was not statistically significant (This is the first study to demonstrate the antiplatelet efficacy and safety of switching from clopidogrel to maintenance dose of prasugrel (3.75 mg) among Taiwanese ACS patients who have undergone PCI. The Switch Study population is comparable to prior Japanese ACS cohorts reported in literature . In addip = 0.056). The smaller sample size of UA patients relative to the entire group of patients with myocardial infarction may have led to this observation. Only 3.0% of study subjects were found to have HPR (per the protocol definition) at Week 4, leading us to adopt the more stringent post-switch HPR definition (PRU > 208) in the exploratory analysis. Even at the more stringent level, only 10% of the study population had Week 4 PRU > 208, which was associated with HPR at baseline, age ≥ 65 and BMI ≥ 25 . These were comparable to bleeding outcomes in PRASFIT-ACS, including TIMI major bleeding (1.9%); non-major, non-minor clinically relevant bleeding (4.2%); and TIMI minor bleeding (3.9%); whereas rates were lower in the Switch Study in terms of BARC 2, 3, and 5 bleeding (8.4% vs 17.0%) [Throughout study Period 1 and 2, both the occurrences of MACE (1.0%); TIMI major bleeding (2.0%); and BARC 2, 3, and 5 bleeding (7.9%) were relatively low, accompanied by acceptable TIMI minor (6.4%) and non-major, non-minor clinically relevant bleeding (3.0%) among the prespecified subgroups  . One patDespite the lower prevalence of HBR cases in our study cohort compared with prior studies , we obseCYP2C19 loss-of-function carriers in East Asians, this study provides meaningful snapshots of pharmacodynamic responses of both prasugrel and clopidogrel in Taiwanese patients with ACS treated with PCI. The study proved that maintenance dose of prasugrel delivers significant PRU reduction after a switch from clopidogrel, and at the same time preserves the modest safety profile as revealed in the 48-week observation. This provides a choice for Taiwanese physicians who face the dilemma to balance between high ischemic  and bleeding  risks, as well as other conditions . Partly concordant with the post hoc risk factor analysis performed in PRASFIT and PRASFIT-Practice I and II studies [Given the high prevalence of  studies , 24, we  studies , 25–28; As a result of the study design, our safety observation only started at the point of switching until the end of treatment . However, pre-switching safety outcomes have been included in our analysis for the purpose of comprehensiveness. Several previous studies reported demographic factors that may be predictive of P2Y12 inhibitor antiplatelet effects –31, but Switching to prasugrel induced a significant PRU reduction beyond that achieved by maintenance dose of clopidogrel (75 mg) while maintaining a good safety profile and high compliance. Similar to PRASFIT-ACS, the current results suggest that switching to a maintenance dose of prasugrel (3.75 mg) shows good long-term reduction of platelet reactivity across patient populations, without evidently increasing the risk of excessive bleeding and inconsistent antiplatelet effects in post-PCI patients with ACS.Supplementary file1 (DOCX 193 kb)Below is the link to the electronic supplementary material.


Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition.IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms. Iron is a vital element required by every living organism that functions as a cofactor in numerous cellular processes, such as the tricarboxylic acid cycle, electron transport chain, and oxidative phosphorylation, or the biosynthesis of vitamins, antibiotics, toxins, and other Fe-containing aromatic compounds . It also3+ ligands; thus, they can be classified as hydroxamate, hydroxycarboxylate, or catecholate type siderophores, respectively  based on its antifungal activity against agronomically important fungal phytopathogens    , and tomacearum) . More regar beet  and tomagar beet , 32. Seresponses , includiesponses .B. amyloliquefaciens MBI600 to produce and secrete the catecholate siderophore bacillibactin is demonstrated. In parallel, bactericidal efficacy evaluation experiments showed that iron starvation induced production of bacillibactin, which significantly enhanced, in vitro and in planta, the bactericidal activity of MBI600 against the phytopathogenic species Pseudomonas syringae pv. tomato. It also broadened or enhanced the antibiotic activity against phytopathogenic fungi. Finally, it is shown that under iron starvation conditions, the expression of genes involved in bacillibactin biosynthesis is coordinated with that of genes involved in the synthesis of secondary metabolites as well as with antibiotic activity, cell mobility, and chemotaxis.In this study, the ability of 3+ stress levels  using the chrome azurol S (CAS) assay (3+ (max of 510 nm in the supernatants with 0 μM Fe3+), as shown in We screened for siderophore production in the supernatants of MBI600 cultures grown in chemically defined low-iron medium (CDLIM)  at varioS) assay . Under sssay . Optical densities at 600 nm were measured for 48 h at 37°C using a multidetection microplate reader and automatically recorded for each well every 29 min. Each ribbon depicts growth under a specific Fe3+ concentration; full line represents mean values and bars represent SEs from three independent biological experiments. Download FIG S7, EPS file, 0.5 MB.Copyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 3+ stress conditions (0 μM Fe3+) was verified through a two-stage reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled with nuclear magnetic resonance (NMR) analysis. A clear peak at 315 nm and 19.72 min, corresponding to bacillibactin standard, was detected in the chromatograms of the first preparative column and only in the 0 μΜ Fe3+ supernatants  The corresponding NMR spectra. Download FIG S1, EPS file, 0.3 MB.HPLC chromatograms of the purified peak of bacillibactin from 0 μM FeCopyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 1H and 13C NMR spectroscopy. In the 1H one-dimensional (1D) NMR spectrum of the purified peak of 0 μM Fe3+ supernatant extract  (A) and 1H-13C HSQC-DEPT  (B) NMR spectra of bacillibactin extracted from 0 μM Fe3+ supernatants of MBI600, in deuterated methanol, at 305 K. In HSQC-DEPT spectrum, methylene protons are colored red, while methine and methylene protons are black. Download FIG S2, EPS file, 0.1 MB.Copyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In vitro bactericidal efficacy of cell-free culture filtrate.(i) P. syringae pv. tomato was found to be marginally nonsusceptible to MBI600 in broth and plate susceptibility testing  extracted from cultures grown under a range of iron starvation conditions  . It was only negatively affected by the 25% treatment with MBI600 CFCF extracted from iron-limiting conditions (0 μM and 18.4 μM Fe3+). However, under all iron conditions, except 18.4 μΜ Fe3+ , results were not statistically significant compared to those for control (740 mM) and mock (LB) conditions, at both time points tested  or mock  conditions. The effect of iron starvation was also confirmed in experiments using a different carbon source (succinic acid)  . P. syris tested . The 50%cid) see  to exclulawn see . The inh10.1128/mSphere.00376-21.3FIG S3In vitro bactericidal efficacy of B. amyloliquefaciens MBI600. (Left) P. syringae pv. tomato broth susceptibility was tested against increasing MBI600 cell free culture filtrate (CFCF) concentrations . CFCF was extracted from MBI600 cells grown in LB medium. Observed effects were evaluated based on OD600 readings at 0, 24, and 48 hpi in microtiter plates. Estimated marginal means and their standard errors are plotted. (Right) No inhibition zone was formed around MBI600 culture grown on 9-mm Whatman discs that were placed on agar medium inoculated with P. syringae pv. tomato. Download FIG S3, EPS file, 1.4 MB.Copyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00376-21.4FIG S4Pseudomonas syringae pv. tomato (Pto) in LB medium supplemented with 10% (a), 25% (b), or 50% (c) B. amyloliquefaciens (Bam) CFCF (cell-free culture filtrate) supplemented with different iron concentrations . Optical densities at 600 nm were measured for 48 h at 28°C using a multidetection microplate reader. Estimated marginal means and their standard errors are plotted. Download FIG S4, EPS file, 2.4 MB.Effect on growth of Copyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00376-21.5FIG S5Pseudomonas syringae pv. tomato (Pto) agar cultures in the presence of B. amyloliquefaciens (Bam). Pto inoculum suspension was adjusted to 109 CFU/ml and allowed to grow in agar medium containing increasing iron concentrations . Values represent the averages of 24 inhibition zone measurements for each iron concentration. Different letters represent statistically different data points at a P value of ≤0.05 according to Tukey’s post hoc comparisons. Download FIG S5, EPS file, 0.7 MB.Inhibition zones formed in Copyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. syringae pv. tomato. Inhibition clear zones were observed and measured at 0.39 and 0.47 cm in the case of 0 and 18.4 μM Fe3+ conditions, respectively . Both treatments leading to bacillibactin production  led to statistically significant bactericidal efficacy compared to that with both 370 and 740 μM Fe3+ and controls.We tested the bactericidal efficacy of ethyl acetate siderophore extracts in agar cultures of ectively . When thIn planta bactericidal activity of CFCF was based on P. syringae pv. tomato total population in leaves of tomato plants sprayed with MBI600 CFCF over a period of 16 days postinoculation (dpi). At 1 dpi, the P. syringae pv. tomato population was significantly reduced after treatment with CFCF from iron-depleted media (0 and 18.4 μM Fe3+) compared to that in the controls (370 and 740 μM Fe3+ and the succinate control). Such differences were amplified at 4 dpi and persisted for several days before disease development  at the 50% (vol/vol) dose (nominated LB treatment). Susceptibility of all species was then tested against three doses/treatments  of CFCF extracted from MBI600 cultures grown under 18.4 or 740 μΜ Fe3+ conditions as well as the LB treatment. Effects were attributed to bacillibactin only if comparisons between the 18.4 μM Fe3+ condition and each of the three controls  were statistically significant. We found that iron starvation resulted in an inhibition of fungal growth, which was positively correlated with treatment dose and time  or LB treatments but not with 50% 740 μM Fe3+ , we selected 7 strains of nonsusceptible fungal species see  as well and time . Fungal nd 7 dpi . In the Fe3+ see .10.1128/mSphere.00376-21.6FIG S6+3 (ref) or 18.4 μM Fe+3 conditions, and 50% CFCF of MBI600 grown in LB. Values represent the estimated marginal means and corresponding standard errors of inhibition zone measurements. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ·, P < 0.1 according to Tukey’s post hoc comparisons. Download FIG S6, EPS file, 0.9 MB.Iron starvation expands the range of antifungal activity of MBI600. Radial growth of 4 fungal species in minimal medium (MM) agar plates and in response to the following treatments: no treatment (control), 10% and 50% CFCF of MBI600 grown under either 740 μM FeCopyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00376-21.8TABLE S1Table S1, DOCX file, 0.1 MB.List of bacterial, fungal, and oomycete strains used in this study. Download Copyright © 2021 Dimopoulou et al.2021Dimopoulou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the dhbC) is activated in iron-depleted cultures found in late static phase (48 h) but not under the 740 μM Fe3+ condition (3+ exceeds the threshold for iron deficiency (B. amyloliquefaciens competence (comK), chemotaxis (cheC), and swarming (swrA) were significantly downregulated under all conditions compared to expression in the control at 24 h postinoculation (hpi). At 48 hpi, all 3 genes showed a significant upregulation under all iron conditions compared to expression in the control (baseline log2 fold change [FC])  and remained elevated at 48 hpi only in the case of the 0 μM Fe3+ condition. The upregulation of yczE occurred later (48 hpi) under all conditions compared to expression in the control, while the regulatory gene degU was not activated upon iron depletion.Using the aforementioned iron-depleted media, we showed that bacillibactin synthase  . A 2-folBacillus amyloliquefaciens is known to produce a broad range of antimicrobial metabolites  (Pseudomonas syringae pv. tomato and that its antibiotic function is intercellular.The combination of agar and broth tests suggested that avenging . We suggophores) . It is, in vitro, with the maximum correlation to in planta outcomes, it is necessary to test them in different minimal media in which both BCA and pathogens can grow. Such media would best mimic conditions of the nutrient-poor phyllosphere but also allow antagonists to fully exploit their antimicrobial arsenal. This hypothesis was confirmed in planta. Growth of MBI600 cultures under iron starvation conditions induced bacillibactin synthesis and secretion to a CFCF that was capable of reducing P. syringae pv. tomato population and delay the infection of tomato plants. Siderophores have already been hypothesized to contribute to the antifungal activity of B. subtilisin planta  genes involved in secondary metabolite synthesis  and (ii) comΚ (competition), cheC (chemotaxis), swrA (swarming), deqU (positive regulator of bacillomycin [iturin]), and yusV (probable siderophore transport system) confirmed the activation of the metabolic network responsible for B. amyloliquefaciens antibiosis, competence, and mobility.The enhanced susceptibility of Bacillus subtilis has a strong antifungal activity due to the synergistic actions of surfactin, bacillomycin D, and fengycin  and fengycin synthase C (fenC) was enhanced at 48 hpi. In support, yczE, a positive regulator of bacillomycin, was also activated at 48 hpi under low/depleted-iron conditions. These data suggest that MBI600 coordinately induces its offense to prevail against other organisms in iron-depleted habitats. Such a hypothesis is supported by the activation of comK during stationary phase, as previously reported  agar and tryptic soy broth  media at 37°C. Pseudomonas syringae pv. tomato was routinely grown on LB and nutrient agar (NA) media at 28°C for 48 h. Fungal and oomycete strains were grown on solid (12 g · liter−1 agar) minimal medium (MM) that consisted of salt solution supplemented with glucose (10 g · liter−1) and ammonium tartrate (1 g · liter−1) as carbon and nitrogen sources, respectively  used for growth medium and reagent preparation were cleaned, prior to use, with 6 N HCl to remove residual iron, rinsed with deionized water, and oven dried.−1): KHSO4, 1.5; K2HPO4, 3; NaCl, 1; and NH4Cl, 5; pH 6.5. The medium was supplemented with 2 mg · liter−1 thiamine and 2% (wt/vol) glucose as an energy source : CaCl2·2H2O, 100; MgSO4·7H2O, 80; ZnSO4·7H2O, 2; MnSO4, 0.0035; CuSO4, 0.005. CDLIM was further supplemented with various final Fe3+ concentrations  using FeCl3. The MBI600 inoculum for siderophore production  was prepared from 10 ml of an overnight culture in LB that was pelleted, washed once, and resuspended in 10 ml CDLIM. Flask cultures were incubated at 28°C and 200 rpm for 48 h. Cell mass was separated by centrifugation at 4,000 rpm for 15 min, and the supernatant was aspired for further treatment.Chemically defined low-iron medium (CDLIM) consisted of  method as described by Schwyn and Neilands  and quanA modified version of the siderophore extraction protocol developed by Patel et al.  was used18-10, 25 cm by 10 mm, 10 μm; SUPELCO Analytical) in 250-μl batches and a 4.5-ml/min flowrate. The elution gradient was applied follows: 0 to 10 min, 100% solution A (sol.A) ; 10- to 30-min linear gradient from 0% to 40% solution B (sol.B) . The principal sample peaks from the various runs that corresponded to bacillibactin standard (detection at 315 nm) were collected and concentrated to dryness in a rotary evaporator and resuspended in HPLC-grade methanol. The concentrate from the preparative column was further purified in 20-μl batches on an analytical column  at a flowrate of 0.8 ml/min. The elution gradient was applied as follows: 0 to 6.5 min with sol.A followed by a linear gradient (6.5 to 26.5 min) from 0% to 25% sol.B. Bacillibactin, eluted as a highly symmetrical peak, was collected, concentrated, and stored in HPLC-grade methanol at −20°C until NMR analysis.Bacillibactin was quantified and purified from the various culture supernatants through reverse-phase HPLC on an Agilent 1220 Infinity LC system using a two-step purification protocol and an HPLC-grade bacillibactin standard . The concentrated aqueous product from the ethyl acetate extraction was initially fractionated in a preparative column  was recorded for a spectral width of 10 ppm with 2,000 data points in F2 dimension and 150 increments of 32 repetitions each. 1H-13C single bond correlations were detected in a HSQC-DEPT spectrum  recorded with the standard parameters (1JH-X = 145) for spectral widths of 12 ppm for 1H and 185 ppm for 13C nuclei, 2,000 data points in F2 dimension, and 150 increments of 32 repetitions each. The solvent signal was used for axis calibration. Data manipulations were performed with the software TopSpin v3.1 .NMR spectra were recorded on a Bruker AVANCE III 600 spectrometer  operating at 600.11 MHz for B. amyloliquefaciens MBI600 cells were allowed to grow in LB or siderophore-producing medium, CDLIM or succinic acid  as previously described  containing each test concentration were also included in duplicates. Plates were incubated at 30°C for 48 h. Three independent biological experiments  were performed. Absorbance measurements were recorded at 0, 24, and 48 hpi at 30°C using triplicate readings from a multidetection microplate reader  at 600 nm.The escribed , 74. In 8 CFU · ml−1 and added to 180 ml LB agar before the mixture was poured into petri dishes and incubated overnight at 30°C. A 40-μl MBI600 suspension, adjusted to 5 × 106 cells, was then pipetted on 9-mm antibiotic assay discs (Whatman) that were placed on the agar cultures of each pathogen. As a control, sterile LB was used in place of the MBI600 suspension. Each plate was quadruplicated in 3 independent experiments and incubated at 30°C for 2 days before the diameter of the clear halo surrounding the disk was measured on three different axes. A similar protocol was developed to evaluate the effect of siderophore extracts, only this time, a 20-μl aliquot of concentrated siderophore extract was pipetted directly onto LB agar plates containing P. syringae pv. tomato pathogen suspension. As a control, a noninoculated liquid CDLIM for each Fe3+ concentration was used.To prepare dual cultures, 20 ml of overnight-grown naive stationary-phase cultures of each bacterial pathogen were adjusted to a concentration of 103 concentrations (18.4 or 740 μM Fe3+) were mixed with MM agar in ratios of 1:2 or 1:10 (vol/vol) and poured into petri dishes. MM agar was used as control. MM agar containing 10% or 50% MBI600 CFCF grown in minimal medium supplemented with 740 μM Fe3+ was used as the reference (ref). The technique of point inoculation was used for both treatment plates and controls  at 37°C and 200 rpm for 48 h. Total RNA was extracted from each sample using TRI reagent (Ambion) according to the manufacturer’s instructions. The concentration and purity of RNA were estimated using a P330 nanophotometer (IMPLEN). DNase-treated RNA  was reverse transcribed into cDNA using Superscript II reverse transcriptase and oligo(dT) as primers (Invitrogen). Reverse transcription-quantitative PCR (RT-qPCR) was performed as previously described  Broth microdilution method. Broth microdilution method analysis was performed using the method described in previous works  or mycelial growth  susceptibility, radial growth was modeled using linear mixed models (LMMs) in R language . In the in planta was assessed as previously described . The times after inoculation and treatment as well as their interaction were modeled as fixed factors, while plant individuals nested within a block comprised the random factor. Tukey’s tests were applied for post hoc comparisons of estimated marginal means.Pathogen growth escribed , 77. CFUTC) values were modeled in the context of a LMM and comprised the independent variables, while time, gene, Fe concentrations, and their interactions were included as the dependent variables. Samples nested within experiments were the random factors of the model. Contrasts were formulated according to equation 5 reported by Steibel et al. (gyrA) and reference treatment  represented the baselines of all relevant comparisons.RT-qPCR analysis was performed as previously described , 34. Thrl et al. , so that