V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2V617F mutation and its effects on downstream signaling pathways in cMPNs.Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes.Specimens of Jak2V617F positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2V617F positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2V617F-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest.Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2V617F cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2The online version contains supplementary material available at 10.1186/s12885-021-08915-0. V617F) is the most important pathogenesis of cMPNs [V617F mutation and DNMT3a deletion, which leads to the activation of inflammatory signals driven by hematopoietic stem and progenitor cell (HSPC) enhancers [Classical myeloproliferative neoplasms (cMPNs) are characterized by the overproduction of terminally differentiated blood cells , includiof cMPNs . DNA metof cMPNs  which acof cMPNs –7. JacquV617F mutation, and its effects on downstream signaling pathways in cMPNs still needs further research.These above studies indicated that as well as the JAK2 gene, the DNMT3a gene is also important in the pathogenesis of cMPNs, but their relationship in cMPNs is still a mystery. Few research has answered the question how the Dnmt3a is regulated by Jak2V617F-positive BaF3 cells (hereinafter referred to as Jak2V617F BaF3). Further research showed that the binding of Stat5a protein  with Dnmt3a promoter is the mechanism of this downregulation. The gamma-activated sequence (GAS) motif of Stat5a at the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter is the specific site of the binding. Dnmt3a significantly affected the cell proliferation capacity and cell cycle distribution and mediated G0/G1 arrest by suppressing the Cdkn1a via miR-17-5p in Jak2V617F BaF3 cells. This Stat5a/Dnmt3a/miR-17-5p/Cdkn1a regulation axis identified in this study might play an important role in cMPNs and might serve as a potential treatment target for cMPNs.In order to answer the question, we carried out the present study. Firstly, we found that Dnmt3a was downregulated at the transcriptional and translational levels in Jak2V617F-positive cMPNs patients and six normal controls) were recruited from Tongji hospital of Tongji University  and were characterized using Short Tandem Repeat (STR) markers. HEL, U937, NB4, THP1 and BaF3 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium , and wild-type BaF3 cells were grown with 1 ng/mL IL-3. 293 T/17 cells were grown in Dulbecco’s modified Eagle’s medium . K562 and KG1α cells were grown in Iscove’s Modified Dulbecco’s Medium . All media were supplemented with 1% penicillin-streptomycin  and 10% fetal bovine serum . All cell lines were cultured in a humidified atmosphere of 5% COV617F BaF3 cells with growth factor independence by ectopic expression of Jak2V617F, resulting in constitutive phosphorylation of Jak2 as well as downstream targets, such as Stat5a. The ectopic expression plasmid of Jak2V617F was generated by inserting the full-length Jak2 with Jak2V617F mutation, which was generated by site-directed mutagenesis and confirmed by sequencing, into the pLVX-zsGreen vector . The wild type BaF3 cells transformed empty vector were used as control. The Stat5a overexpression plasmid was generated by inserting the Stat5a coding DNA sequence (CDS) into the pLVX-Puro vector. The Dnmt3a overexpression plasmid was generated by inserting the Dnmt3a CDS into the pLVX-zsGreen vector . shRNA lentiviral plasmids targeting Stat5a, Dnmt3a and Cdkn1a were constructed by inserting annealed shRNA template DNA sequences into pLVX-shRNA  vector containing either a puromycin resistance cassette or ZsGreen, respectively. The effective targeting shRNA sequences of the Stat5a gene were 5′-GCCAGATGCAAGTGTTGTA-3′ and 5′-GCACCTTCAGATCAACCAA-3′. The effective targeting shRNA sequences of the Dnmt3a gene were 5′-CCAGATGTTCTTTGCCAATAA-3′ and 5′-GCAGACCAACATCGAATCCAT-3′. The effective targeting shRNA sequences of the Cdkn1a gene were 5′-CCGAGAACGGTGGAACTTT-3′ and 5′-GCAAAGTGTGCCGTTGTCT-3′. The sequence 5′-GCGCGCTTTGTAGGATTCG-3′, which is unrelated to any sequence in humans and mice, served as a negative control (shRNA-Ctrl). To produce lentiviral particles, all of the aforementioned plasmids  were cotransfected into 293 T/17 cells via the calcium phosphate precipitation method  according to the manufacturer’s instructions. Cell transfection with lentivirus supernatant was performed with 10 μg/ml polybrene, and GFP or puromycin resistance expression in cells was tested after 48–72 h. As a high level of Stat5a depletion would result in significant inhibition of cell growth and massive cell death, we repeated the construction of stable-transfected cells for each assay. For luciferase reporter plasmids, different promoter regions of Dnmt3a  and LY2784544 (cat. no. S2179) were obtained from Selleckchem (USA) and dissolved in DMSO . Primary antibodies against Jak2 (cat. no. 3230), p-Jak2 (cat. no. 3771), Stat5a (cat. no. 94205), p-Stat5a (cat. no. 4322), Dnmt3a (cat. no. 3598), β-actin (cat. no. 8457) and Cdkn1a (cat. no. 64016) were purchased from Cell Signaling Technology (USA). Mmu-miR-17-5p (hereinafter referred to as miR-17-5p) mimic, related mimics control, inhibitor and related inhibitor control were got from GenePharma (China). The sequences of miRNA were listed in Table Total protein was extracted with RIPA buffer  containing protease inhibitor cocktail  was used to detect the protein concentration. Equal amounts of total protein (30 μg) were separated by protein electrophoresis on a 10% SDS-PAGE gel and transferred onto PVDF membranes . The membranes were probed with the indicated antibodies. Protein bands were detected and quantified by Amersham Imager 600 .293 T/17 cells were co-transfected with different luciferase reporter plasmids and a Stat5a overexpression plasmid for 48 h for the luciferase reporter assay of the Dnmt3a promoter. Luciferase activities were measured with the Dual Luciferase Reporter Gene Assay Kit . Data were normalized by calculating the ratio between firefly and Renilla luciferase activity.2 at 37 °C. A total of 5 groups of 3 wells each were counted. The cells were counted in one group every three days from the 4th day of incubation. The cell growth curve was plotted as a line graph based on the count of each group of cells.Cells in good growth conditions to be tested were centrifuged, the supernatant was removed, new medium was added, and the cells were resuspended by gentle blowing, and cell counting was performed. Fifty cells were inoculated in each well of a 24-well plate and incubated in a humidified atmosphere of 5% CO3 cells/mL (100 cells per well). Then, 0.2 mL of the cytosol, 2 mL of 2× cell culture medium containing 20% FBS and 2 mL of 0.7% agarose were added to each tube, mixed well and spread on the bottom agar layer . After solidification at room temperature, the 6-well cell culture plates were incubated at 37 °C for 14 d. Cells were observed under a microscope after continuous culture for 14 d. The number of colonies with more than 10 cells was counted.The 1% agarose solution was prepared with ultrapure water, autoclaved at 121 °C for 15 min, placed in a water bath at 42 °C, mixed with 2× complete medium containing 20% FBS in equal volumes to make the final concentration of agarose 0.5%, and poured rapidly into 6-well cell culture plates at 4 mL per well. Equilibrate the 6-well cell culture plates in a 37 °C incubator for more than 30 min before use. The prepared 0.7% agarose solution was placed in a 42 °C-water bath. The cells were resuspended in single-cell suspensions with culture medium, counted, and diluted to 1 × 102B4O7 for 2 min. The cells were washed again and resuspended in 50 μl of the anti-BrdU APC antibody solution  and incubated for 30–60 min. Then, 5 μl of PI staining solution was added and incubated for 10 min. The cell cycle distribution was determined by a flow cytometer (BD Biosciences) and analyzed by FlowJo V10.For cell cycle distribution analysis, BrdU solution was added to the medium at a final concentration of 10 ng/ml. After culturing at 37 °C for 2 h, the cells were collected, washed twice with PBS and fixed with 95% ethanol overnight at 4 °C. The next day, the cells were washed twice with PBS buffer . The cells were resuspended in 2 ml of PBS buffer (2 N HCl and 0.5% Triton X-100) and incubated in the dark for 30 min at room temperature. The cells were washed once with 1 ml of PBS and resuspended in 0.5 ml of 0.1 M Na-ΔΔCt method [-ΔΔCt method using U6 small nucleolar RNA gene as internal reference. The forward primer of miR-17-5p is 5′-GCTTCGCAAAGTGCTTACAGTGC-3′, and the forward primer of U6 is GCTTCGGCAGCACATATACTAAAAT. The reverse primer of qPCR for miRNA is provided in the TransScript miRNA First-Strand cDNA Synthesis SuperMix named as Universal miRNA qPCR Primer.Total RNA was extracted with a Quick-RNA MicroPrep RNA Extraction Kit , cDNA of coding genes was produced by PrimeScript RT Master Mix , and cDNA of miRNAs was produced by TransScript miRNA First-Strand cDNA Synthesis SuperMix . The ChamQ Universal SYBR qPCR Master Mix Kit  was used to detect relative mRNA expression by a LightCycler 96 system  with 40 cycles of PCR thermocycling. The primer sequences were as follows: For BaF3 cells: β-actin forward, 5′-GTGACGTTGACATCCGTAAAGA-3′ and reverse, 5′-GCCGGACTCATCGTA CTCC-3′; Dnmt3a forward, 5′-GATGAGCCTGAGTATGAGGATGG-3′ and reverse, 5′-CAAGA CACAATTCGGCCTGG-3′; Stat5a forward, 5′-CAGATGCAAGTGTTGTATGGGC-3′ and reverse, 5′-GCTGGCTCTCGATCCACTG-3′; Cdkn1a forward, 5′-CCTGGTGATGTCCGAC CTG-3′ and reverse, 5′-CCATGAGCGCATCGCAATC-3′. For specimens of cMPN patients and normal controls: DNMT3a forward, 5′-GGAGGACCGAAAGGACGGA-3′ and reverse, 5′-CCCCATTGGGTAATAGCTCTGAG-3′; β-actin forward, 5′-CATGTACGTTGCTATCCAG GC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′; Cdkn1a forward, 5′-TGTCCGTCAGAACCCATGC-3′ and reverse, 5′-AAAGTCGAAGTTCCATCGCTC-3′. The mRNA expression of the target gene was calculated by the 2ChIP assays were performed using a ChIP assay kit  according to the manufacturer’s instructions. Briefly, cells were fixed for 8 min with 1% free formaldehyde  and then disrupted in SDS lysis buffer. Chromatin was sonicated by an M220 Focused-ultrasonicator  to shear DNA to an average length ranging from 200 to 1000 bp, as verified by agarose gel electrophoresis. Next, chromatin was immunoprecipitated with antibodies (2 μL) directed against Stat5a, and 0.8 μL of nonimmune IgG was used as the negative control. Final DNA extractions were quantitative-PCR amplified using primer pairs that cover the sequence in the Dnmt3a promoter region (− 2860 bp to + 140 bp). Sequences of the primers for quantitative PCR are listed in Table p-value cut-offs: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05) and the Type I error for all statistical tests is α = 0.05.The statistical analyses were performed with GraphPad Prism 7 software. The results were from triplicate experiments, and the data are presented as the mean ± SD (standard deviation). The significance of mean values was determined by unpaired two-tailed Student’s t test . The gamma-activated sequence motif (GAS) which was well reported to be binded with STAT5a protein [To investigate the specific site of the Dnmt3a promoter at which Stat5a binds to, we predicted it using the JASPAR database  Jak2V617F BaF3 cells had significantly increased S-phase cell proportion and decreased G0/G1-phase cell proportion, while Dnmt3a overexpression (Dnmt3a OE) Jak2V617F BaF3 cells had significantly increased G0/G1-phase cell proportion and decreased S-phase cell proportion. These results indicates that Dnmt3a can inhibit the cell cycle progression of Jak2V617F BaF3 cells, mediates G0/G1 arrest and results in the suppression of the proliferation of Jak2V617F BaF3 cells at last.To clarify how Dnmt3a suppresses the proliferation capacity of Jak2V617F BaF3 cells compared with control BaF3 cells , the Jak2V617F BaF3 with Dnmt3a overexpression (Jak2V617F + Dnmt3a OE) cell line, and the Jak2V617F BaF3 with both Dnmt3a OE and Cdkn1a knockdown (Jak2V617F + Dnmt3a OE + Cdkn1a KD) cell line  showed no significant differences in cell cycle . Thus we suspect that miRNA may play a role in the regulation of Cdkn1a by Dnmt3a. To confirm the hypothesis, we detected the expression level of miR-17-5p in Jak2V617F BaF3 cells, control BaF3 cells, Jak2V617F BaF3 cells with Dnmt3a overexpression and Jak2617F BaF3 cells with Dnmt3a knockdown. It was observed that the expression of miR-17-5p was higher in Jak2V617F BaF3 cells than in control BaF3 cells  with echinomycin impairs the growth and survival of Jak2V617F-positive 32D cells [V617F mutation also enforced Mcl-1 transcription to sensitize myelodysplastic syndrome cell lines to apoptosis induced by a Bcl-xL/Bcl-2 inhibitor [V617F and DNMT3a deletion, how the Dnmt3a is regulated by Jak2V617F mutation still needs research. Here, we found that in Jak2V617F BaF3 cells, the expression of Dnmt3a was inhibited compared with the wild-type BaF3. Further investigation of Stat5a inhibitor and Stat5a knockdown or overexpression found that the reduction in Dnmt3a in Jak2V617F BaF3 cells was mediated by abnormal activation of Stat5a. Subsequently, we overexpressed and knocked down Stat5a in wild-type BaF3 cells and found that as the expression of the Stat5a protein increased or decreased, the expression of Dnmt3a decreased or increased, respectively. Therefore, our research shows that the inhibition of Dnmt3a expression is mediated by abnormally activated Stat5a protein, indicating that Dnmt3a is a target gene of Stat5a in Jak2V617F BaF3 cells.The somatic JAK2nhibitor . While tSignal transducer and activator of transcription (STAT) proteins reportedly bind to palindromic sites to regulate target gene expression , 20, andV617F BaF3 cells significantly inhibited cell proliferation and induced G0/G1 phase cell cycle arrest. Our results showed that Dnmt3a might mediate G0/G1 phase cell cycle arrest by positively regulating the G1/S checkpoint marker Cdkn1a. It has been reported that compared with AML patients with FLT3 mutations alone, AML patients with mutations of both DNMT3A and FLT3 showed homeobox gene overexpression and enhancer hypomethylation, which resulted in a lower enrichment score of G2/M checkpoint genes [V617F cMPNs, Dnmt3a positively regulates the expression of Cdkn1a, which induces G0/G1 cell cycle arrest, indicating that Dnmt3a also serves as a tumor suppressor in cMPNs by regulating the cell cycle through Cdkn1a, which in turn affects the proliferative capacity of Jak2V617F BaF3 cells. Although Dnmt3a is a de novo methyltransferase [V617F BaF3 cells, which was regulated by the expression of Dnmt3a. Futhermore, we found that miR-17-5p inhibits the expression of Cdkn1a and Dnmt3a downregulats miR-17-5p in Jak2V617F BaF3 cells, thus the mechanism under which Cdkn1a expression is changed in Dnmt3a expression-altered cells might be Dnmt3a attenuate the inhibition of Cdkn1a mediated by miR-17-5p. However, the direct regulation mechanism between Dnmt3a and miR-17-5p has not been explored, which is one of limitation of the study, will be followed up persistently in our future study. The deficiency of lack of in vivo validation will also be addressed in our future studies.DNMT3a belongs to a family of highly conserved DNA methyltransferases that catalyze 5-methylcytosine methylation and methylate DNA at unique sites as well as at repetitive elements . It was nt genes . Our stunsferase  and usuansferase , in somensferase , 27. Thensferase . Our stuV617F Baf3 cells through Cdkn1a, which is down-regulated by the miR-17-5p. Our discovery of the Stat5a/Dnmt3a/miR-17-5p/Cdkn1a axis existing in JAK2V617F cells might shed new light on the pathogenesis of JAK2V617F-positive cMPNs and will inform new approaches to treatment.In conclusion, our research reveals that Dnmt3a is a downstream target of Stat5a, it is negatively regulated by the Stat5a through occupance of the promoter region of Dnmt3a, and that Dnmt3a induces cell cycle arrest at the G0/G1 phase in Jak2Additional file 1: Table S1. Clinical characteristics of 12 samples , from which bone marrow mononuclear cells were isolatedAdditional file 2: Table S2. Primer sequences of different promoter regions of Dnmt3a (− 2860 bp to + 140 bp)Additional file 3: Fig. S1. Uncropped images of Fig. Additional file 4: Fig. S2. Uncropped images of Fig. Additional file 5: Fig. S3. Uncropped images of Fig. Additional file 6: Fig. S4. Uncropped images of Fig. Additional file 7: Fig. S5. Uncropped images of Fig. Additional file 8: Fig. S6. Uncropped images of Fig. Additional file 9: Fig. S7. Uncropped images of Fig. Additional file 10: Fig. S8. Uncropped images of Fig. Additional file 11: Fig. S9. Uncropped images of Fig. Additional file 12: Fig. S10. Uncropped images of Fig. Additional file 13: Fig. S11. Uncropped images of Fig. Additional file 14: Fig. S12. Uncropped images of Fig. Additional file 15: Fig. S13. Uncropped images of Fig. Additional file 16: Fig. S14. Uncropped images of Fig. Additional file 17: Fig. S15. Uncropped images of Fig. Additional file 18: Fig. S16. Uncropped images of Fig. Additional file 19: Fig. S17. Uncropped images of Fig. Additional file 20: Fig. S18. Uncropped images of Fig. Additional file 21: Fig. S19. Uncropped images of Fig.


Leptosphaeria maculans isolates (HCRT75 8-1 and HCRT77 7-2) and two Brassica napus genotypes (Surpass400 and 01-23-2-1) were selected. The selected B. napus genotypes displayed intermediate and resistant phenotypes. The inoculated seedlings were tested under three temperature conditions: 16 °C/10 °C, 22 °C/16 °C and 28 °C/22 °C (day/night: 16 h/8 h). Lesion measurements demonstrated that the necrotic lesions from the 28 °C/22 °C treatment were enlarged compared with the other two temperature treatments . The results of expression analysis indicated that the three temperature treatments displayed distinct differences in two marker genes (PATHOGENESIS–RELATED (PR) 1 and 2) for plant defense and one temperature-sensitive gene BONZAI 1 (BON1). Additionally, seven dpi at 22 °C/16 °C appeared to be the optimal pre-condition for the induction of PR1 and 2. These findings suggest that B. napus responds to temperature changes when infected with L. maculans.Temperature is considered one of the crucial environmental elements in plant pathological interactions, and previous studies have indicated that there is a relationship between temperature change and host–pathogen interactions. The objective of this research is to investigate the link between temperature and the incompatible interactions of the host and pathogen. In this study, two  Arabidopsis thaliana–Pseudomonas syringae pv. tomato DC3000, the elicitation of innate immunity induces salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) responsive genes [Brassica napus was also found to express hormone/ROS-related signals when coping with the fungal pathogen Leptosphaeria maculans [Plants develop sets of mechanisms to combat the threat from phytopathogens. Plants secrete a set of metabolites, proteins and gene factors after the triggering of defense responses. According to two studies on the ve genes ,2; Brassmaculans ,5. Plant disease epidemics are affected by various environmental factors, including the temperature, humidity and wind, and these factors can be crucial elements influencing the development of disease in nature. Among these factors, temperature is an important element which influences both hosts and pathogens. Moreover, there is molecular evidence suggesting that these organisms  have developed various adaptive genetic backgrounds to interact with changes in temperature by evolution; as such, certain genes related to infection/defense can be regulated to change the general physiology of an organism when temperatures reach specific levels. Puccinia striiformis is less effective when the temperature increases (starting at 15.4 °C), and the pathogen is unable to infect seedlings of wheat when the temperature is over 21 °C. Shayka et al., (2015) [Phytophthora infestans within a temperature range of from 16 to 23 °C in Spain, and the optimal temperature for oomycete formation was 21 °C. In the Brassica napus–Leptosphaeria maculans pathosystem, an alteration in the temperature could also change the interaction between the host and fungus. One study based on the Effector–Triggered Immunity (ETI) between Arabidopsis thaliana and Pseudomonas syringae revealed that the hypersensitive response (HR) was suppressed at an elevated temperature of 28–30 °C compared with the ambient temperature (21–24 °C) [B. napus–L. maculans pathosystem, Huang et al. (2006) [PR1 gene expression. Compared with a lower temperature (22 °C), both free SA and conjugated SA were increased and PR1 expression was induced, which was able to cause necrotic lesions [Arabidopsis, the gene BONZAI1 (BON1) has been found to modulate the plant defense in a temperature-sensitive manner. The BON1 gene is part of the COPINE gene family, which supports plant growth and development, and negatively regulates plant defense and programmed cell death (PCD) [BON genes consists of three homologs: BON1, 2 and 3. BON1 plays a major role while the other two BON genes are more redundant. Moreover, the triple mutant of all three BON genes (bon1bon2bon3) of Arabidopsis also displays difficulty in germination [BON1 positively regulates plant growth at lower temperatures, and the mutation of BON genes induces excessive cell death at 22 °C [BON1 also represses an R gene named SNC1 , and the BON1 mutant bon1–1 induces constitutive resistant responses [B. napus against the blackleg pathogen. In nature, there is plenty of evidence indicating that a change in temperature alters disease epidemics by changing the infectivity (from pathogens) and defense (from hosts). For example, the rust pathogen , 2015) , by stud, (2015) , found t. (2006) , suggest. (2006)  suggeste lesions . In Arabth (PCD) ,12,13,14 , by stuesponses ,18. In tesponses . By consL. maculans races on B. napus genotypes at the seedling stagea set of experiments were conducted to explore the effects of temperature upon HR resistance against . By analyzing the inoculated B. napus seedlings at three different growing temperatures, distinct patterns of fungal development were observed and molecular evidence suggested that the intrinsic signaling also responded differently at those temperatures.In this study, L. maculans isolate HCRT75 8-1  induced incompatible interaction upon Surpass400 (BLMR1/LepR3 and BLMR2/LepR2) by AvrLepR2-BLMR2/LepR2 interaction, and upon 01-23-2-1 (Rlm7) by AvrLm4-7–Rlm7 interaction, respectively. The L. maculans isolate HCRT77 7-2  induced incompatible interaction upon Surpass400 (BLMR1/LepR3 and BLMR2/LepR2) by AvrLm1–BLMR1/LepR3 and upon 01-23-2-1 (Rlm7) by AvrLm4-7–Rlm7 interaction, respectively [By measuring the lesions on the cotyledons after three temperature treatments, both Surpass400–H75 8-1/H77 7-2 and 01-23-2-1–H75 8-1/H77 7-2 exhibited phenotypes of incompatible interactions; as such, brownish necrotic lesions formed around the sites of inoculation . The L. ectively ,21,22. GBON genes are related to the growth/defense balance at a low temperature (22 °C), and BON1 has been found to play a dominant role in repressing the plant defense [BON1 in BON1 was more pronounced at the 22 °C/16 °C condition compared with the other two conditions at 7 dpi. Similar to at 7 dpi, BON1 was still induced at 11 dpi when the plant was treated with the 28 °C/22 °C condition , suggesting that this gene might have the function of defense suppression in some conditions, such as temperature [PR1 expression in PR1 appeared to be at 11 dpi, at 28 °C/22 °C. It seemed that PR1 expression in 01-23-2-1 synchronized with the expression of BON1, in which the trends of up- and down-regulation between these two genes were similar (both 7 and 11 dpi). On the other hand, PR1 and BON1 generally exhibited an antagonistic relationship in Surpass400, which matched with previous studies in Arabidopsis.According to previous studies on perature . LookingPR gene tested was PR2. PR2 encodes an enzyme called beta-1, 3 glucanase 2. It is SA-responsive and functions as a regulator of sugar metabolism and fungal cell wall degradation [PR2 expression. Except for Surpass400–H77 7-2 (at 7 dpi) and 01-23-2-1 – H77 7-2 (11 dpi), PR2 was highly induced at 22 °C/16 °C, suggesting that, unlike PR1, PR2 expression was only induced at 22 °C/16 °C. It seemed that PR2 had no apparent connection with BON1, but it had generally lower expression in the 28 °C/22 °C condition, which indicated that PR2 expression might be affected by other factors besides temperature and/or BON1 regulation.Another radation ,24,25. ABON1, PR1 and PR2 were distinct among three temperature conditions. The temperature 22 °C/16 °C is generally optimal for PR1/2 to express. The PR1/2 was also induced (higher than water inoculation) when the temperature treatment reached 28 °C/22 °C.Our experiments suggested that the HR lesions were increased at the higher temperature treatment (28 °C/22 °C). The expression profiles of PR1 expression in tobacco leaves (inoculated with tobacco mosaic virus (TMV)) when the temperature was increased to 32 °C from 22 °C; on the other hand, a higher temperature  enabled the virus to replicate and infect the host. Other signaling regulators, such as BON family genes, are involved in temperature-dependent regulation of plant defense.Previous evidence has suggested that a change in temperature is able to alter the general defense in plants. Malamy et al., (1992) , observeArabidopsis, BONZAI1 (BON1) is expressed at a lower temperature (22 °C) to regulate the general plant defense [BON genes appear to regulate SA-related signals, such as PR1, and the expression of BON1 represses SA-related pathogenesis-related proteins  [ SNC1 , as an R gene in Arabidopsis, is repressed by BON1 on its promoter region [SNC1 has been found to play a role in defense against Nicotiana benthamiana, together with the N gene (another R gene specific for N. benthamiana). Moreover, the nuclear accumulation of SNC1 and N genes is reduced when the temperature is elevated [SCN1 is also suppressed by factors other than BON1 at a higher temperature. In Arabidopsis, HOPZ-ETI-DEFICIENT 1 (ZED1) and ZED1-related kinases (ZRKs) suppress SNC1 expression at a temperature of 25 °C, and the mutation of ZED1 was shown to activate defense genes PR1 and PR2 at 25 °C [According to previous studies on  defense ,15. BON and PR5) ,15. The and PR5) ,19,26,27r region . SNC1 haelevated . SCN1 isat 25 °C .BON1, PR1 and PR2 were found to react to the thermal changes by observing the transcriptional analyses; however, some changes did not follow the indicated rules set out in previous studies. One remarkable discrepancy is that both BON1 and PR1 displayed a high level of induction at 28 °C/22 °C. Conversely, in previous research, the expression of these genes was shown to be lower than in the other low-temperature conditions.According to the qPCR results, the genes BON1 and PR1 appeared to be shown. PR1 displayed a very high expression when the expression of BON1 was not very high, it was more obvious from Surpass400 , and this finding matched that of the repression of BON1 upon defense genes and cell death [PR1 and 2 were induced at 7 dpi and repressed in 11 dpi, which synchronized with the lower expression of BON1 at 7 dpi and its high expression at 11 dpi.In the 22 °C/16 °C condition, an antagonistic relationship between ll death ,15. In tBON1 and PR1 were induced at 11 dpi. By considering the larger lesion size in this condition for both genotypes, the high expression of PR1 can be explained as the physiological response towards more severe infectious situations, similar trends also happened to 01-23-2-1 at 22 °C/16 °C. The infected hosts with compatible interactions were found to have a high level of induction of defense-related genes at a later stage of infection compared with cases of incompatible interactions [BON1 was also observed, which did not display its negative regulation upon PR1, as mentioned in previous studies. It is possible that, in a high-temperature growth condition, the resistant B. napus genotypes obtain a homeostatic status, where both the activation and repression of defense mechanisms occur.Surprisingly, in the 28 °C/22 °C condition, both ractions . On the PR2 did not follow the presumed pattern of expression when changing the temperature, and other factors may affect the expression of downstream proteins like PR proteins. One possible explanation for this unusual situation is the homeostatic regulation between plant growth and defense. A plant body may suppress excessive defensive activities when there are few pathogens present inside. Plants may develop certain mechanisms, such as a guard model, to activate their resistance when a large amount of pathogen inoculum are recognized, since R gene-related defense is destructive to plant bodies [There are still many questions that need to be answered in order to explain the relationship between the regulation of plant defense and changing of temperature. This signaling system may be more complicated and depends on different species. In this article, t bodies . In addit bodies .Brassica napus genotypes (Surpass400 (BLMR1/LepR3 and BLMR2/RlmS) and 01-23-2-1 (Rlm7)) were grown under three temperature conditions: 16 °C/10 °C (day/night: 16 h/8 h); 22 °C/16 °C (day/night: 16 h/8 h); 28 °C/22 °C (day/night: 16 h/8 h). All flats with seedlings were grown under 22 °C/16 °C (day/night: 16 h/8 h) first, and the seedlings to be tested under the other two conditions were moved into the growth cabinets 24 h before inoculation.Two L. maculans isolates were selected for inoculation: HCRT75 8-1  and HCRT77 7-2 .Two B. napus cultivars were inoculated seven days after sowing (cotyledon stage) by puncture inoculation. Each lobe of cotyledons was punctured by a sterile needle twice from each side, to have four inoculation points on each seedling of the canola plant.The cotyledons of The cotyledons at 11 days post-inoculation (dpi) were scanned and the lesion size was measured by ImageJ .® Green Supermix . The experiments were based on three biological replicates. The RT-qPCR experiments were run by Touch Real-Time PCR System .Frozen cotyledons (7 and 11 dpi) were ground in liquid nitrogen with pestles and mortars. The total RNA was extracted with TRI reagent (Sigma-Aldrich) . According to the manual, the total RNA was purified by DNaseI treatment with a recombinant DNaseI, RNase-free kit (Roche). Purified RNA was used to synthesize cDNA by employing the GOScript Reverse Transcription System (Promega). The cDNA stock solution was diluted to 100 ng/µL. Quantitative-PCR was performed by loading 1 µL of cDNA (100 ng) into the 10 µL reaction system of the IQTM SYBRCOI1 and ACO1) was 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s and 60 °C for 20 s. This was followed by a melting curve analysis.The qPCR program used for all of the analysed genes , both BON1 and PR1 were triggered at 7 and 11 dpi, and were presumed to have an antagonistic relationship with each other based on previous studies on Arabidopsis. The results indicated that, at a higher temperature, B. napus seems to display a balance between the plant defense and growth mechanism, at the same time as exhibiting incompatible interactions, from which the expression of defense-repressing factor BON1 and defense gene PR1 coincides during a pathogen attack.Taken together, the evidence displayed in this article suggests that infected genotypes growing at a higher temperature cause


The segmented acquisition minimises the effective TE and readout duration compared to a single-shot EPI scheme, reducing geometric distortions to provide high quality T1 maps with a narrow point-spread function. The precision and repeatability of MS-IR-EPI T1 measurements are assessed using both T1-calibrated and T2-calibrated ISMRM/NIST phantom spheres at 3 and 7 T and compared with single slice IR and MP2RAGE methods. Magnetization transfer (MT) effects of the spectrally-selective fat-suppression (FS) pulses required for in vivo imaging are shown to shorten the measured in-vivo T1 values. We model the effect of these fat suppression pulses on T1 measurements and show that the model can remove their MT contribution from the measured T1, thus providing accurate T1 quantification. High spatial resolution T1 maps of the human brain generated with MS-IR-EPI at 7 T are compared with those generated with the widely implemented MP2RAGE sequence. Our MS-IR-EPI sequence provides high SNR per unit time and sharper T1 maps than MP2RAGE, demonstrating the potential for ultra-high resolution T1 mapping and the improved discrimination of functionally relevant cortical areas in the human brain.An efficient multi-slice inversion–recovery EPI (MS-IR-EPI) sequence for fast, high spatial resolution, quantitative T T1 relaxometry removes the confounding effects of sequence-related variations in the signal intensity in T1-weighted images – which are also weighted by proton density and T2*– facilitating comparison across subjects, scanners and time to characterize the underlying physical composition of tissue, useful clinically in a wide range of applications  and improvements in image acquisition are required to provide adequate ultra-high resolution quantitative T1 maps with a narrow point-spread function, maximal SNR per unit time, and low SAR.In systems neuroscience, quantitative Tal areas . Ultra-hal areas ; for a ral areas . This inal areas . T1 mapssections . Other slination , paving 1 mapping. The inversion recovery (IR) method is considered the most accurate approach to measure T1, acquiring multiple lines of k-space or images at a range of values of the time TI after a 180° inversion in order to sample the longitudinal magnetization recovery curve and derive an estimate of T1 by fitting the data to a mono-exponential function. However, the main disadvantage of this method is its temporal inefficiency. The gold standard sequence combines an IR with a spin-echo readout of a single line of k-space to limit the effect of B1 and B0 inhomogeneities, but the long acquisition time makes this impractical for human studies. Alternatively, the 2D approach can use an echo-planar imaging (EPI) readout to collect an image or series of multi-slice images after a given TI. While this significantly reduces the scan duration, spatial distortions can occur which are intrinsic to the use of single-shot EPI at high spatial resolution. Several methods have been proposed to improve the efficiency of T1 mapping including Look-Locker (LL)  strategies can now accelerate such acquisitions. A faster multi-slice inversion recovery variant was introduced   methods ker (LL)  or EPI (ker (LL)  readoutstroduced  to allowch slice . If the ion time .1 maps collected using single shot EPI are prone to geometric distortions due to the low bandwidth in the phase-encode direction and B0-field inhomogeneity. The MS-IR-EPI work presented in this manuscript aims to provide an alternative to the FLASH-based sequences for 'distortion-free' T1 mapping; however it should be noted that for some fMRI applications single-shot IR-EPI T1 maps are collected by design and intent to provide anatomical data with matched distortions to the fMRI data (1 maps can be acquired with submillimeter (0.7 mm) spatial resolution over a small field of view to improve the accuracy of cortical depth definitions in fMRI data space ’ sequence (1-weighted and the second PD-weighted) within a single repetition time to calculate a quantitative T1 map based on the T1-weighted combination image, which is free from bias-field effects, and the use of a look-up table. A non-selective adiabatic inversion pulse is used to minimize sensitivity to the inhomogeneity of transmit B1 field (B1+) at 7 T  and long total scan duration for sufficient SNR. When MP2RAGE is used at nominal spatial resolution of the order of 0.3 mm to depict intracortical details, these invariably appear blurred, even on trained subjects when no apparent motion is detected or when motion is prospectively corrected  at 7 T . Deviatiare used  as requi mapping , enablin  between the free water proton pool and the macromolecule-bonded proton pool  acquisition techniques to improve spatial coverage.In this present study, we aimed to implement a fast multi-slice multi-shot inversion-recovery 2D-EPI Tton pool . The fea1 maps at 7 T with accuracy and repeatability comparable to MP2RAGE and multi-shot single slice IR-EPI, but in a faster time; (b) MS-IR-EPI provides T1 maps with higher SNR per unit time than MP2RAGE; (c) the MT effects due to spectrally selective fat suppression pulses in MS-IR-EPI can be effectively removed for accurate T1 quantification; (d) MS-IR-EPI T1 maps yield sharper images than MP2RAGE with the same nominal spatial resolution, and can be used for intracortical mapping.We hypothesise that: (a) MS-IR-EPI produces T22.11, is used to provide a well-defined inversion time for each segment and consequently a narrow point-spread function, while multiple shots are required to sample the entire k-space plane. This sequence can be used with in-plane acceleration and SMS excitation pulses to achieve whole brain coverage even more quickly (acq) are collected in which the slice order is shifted by a constant offset number, resulting in a set of equally spaced distinct inversion times for each segment  = S0[1–2exp(−TI/T1)+exp(-TR/T1)], which can easily be fit using a least squares regression. The S0 image provided by the fit represents the proton density weighted by the effective transverse relaxation time T2*. quickly . Multipl2.21 map from the different inversion times experienced by each slice, care must be taken to ensure that the sampling of the inversion times across all slices provides consistent T1 values. The minimum TI each slice experiences has a key impact on the measured T1, especially for lower T1 values, and this can lead to banding artefacts observed in the slice direction when an insufficient number of acquisitions are performed . To test the robustness of the T1 fitting to the range of acquisition strategy and noise level, a Monte Carlo (MC) simulation of the entire fitting algorithm (including the phase correction) was implemented. Each MC run included 1024 samples with gaussian distributed noise added to the complex signal, at SNR levels of 10 and 25, and target T1s of 1000 and 1800 ms. The MC simulation was used to determine the minimum number of acquisitions (Nacq and thus inversion times) needed to obtain homogenous T1 maps across the multiple slices of the data set (acq for whole brain 0.7 mm isotropic resolution at 7 T (for a TR of 5 and 3.2 s with SMS factors of 2 and 3 respectively), and whole brain 1 mm isotropic resolution at 3 T (for a TR of 4.5 s with SMS factor 2) (See 1 mapping data acquisition’ section).Since the MS-IR-EPI method computes a T2.32 with 0.7 mm isotropic resolution using protocols MS1 and MS2 outlined in Scanning was performed on a 7 T Philips Achieva system and 3 T Philips Ingenia system, both equipped with a 32-channel receive array NOVA coil. The 7 T whole head MS-IR-EPI protocol comprised a field of view (FOV) of 192(AP) x 164(RL) mm3) were acquired using protocol MS5 parameters with a SMS factor of 2 x252(RL)x198(FH) mm3, SENSE factor=2 (RL), FA=8°, TE=1 ms, TR=2.3 ms, Turbo-Field-Echo factor=890, acquisition bandwidth=5351 Hz, 2.2 s acquisition time) to allow computation of the effective excitation flip angles in the MP2RAGE trains for improved accuracy of the T1 fit (MP2RAGE) was formed based on the combination of the two complex images, SMP2RAGE=(STI1xSTI2*)/(STI12+STI22), where the asterisk indicates the complex conjugate. A look-up table was used in order to estimate T1 maps from the SMP2RAGE values  calibrated T1-spheres, with T1 values ranging from 1998 ms (T1_1) to 89 ms (T1_10) at 3 T  T2-spheres ranging from 3025 ms (T2_1) to 293 ms (T2_10). Note that the reference T1 values provided by the manufacturer for spheres T2_1 and T2_5 reflect measurements known to be deviant from the MnCl2 nominal solutions due to a problem at the time of the manufacturing process of our phantom. Due to the reduced size of the 7 T NOVA head coil compared to a 3 T head coil, home-built casings were manufactured to fit each plate of T1-spheres and T2-spheres within a separate 160 mm diameter spherical phantom that would fit within the 7 T NOVA head coil.The precision and repeatability of the optimized MS-IR-EPI sequence were assessed using the International Society for Magnetic Resonance in Medicine (ISMRM)/National Institute of Standards and Technology (NIST) fiducial T spheres . The NIS) at 3 T  were use1 in the short T1_7 to T1_14 spheres  of the brain tissues, were considered for analysis, in line with recent studies at 3 T  a gold-standard IR-TSE sequence using the ISMRM/NIST recommended parameters: a single 6 mm slice with 0.98 × 0.98 mm2 in-plane resolution, TR/TE= 4.5 s/7 ms repeated for a range of inversion times TIs (ms) = , 35 min acquisition, (ii) MS-IR-EPI, and (iii) an MP2RAGE acquired at 1 mm isotropic resolution (parameters in 1-spheres phantom (6 and 9 months apart) and a second scan session (1 week apart) for the T2-spheres phantom. The temperature of each phantom (in the solution surrounding the spheres) was measured across the scan sessions. The mean T1 values across scan sessions was computed using each of the T1 mapping methods and compared to the NIST reference T1 values.To compare the accuracy of the MS-IR-EPI method with other T1-spheres phantom and 4 measurements  were collected for the T2-spheres phantom, again temperature was measured in the phantoms after each scan session. Repeatability was characterized by the coefficient of variation (CoV), defined as the ratio between the standard deviation and the mean T1 values across all repeat measurements  a single slice multi-shot IR-EPI acquisition at a range of TIs (ms) = with other acquisition parameters matched to the MS-IR-EPI, (ii) MS-IR-EPI (TR 5 s) and (iii) MP2RAGE sequences  using SMS factor 1 (92 slices) and SMS factor 2 (184 slices). To estimate image SNR, T1 maps generated from each individual run were subtracted to provide a ‘noise’ map. For each region of interest (ROI), SNR was estimated as the mean across voxels (in both T1 maps) divided by the standard deviation of the noise map. For each sequence, SNR per unit time was computed by dividing the image SNR by the total acquisition time (in minutes).Further scan sessions were performed in which two measurements of sequences (ii) and (iii) were collected for the NIST T2.5Five subjects ) were recruited to the study. Experimental procedures for all studies were approved by the University of Nottingham Medical School's Ethics Committee. All subjects gave written informed consent. None of the subjects had a history of neurological disorders.2.5.11 of shortening the TR, a subject was scanned at 7 T using MS-IR-EPI protocols with TR=5 s and TR=3.2 s  protocol were repeated with varying number of slices, ns=. For this, the coverage in the slice direction across the different acquisitions was maintained by varying the slice gap: sg= mm for ns= respectively. A single slice IR-EPI acquisition (with parameters matched to the MS-IR-EPI acquisition) was also collected with TI values = ms for comparison with the central slice of the MS-IR-EPI acquisitions. All data sets were acquired without using spectrally selective fat suppression pulses.To assess the effect on the measured T2.5.21 (r), for a range of nominal flip angles (FA) = of the SPIR  fat suppression pulse. In addition, a dual-TR (1-mapping sequence (FOV=205(AP)x179(RL)x132(FH) mm3, FA=60°, TR1=23 ms, TR2=103 ms, TE=5 s, acquisition bandwidth= 4271 (Hz), 3.2 × 3.2 × 4 mm3 resolution, 3 min 3 s acquisition time) was used to measure the B1-field distribution. A further experiment was performed in one subject where data were acquired with a set of SPIR pulses of nominal FA=. In addition, for a FA=90° two data sets were acquired, one in which the SPIR pulse was applied prior to each RF acquisition and one in which a SPIR pulse was applied prior to every alternate RF acquisition (hence using 50% of the number of FS pulses).Experiments were performed in three subjects using the 0.7 mm isotropic resolution MS-IR-EPI MS1 protocol  to measu dual-TR  B1-mappi1(r)R was modelled as a weighted sum of the relaxation rates due to macromolecular relaxation and its modulation by the fat suppression pulses, which is a function of the nominal flip angle of the SPIR fat suppression pulse, FA, the RF field distribution, X(r), and myelin density m(r):a and b are constants. This model assumes that (i) the longitudinal relaxation of water protons in brain tissue is mono-exponential R versus FA.X(r) was performed to estimate α=a.m(r) and β=b.m(r) from a least-squares regression, and thus evaluate the constant (b/a) for each voxel of the brain. This constant was then used to form a corrected R1 map , by removing the contribution of magnetization transfer arising from a SPIR fat suppression RF pulse with amplitude FA:A linear fit of 1 maps were generated in two ways, using a voxel specific (b/a) parameter (voxel-wise correction) or using a constant (b/a) value for every voxel , defined by the peak of the histogram given by the distribution of b/a values across the entire brain.Corrected R2.5.31 maps corrected for B1-inhomogeneities, and T1 maps obtained using a single slice IR-EPI acquisition  repeated for multiple inversion times (TI(ms)=) in a 4 min 50 s acquisition time.Whole head images were obtained at 7 T, with 0.7 mm isotropic resolution ((b/a) correction of the T1 maps performed to remove MT effects due to the FS pulses. An additional acquisition using a SPIR fat suppression FA=40° pulse was performed for Subjects 4 and 5, who did not participate in the experiment to model the impact of FS pulses (see Section 2.2.1), to compute subject-specific global (b/a) parameters .Five subjects were scanned at 7 T. A SPIR fat suppression FA=70° pulse was applied, and a global 1 maps derived from the MS-IR-EPI and MP2RAGE sequences, the MP2RAGE and MS-IR-EPI protocols were repeated on Subject 1 at 7 T. In addition to the whole head MS-IR-EPI protocol , additional pairs of MS-IR-EPI acquisitions were performed using SMS=1 (two repeats with and without FS pulses).In order to evaluate the SNR per unit time of T2.5.41 mapping, a 0.5 mm isotropic resolution sequence with whole head coverage (FOV=164(RL)x192(AP)x168(FH) mm3) was implemented at 7 T  positioned over the visual cortex. MS-IR-EPI data  comprised 35 slices. Data were acquired with 5 slice offsets (5 TIs) with 3 repeats of each offset, resulting in a total scan time of 11 min 8 s. An MP2RAGE acquisition (protocol MP2 in Table1) was performed with the same slice prescription as the MS-IR-EPI . Notice these T1 maps were not corrected for MT effects of the fat suppression pulses, as their spatial fidelity rather than their quantitative precision was key here. Similarly, MP2RAGE data were not corrected for B1+ profile inhomogeneities in this comparison.To demonstrate the potential of MS-IR-EPI for high spatial resolution T1 maps for MS-IR-EPI and MP2RAGE acquisitions over the visual cortex were generated both before and after denoising, using a local complex principal component analysis (LCPCA) technique , all maps were re-sampled to an in-plane resolution of 0.11 × 0.11mm2 using nearest neighbour interpolation; FSL's FAST algorithm was run on the re-sampled ‘de-noised’ T1 maps to provide WM/GM and GM/CSF boundaries which were then used to derive 20 cortical depths using the equivolume approach (https://doi.org/10.5281/zenodo.3514297). This cortical depth segmentation was used to bin T1 (and S0) values into specific cortical depths.To plot the cortical profiles of the Tapproach  implemen33.11 maps using a TR of 5 s is shown in 1 is independent of the number of TIs used in the fit for greater than 4 TIs chosen, but that the standard deviation of the fitted T1 varies across slices. As the number of TIs used to derive T1 decreases, and with fewer permutations the minimum TI will vary significantly by slice, the variation in the standard deviation across slices increases, which can be visualized as ‘banding’ in the 3D reconstruction of the T1 map generated using 4 TIs but not for maps generated using 8 TIs, as shown in The effect of the number of acquisitions used to derive the T3.21 values measured in the NIST T1 and T2 spheres at 3 and 7 T for each T1 mapping method, along with their respective NiCL2 and MnCl2 concentration and reference 3 T T1 values provided by ISMRM/NIST.Supplementary material Tables 1 and 2 provide the mean T1 values measured at 3 T for the single slice IR-TSE, MS-IR-EPI and MP2RAGE sequences against the reference T1 value for the T1-spheres and T2-spheres. For the T1-spheres, the maximum deviation from the NIST reference T1 values was 4.7% (sphere T1_2) for IR-TSE, and 5.1% (sphere T1_3) for MS-IR-EPI, while for MP2RAGE the accuracy was 17.3% for sphere T1_5, but 8% or better for the other spheres. The mean temperature of the T1-spheres phantom was 21.2 ± 0.4 °C and 20.0 ± 1.3 °C for the T2-spheres phantom. Linear regression analysis of the different T1 measures versus the reference T1 values showed a very strong linear correlation  for the T1 spheres. For the T2-spheres (right), T1-measurements were shorter than the NIST reference T1 values, particularly for the MP2RAGE measurement of sphere T2_1 (24% shorter compared to 10% and 7.6% for IR-TSE and MS-IR-EPI respectively). The accuracy was better than 9% for all spheres when using MS-IR-EPI. Since T2_10 was below 300 ms (hence below the range of T1 in human brain at 3 T), this sphere was excluded from further analysis. Linear regression had a strong linear correlation  for the T2-spheres.1 was used as the reference to compare T1 values from MS-IR-EPI and MP2RAGE methods  than at 3 T. In general, MS-IR-EPI slightly overestimates T1 across all spheres, but accuracy is better than 5% for all spheres except the three T2-spheres with shortest T1 values. MP2RAGE T1 measurements in these spheres with short T1-values are also overestimated by more than 5%, while spheres with the longest T1 values  are underestimated by ~14%, 7% and 8% respectively, showing poor accuracy compared with MS-IR-EPI . The mean temperature of the T1-spheres phantom was 20.5 ± 1.3 °C and the T2-spheres phantom was 20.4 ± 0.9 °C. The repeatability of T1 measurements is characterized as the CoV , while the CoV for the T1 spheres was 2.2% (T1_3) or less. For MS-IR-EPI, the highest CoV was measured for spheres T2_9 (6.1%) and T1_5 (4.3%), but for T1s> 400 ms the CoV was below 3.5%. The CoV of T1 values measured with MP2RAGE was the largest, larger than 5% for several T2-spheres , and the largest CoV was 3.6% (T1_6) for the T1-spheres.At 7 T, the average single slice IR-EPI T2 and MnCl2 at both field strengths, the longitudinal relaxation rates (R1) measured with each T1 mapping method were linearly fit versus the NiCl2 (for the T1-spheres phantom) and MnCl2 (for the T2-spheres phantom) nominal concentrations. Spheres T2_2, T2_9 and T2_10 were excluded from the fit, given the poor accuracy for MP2RAGE measurements . Plots for MS-IR-EPI sequence are shown in 2 relaxivity at 3 T was found to be 0.66±0.01 (R2=0.9999), 0.63±0.01 (R2=0.9997) and 0.65±0.01 (R2=0.9529) s−1mM−1 using single slice IR-TSE, MS-IR-EPI and MP2RAGE respectively, whereas at 7 T the NiCl2 relaxivity was measured to be 0.86±0.01 (R2=0.9999), 0.81±0.01 (R2=0.9998) and 0.79±0.01 (R2=0.9997) s−1mM−1 respectively. For MnCl2, similar relaxivity values were obtained at 3 T and 7 T; 8.20±0.34 (R2=0.9997) and 7.96±0.33 (R2=0.9999) s−1mM−1 for single slice IR-TSE (EPI), 7.10±0.36 (R2=0.9999) and 7.29±0.34 (R2=0.9999) s−1mM−1for MS-IR-EPI, and 9.42±0.23 (R2=0.9981) and 7.00±0.39 (R2=0.9999) s−1mM−1 for MP2RAGE.In order to determine the relaxivity of NiCl3.31 histogram generated from data acquired with (dash lines) and without (solid lines) SMS for a TR of 5 s (blue) and 3.2 s (red). The T1 histograms for data acquired with SMS=1 and SMS=2 (TR=5 s) are very similar, whilst the T1 histogram for data acquired with SMS=3 is broader than that for the SMS=1, with a slightly lower peak for GM. Data acquired with SMS=1 shows that the reduced TR of 3.2 s (solid lines) yields shorter T1 values than for a TR of 5 s, particularly for GM. 1 histograms are very similar for a central slice of the MS-IR-EPI acquisition (TR=3.2 s (SMS=1)) acquired with a different number of slices and a single slice IR-EPI data set.3.42>0.99) for each of the data sets acquired with different fat suppression levels  (b/a parameter obtained by voxel-wise linear regression of R1 versus SPIR FS flip angle for GM (dark grey), WM (light grey) and total (WM+GM) tissue (black) for the three subjects scanned. The distribution of b/a is very similar for GM and WM, hence the distribution of total GM and WM tissue values across the brain was fit to a two Gaussian mixture model (red line), and the mode of highest amplitude Gaussian used as a constant parameter for the global correction method; this was found to be very similar across subjects . The voxel-wise correction introduces propagated noise in the corrected R1 maps. Given that the measured voxel-wise b/a correction factor shows a very slow spatial variation compared with structural details, we were able to improve the SNR of the corrected R1 maps when using the voxel-wise correction by spatially smoothing the b/a parameter maps with a 2 mm Gaussian kernel. Larger kernels did not further reduce the standard deviation in the corrected R1 maps.The model of the effect of fat suppression on longitudinal relaxation rate Eqn. assumes n levels A. There =0.9954) A inset. 1 histogram increases as the SPIR fat suppression flip angle increases (solid colour lines) as compared to no fat suppression (black line). 1 values at the histogram peaks in GM and WM for data acquired with no fat suppression and with 70° flip angle SPIR pulses. When we used 70° flip angle SPIR pulses, GM T1 values were shortened by 17% (Subject 3) to 23% (Subject 1) and WM T1 values by 21% (Subject 2) to 25% (Subjects 1), compared with no fat suppression. R1 maps collected across SPIR flip angles were linearly fit to b/a parameter map. Histograms of R1 maps corrected . The R1-corrected histograms (dash lines) are similar across fat suppression levels and match the data acquired with no fat suppression (black line), with a narrower distribution  than the uncorrected data, particularly for the global correction method (see 1-corrected data are closer to the reference (no fat suppression data), with slightly longer T1 values 7.1 and 5.6% of (GM) and 10.7 and 3.5% (WM) (across subjects) for voxelwise and global correction respectively. The use of a reduced dataset of two R1 maps with SPIR FAs of 40° and 70° to derive the global b/a correction parameter yields results (1 (compared to correction using the full data set) was 1.8% for GM and 1.1% for WM in Subject 2  with R1 map after correction with a global constant b/a (third and fourth rows for regression using data with 5 and 2 FS flip angles respectively). The corrected R1 maps are generally similar to those acquired with no fat suppression . The R1 histograms for the global correction display a higher peak and narrower FWHM, reflecting the increased SNR compared to the voxel-wise method. It should be noted, however, that when a B1-map is unavailable the voxel-wise method can be used to remove spatial variations due to both an inhomogeneous RF transmit field and attenuation of the water signal by the spectrally selective FS pulses in areas of inhomogeneous B0 . MP2RAGE gives a WM peak T1 similar to MS-IR-EPI (with FS) whereas the GM peak is shorter. This pattern is repeated for the whole brain volume (1 of 1170±38 ms and 1140±25 ms measured with MS-IR-EPI and MP2RAGE sequences respectively (n = 5), and mean GM T1 of 1783±60 ms for MS-IR-EPI compared to 1653±32 ms for MP2RAGE. 1 maps generated with MS-IR-EPI and MP2RAGE in axial, sagittal and coronal views. The red arrow indicates the effect of the large B0 field gradient in regions near air-tissue interface, causing larger distortions in MS-IR-EPI than MP2RAGE.Tn volume , with me1 at 7 T in the NIST/ISMRM spheres and in-vivo are shown in 1 in GM tissue measured with MS-IR-EPI for SMS=1 but is reduced below the level of MP2RAGE when using SMS=2 with FS and for all MS-IR-EPI measures in WM.The SNR-per-unit-time for MS-IR-EPI and MP2RAGE measures of T3.63) T1 maps of the visual cortex generated with MS-IR-EPI and MP2RAGE are shown in 1 maps generated with MP2RAGE (top row) and MS-IR-EPI (middle row), as well as in the S0 parameter fit for MS-IR-EPI (bottom row). The visual appearance of the MP2RAGE data is in line with previous work .High spatial resolution  and T2-spheres (R2=0.9767) provided by NIST. We report, for the first time, quantitative T1 values of the ISMRM/NIST T1-sphere system and T2-sphere system at 7 T. At 7 T, both the T1 mapping accuracy (with respect to the single slice IR measure) and repeatability of MS-IR-EPI were superior to that of MP2RAGE. In particular, for long T1 values (>1800 ms) MS-IR-EPI provides good accuracy while MP2RAGE underestimates T1, suggesting that longer TRshot is required for MP2RAGE to accurately measure T1 in grey matter tissue. The accuracy of both MS-IR-EPI and MP2RAGE decreases for low T1 values (<500 ms), which are below the physiological range of T1 in the human brain at 7 T.T1 values at 7 T as compared with 3 T, which at first may seem inconsistent with the commonly expected increase in T1 with field strength (r) of NiCl2 between 3 T (~0.63 s−1mM−1) and 7 T (~ 0.81 s−1mM−1) measured with MS-IR-EPI. This is consistent with previous work showing a relaxivity of NiCl2 at 7 T of 0.93±0.23 s−1mM−1 (r of 0.620 s−1mM−1 (2 were similar at 3 and 7 T (7.10±0.36 and 7.29±0.34 s−1mM−1 respectively for MS-IR-EPI) and comparable to r values for MnCl2 reported in the literature: 6.397 s−1mM−1  was better for the T1-spheres than the T2-spheres, the T2-spheres are better suited for 7 T imaging as they offer a wider range of T1-values which more closely match those found within the human brain compared to the T1-spheres, due to the increased relaxivity of NiCl2 at 7 T.It is worth noting that although we show a reduction in the sphere Tstrength , we have s−1mM−1  compared s−1mM−1 . We foun s−1mM−1  and 7.4   which were considerably shorter than those measured with MP2RAGE . Note that the degree of reduction in T1 values may vary across MR vendors as the fat suppression implementation is not identical across vendors. This discrepancy above can be explained by the small number of fat suppression pulses used (4.6 pulses per second) by Polders et al. compared to the Kashyap et al. study (11.7 pulses per second) and the multi-shot MS-IR-EPI sequences used in the present study . This is consistent with MS-IR-EPI data were collected using a range of SPIR fat suppression flip angles. Our results show that spectrally selective fat suppression pulses decrease the apparent value of TS pulses . It has S pulses  that frent study  reportin1 value  the myelin density, (ii) nominal flip angle of the spectrally selective FS pulses and (iii) the RF field distribution. Our model assumes that the R1 of water protons in brain tissue is mono-exponential. 1 component, associated with myelin water  histogram. Both methods worked well  although T1 values were slightly longer in WM (6.5%) using the voxel-wise correction  are close to those reported in the literature  are in line with values reported in the literature, ranging from 1074/1120 ms measured using MS-IR-EPI (1 component (1349 ms) measured with IR-SE  largely agree, with very slightly longer T1 (2.5% and 6.9% in GM and WM respectively) measured with MS-IR-EPI. Thus, the proposed correction method appears to enable accurate T1 quantification with MS-IR-EPI when using spectrally selective fat suppression pulses.Our correction of Ra values , which wked well  to correrrection . Our T1 terature  and to tS-IR-EPI , 1220 msS-IR-EPI  and 1284S-IR-EPI , but shoth IR-SE . Direct 4.2.1b/a FS correction method produces artefact-free maps, as expected, regardless of the power of the FS pulses used in the b/a parameter estimation. Since a SPIR FS pulse with FA=70° is required to completely remove the fat ring image artefact , indicating that the accuracy of the global correction method is not compromised when fewer datasets are used. This is in contrast with the voxel-wise correction, where fewer data sets results in a wider histogram of R1 values , rather than by iron or brain proteins for example. This assumption is supported by recent work  and T1=57 ms (13%) at 3 and 7 T respectively. They showed, however, that for sequences that acquire data using multiple inversion times, as performed for the proposed MS-IR-EPI sequence, a mono-exponential model is precise for measuring the long T1 component.There has been much discussion of the accuracy of Trameters , the fitrameters  and by srameters  giving rM tissue  has beenter work  using IR1 component on T1 estimation using MP2RAGE, since the signal equations used to convert the intensity of a composite MP2RAGE image into a T1 value assume a mono-exponential T1 recovery. They found that the effect of the short T1 component cannot be mitigated as easily as in sequences which use multiple IRs, with significant differences (90 and 125 ms at 3 and 7 T respectively) with respect to the T1 long component.Interestingly, 4.31 values measured with MS-IR-EPI were similar (2.6% longer) in WM but longer (7.9%) in GM. However, our MP2RAGE T1 measure for GM  is shorter than reported in other studies using MP2RAGE, where values of 1800 ms (shot (6 s) than the 5 s used in the present study, which could potentially result in longer T1 estimates. In our study, the 5 s time between inversion pulses was chosen to match the TR for the MS-IR-EPI sequence. A recent study using the same TRshot=5 s for MP2RAGE reported a very similar T1 value (1660±35 ms) for GM tissue to results reported in this manuscript. These observations, taken together with our phantom results suggest that the MP2RAGE sequence underestimates T1 values > 1800 ms, as a TRshot of 5 s is too short to accurately measure GM. In contrast, for the MS-IR-EPI sequence, T1 could be accurately measured in GM (yielding comparable measures to single slice IR-EPI). It should be noted that the longitudinal magnetization has more time to freely recover in MS-IR-EPI compared with MP2RAGE, where the longitudinal magnetization is continuously sampled over the long GRE trains.In comparison with MP2RAGE, T 1800 ms , 1959 ms 1800 ms  and 1870 1800 ms  are stat 1800 ms  used the1-field inhomogeneities than other sequences, such as the Variable Flip Angle method  when the efficiency is 90%, increasing to ~30 ms (1.5%) when efficiency is further reduced to 70%, and this error decreases for shorter T1 targets . Prior to implementation of the study we optimized the RF pulses to optimize slice profile whilst minimizing side-bands, as imperfect thin slice profiles can leave some signal un-acquired in multi-slice 2D imaging. For the NIST spherical phantom, we measured higher SNR per unit time for T1 maps, without SMS acquisition, both with and without fat suppression, but with an SMS factor of two slices, and with fat suppression, it was slightly lower than that of MP2RAGE. Note that head motion between scans can result in errors in the estimation of the noise, measured as the difference between the two scans. In SMS acquisitions, SNR is degraded by the g-factor reconstruction noise, which varies considerable between scanners and vendors. In addition to the g-factor noise penalty, non-perfectly square slice profiles can cause signal loss within a slice due to partial saturation resulting from RF excitation meant for an adjacent slice Ideal slice profiles are harder to achieve for thinner slices, particularly when using SMS excitation. Another noise contribution is introduced by the fitting procedure required for the T1 mapping in MS-IR-EPI, compared with MP2RAGE which uses a look-up table. To reduce this source of noise, a look-up table could also be used to estimate T1 in MS-IR-EPI, as previous studies have shown  reported for quantitative T1 maps of comparable SNR generated with 0.5 mm isotropic resolution using MP2RAGE at 7 T  and extra-striate cortex from T1 maps generated using MS-IR-EPI are distinct, showing a clear dip in T1 indicating the presence of the Stria of Gennari, whereas this is not clearly evident for the T1 map generated using MP2RAGE  and hence less T1-induced blurring.It has been suggested that a high spatial resolution, ideally to 0.3 mm, will be required to reliably distinguish the cortical areas solely based on intra-cortical features . For thi MP2RAGE B. This f2*, due to the lines of k-space being acquired at different times. In our implementation this effect is minimal due to the short acquisition window used, resulting from a high readout bandwidth and low EPI factor . It is also worth noting that the use of motion correction across the individual MS-IR-EPI volumes may have introduced additional image blurring ; however, even with these additional sources of blurring, the effective resolution of MS-IR-EPI was superior to that of MP2RAGE. The improved sharpness of MS-IR-EPI and efficiency (faster than MP2RAGE) highlight the feasibility of MS-IR-EPI for improving the spatial resolution of T1 mapping for the purpose of in-vivo cortical parcellation using myeloarchitecture.MS-IR-EPI images can also be prone to blurring in the phase encoding direction, associated with the relatively short Tctor see , though 1 analysis, a long TR (at least 5 s) is required. However, for the purpose of cortical parcellation, where the relative T1 profile across the cortex is of greatest interest, a shorter TR could be used to speed up the acquisition. Note, however, that a longer TR can be used to improve SNR, in place of signal averaging. For the purpose of cortical parcellation MS-IR-EPI presents an additional advantage as it also provides a S0 parameter fit, which is a map of proton density , thus providing an additional contrast that could be used in conjunction with the T1 map to assist in cortical parcellation.For fully quantitative T1 maps with excellent spatial fidelity, it should be noted that this MS-IR-EPI scheme can be adapted to acquire T1 maps which are distortion-matched to fMRI data acquisitions, by adjusting the acquisition bandwidth. This can provide 0.3 mm isotropic resolution T1 structural maps useful in cortical depth-dependant fMRI applications in fMRI space, especially important given the role of such maps in intra-cortical parcellation.Although the aim of this study was to collect very high resolution T4.51 maps with such spatial resolution as high as 0.3 mm isotropic, prospective motion correction methods, using either an optical tracking system  for MS1/MS2 sequences, but larger (22%) for the 0.5 mm isotropic acquisition  with respect to the single slice excitation acquisition. The RF pulse duration (9.6 ms) required for 0.5 mm thick slices is significant longer than the pulse duration for a single slice acquisition (3.6 ms), leading to increase TE (14 instead of 12 ms) and making sharp slice profiles harder to achieve. In order to minimize interference from neighbouring slices due to non-perfectly square slice profiles, interleaved slice excitation schemes, which can be implemented with SMS imaging  to studclerosis  or multiclerosis ), and toRosa M. Sanchez Panchuelo: Data curtion, Formal analysis, Methodology, Conceptualization, Funding acquisition, Writing – original draft. Olivier Mougin: Data curtion, Formal analysis, Methodology, Conceptualization, Writing – review & editing. Robert Turner: Methodology, Conceptualization, Writing – review & editing. Susan T. Francis: Methodology, Conceptualization, Funding acquisition, Writing – review & editing.None.


Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca Annexin A1 (ANXA1) is a 38-kDa protein which belongs to the annexin family of calcium-dependent phospholipid-binding proteins . The C-tIn this study, we characterize the binding region of a therapeutic antibody candidate binding to ANXA1 in complex with calcium . The humEpitopes of therapeutic monoclonal antibodies (mAbs) are often discontinuous and are only recognized by the mAb in their native conformational state . A detaiE. coli and Chinese hamster ovary (CHO) cells, respectively. Their identity and purity were confirmed by HPLC-MS (2+) were measured by surface plasmon resonance (SPR). For the Ca2+-bound holo-state of ANXA1, an equilibrium dissociation constant (KD) of 2.66 ± 0.02 nM was determined  and thereby avoid a false EX1 signature, the KD value was used to calculate the antibody and antigen concentrations.The antigen and the humanized murine anti-ANXA1 antibody (IgG1) were produced recombinant in  HPLC-MS . Bindingtermined a, whereatermined b. This s2O. Thereafter, only a very low increase in deuteration was observed for this region. In contrast, peptic peptides covering highly structured domains, starting with the first α-helix of repeat I showing a slow deuteration rate. During the first 0.5 min, in which all the easily solvent-accessible amide hydrogens were exchanged, only an average relative deuterium uptake of 21% was observed. The deuteration kinetic of repeat III is faster compared to the other core domains. Even after 24 h of deuteration, no saturation was reached and some peptides still showed an ongoing uptake of deuterium . All synthetic peptides used for determination of the HDX back exchange were obtained from Intavis .First, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic free acid (HEPES) was obtained from VWR Life Science . Formic acid was obtained from LGC Standards . Tris(2-carboxyethyl)phosphine (TCEP), calcium chloride, sodium chloride, and guanidinium hydrochloride were obtained from Carl Roth . Ammonium formate was obtained from Fluka . Pepsin from porcine gastric mucosa (3200–4500 U/mg), ethylenediaminetetraacetic acid tetra sodium salt dihydrate (EDTA), urea-d4 (98 atom %D), and 99.9% deuterium oxide were obtained from Sigma-Aldrich . Pierce immobilized porcine pepsin was obtained from Thermo Scientific . PNGase F was obtained from R&D Systems GmbH . The human monoclonal anti-ANXA1-antibody (IgG1) was obtained by humanization of a murine IgG2b antibody that was obtained from mice by genetic immunization with human ANXA1, as previously described . The murE. coli BL21(DE3) cells obtained from New England Biolabs . Purification of the GST-ANXA1 from soluble E. coli extract was achieved by affinity chromatography using GSTrap FF  according to the manufacturer’s instructions. After buffer exchange into PreScission protease cleavage buffer using a HiTrap desalting column , the GST tag was cleaved off upon incubation with PreScission protease . Subsequently, GST was removed by affinity capture using Glutathione Sepharose 4B . Residual amounts of GST were removed by incubation with GST Trap resin . Finally, the buffer comprising cleaved ANXA1 was exchanged to 20 mM HEPES pH 6.0, 150 mM NaCl by dialysis using D-Tube Dialyzer . Purified ANXA1 was stored at a concentration of 1.6 mg/mL at −78 °C. Purity and integrity of produced ANXA1 were examined by SDS PAGE followed by Coomassie staining and mass spectrometry.ANXA1 cDNA was cloned into a pGEX-6P-1 expression vector. The protein was expressed as GST-ANXA1 fusion protein in First, 1.6 µg (4 µL) of ANXA1 and PNGaseF-deglycosylated antibody, respectively, was analyzed by LC-MS using a UHPLC system  coupled to a QTOF-type mass spectrometer . Reversed-phase chromatography  was applied for desalting. The solvents were 0.1% formic acid in (A) water  and in (B) acetonitrile , respectively. A stepwise linear gradient was run at 75 °C at a flow rate of 150 µL/min  followed by wash steps. Mass spectrometer parameters were adapted to the size of the molecule and the LC flow rate.v/v) P20 surfactant) at 25 °C. Kinetic data were collected at five different concentrations of ANXA1 with or without calcium, 25, 12.5, 6.25, 3.125, and 1.563 nM, through all flow cells to record the association phase. The analyte was allowed to associate for 5 min, followed by a dissociation phase in buffer for 10 min at 30 µL/min flow rate. At the end of each binding cycle, the sensor surface was regenerated by 8-min injections of 10 mM glycine HCl, pH 2.0, at a flow rate of 10 µL/min, followed by 5-s injections of 50 mM NaOH at a flow rate of 60 µL/min. The data were fitted to a 1:1 binding with drifting baseline using the BIAevaluation 4.1 software . Kinetic rate constants were determined from the fits of the association and dissociation phases, and the KD was derived from the ratio of these constants.SPR interaction analysis was performed on a Biacore 3000 system . For ANXA1 interaction analysis, purified goat antihuman IgG Fc  was immobilized to achieve a level of 13,000 response units (RU). Immobilization of the capturing antibody was performed on a CM5 chip  using amino coupling chemistry. Anti-ANXA1 IgG was captured by the anti-human IgG at a concentration of 7 nM with a pulse of 60 s at a flow rate of 10 µL/min to achieve a binding level of 300 (±50) RU on the measuring flow cells. Binding experiments were carried out in HEPES-buffered saline  prepared with D2O. The final concentrations in the exchange reaction were 2.0 and 1.8 µM for ANXA1 and antibody, respectively. To ensure a minimum of 89% of complex formation, the molar ratio of antigen to antibody was calculated as per [D) of 2.66 nM determined by SPR analysis. The final D2O concentration was 90%. After 0.5, 5, 50, 500 min, and 24 h at 20 °C, aliquots of 20 µL were taken and quenched by adding 20 µL ice-cold quenching solution (0.4 M TCEP and 0.8 M guanidine HCl in 100 mM ammonium formate solution pH 2.2), resulting in a final pH of 2.5. The HDX of the bead-based digest was followed for 5 and 500 min. The immobilized pepsin was prepared by adding 40 µL of 50% slurry (in ammonium formate solution pH 2.5) to a tube and dried by centrifugation at 1000× g for 3 min at 0 °C and discarding the supernatant. Quenched samples were immediately snap-frozen. Before injection, aliquots were thawed and either 0.45 µL pepsin (100 µM) was added to the quenched samples (in-solution digest) or the samples were added to the dried pepsin beads (bead-based digest). Proteolysis of the in-solution and bead-based digest was performed for 10 min or 1 min, respectively, in a water ice bath. The samples of the in-solution digest were injected immediately into an LC-MS system. The samples prepared with the bead-based digest were added to a 22-µm filterand the beads were separated by centrifugation at 1000× g for 30 s at 0 °C and were afterwards also applied to an LC-MS system. Undeuterated control samples were prepared under the same conditions using HEPES-buffered saline prepared with H2O instead of D2O. All experiments were performed in triplicate. A high-deuterated control was prepared for 24 h at 20 °C. Labeling was performed using HEPES-buffered saline  in D2O with addition of 6 M urea-d4 to the deuteration buffer. Samples were prepared and analyzed as described above. Samples were quenched by adding 1:1 (v/v) ice-cold quenching solution (0.2 M TCEP in 100 mM ammonium formate solution pH 2.6) to pH 3.0. Digest and analysis were performed as described above.ANXA1 (5 µL) was incubated with and without anti-ANXA1 (5 µL) at 20 °C for 10 min. Deuterium exchange of the pre-incubated antibody–antigen complex was initiated by dilution with 90 µL HEPES-buffered saline  incubation at 20 °C, deuteration was quenched by adding 10 µL of quenching solution to each aliquot. The proteolysis step was simulated by storing the samples on ice or at 20 °C for 2, 5, and 10 min. The samples were injected into the LC-MS system immediately after incubation.The overall deuterium recovery of the method was determined using 14 synthetic peptides with different lengths and compositions (see supplement). A stock solution was prepared containing 1 mg/mL of each peptide. Aliquots of 1 µL of the stock solution were diluted 1:10 in PBS and freeze-dried. Deuteration was initiated by dissolving each aliquot in 10 µL, 99.9% DHDX data acquisition and analysis was performed as previously described . For dat


The applications demand maximizing Eu3+ photoluminescence response. Eu-doped ZnO, TiO2, and Lu2O3 thin films were deposited by Pulsed Laser Deposition (PLD). Pulsed UV Laser Annealing (PLA) was utilized to modify the properties of the films. In situ monitoring of the evolution of optical properties (photoluminescence and transmittance) at PLA was realized to optimize efficiently PLA conditions. The changes in optical properties were related to structural, microstructural, and surface properties characterized by X-ray diffraction (XRD) and atomic force microscopy (AFM). The substantial increase of Eu3+ emission was observed for all annealed materials. PLA induces crystallization of TiO2 and Lu2O3 amorphous matrix, while in the case of already nanocrystalline ZnO, rather surface smoothening0related grains’ coalescence was observed.Eu The Eu3+-doped thin films are fabricated by a variety of methods [Euillators ,9,10,11. methods , plasma- methods ,14, elec methods , hydroth methods ,16, chem methods ,18, sput methods ,20,21, e methods ,23, puls methods ,25,26,27 methods , and sol methods ,31,32,33Post-deposition thermal treatment is usually required to activate the Eu ions and optimize the photoluminescence (PL) response ,30,32,333+-doped films may profit from numerous advantages [high-tech electronic, optoelectronic, photonics, and sensing devices.The utilization of Pulsed Laser Annealing (PLA) to obtain and tune optically active Euvantages ,35, i.e.FL), repetition rate (frep), number of shots, and ambient atmospheres [3+ PL response, a number of testing samples will be necessary. In situ monitoring of PLA can be an effective way to reach the optimal conditions accurately and fast.PLA technique offers a great flexibility in the number of control parameters available, i.e., laser wavelength, fluence  and KrF laser . The targets were Eu2O3:ZnO (1% at. Eu), Eu2O3:Lu2O3 (3% at. Eu), and Eu2O3:TiO2 (1% at. Eu). Depositions were performed in an oxygen ambient. The vacuum chamber was pumped down to an ultimate pressure of 5.10−4 Pa. The films were grown on fused silica substrates maintained at room temperature. The distance between substrate and target was 50 mm. The deposition conditions are listed in ZnO:Eu, Lufrep = 1 Hz. The signal for in situ PL was taken from the sample edge using an optical fiber connected to a spectrometer Horiba JY Triax iHR550  equipped with LN cooled CCD Symphony. The same ArF laser was used for spectra excitation at lowered FL = 10 mJ⋅cm−2 with frep = 5 Hz. Optical transmittance was in situ probed perpendicularly using light generated by a DH 2000 source  and analyzed by a spectrometer. The PLA conditions are listed in PLA was performed using an ArF laser  Compex PRO 205 F . To perform PLA the laser beam was focused using converging lens on the sample at the angle of 70°. The optical properties were analyzed sequentially after each PLA pulse or burst of pulses that were fired at ® PC Micro-XRF spectrometer , Rh X-ray tube, 40 kV, polycapillary optics, a diameter of 30 µm of incident beam, and a silicon-drift detector with resolution FWHM~130 eV/Mn. The thickness measurement took place on an Alphastep IQ device  with a scan length of 5 mm, stylus force of 5.5 mg, and scan speed of 50 µm·s−1. Each line was measured two times. The stylus diamond tip had a radius of 5 µm and an angle of 60°.The structure of the films was characterized by X-ray diffraction (XRD) using an X-Ray diffractometer  with Cu anode (λ = 1.5406 Å) by grazing incidence (omega = 0.85°) and Bragg–Brentano measurements at room temperature. Morphology was examined by atomic force microscopy (AFM) . Chemical composition was determined by Scanning electron microscopy-Energy-dispersive X-ray spectroscopy (SEM-EDX)  and the Orbis3+ PL 5D0→7Fj  emission related to f-f transition. The observation of the PL emission confirmed that the energy transfer processes in Eu3+ excitation must take place considering excitation wavelength λ = 193 nm (6.42 eV). It can be mainly related to host absorption and marginally in case of Lu2O3:Eu also to the O2−–Eu3+ charge transfer band (CTB) [5D0→7F2, its Stark splitting and 5D0→7F2/5D0→7F1 (magnetic dipole transition) emission ratio (asymmetry ratio), we could correlate the effect of PLA on the structural properties’ modification. It provided information related to the surrounding defects and disorder around the Eu3+ ions. The 5D0→7F1 transition was not considerably influenced by the surrounding of Eu3+ in the particular host. The transition 5D0→7F2 dominated all emission spectra from Eu3+,, suggesting the local symmetry around Eu3+ was low and deviated from an inversion center. The 5D0→7F0 transition was only allowed, taking into account the electric dipole selection rule, in the following 10 site symmetries: Cs, C1, Cn, and Cnv  [The deposited films of all investigated materials exhibited characteristic Eund (CTB) ,40. Foll3, 4, 6) .g listed in 1/m against photon energy (hν), where m is a parameter related to the type of transition, m = ½, and 2 corresponds to direct allowed transition and indirect allowed transition, respectively. TiO2 in anatase phase is considered as an indirect semiconductor, where m = 2 [2O3 is considered as a direct ultrawide band gap semiconductor (m = ½) [g values were obtained with error of ±0.02 eV.The values of Ere m = 2  while Znre m = 2  and Lu2O (m = ½) . The Eg As for the chemical composition of the films, Eu dopant concentrations obtained by EDX and XRF analyses corresponded to those of the targets and any substantial variation after PLA was not detected.2, and Lu2O3 by PLA, in more details.In the following, let us discuss the properties’ modifications of each material, Eu-doped ZnO, TiO3+ red emission was detected at 587.3 nm and 593.8 nm (5D0→7F1), 609.9 nm and 618.8 nm (5D0→7F2), 654.8 nm (5D0→7F3), and 691.5 nm, 692.8 nm and 704.4 nm (5D0→7F4). The spectra in 3+ ions in ZnO:Eu films generally occupy the substitutional sites of Zn2+ ions. This fact turns 5D0→7F1 and 5D0→7F2 transitions to be allowed. The observation of 5D0→7F2 transition splitting suggests that Eu3+ can be at a substitutional Zn site (C3v symmetry). We could not observe clear evidence of 5D0→7F0 transition whose appearance in PL spectra of ZnO:Eu was related with Eu3+ presence in other sites  [3+ in the sample located at ZnO nanocrystals’ surface, Eu3+ emission lines’ intensities and asymmetric ratio would increase with the increasing number of PLA shots, while the asymmetric ratio saturated at N# = 400, as it is depicted in 3+ PL intensity at 609.9 nm by a factor of 4.1× was obtained. The PL signal intensity and asymmetric ratio variations might be related to microstructure modification, as displayed in 3+ ions should play a more important role toward PL efficiency enhancement than the energy transfer process from ZnO matrix to Eu3+ ions [The evolution of optical properties (PL emission and transmittance spectra) of ZnO:Eu films with increasing number of PLA shots is shown in rystals) ,45,46. Au3+ ions .g~3.28 eV to 3.25 eV. Recently, we observed similar Eg values and trends at ZnO:Eu film PLD deposited at a substrate temperature of 300 °C [The transmittance spectra shown in f 300 °C .L ≥ 140 mJ/cm2, where coalesced nanoclusters kept the wurtzite crystalline structure [AFM images of the film surface presented in tructure . The PLAtructure  and ZnO:tructure  films of2O3, was not detected. Considering the larger ionic radius of Eu2+ (1.13 Å) than Eu3+ (0.95 Å) in comparison with Zn2+ (0.74 Å), substitution of Eu3+ ions at Zn2+ sites seemed to be more favorable than that of Eu2+ ions but charge compensation must take place [XRD patterns of as-deposited and PLA-treated ZnO:Eu film, shown in ke place .2:Eu films, i.e., PL emission and transmittance spectra, on the number of PLA shots is presented in 3+ for the as-grown, which is characteristic for the amorphous phase [3+ PL intensity accompanied by sharpening lines of split 5D0→7F2 transition, as depicted in 2 matrix. The significant increase of Eu3+ PL intensity (~5×) was detected at N# = 50, where transmittance decreased and further remained constant. TiO2 nanocrystallites formed by PLA may act as sensitizers absorbing the excitation energy more efficiently [5D0→7F2 transition to peaks located at 611.1 nm, 614 nm, 616.9 nm, 624 nm, and 626.1 nm. The splitting may indicate Eu3+ located in D2 and S4 symmetry sites of TiO2 [3+ PL intensity of 5D0→7F2 transitions at 616.9 nm and 624 nm after N# = 200 can be attributed to the creation of well-crystalized anatase TiO2 matrix [5D0→7F1 transition was weak, it made it impossible to conclude on its splitting. The 5D0→7F0 transition could not be followed at all, which may exclude Eu3+ occupying C2v symmetry sites. Concerning transmittance, we must notice that its variation within PLA processing was generally rather weak. Its relative variation was less than 2%, which is slightly above the measurement error.The dependence of optical properties of TiOus phase . The inciciently ,49. We c2 matrix ,50. Becag value after PLA, as listed in g~3.22 eV for TiO2 material [2 films [2:Eu films deposited by the spray pyrolysis technique. Our Eg values suggested we did not introduce defects in the films since lowering Eg from 3.03 eV to 2.52 eV was observed after aerodynamic levitated laser annealing of defect-rich (oxygen vacancies) TiO2 nanoparticles [g~2.7 eV was also reported for TiO2:Eu films prepared by MAPLE, where Eg value shift was related to interstitial Eu doping [We did not obtain any shift in Ematerial  and anat[2 films  were reparticles . Lower Eu doping .An AFM image of the film surface, shown in 2, as referred in the JCPDS Card NO: 96-500-0224 file. Peak positions and reflections’ planes derived from XRD patterns were 25.38° (011), 36.78° (013), 37.68° (004), 48.03° (020), 53.68° (015), and 55.08° (121). Crystallites’ size was derived using Sherrer’s formula and was ≈ 46.1 nm. Anatase TiO2 phase was also obtained by KrF PLA of nanoparticles at FL = 34 mJ/cm2, while at a higher FL a rutile phase appeared [3+ and Ti4+, 0.95 A and 0.68 A, respectively, may cause a crystal structural distortion and also make difficult the substitution of Ti4+ by Eu3+ within the anatase lattice [XRD analyses, shown in appeared . The cry lattice .2O3:Eu film, shown in 3+ attributed to transition 5D0→7Fj (j = 0–4) as well as a lower intensity signal from transition 5D1→7Fj (j = 1–2). The excitation efficiency by ArF laser at λexc = 193 nm profited from Lu2O3 host lattice broad absorption band centered around λ = 210 nm [5D1→7Fj (j = 1–2) transitions is much less common in Eu3+-doped materials. These transitions could be observed in compounds with low-lattice phonon energy, e.g., Lu−O that leads to multiphonon relaxation [5D0→7Fj (j = 0–2) transitions, the PL spectrum revealed similar features as the spectrum (excited at λexc = 254 nm) of PLD-prepared Lu2O3:Eu film with 5% Eu content [5D0→7F0 (Eu3+ in the noncentrosymmetric C2 site [5D0→7F1 (Eu3+ in the C2 and S6 (centrosymmetric) sites [5D0→7F2 ; 582.2 c) sites ,58); 611 C2 site ,58); 650uioxides ,40.3+ intensity emission and optical transmittance was obtained. The Eg value shifted from 5.47 eV to 5.51 eV, where the latter values corresponded to those reported for evaporated Lu2O3 films [# = 2, which is reflecting the site symmetry decreasing related with film structure modification. The asymmetric ratio value was then decreasing to 9 and not changing for N# = 4–7. PLA led to the maximum enhancement of 2.8 times for Eu3+ intensity emission at 611.0 nm. The energy transfer from Eu3+ residing in S6 to Eu3+ in C2 may take place because the levels of Eu3+ in the site S6 lie above those for site C2. It was reported, based on following the 5D0→7F1/5D0→7F0 ratio, that the rate of the energy transfer may change with the crystallites’ sizes  or because the location of Eu3+ in C2 site is more common for small crystallites [We noticed the most substantial change in the film optical properties, shown in O3 films ,59. Thistallites ,60. We dq = 3.7 nm. Slight surface smoothing was observed after PLA and Sa decreased from 2.04 nm to 1.48 nm.AFM analyses, shown in 2O3:Eu films deposited by PLD at a substrate temperature higher than 400 °C [2O3, according to JCPDS Card NO: 96-101-0596 file. The Eu2O3 phase was not detected in the patterns, confirming the Eu dopant was substituting Lu in Lu2O3 host, which was supported by the closed values of the ionic radii of 0.95 Å and 0.85 Å for Eu3+ and Lu3+, respectively [A substantial improvement of the crystalline structure after PLA treatment was evidenced by XRD diffraction patterns, depicted in n 400 °C  and by mn 400 °C . The refectively . The bro2, and Lu2O3 films were fabricated by PLD at room temperature. The system allowed us to find quickly the optimal PLA conditions  for maximizing Eu3+ photoluminescence. The Eu3+ emission enhancement of 4.1×, 5× and 2.8×, after 800, 400, and 7 PLA shots, for ZnO, TiO2, and Lu2O3 matrix was obtained, respectively. Transmission was markedly modified at Lu2O3:Eu film, where also Eg increased to 5.51 eV. On the other hand, ZnO:Eu and TiO2:Eu films exhibited lowering Eg to 3.25 eV and 3.23 eV, respectively. TiO2:Eu and Lu2O3:Eu as-deposited films exhibited an amorphous character that was modified by PLA to anatase and cubic nanocrystalline phase, respectively. In the case of ZnO:Eu, the application of PLA induced rather surface smoothening-related grains’ coalescence.The in situ monitoring system of optical properties (photoluminescence and transmission) at pulsed laser annealing processing of Eu-doped oxide thin films was demonstrated. Eu-doped ZnO, TiO