DA1 and DAR1 are the ubiquitin receptors that function as the negative regulators of cell proliferation during development in Arabidopsis. An arginine to lysine mutant at amino acid site 358 could lead to the da1‐1 phenotype, which results in an increased organ size and larger seeds. In this study, the mutated DA1Zm (Zmda1) and mutated DAR1Zm (Zmdar1) driven by the maize ubiquitin promoter were separately introduced into maize elite inbred line DH4866. The grain yield of the transgenic plants was 15% greater than that of the wild‐type in 3 years of field trials due to improvements in the grain number, weight and starch content. Interestingly, the over‐expression of Zmda1 and Zmdar1 promoted kernel development, resulting in a more developed basal endosperm transfer cell layer (BETL) than WT and enhanced expression of starch synthase genes. This study suggests that the over‐expression of the mutated DA1Zm or DAR1Zm genes improves the sugar imports into the sink organ and starch synthesis in maize kernels.Grain weight and grain number are important crop yield determinants.  In recent decades, genetic engineering has been shown to represent a powerful means of improving grain yields in various crops, such as rice, wheat, and maize  with an arginine to lysine change at the conserved amino acid position 358 (DA1R358K) leads to a da1‐1 phenotype, and these mutants have larger leaves, flowers and seeds than the wild‐type (WT) , which are the characteristic domains of the DA1 gene family. Most of the DA1 gene family members in maize and ns Table .ZmDA1 encodes a 507‐amino acid protein, and ZmDAR1 encodes a 503‐amino acid protein. Interestingly, AtDAR3 to AtDAR7 was a separate clustering in a clade in the evolutionary tree; however, ZmDA1 and ZmDARs had a closer relationship with AtDA1, AtDAR1 and AtDAR2. The expression patterns of ZmDA1 and ZmDAR1 were examined using qRT‐PCR. ZmDA1 and ZmDAR1 are ubiquitously expressed in maize plants. ZmDA1 is primarily expressed in leaves and immature tassels and ears, while ZmDAR1 is primarily expressed in kernels at DAP10 to DAP15; embryos show low expression at DAP15  and ZmBt2 (ZmBrittle2) genes) to yield ADP‐glucose, which is the key substrate for starch synthesis. Through the action of starch synthase and branching enzymes, the glucose from this high‐energy intermediate is polymerized. The starch synthase enzymes can be divided into two categories: soluble enzymes and enzymes bound tightly to the starch grain. The ZmGBSSI (ZmWx1) gene encoded starch granule‐bound ADP‐glucose glucosyltransferase (starch synthase I), which is responsible for amylose synthesis and influences the starch content in the endosperm. ZmGBSSI and ZmBt2 played a very important role in starch synthesis and showed a significant difference between the transgenic and WT. The relative expression levels of the genes were measured using qRT‐PCR. Both genes were up‐regulated in the Zmda1 and Zmdar1 transgenic lines and down‐regulated in the ZmDA1 and ZmDAR1 over‐expression lines  plays an important role in transfer cell development. We detected the relative expression level of ZmMRP1 in various transgenic lines and the WT. As shown in Figure ZmMRP1 in Zmda1 or Zmdar1 transgenic plants was up‐regulated relative to that in the WT. Thus, the expression of Zmda1 or Zmdar1 enhanced the expression of ZmMRP1 and transfer cell development. When we measured the soluble sugar content in the developing kernels using anthrone‐sulphuric acid colorimetric method, the soluble sugar levels were different between transgenic plants and the WT. Compared with the WT, the sugar content was higher in the Zmda1 or Zmdar1 transgenic plants and lower in the ZmDA1 or ZmDAR1 over‐expression line at DAP5 and DAP10  in developing kernels. Moreover, ZmDA1 or ZmDAR1 may play a role in the regulation of sugar transport and starch synthesis.In conclusion, we successfully obtained maize Arabidopsis were obtained from TAIR (http://www.Arabidopsis.org/). The sequences were used as bait to acquire maize DA1 and DARs proteins. BLASTP was performed in the Gramene database (http://www.gramene.org/). Amino acid sequences of the proteins in Arabidopsis and maize were aligned using ClustalW  .Sequences of the eight members of the DA1 gene family proteins in ZmDA1 and ZmDAR1 were used to detect the gene expression pattern. The maize ACTIN1 gene served as the reference control. The decimal dilution cDNA was used as the template. All of the primer pairs used in this study are listed in Table ®Premix Ex Taq  systems. The fold changes in the target genes were calculated using the 2−▵▵Ct method.The seeds of the maize elite inbred DH4866 line were grown in the field. The developing organs and kernels were harvested and frozen with liquid nitrogen and stored at −80 °C for RNA extraction. Total RNA was extracted using a TRIzol kit . cDNA was synthesized using a reverse transcription kit  with 500 ng of total RNA as the template. The specific primer pairs of ZmDA1 and ZmDAR1 were cloned using PCR and cDNAs from maize inbred line B73 with primer pairs ZmDA1F and ZmDA1R as well as ZmDAR1F and ZmDAR1R. ZmDA1F and mZmDA1r as well as mZmDA1f and ZmDA1R bridge PCR primer pairs were used in the first PCR amplification to introduce a G to A single‐base mutant in two fragments. ZmDA1F and ZmDA1R were used to join the two fragments together. A similar method was used to introduce a G to A single‐base mutant at the 966 bp site in ZmDAR1. The first PCR amplification was performed with the following parameters: 95 °C for 5 min; 35 cycles of 95 °C for 1 min, 56 °C for 1 min, 72 °C for 1 min; and 72 °C for 5 min. The second PCR amplification was similar to the first but with extension duration of 2 min. The fragments of Zmda1, Zmdar1, ZmDA1 and ZmDAR1 were cloned into a donor vector that cascaded with a bt gene and a bar gene; the fragment was then recombined with the pB7WG2.0 vector using a LR gateway reaction with the LR Clonase enzyme kit .Full‐length fragments of Agrobacterium‐induced maize shoot‐tip transformation followed a previously described protocol . The PCR product of bar gene fragment was labelled with digoxigenin‐dUTP (Roche). Probe hybridization and detection were using the DIG NBT/BCIP kit for nucleic acids (Roche) following the manufacturer's instructions.Totally 45 μg genomic DNA of the T3 transgenic maize and wild‐type leaves was digested by To obtain the yield parameters of the transgenic lines, field trials were carried out in the field in Jinan  in 2013 to 2015. T3 and T5 generation plants were grown in three replicates in four‐row plots (48 plants per plot). The plot was 2.5 m in length and 2 m in width with 20 cm between the plants in each row. The measured values were collected from three replicates with six plants per replicate. The mature ears were harvested and dried to a constant weight at 48 °C; the ears were measured for length, the kernel number and rows were counted, and the ears were weighed. The 100‐grain weight was determined in three replicates for each plot. Ten seeds from each ear were sampled to measure the weight of the endosperm and embryo with three replicates of each.To determine the cell size and number, the fully expanded 7th leaf of the maize plants at the V9 stage was obtained. The samples were placed in a destaining solution (75% ethanol and 25% acetic acid) overnight at room temperature. The samples were then transferred into a basic solution (7% NaOH in 60% ethanol) for 15 min at room temperature. An ethanol series  was used for rehydration for 15 min. The leaves were then placed in 5% ethanol and 25% glycerol for 30 min. Samples were mounted in 50% glycerol and photographed using a microscope . For the cell area count, at least 200 cells were measured in one biological replicate using ImageJ  for 24 h. The samples were dehydrated in an ethanol gradient series from 50% to 100%. The samples were then cleared using xylene and were embedded in paraffin. The samples were sectioned at 10 μm thickness, stained with toluidine blue‐O and observed with an Olympus BX51 microscope. To observe the starch content during the grain‐filling stage (12 DAP), starchy endosperm cells were counterstained using Schiff's reagent and fast green.et al., The kernels of the transgenic and wild‐type plants were obtained at DAP12. The samples were dissected (3 mm in diameter) to keep the bottom of the kernels. The samples were prepared and fixed as previously described  after self‐pollination and were then dried to a constant weight at 80 °C in a drier. The soluble sugar was measured as previously described . The protocol was completed as previously described . The positive clones were screened by the ‐Leu–Trp and ‐Ade‐His‐Leu–Trp dropout supplements , and the positive clones were coloured by X‐α‐Gal .The full‐length cDNA sequences of The kernels were harvested from the middle site of the ears at DAP15. Paraffin‐embedded kernels were cut into 10 μm thickness and were stained by Schiffer's reaction. The cell number and the section area of kernels were measured in WT and transgenic kernels by ImageJ . Standard errors are provided for statistical diagrams as appropriate.Comparisons between the transgenic and WT plants were made using Duncan's multiple‐range test with a one‐way Figure S1 The phenotype of the wild‐type (WT) and T4 generation plants during the vegetative growth stage.Click here for additional data file.Figure S2 The distribution of cell length and width in transgenic and the wild‐type plants.Click here for additional data file.Figure S3 The agronomic traits of the wild‐type (WT), Zmda1 and Zmdar1 over‐expression plants in the field.Click here for additional data file.Figure S4 The agronomic traits of the wild‐type (WT) and ZmDA1 and ZmDAR1 over‐expression plants in the field.Click here for additional data file.Figure S5 The agronomic traits of the wild‐type and transgenic plants.Click here for additional data file.Table S1 The expression pattern of DA1 gene family of maize and Arabidopsis.Click here for additional data file.Table S2 Agronomic traits of WT and transgenic maize in the field in 2013 and 2015.Click here for additional data file.Table S3 The primer pairs used in this study.Click here for additional data file.


DNA generates a physical barrier to replication fork progression and creates topological stress. By allowing replisome rotation, absence of the Tof1/Csm3 complex promotes the conversion of impending topological stress to DNA catenation and causes camptothecin hypersensitivity. Through synthetic viability screening, we discovered that histone H4 K16 deacetylation drives the sensitivity of yeast cells to camptothecin and that inactivation of this pathway by mutating H4 K16 or the genes SIR1‐4 suppresses much of the hypersensitivity of tof1∆ strains towards this agent. We show that disruption of rDNA or telomeric silencing does not mediate camptothecin resistance but that disruption of Sir1‐dependent chromatin domains is sufficient to suppress camptothecin sensitivity in wild‐type and tof1∆ cells. We suggest that topoisomerase 1 inhibition in proximity of these domains causes topological stress that leads to DNA hypercatenation, especially in the absence of the Tof1/Csm3 complex. Finally, we provide evidence of the evolutionarily conservation of this mechanism.Camptothecin‐induced locking of topoisomerase 1 on  Saccharomyces cerevisiae cells lacking Top1 can fully replicate their genome because in the absence of Top1, positive supercoils can either be relaxed directly by Top2 Separation of the two parental DNA strands during DNA replication creates positive supercoiling ahead of the replication fork. Such over‐winding hinders replisome progression and must be removed for DNA replication to be completed. In eukaryotes, two DNA topoisomerases, Top1 and Top2, cooperate to allow DNA replication and segregation. The main DNA topoisomerase that relaxes positive supercoiling during DNA replication is considered to be Top1, a type‐IB topoisomerase, while Top2 activity seems to be concentrated behind replication forks tof1∆ strains, similar to mrc1∆ strains, show synergistic phenotypes in combination with loss of Rad9—the other major checkpoint adaptor protein in S. cerevisiae—suggests that the Tof1/Csm3 complex recruits Mrc1 for the purpose of Rad53 activation To reduce the requirement for decatenation, replisome rotation is normally restricted by the Tof1/Csm3 complex tof1∆ and csm3∆ yeast strains were shown to be hypersensitive to high doses of camptothecin tof1∆ and csm3∆ strains might arise through topologically stressed DNA structures generated by Top1 inhibition rather than from DNA damage per se Despite the above findings, certain results have remained unexplained, and the precise roles of the Tof1/Csm3 complex have remained elusive. For instance, tof1∆ and csm3∆ cells, suggesting that the increased sister chromatid catenation generated in the absence of these proteins promotes camptothecin toxicity. Finally, we show that the role of sirtuins in driving camptothecin sensitivity in S. cerevisiae is evolutionarily conserved in the yeast Schizosaccharomyces pombe and in human cells.Here, we show that histone H4 K16 deacetylation by the yeast sirtuin complex drives the sensitivity of wild‐type cells to camptothecin. Our results also show that the disruption of chromatin domains bearing deacetylated H4 K16 rescues the camptothecin hypersensitivity of TOF1‐ or CSM3‐deleted cells. This hypersensitivity arises from the well‐established trapping of Top1 in a covalent complex with DNA, as shown by the fact that it was rescued by TOP1 deletion  or IRC15. However, ensuing studies established that neither IME2 nor IRC15 deletion suppressed the camptothecin hypersensitivity of tof1∆ cells , which is found in an acetylated state through much of the transcriptionally active yeast genome. In S. cerevisiae, deacetylation of this residue by Sir2 allows binding of Sir3, thus recruiting further Sir2 that removes acetylation marks from flanking H4‐K16 residues, a process that is then propagated to produce a transcriptionally silent heterochromatic state tof1∆ cells was H4‐K16, we mutated this residue to glutamine (Q), a residue that mimics a constitutively acetylated lysine and abrogates Sir3 binding hhf‐K16Q mutation suppressed the camptothecin hypersensitivity of a tof1∆ strain, and at higher doses also reduced the camptothecin sensitivity of a wild‐type strain .To further explore how tof1∆ cells. Conversely, if broad acetylation of the chromatin template was required to rescue the tof1∆ phenotype, sirtinol should lead to suppression of extended mitotic delay only if tof1∆ cells were pre‐grown in the presence of sirtinol. To discriminate between these two hypotheses, we grew hml and hml, tof1∆ cells either in the presence or in the absence of sirtinol, and then synchronised them in G1 by addition of alpha‐factor  loci HML mutation did not affect camptothecin sensitivity). We then released the G1‐synchronised cells into S phase in the presence of camptothecin alone, or in the presence of camptothecin plus sirtinol. While addition of sirtinol after the G1 release was not sufficient to rescue the mitotic delay of tof1∆ cells , the so‐called rap1‐17 allele. This mutation completely disrupts transcriptional silencing at telomeres (telomere position effect) and partially affects silencing of the cryptic mating‐type locus HMLrap1∆663 allele grew slower than wild‐type strains, presumably due to the role of Rap1 in regulating transcription of genes involved in ribosome formation and glycolysis rap1∆663 mutation also failed to suppress the camptothecin hypersensitivity of tof1∆ cells ‐mediated recruitment of Sir1, which then attracts the SIR complex via an interaction with Sir4 SIR1 results in partial loss of silencing at the cryptic mating‐type loci, but does not affect telomeric or rDNA silencing SIR1 deletion strongly alleviated the camptothecin hypersensitivity of a tof1∆ strain  for every ORF in the yeast genome. While the majority of all ORFs essentially had a null SIR score (indicative of no enrichment of SIR complex binding), we found that 82 of them showed an enrichment of Sir2/3/4 and concomitant loss of H4‐K16 acetylation along more than 20% of their sequence . Of thesloci Fig . Taken tHM loci requires its interaction with the bromo‐adjacent domain (BAH) region of Orc1 tof1∆ cells by preventing ORC‐mediated recruitment of Sir1. In line with this hypothesis—and in contrast to what we had observed upon deleting the HM locus—deleting the BAH domain of Orc1 markedly suppressed the camptothecin hypersensitivity of tof1∆ cells . In agreement with the proposed role of Orc1 BAH domain in recruiting the SIR complex, deletion of SIR2 did not further enhance camptothecin resistance of orc1∆BAH cells, either in a wild‐type or a tof1∆ background . We established that, rather than by reducing camptothecin action, deletion of these SIR genes removes a factor that hinders cell proliferation in the presence of camptothecin in wild‐type cells and that is particularly toxic to cells lacking the Tof1‐Csm3 replication‐pausing complex. Camptothecin promotes the accumulation of positive supercoiling during DNA replication by locking topoisomerase 1 on DNA in a non‐functional state SIR genes.By using a synthetic viability screening approach tof1∆ and csm3∆ cells, as deletion of TOP1 is not toxic to tof1∆ cells. Instead, camptothecin‐mediated locking of Top1 on DNA could be the source of topological stress, either directly by creating topologically closed domains, or indirectly by preventing Top2 action—similar to what is thought to happen when a catalytically inactive Top2 that still retains an ability to bind DNA is expressed While camptothecin is a Top1 inhibitor, lack of Top1 activity is not sufficient to explain the hypersensitivity of tof1∆ cells. Importantly, we have established that the cell cycle delay observed in camptothecin‐treated tof1∆ cells can only be suppressed if Sir2 activity is inhibited prior to camptothecin treatment, suggesting that it is the state of the chromatin template itself that becomes toxic to cells when replicated in the presence of camptothecin.Lack of Sir2, Sir3, or Sir4 leads to loss of histone H4 lysine 16 (H4‐K16) deacetylation and subsequent impairment in heterochromatin formation. We have observed that inhibition of Sir2 deacetylase activity or mutation of H4‐K16 to glutamine (a residue that mimics an acetylated lysine) also increases the camptothecin resistance of both wild‐type and HML and HMR; and of these, only the cryptic mating‐type loci require Sir1 for their silencing SIR1 deletion also suppresses the camptothecin sensitivity of tof1∆ cells initially suggested to us that HML and HMR represent the chromatin templates that are toxic to tof1∆ cells in the presence of camptothecin. However, we did not observe a reduction in tof1∆ sensitivity to camptothecin by deleting HML and HMR, meaning that these two genomic loci alone are not responsible for the strong camptothecin sensitivity phenotype displayed by tof1∆ cells.Yeast genomes contain three well‐characterised regions of transcriptionally silenced chromatin: the ribosomal DNA, subtelomeric regions and the cryptic mating‐type loci tof1∆ cells. Another possibility is that SIR‐mediated genomic loci could be particularly prone to replication‐induced topological stress and would therefore be more frequently targeted by Top1 and more susceptible to camptothecin‐induced DNA damage. In this context, replisome instability caused by TOF1 deletion would increase the chance of fork breakdown or failure to rescue fork reversal events.Analysis of publicly available ChIP‐seq data allowed us to identify various genomic loci that exhibit enhanced localisation of Sir2, Sir3 and Sir4 as well as H4‐K16 under‐acetylation. Notably, we found that these genomic loci colocalise with confirmed ORFs and are located closer to sites of ORC binding than the average yeast ORF. Indeed, we found that many of these sites colocalise with genomic loci that were previously shown to bind ORC despite not having replication origin activity S. pombe, either loss or inhibition of Sir2, the fission yeast SIR2 ortholog, results in camptothecin resistance. As in S. cerevisiae, fission yeast Sir2 is involved in the heterochromatin assembly within the mating‐type locus, subtelomeric regions and centromeric DNA We have also provided evidence that the role of sirtuins in driving camptothecin sensitivity is evolutionarily conserved from yeast to humans. In the case of Inhibition of topoisomerase 1 is a widely used therapeutic strategy to selectively kill proliferating cancer cells, with camptothecin analogues being part of the standard of care provided by many cancer clinics worldwide. Various mechanisms of camptothecin resistance have been observed, ranging from overexpression of drug‐efflux transporters, which actively reduce intracellular drug concentration hht1‐hhf1∆::KanMX6 hht2‐hhf2∆::HPH) with plasmids obtained by site‐directed mutagenesis of plasmid pMR206 . Isolation of suppressor strains was carried out as previously described S. pombe strains used were 49 (h+ ade6‐M210 leu1‐32 ura4‐D18) and 34 (h+ sir2::kanMX6 ade6‐M216 leu1‐32 ura4‐D18).Yeast strains used for this work are haploid derivatives of W303 unless otherwise indicated and are listed in Whole‐genome paired‐end DNA sequencing and data analysis were performed as previously described n ≥ 2).Overnight‐grown saturated cultures of the indicated strains were serially diluted (10 fold) in water; 10‐μl drops of each dilution were deposited on each plate. Images were scanned 2–3 days after plating and growth at 30°C. Each experiment was repeated at least twice  were synchronised in G1 by addition of 5 μg/ml alpha‐factor for 2 h. G1‐synchronised cultures were then transferred to fresh YPD and released into S phase in the presence or in the absence of camptothecin and/or sirtinol; 45 min after the release, 20 μg/ml alpha‐factor was added to allow quantification of G1 cells by preventing re‐entry into the cell cycle. To detect Clb2, trichloroacetic acid protein extracts were separated on 10% polyacrylamide gels and Clb2 detection was carried out using anti‐Clb2 antibodies (Santa Cruz sc9071).Esir2 > 1.75 and Esir3 > 1.5 and Esir4 > 2 and EGFP < 3 and EH4‐K16ac < 0.3 and EH3 > 0.75 was exported to a bed file. These values were determined empirically, and small adjustments did not substantially alter the final results. For every ORF, the total number (T) of positions (nucleotides) for which the above conditions held was calculated by querying the bed file. The final SIR score was obtained by dividing this number (T) by the length of the ORF.Reads were aligned using BWA‐MEM, and duplicates marked with Picard. For each genomic coordinate, coverage was calculated using samtools and bedtools (samtools view ‐q10 ‐b $filename|genomeCoverageBed ‐d ‐ibam stdin ‐g) and normalised using the genomewide median of each sample. For each coordinate, the enrichment (E) was calculated as the ratio of the normalised coverages of IP and input samples. Every genomic position showing 3)2.hTERT RPE‐1 cells were cultured in Dulbecco's modified Eagle's (DME)/F12 1:1 medium (Sigma‐Aldrich) supplemented with 10% foetal bovine serum (BioSera), 2 mM L‐glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma‐Aldrich) and buffered with 0.2% Na(COCells were treated for 48 h with 50 μM sirtinol (Tocris), with camptothecin 30 nM for 24 h or pre‐treated with sirtinol for 24 h and then incubated with camptothecin and sirtinol for another 24 h as indicated. Cells were then washed three times with PBS and left to form colonies for 7–14 days. Colonies were stained with 0.1% (w/v) crystal violet in 20% (v/v) ethanol for counting. Results were normalised to plating efficiencies of untreated cells.Flow cytometry assays were performed as described in et alChIP‐seq data were downloaded from the Sequence Read Archive (NCBI) using accession numbers specified in in vivo experiments and analyses of ChIP‐seq data were designed and carried out by FP; experiments in S. pombe and human cells were carried out by IS. The manuscript was largely written by FP, IS and SPJ, with contributions made by all the other authors.The initial project was conceived by FP, VC and SPJ. Screening for suppressors and DNA extractions were carried out by FP and NJG. Analysis of whole‐genome sequencing data was carried out by MH and FP. Subsequent The authors declare that they have no conflict of interest.Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Table EV3Click here for additional data file.Table EV4Click here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 3Click here for additional data file.


B1+) field inhomogeneities. B1+ field information is required in order to correct these. The use of a generic, coil‐specific B1+ template is proposed and tested.Dynamic contrast‐enhanced MRI is the workhorse of breast MRI, where the diagnosis of lesions is largely based on the enhancement curve shape. However, this curve shape is biased by RF transmit  metrics were calculated between individual simulations and the template, and between individual measurements and the template. The agreement between the proposed template approach and a B1+ mapping method was compared against the agreement between acquisition and reacquisition using the same mapping protocol.Finite‐difference time‐domain simulations for B1+ mapping method was only slightly worse than the agreement between two consecutive acquisitions using the same mapping protocol in one volunteer: the range of agreement increased from ±16% of the nominal angle for repeated measurement to ±22% for the B1+ template.RMSE values  comparing individual simulations with the template were in the range 2.00‐4.01%, with mean 2.68%. RMSE values comparing individual measurements with the template were in the range8.1‐16%, with mean 11.7%. The agreement between the proposed template approach and a B1+ fields of the breast are comparable to the accuracy of B1+ mapping methods, even at 7 T. Consequently, a single generic B1+ template suits subjects over a wide range of breast anatomies, eliminating the need for a time‐consuming B1+ mapping protocol.With local RF transmit coils, intersubject differences in  B1+RF transmit fieldDCE‐MRIdynamic contrast‐enhanced MRIDESPOT1T1driven‐equilibrium single‐pulse observation of DREAMdual refocusing echo acquisition modeRMSEroot mean square errorSARspecific absorption rateSDstandard deviationT1NRT1‐to‐noise ratio.1Dynamic contrast‐enhanced MRI (DCE‐MRI) is the workhorse of clinical breast MRI examinations. Since its introduction in the 1980s, it has become a standard in breast MRI examinations due to its robustness and ability to detect tumor malignancy.Taking breast DCE‐MRI to higher field strengths such as 7 T is currently being investigated, showing potential for earlier and more accurate diagnosis.B1+) field variations, potentially shifting the curve of a tumor that should have caused a wash‐out shape into a more stable curve. This can be conceptually understood by considering the fact that at different B1+ levels there are different amounts of T1 saturation, and the effects of a change in T1 (due to contrast administration) will differ. If we define B1+ induced image intensity bias as measured intensitytrue intensityB1+=100%, then for fast RF spoiled gradient echo sequences bias=sinB1+θnom1−e−TRT1cosθnomsinθnom1−e−TRT1cosB1+θnom. Observe that this bias depends not only on B1+, but also on T1, which in DCE‐MRI is not constant in time, and the image intensity bias will change over the dynamic series. Generally, for any B1+ below 100%, the bias increases with increasing T1; consequently, when the T1 of tumor tissue drops due to contrast injection, the DCE curve's wash‐in is reduced due to the counteracting effect of the intensity bias. The opposite effect occurs when due to contrast wash‐out the tumor's T1 rises again, leading to a compromised curve, shifted to appear more stable than the true curve.A fast wash‐out rate is a typical indication of a malignant tumor, while a stable curve or continued wash‐in often reflects benign lesions. However, the curve shape can be compromised by RF transmit . The breasts, however, contain high amounts of fat, which has a relative permittivity that is an order of magnitude lower than that of most other tissues. This leads to a longer RF wavelength inside the breasts, approximately 40 cm assuming a relative permittivity around 10. Such a wavelength is usually larger than the size of the imaged anatomy. In this case, the B1+ field distribution within the breast will depend mainly on the local transmit setup used, and hardly at all on the individual anatomy. Therefore, we hypothesize that one generic, coil‐specific B1+ template will suit a wide range of subjects in the case of breast examinations with local transmit coils. We set out to test our hypothesis at 7 T, where local transmit is a commonly used strategy to overcome RF inhomogeneity issues.B1+ template in a clinical setting are twofold. It eliminates the need to acquire a B1+ field map, saving scan time. Furthermore, B1+ mapping techniques are known to be prone to noise and many are only reliable within a certain range.B1+ inhomogeneities present. Though other techniques to estimate B1+ in breast without acquiring field maps exist, these methods often rely on fat as a reference tissue (with a fixed T1) in order to estimate B1+ in the parenchyma.B1+ distributions can be deducted regardless of the sequence used. This method is limited only by the availability of a B1+ template of the coil design used.The advantages of using a generic B1+ template by investigating the inter‐subject differences in B1+ inhomogeneity. The work comprises both simulated field maps and measured ones, in order to compare template performance with the accuracy of B1+ mapping.The present work aims to explore the feasibility of using a generic 2B1+ template will suit a wide range of subjects when performing breast MRI with local transmit coils, a number of experiments were performed. First, RF simulations from previous work were used to create the template , presented in previous work, were used to investigate inter‐subject differences in B1+ distribution at 7 T when using this local transmit coil setup.B1 field distributions were calculated from personalized breast segmentations obtained from T1w Dixon scans fused with Virtual Family model Ella, and a 3D model of the relevant MR equipment.−12 s (one Larmor period) with a mesh of 2 × 2 × 2 mm3, assuming perfectly absorbing boundary layers. Convergence was assessed by visual inspection. Coil losses were not considered, since these have no impact on B1+ distributions. Vitamin tablets were attached to the coil elements using adhesive tape, in order to identify their position in the T1w scans. Though a limited number of volunteers were used to conduct the simulations, volunteers were selected to represent a reasonably wide range in breast anatomies, as can be appreciated from their T1w gradient echo scans in Figure Finite‐difference time‐domain simulations of B1+ and B1‐ fields, which did not share a common coordinate system, all simulated field distributions were aligned using multi‐resolution intensity‐based rigid registration in elastix.B‐spline interpolation and an adaptive stochastic gradient descent optimizer were used. This allows transmit coil alignment, since these coils are present in the simulations as hyperintense fields closest to the coil conductors. Differences in RF amplification settings between volunteers were overcome by normalizing all simulations relative to the simulation of V5 (arbitrarily chosen). In order to do so, the following intensity scaling factor was applied to the simulated field distribution for volunteers V2‐V5:i is the volunteer number and r→=xyz the position in the simulation. Note that the median was preferred over the mean to ensure robustness against outliers.In order to directly compare the simulated B1+ and B1‐ templates. Differences between volunteers were assessed per volunteer by comparing each individual simulation result with the B1+ template. The comparison was quantified using mean difference, standard deviation (SD) of difference and root mean square error (RMSE) metrics. All metrics were calculated over all voxels within the breast region of interest, which was determined previously.B1‐ template was created to facilitate B1+ template scaling using information obtained from the power optimization phase.Subsequently, the average of the rescaled simulation distributions of volunteer V2‐V5 was taken, we denote these the 2.2B1+ mapping, a validation set of 15 additional healthy female volunteers (S1‐S15), mean age 39 years  technique with the following parameters: pulse repetition time 4.0 ms, stimulated echo time 1.49 ms, free induction decay echo time 1.97 ms, 2.5 mm isotropic resolution, preparation angle 55°, imaging angle 25° and turbo field echo factor 32.B1+ acquisition was repeated, to compare the accuracy of the proposed template method with the variation between repeated measures. 3D T1‐weighted gradient echo images were acquired at four flip angles  using Dixon water‐fat separation with the following parameters: in‐phase echo time 1.97 ms, out‐phase echo time 4.4 ms, repetition time 6.0 ms and isotropic resolution 1.5 mm.B1+ map and variable flip angle images allowed the calculation of T1 maps using the driven‐equilibrium single‐pulse observation of T1 relaxation (DESPOT1) techniqueT1 mapping section for more details and the rationale behind the choice of angles.In order to prospectively compare the accuracy of the template approach with 2.3Estimates of breast volume and composition were calculated for all volunteers. Since the pectoral muscle was not visible for all volunteers due to the limited range where the coil transmits and receives sufficient signal, estimates of breast volume were obtained using the method described by Katariya et al.2.4Q‐factor measurements. Out of 15, nine volunteers were able to participate but in one volunteer the measurement failed, leading to eight useable data points. Measurements were made using a purpose‐built coil that contained a replica of the innermost element of the coil that was used in the MR experiments. The mechanics of the setup were identical to those used in the scanner. The Q factor  was determined using a network analyzer. Volunteers were asked to lie down in the prone position on the setup as they did in the scanner, positioning the arms on their back. Values for Q both with and without loading were recorded for each volunteer; the ratio Qunloaded/Qloaded was calculated as a measure for coil loading. The ratios were plotted against breast volume and volumetric glandular percentage for each volunteer, and a trend line was calculated using analytical ordinary least squares estimation.In order to check individual differences of coil loading, all volunteers (S1‐S15) were asked to return on a different day for additional 2.5B1+ template to facilitate direct comparison with the measured B1+ map for every volunteer. The map was masked before registration to exclude regions where a B1+ reconstruction was not available. The template was masked by thresholding to exclude values corresponding to flip angles below 20% and above 100% of the nominal flip angle. All values higher than 100% are very close to or in coil elements; the bottom cut‐off of 20% was empirically chosen to avoid registration of the edge of the map to the edge of the template. In the resulting binary image, a 3D connected components algorithm using a 6‐connected neighborhood was used to find connected regions. The largest connected component was selected as the mask. Multi‐resolution intensity‐based rigid registration was applied in elastix, using a mutual information similarity metric, B‐spline interpolation and an adaptive stochastic gradient descent optimizer.Rigid registration was applied to the B1+ values of the registered template were intensity scaled using information from the scanner's power optimization phase. During this phase, a global B1+ level (PO B1+) for the sample is measured. Using scanner log data from the power optimization phase for all volunteers, a calibration line was determined between this global PO B1+ and an average B1+ value determined from the registered template, scaled to match the measured B1+ map . Taking into account the global nature of the PO B1+, the average B1+ value was weighted with both B1+ and B1−: weightedB1+=∑∀r∈MB1−rB1+rB1+r∑∀r∈MB1−rB1+r, where M is a mask created by thresholding the survey scan using Otsu's method.B1+ template, independently of its measured B1+ map.Subsequently, the B1+ map and the registered and scaled template for all 15 volunteers (S1‐S15). As suggested by Bland and Altman, the same kind of plot was created for two repeated DREAM‐based B1+ mapping measurements to study repeatability.B1+ mapping procedures for volunteers S13‐S15.The map and the registered template were compared on individual bases through calculation of the RMSE, mean error and SD of the error per volunteer and a total mean absolute error for the validation set. Additionally, a Bland‐Altman density plot was created, showing the agreement between the measured 2.6B1+ maps to correct for the effects of B1+ inhomogeneities using a variable flip‐angle T1 mapping method.T1‐weighted gradient echo scans at different flip angles to estimate the T1 value at every recorded voxel by performing a fit of the signal equation, which is a function of the applied flip angle. Since this is a voxel‐wise method, B1+ correction can be easily applied by fitting the function while using the actual flip angle as the independent variable, i.e. the nominal angle multiplied by the value in the B1+ map for that voxel.As is commonly done in quantitative DCE‐MRI, we used T1 measurement over the wide B1+ range  and the wide T1 range (600‐2200 ms) present in the breast. To determine the best flip‐angle combination, all combinations of four integer angles in the range 1‐90° were tested and the T1‐to‐noise ratio (T1NR) was calculated for every combination of angles with T1 set to either 600 or 2200 ms and B1+ set to either 50% or 120% of the nominal angle. The sum over all four combinations of B1+ and T1 for T1NR determined the suitability of every combination of angles. Figure T1NR using the selected flip angle combination  over a wide range of T1 values for three levels of B1+.The flip‐angle combination was chosen by taking into consideration the notions put forth by Deoni et al.,T1 maps were calculated from the data using the DESPOT1 method.T1 maps that are not corrected for B1+, corrected by the DREAM B1+ data or corrected by the generic B1+ template respectively. The SD of the T1 estimate was calculated in every voxel, following the methodology described in Reference T1 estimate was larger than 100 ms were  considered unreliable and excluded .T1 maps were analyzed by comparing the measurements corrected using the measured map versus using the template. The T1 estimates were compared on individual bases through calculation of the RMSE, mean error and SD of the error.The obtained 33 and glandular percentage from 17 to 44%; in the validation set, breast volume ranged from 129 to 1032 cm3 and glandular percentage from 7 to 87%.Table B1+ in each individual. As Table The difference between the constructed generic template and every individual volunteer's simulation is shown in Figure Qunloaded/Qloaded ranged from 2.8 to 5.2. Measurements per volunteer are reported in Table Measurements for R2 of 0.825. The  generic template and the measured B1+ map are similar, as can be appreciated visually from Figure B1+ than areas with high B1+. Figure B1+ mapping for the same volunteers (S13‐S15); note that the limits of agreement are 12% wider in B than in C.The calibration line used in power‐optimization‐based scaling of the template is shown in Figure T1 estimates calculated with DESPOT1 and either map‐based or template‐based B1+ information are close, as can be appreciated visually in Figure 4B1+ distributions in the breast between volunteers covering a wide anatomical range are generally very small, only of the order of 2‐3%. This amount of variation is of the same order of magnitude as the accuracy of popular B1+ mapping methods.Our results from the simulations show that differences in B1+ estimation stronger.The width of the anatomical range of the validation set, quantified in Table B1+ maps show that a generic template can accommodate volunteers over a wide range of breast anatomies. The Bland‐Altman analysis in Figure B1+ is low, where the measured maps are unreliable.B1+ template. The bias that can be observed in Figure Our results from the measured B1+ corrected DESPOT1‐based T1 maps are substantially more homogeneous in both lipids and glandular tissue, irrespective of whether the B1+ information is from a map or the template. When comparing the analyses in Table B1+ propagate into mean errors in T1 estimates. In all cases but one, the SD of the error distribution is bigger than the mean error, which means that the two measurements of T1 do not significantly differ.It is clear from Figure B1+ distributions and show high agreement between measured and simulated field maps.Q measurements also contribute to this conclusion. While they show that tissue load is dominant , they also show that, even with breast volume changes by up to a factor of 5, load variations were all within 30% of Qunloaded/Qloaded = 4. This means that the biggest tissue load is caused by the rest of the body and that the Q variance over breast anatomies is limited, analogous to our results in B1+. Probably the RF eddy currents that occur in the rest of the body predominantly have a local B1+ effect that is either of insufficient strength to affect the B1+ in the breast, or hardly differs from the effects observed in the Virtual Family model (Ella).A limitation of this study is the fact that we have to rely on RF simulations to be able to construct the template. Several studies, however, have shown that these kinds of simulation are able to accurately predict B1+ is a reasonable assumption.Though this paper only demonstrates the use of a template for a unilateral breast coil, we believe that this can be extended to bilateral cases. Hardware developments in high‐field MRI tend to go towards parallel multi‐transmit systems, where amplitude and phase of all coil elements can be steered individually. In such setups, regarding each breast independently in terms of B1+ distributions is often used to validate predictions in specific absorption rate (SAR). While our work shows that B1+ distributions in the breast are very similar from person to person, the same does not necessarily hold for SAR. In fact, in recent work by Alon et al., it was demonstrated that B1+ distributions tend to be correlated over samples, but the same did not hold for SAR.B1+ distributions to validate SAR predictions should be done with caution. The present work serves as further proof of the statement that B1+ distributions tend to have high correlations between subjects; this is the very phenomenon we exploit when constructing and using a B1+ template.It is of note that demonstrating agreement between measured and simulated B1+ distribution. As reported in Table B1+ map, this SD reduces to 1.78% of the nominal angle. The main reason for the reduced performance  might be that the B1+ measured during the power optimization procedure is non‐localized. Therefore, it was assumed that the measured B1+ level during the power optimization was a weighted average over the entire imaged region that contained tissue. The differences in mean error between volunteers may be further reduced when a localized power optimization method is employed for template scaling.Implementation of the generic template approach in a clinical setting requires knowledge of both the position of the transmit coil in the image and the amount of template scaling that is needed for each subject. In many setups, the position of the coil is fixed on the bed, eliminating the positioning problem altogether. We have solved the scaling issue by using readily available information from the power optimization phase and the survey scan, information that will be present in any clinical protocol. This strategy brings a dependence of the template's performance on the goodness of fit of the calibration line of Figure B1+ fields of the breast at 7 T are comparable to the accuracy of popular B1+ mapping methods reported in literature. Consequently, we have shown that, at the cost of a small loss in accuracy , using a generic B1+ template to account for substantial RF transmit inhomogeneity in T1 mapping may be feasible across a wide range of volunteers.In conclusion, simulations show that inter‐subject differences in


The metamagnetic transition moves to higher fields for lower temperatures and splits into a double anomaly at critical fields Hc1 = 2.3 T and Hc2 = 2.8 T, respectively. Here, we report a detailed study of the different components of the magnetization vector as a function of temperature, applied magnetic field, and varying angle in Sr4Ru3O10. We discover for the first time a reduction of the magnetic moment in the plane of rotation at the metamagnetic transition. The anomaly shifts to higher fields by rotating the field from H ⊥ c to H || c. We compare our experimental findings with numerical simulations based on spin reorientation models taking into account magnetocrystalline anisotropy, Zeeman effect and antisymmetric exchange interactions. While Magnetocrystalline anisotropy combined with a Zeeman term are sufficient to explain a metamagnetic transition in Sr4Ru3O10, a Dzyaloshinskii-Moriya term is crucial to account for the reduction of the magnetic moment as observed in the experiments.Metamagnetism occuring inside a ferromagnetic phase is peculiar. Therefore, Sr This class of metallic compounds caught much attention in recent years due to its rich variety of ground states. Sr2RuO4 the n = 1 member, is discussed as an example of rare p-wave superconductivity1. A quantum critical endpoint covered by a high entropy phase was found in the n = 2 layer system Sr3Ru2O72. The compound Sr4Ru3O10 (n = 3) discussed here shows ferromagnetism below TC = 105 K3. Neutron diffraction experiments in zero magnetic field reveal ordering of the Ru moments along the c-axis. No ferromagnetic (FM) or antiferromagnetic (AFM) correlations are observed in the ab-plane5. Sr4Ru3O10 contains four inequivalent Ru sites with two different magnetic moments of 0.9 μB and 1.5 μB sitting on outer and inner RuO layers, respectively. The magnetic unit cell contains 8 of the smaller and 4 of the larger magnetic moments averaging to 1.1 μB per Ru. The higher order ruthenate SrRuO3 with n = ∞ also orders FM at a Curie temperature of 165 K7. The Srn+1RunOn+13 are strongly 2-dimensional electron systems with the trend to become more isotropic for higher n, because of their layered structure. Two-dimensionality is reflected in anisotropic transport properties as seen for Sr4Ru3O10 in the ratio of the electrical resistivity 8 and confirmed by optical conductivity experiments9.Sr12, but also changes of the band structure in itinerant electron systems. Latter scenarios are in the vast majority described on the basis of the well-known Stoner model13 and refined16, to accommodate special cases, e.g. Fermi surface reconstruction18 or in the vicinity of a quantum critical point20. In a general description, metamagnetism is a phase transition or crossover from a magnetically disordered or ordered state with small net magnetization to a field polarized (FP) or partially FP state. In the case of Sr4Ru3O10 the term metamagnetism refers to the sudden increase in the magnetization when the field applied in the (ab) in-plane of this layered compound exceeds 2 T. Magnetism is hidden only because the spontaneous moment is mainly aligned with the easy c-axis at smaller fields; nonetheless, we continue to refer to this as metamagnetic (MM) transition. Interestingly, the saturation magnetization above the MM transition (H ⊥ c) is about 10% smaller than the saturated moment for H || c21, which points to a more complex underlying scenario for the metamagnetism than just a simple spin flip transition. While the MM transition in Sr4Ru3O10 was discovered from early on in flux grown single crystals21, it took more than a decade to improve the crystal quality to a level to see a double step in the magnetization at the MM transition22. This strong dependence of physical properties on the crystal purity is a characteristic signature of strontium ruthenates Srn+1RunOn+13 and was also observed in the sister compound Sr3Ru2O725. The MM transition in Sr4Ru3O10 develops below 68 K as a double-transition close to zero field and shifts gradually to about 2.5 T with temperatures down to 1.7 K22. Carleschi et al.22 speculate that the double transition originates either in the ordering of Ru magnetic moments on two inequivalent crystallographic sites or in the presence of two van Hove singularities in the density of states close to the Fermi level. A transport study based on electrical resistivity27 reveals steps in the magnetoresistance at various critical fields around Hc accompanied by pronounced hysteresis. Fobes et al.27 interpret the transport data as domain movement of regions with high and low electronic spin polarization. Anomalous behavior at the MM transition was also observed in specific heat experiments28 up to 9 T and in thermopower investigations29. Neutron diffraction experiments up to 6 T reveal a change of lattice parameters at the critical field Hc4. Field and pressure dependent Raman measurements30 as well as a recent study of thermal expansion and magnetostriction31 confirm strong magnetoelastic coupling in Sr4Ru3O10.Metamagnetism is a phenomenon observed in magnetic materials, where hidden magnetism is suddenly uncovered by the application of an external field. The origin of metamagnetism can be spin flip transitions in antiferromagnetsd oxides in general and the peculiar MM transition inside the ferromagnetic order of Sr4Ru3O10 in particular. We carry out magnetization measurements up to 7 T and down to lowest temperatures of 0.46 K and under rotational fields between the c-axis and the ab-plane as well as (ab) in-plane rotation. Our investigations include a detailed analysis of the behavior of the magnetization modulus M at the MM transition and its individual components Mab and Mc simultaneously. In the following, we analyze and interpret our data in a localized picture, meaning the magnetic moments are mainly confined on the Ru4+ sites in the crystal structure of Sr4Ru3O10. This scenario is supported by neutron diffraction experiments which have determined the spin and orbital momentum distribution in great detail5. Our main discovery is the observation of a reduced measured moment at the MM transition caused by a spin component pointing out of the rotational plane which we assert can best be explained by significant anisotropic exchange interactions in Sr4Ru3O10.This work aims to increase our understanding of MM phenomena in 4H ⊥ c at temperatures below 2 K. Figure Hc1 = 2.3 T and a second anomaly at Hc2 = 2.8 T for increasing field as observed by Carleschi et al.22. Our new experimental data down to 0.46 K clarify that the transition neither sharpens to lower temperatures nor is there splitting into more distinct anomalies. The inset in Fig. H − T phase diagram with near-vertical phase boundaries for T → 0 at the MM transition. Both anomalies are shifted by −0.3 T for measurements in decreasing magnetic field. The size of the hysteretic region, marked as striped pattern, remains similar for all temperatures below 1.8 K.At first, we focus on the magnetization measured for mentclass2pt{minimMlong and transversal component Mtrans of the sample magnetization in respect to the applied magnetic field Mperp occurring perpendicular to the rotational plane is not recorded during the measurements. This geometry allows us to calculate the magnetization θ with respect to the magnetic easy axis c in the rotational plane. We can now calculate Mab = Mrot sin θ and Mc = Mrot cos θ, the magnetization occurring in the plane of rotation. Note, we follow closely the notation of angles used for magnetic anisotropic materials. Figure ψ = 81.6° taken at 1.8 K. Prominent feature is the hysteresis loop around ±1 T, caused by FM domain dynamics. The longitudinal magnetization Mlong increases moderately in small fields and shows a sudden rise at Hc1,2 Mtrans on the other hand consists mainly of the Mc component with a sudden decrease of the magnetization at the same critical fields Hc1,2. Hc1,2 indicates incomplete field polarization meaning that Mrot in the plane of rotation is depicted as black line in Fig. μB slightly higher than obtained in neutron experiments5, but in good agreement with previous magnetization studies32. Most peculiar is that Mrot drops suddenly below 1.2 μB at the MM transition and only recovers partially to 1.2 μB up to maximum applied field of 7 T. This missing component of the magnetic moment in Sr4Ru3O10 was never recognized before. Furthermore, we observe strong hysteresis at the MM transition between up and down measurements as reported in previous investigations32.The operation mode of the SQUID magnetometer allows the simultaneous collection of longitudinal entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaMab as a function of the field component in the ab-plane Hc1,2 change with ψ. In contrast to previous results by Jo et al.33 obtained by torque magnetometry, we observe a clear simultaneous increase of both critical fields Hc1,2 to higher values while rotating from H ⊥ c to H || c. In fact, Hc1,2 move out of the observable field range of H ≤ 7 T for ψ ≲ 72°. A similar shift to higher critical fields was observed in measurements of the longitudinal magnetoresistance for currents j || c and j ⊥ c as a function of rotating magnetic field as reported by Fobes et al.27.Geometrical effects can distort magnetic properties during magnetization experiments. To avoid this problem, we plot in Fig. Hc1,2 in the H − ψ phase diagram for field up and down sweep measurements. The difference Hc1 − Hc2 increases slightly with smaller ψ. As mentioned above, the double anomaly is accompanied by significant hysteresis. The inset of Fig. Mab of both MM transitions for decreasing ψ which were extracted from the curves in Fig. Mrot recorded in the plane of rotation for ψ between 85.3° and 70.5° is plotted in Fig. μB to below 1.2 μB at the critical field of the main anomaly Hc1 followed by a minimum and a small step at the second anomaly at Hc2. The described features are marked in Fig. ψ = 85.3°. The MM anomaly broadens and moves to higher fields for decreasing angles ψ.Figure Hc1 and Hc2 of the double step in Mab are intimately connected to the missing moment and represent basically the same critical magnetic fields, where the magnetization drops and partially recovers. Please compare Figs et al. found out in a temperature study22 that the double transition occurs right below the ferromagnetic ordering temperature TC = 105 K. This observation makes us believe that the effect of a missing moment occurs right below TC as well with some temperature broadening.So far, we only carried out magnetization measurements under rotational fields at 1.8 K and did not expand to higher temperatures. Therefore, we cannot conclude with certainty, how the “moment loss” changes with increasing temperature. We have shown, however, that Mperp occurring perpendicular to the rotation . The first scenario can be excluded based on neutron experiments where no short or long range AFM coupling neither in zero nor in magnetic fields H > Hc1,2 was observed in the ab-plane5. The second scenario is rather unexpected since magnetic moments tend to align with field and stay within the rotational plane, if no further coupling is present. We want to focus in our discussion on two mechanisms that potentially lead to a Mperp component in the magnetization. First one is based on general magnetocrystalline anisotropy in tetragonal symmetry, with an easy c-axis and 4-fold in-plane anisotropy. The second mechanism is antisymmetric exchange between spins, also called Dzyaloshinskii-Moriya (DM) interaction, causing a canting of the spins The “loss” of magnetic moment in the rotational plane can be explained either by partial AFM alignment or by a moment 4Ru3O10 in order to understand and model its magnetic anisotropy caused by spin-orbit coupling. Sr4Ru3O10 crystals consist of three layers of corner sharing RuO6 octahedra separated by a double layer of Sr-O. Primary Bragg reflections in synchrotron experiments can be indexed assuming a tetragonal unit cell with space-group I4/mmm, but a more detailed analysis of secondary reflections reveals orthorhombic Pbam symmetry3. The lower symmetry originates in c-axis rotation of the RuO6 octahedra that are correlated between different layers, meaning +11.2° clockwise rotation for inner and −5.6° counterclockwise rotation for outer layers.We have to have a closer look at the crystal structure of SrF accounting for magnetocrystalline anisotropy in a tetragonal lattice, can be modeled34 byF0 is a constant background contribution independent of θ, φ), with θ = 0 along the crystal c-axis and φ = 0 defining the in-plane hard axis for K3 < 0. Here, K1 > 0 defines the easy direction and the Zeeman term FZ can be written asψ, ω), with ω = 0 corresponding to field rotation from the c-axis to the in-plane hard direction and ω = π/4, field rotation to the easy direction. We used numerical minimization of equation . However, uniaxial anisotropy is unable to reproduce any reduction of the magnetization in Mrot, since the moment always stays in the rotational plane. Therefore, we considered in-plane anisotropy with The free energy ψ rotation axis is parallel to one of the principal axes such as [100] or [110]. For 4-fold tetragonal symmetry either one of them would be the intermediate or hard axis, respectively. We consider in the following a projection of ab-plane with ω = 0, because tilting of Note, we do not know precisely the in-plane orientation of our sample. However, the rectangular shape suggests that ψ = 78°, 81°, 84° based on equation  a critical field value of about 2.5 T that increases with smaller ψ, ii) a drop ΔM at Hc1,2 that is comparable in size with the experimental data, and iii) a gradual slope M(H) for H > Hc1,2. The double feature at the MM transition is missing due to the simplicity of the model. We obtain anisotropy parameters K1 = 3.1 K, K2 = 0.1 K and K3 = −2.2 K in Kelvin energy scale which convert to the following values 300 kJ/m3, 10 kJ/m3, and −210 kJ/m3, respectively, in units widely used in magnetic anisotropy tables. The 4th order parameter K2 being more than 10 times smaller than K1 implies that it is irrelevant for the description of the anisotropy in Sr4Ru3O10. For comparison, the 3d FM metal cobalt has anisotropy constants of K1 = 450 kJ/m3 and K2 = 150 kJ/m3, which are of similar size as K1 in Sr4Ru3O1035.Figure ab-plane. Therefore, we rotate the sample by ω = 45° in the plane and measure again M at three different angles ψ as shown in Fig. Mrot shows exactly the same behavior as for the ω = 0 experiments within experimental uncertainty. Even if in both experiments ω = 0 and 45°, the plane of ψ-rotation would not include exactly the principal axes, we would at least expect the observation of a reduced anomaly in Mrot at Hc. Based on our last finding, we exclude general magnetic anisotropy as sole cause for the reduction of moment at the MM transition in Sr4Ru3O10.Despite the reasonable agreement between experiment and model, it is necessary to check in a subsequent experiment our initial assumption of tilting the field towards the magnetic hard axis in the Dzyaloshinsky and Moriya37 to explain weak FM ordering inside an AFM phase in transition metal oxides. Recently, Bellaiche et al.38 pointed out that tilting of oxygen octahedra in perovskites can be described by a rotation (pseudo) vector νi sitting at position i of spin Si. The oxygen octahedra rotation leads to an energy reductionK is a constant. Consequently, we approximate a DM interaction between the in-plane component of the Ru center magnetization and that of the two nearest-neighbor Ru atoms, whose spins are assumed to remain parallel. This gives rise to an effective energy termK3 term of the magnetocrystalline anisotropy. Angle φ is interpreted as the angle between in-plane magnetic moments sitting on one inner and two outer layers with φ = 0 being the direction of in-plane magnetic field. The change in the dependence on angle φ between the magnetization vector and the c-axis prevents an accurate minimization of the total energy. Nonetheless, the approximate solution shown in Fig. ψ = 82° does produce a “missing” portion of the total magnetization, with the minimum moving to higher field with decreasing ψ. The parameter D is approximately 5.3 K and comparable to the energy scale of the magnetocrystalline anisotropy K1.Anisotropic exchange interactions caused by spin-orbit coupling under certain symmetry constraints were first considered by φ between the inner and outer magnetic moments can be seen as AFM order if it occurs periodically along the c-axis. Neutron scattering experiments on periodic holmium-yttrium superlattices39 e.g., were able to distinguish between different types of AFM periodicity along[00l] with increasing in-plane magnetic field, such as helical, helifan-shaped, fan-shaped and FM order. However, the particular crystal structure of Sr4Ru3O10 with three inequivalent layers of RuO6 octahedra connected through a double layer of Sr-O doesn’t give rise to additional periodicity, even if the orientation of the magnetic moments follows a repetitive pattern such as −φ, +φ, −φ along c inside the triple layer. As mentioned before, the inner and outer Ru positions are crystallographic inequivalent and carry different magnetic moment sizes. Consistently, in-plane AFM ordering was excluded by neutron scattering studies4. Another experimental route to gain insight into the nature of the missing moment is the exploration of transversal magnetoresistance ρc as a function of temperature along the c-direction with in-plane magnetic field . In case of a periodic opening 2φ of neighboring spins in different layers, we expect to observe coherent scattering and a decreasing magnetoresistance with lower temperature and in higher magnetic field. In case the missing magnetic moment is caused by domain formation of any sort, ρc should also go down with increasing magnetic field, but stay temperature independent because of scattering on domain walls.We want to point out that the opening of 24Ru3O10 as a function of magnetic field and rotation angle between H ⊥ c and H || c has been studied in great detail by magnetization measurements down to lowest temperatures in a SQUID magnetometer. Our experimental results reveal a reduced magnetic moment in the plane of rotation which was never recognized before. It is robust to (ab) in-plane rotation. We find furthermore that the double step at the MM transition is stable down to lowest temperatures of 0.46 K. Our experimental results are interpreted in a strict localized picture with magnetic moments of different sizes sitting on inner and outer RuO6 layers in the crystal structure. We completed our study with numerical calculations based on energy minimization including Zeeman effect, magnetocrystalline anisotropy and antisymmetric exchange and compared them with our experimental data. We conclude that all three contributions are essential ingredients to understand the behavior of the magnetization and that a Dzyaloshinsky - Moriya like component is crucial to model a reduced magnetic moment in Sr4Ru3O10.In summary, the MM transition in Sr4Ru3O10 single crystals were grown in an image furnace by a floating zone technique40 and characterized by energy dispersive spectroscopy, scanning electron microscopy, electron backscattering diffraction and x-ray diffraction techniques.Sr4He setup for measurements between 1.7 K and 100 K and in an iQuantum 3He insert that fits inside the MPMS sample space for the temperature range 0.46 K to 2 K. Excellent agreement between the 3He and 4He data was observed in the temperature range of overlap. Angular dependent measurements at 1.8 K were obtained with a mechanical rotator mounted inside the MPMS magnetometer. Note that the MPMS operates with a pair of coils for signal detection mounted parallel  and perpendicular  to the applied magnetic field. The rotator is aligned with the rotation axis normal to the plane defined by both SQUID coil axes.Magnetization measurements in fields up to 7 T were carried out in a Quantum Design MPMS SQUID magnetometer equipped with a standard 2 in ab and 0.54 mm along the crystallographic c-direction. We considered demagnetization effects by approximating the sample shape with an ellipsoid41 and estimated small correction fields of −30 mT for H ⊥ c and −140 mT for H || c, which are negligible for the analysis and discussion of our magnetization results close to the metamagnetic transition.The measured single crystal has a rectangular shape of (1.87 × 2.99) mm


Dicentrarchus labrax. Immunohistochemistry showed α-MSH positive cells in the pituitary pars intermedia in all stages examined. As development proceeds, α-MSH content gradually increases; mRNA levels of mc2r and mc4r remain low until first feeding where peak values are observed. Mc1r expression was constant during development, pomc mRNA levels remain low until the stage of flexion after which a significant increase is observed. At the stage of the formation of all fins, whole-body cortisol and α-MSH concentrations responded with peak values at 2 h post stress. Additionally, the stress challenge resulted in elevated transcript levels of pomc, mc2r and mc4r but not in mc1r, with a pattern characterized by peak values at 1 h post stress and a strong correlation with whole body α-MSH concentrations was found. Our data provide for the first time a view on the importance of the α-MSH stress response in early development of European sea bass, an additional and relatively poorly understood signal involved in the stress response in teleosts.Temporal patterns of whole-body α-MSH concentrations and of transcripts of melanocortin receptors during early development as well as the endocrine response  to stress at the end of the larval period were characterized in  The gene is translated into a precursor protein, from which, among others, in the pars distalis adrenocorticotropic hormone (ACTH) and in the pars intermedia alpha-melanocyte stimulating hormone (α-MSH) are derivedDicentrarchus labrax, L.) a single form of a functional pomc gene has been cloned and characterized5In fish, stress leads to the activation of the hypothalamic-pituitary-interrenal (HPI) axis, and stimulates the pituitary corticotropes in the During HPI axis activation, ACTH secreted by the corticotropes is a key regulator of the acute stress response as it stimulates the interrenal cells via MC2R and results in the synthesis and secretion of cortisol which targets a plethora of tissues, if not all cells in an organism179101112There are 4 MCRs which convey MSH signals: MC1R, MC3R, MC4R and MC5R. Interaction of α-MSH and the MC1R plays a key role in the control of the pigmentation. For instance, mutations in the MC1R are responsible for reduced melanization. MC3R and MC4R are considered ‘brain receptors’; the expression of the MC4R is thought to play a role in the regulation of the energy balance in fish through the modulation of feeding behavior1516Sparus aurata) and in rainbow trout (Oncorhynchus mykiss) air exposure, a severe stress for fish, induces an increased α-MSH levels18Oreochromis mossambicus): prolonged netting stress had no effect on ACTH concentrations, but plasma cortisol levels did increase suggesting activity of another corticotrope; when netting was combined with confinement both cortisol and ACTH increasedCyprinus carpio)Alpha-MSH is (at least in the few species studied so far) also involved in the stress response of fishIn teleosts, cortisol acts as glucocorticoid and mineralocorticoid  and is the most commonly used hormonal indicator of stress1pomc, mc1r, mc2r and mc4r genes in European sea bass during defined stages of early development; also we analyzed the response to an acute stressor prior to and after earlier exposure to stress at the end of the larval period.Recently, the cortisol response and its molecular regulation during early ontogeny have been studied in European sea bass26−1) that subsequently increased at first feeding (172 ± 93.5 pg g−1), but these differences were not statistically significant. A significant increase was observed at flexion (307 ± 101 pg g−1) and remained at statistically significant high values onwards to the formation of all fins (408.8 ± 41.1 pg g−1), . All genes of interest are expressed in all developmental stages examined . Expression levels of mc1r . Expression levels of mc4r .European sea bass embryos had low basal α-MSH content  and after  the application of a stressor at the stage of the formation of all fins. A statistically significant effect of the stressor on α-MSH concentrations was observed (−1) followed by a minimum (190.6 ± 31.9 pg g−1) at 24 h post stress . Whole-body cortisol content  after stress, and then returned to basal values at 24 h (10.11 ± 4.3 ng g−1) .observed  with a p content  was 4.58pomc  that dropped to resting values at 24 h post stress. The acute stress application had no effect on mc1r . This expression pattern of mc2r after the acute stress application parallels with the changes observed in whole body cortisol concentrations post stress. Transcript levels of mc4r .Transcripts levels of pomc  showed a on mc1r . The pat1r  that showed a gradual increase of the α-MSH-producing cells in the adenohypophysis293031pomc increases at the stage of flexion and its peak is in line with the first statistically significant elevation of α-MSH levels. Abundance of mc2r remains at low levels until the stage of first feeding where it reaches a maximum and then decreases gradually at the later stages of development. Previous studies of our group have shown that sea bass larvae begin to synthesize cortisol around the stage of first feeding26mc2r. Similar results have been obtained in zebrafish, where the expression of mc2r is upregulated immediately before the rise in whole-body larvae cortisol concentrationsmc1r was not altered depending on the developmental stage, whereas expression levels of mc4r was low in the embryo stage until mouth opening showing an increase at the stage of first feeding and remained at similar levels thereafter till the stage of the full formation of all fins. The acute stress challenge at the stage of the formation of all fins involved an elevation of α-MSH and cortisol levels with a peak at 2 h after application of the stressor. These results are supported by studies in adult gilthead sea bream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss), studies that showed that the application of severe acute stress leads to an increase of α-MSH levels18During ontogenesis of sea bass, temporal changes in whole-body α-MSH levels showed a gradual increase from low levels during the first stages to maximum values at the stages of flexion and development of all fins. Our immunohistochemistry showed that no significant α-MSH positive cells were observed outside of the pomc. We would emphasize that we do not – as of yet – know how the measured transcripts are reflected in peptide levels of ACTH and α-MSH. Expression levels of pomc after the acute stress application appear to be altered at the stage of the formation of all fins where the pattern of pomc stress response is characterized by maximum values at 1 h post stress that gradually drop to resting levels at 24 h , where an up-regulation of pomc mRNA was observed in response to low-water stress, showing peak values at 1 h post stress which at 3 h declined to the level of the control groupα-MSH is a POMC-derived peptide, so to reveal the molecular mechanisms related to the onset of the α-MSH stress response, qPCR experiments were carried out to analyze transcript levels of  at 24 h  and thismc2r which showed an up-regulation after application of the stressor, with peak values at 0.5 h and 1 h post stress to return to basal levels at 2 h and 24 h post stress . MC2R plays a critical role in the HPI axist stress  showing t stress . The obsstressor , whereast stress . It thuspomc, mc1r, mc2r and mc4r genes during early ontogeny. Additionally, sea bass larvae at the stage of the full formation of all fins were exposed to acute stressors and the temporal patterns of whole body α-MSH and cortisol and the expression profiles of pomc, mc1r, mc2r and mc4r genes prior to and 0.5 h, 1 h, 2 h, and 24 h after application of the stressor, were determined. Overall, these data, combined with data on the cortisol response during early ontogeny26In summary, we characterized for the first time in a Mediterranean marine teleost, the European sea bass, the temporal pattern of whole body α-MSH and the expression profile of The laboratories of the Hellenic Centre for Marine Research are certified and obtained the codes for breeding animals for scientific purposes (EL-91-BIO-04). All procedures involving the handling and treatment of fish used during this study were approved by the HCMR Institutional Animal care and use committee following the Three Rs  guiding principles for ethical use of animals in testing, in accordance to Greek (PD 56/2013) and EU (Directive 63/2010) legislation on the care and use of experimental animals.−1 in which both hatching and rearing took place. A biological filter was coupled to the tanks which were initially filled with filtered seawater from a deep well. Water, during embryogenesis, egg hatching, and at the autotrophic larval stage, was re-circulated from the bottom of the tank through the biological filter at a rate of 10% of the tank volume per h and was progressively increased to 70% of the tank volume per h until the end of the trial. Aeration (compressed air) was provided by means of a wooden diffuser located in the tank center at a rate of 150–200 ml min−1. Larvae were held during the whole experimental period under a mean (±SD) water temperature of 18 (±1.6) °C, dissolved oxygen levels of 7.2 ± 0.8 mg l−1, salinity of 36‰ and pH of 7.9–8.2. During hatching and until mouth opening, tanks were kept in complete darkness; a 12D:12 L photoperiod regime (lights on at 08:00 h) was applied during the rest of the experiment. Following mouth opening and eye development, sea bass larvae were exposed to low light intensity conditions (5–10 lux) in the absence of food for a period of 2 to 4 days to ensure normal swim bladder inflation, while the water surface was also kept free from any (food derived) oily film by the use of an air-blower skimmer. Food was delivered only when inflated swim bladder was observed in more than 80% of the population. Exogenous feeding was based on rotifers (Brachionus sp.) at 5 individuals ml−1 enriched with proteins and PUFA  until 10 days post hatching (dph); phytoplankton (Chlorella sp.) was supplied until 10 dph. Enriched Artemia nauplii  were administered from 10 dph until 50 dph at 0.5 to 1.0 individual ml−1. From 30 dph, larvae were offered dry feed  through the use of automated feeders. The trial lasted until individuals completed the formation of their fins at 45 days post hatch (dph).Batches of fertilized European sea bass eggs were obtained from a private fish farm (DIAS S.A.) and transferred to the installations of the Institute of Aquaculture, Hellenic Center for Marine Research . Larval rearing was performed applying the pseudogreen-water technique−1 vs. 150–200 ml min−1) for 90 sec, chased with a net for 20 sec, confined (collection in beakers), and air exposed for 5 sec. Samples for molecular analysis , cortisol (n = 3 pools of ca. 250 mg) and α-MSH (n = 3 pools of ca. 250 mg) were collected with a net, flash frozen in liquid N2 and stored at −80 °C until further analyses. At the stage of the formation of all fins additional samples were also collected at 0.5 h, 1 h, 2 h and 24 h post-stress.Samples were collected at six different points/stages during early life development . Additionally, at the stage of the formation of all fins samples were taken prior to and after the application of an acute stressor: the larvae were exposed to high aeration  HCl/body weight). Whole-body α-MSH concentrations were then measured with a radioimmunoassay; α-MSH was labeled with 2O2 to block endogenous peroxidase activity. Non-specific antigenic sites were blocked with 2% Normal Donkey Serum  followed by overnight incubation with 1:1000 α-MSH antibody2O2.Samples of mouth opening, first feeding, flexion and all fins were fixed in Bouin’s fixative for 6 hours and wash thoroughly in 70% ethanol afterwards. For sectioning, samples were dehydrated trough gradually increasing ethanol series and embedded in paraffin. Sections (7 μm) were cut, mounted on glass slides coated with poly-l-lysine and dried overnight at 37 °C. Next day the sections were deparaffinized in xylene, rehydrated through gradually decreasing ethanol series and incubated with 1% Het al.Whole body cortisol extraction was performed according to Pavlidis ® ND-1000 UV–Vis spectrophotometer , and its integrity was tested by electrophoresis in 1% agarose gels. Reverse transcription (RT) was carried out using QuantiTect Reverse transcription kit  using 1 μg of total RNA, according to the manufacturer’s instructions.Samples of embryos, pre-larvae and larvae were let to thaw on ice, disrupted and homogenized using the TissueRuptor  for 20 s in 600 μl RLT plus buffer . Total RNA was isolated with the RNeasy Plus Mini Kit . RNA yield and purity was determined by measuring the absorbance at 260 and 280 nm using a Nanodropmc4r were as described by Sanchez et al.eukaryotic elongation factor 1 (elf1a) and ribosomal 18S RNA (18S) were used as in previous workmelanocortin 1 receptor (mc1r), melanocortin 2 receptor (mc2r) and pro-opiomelanocortin (pomc) was based on the available sequences with accession numbers FN377856.1mc1r has the sequence 5′ CTCCACCTCATCCTCATC 3′ while the reverse 5′ GAAGCACCAAGAACACAG 3′. In the case of mc2r the forward primer has the sequence 5′ CATCTACGCCTTCCGCATTG 3′ and the reverse 5′ ATGAGCACCGCCTCCATT 3′. The forward primer for pomc has the sequence 5′ CCGGTCAAAGTCTTCACCTC 3′ while the reverse 5′ ACCTCCTGTGCCTTCTCCTC 3′. The products of each primer pair were further checked with sequencing to confirm amplification of the desired genes.Primers for mc1r, mc2r, mc4r and pomc was determined with quantitative polymerase chain reaction (qPCR) assays using the KAPA SYBR®FAST qPCR Kit (Kapa Biosystems), according to the manufacturer’s instructions. The resulting fluorescence was detected with CFX Connect Thermal Cycler (Bio-Rad) under the following cycling parameters: 95 °C for 3 min, 94 °C for 15 sec, 60 °C for 30 sec (for mc2r and mc4r)/55 °C for 30 sec (for pomc and mc1r), 72 °C for 20 sec, 40 cycles. Levels of mc1r, mc2r, mc4r and pomc mRNA were normalized using reference genes 18S and elf1a. A standard curve was constructed for each gene, using 4 serial dilutions (1:5) of a pool of all cDNA samples by graphing the negative log of the dilution factor against the relative cycle threshold value. To be considered suitable for analysis, each primer pair was required to have a linear standard curve with an r2 value above 0.98 and primer amplification efficiency between 90% and 100%. We performed geNORM analysisM which corresponds to the standard deviation of the logarithmically transformed expression values of the compared genes. This stability index together with its decreasing value gives rise to the rank and stability of each gene. In our study 18S and elf1a genes proved have the most stable expression  and the mean of their normalization factors was used for each sample.Relative expression of P < 0.05.All statistical analyses were performed with SigmaPlot 11.0 (Jandel Scientific). Data are presented as means ± standard deviation (SD). Statistical comparisons of α-MSH concentration and gene expression of unstressed specimens (0 h) between the different developmental points/stages and statistical comparisons of temporal patterns of α-MSH and cortisol concentrations and gene expression between the different time points following exposure to a stressor at the stage of the formation of all fins were made using one-way ANOVA. Holm-Sidak’s honestly significant difference test for multiple comparisons was used to determine significant differences among groups. The significance level was set at How to cite this article: Tsalafouta, A. et al. a-MSH and melanocortin receptors at early ontogeny in European sea bass . Sci. Rep.7, 46075; doi: 10.1038/srep46075 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


A binary measurement of type 2 diabetes (T2D) has been found not to influence behaviors. We aimed to examine the influence of other measures of family history such as number of relatives, genetic closeness of relatives, and severity of T2D of family members on nutrition and physical activity behaviors among college students.Students across four colleges in Texas were sampled. Multiple linear regression models, controlling for covariates, were used to model results. Cross-sectional data were used.p = 0.007) and exercise . Having relatives with severe T2D was associated with vegetable consumption  and exercise . Closer genetic relationship with someone with T2D was associated with increased vegetable consumption  and exercise .More number of relatives with T2D was associated with vegetable consumption (β = 0.131, It is likely that the severe complications that may accompany the relatives T2D or having an immediate family member living with T2D may in fact motivate other family members without T2D to modify their attitudes, beliefs, and knowledge about T2D, thus encourage health-protective behaviors. Type 2 diabetes (T2D) has been labeled an “adult onset” disease , 2; yet,Physical activity (PA) and dietary behavior have been highlighted as particularly effective T2D protective factors. PA is associated with lower rates of T2D –9. Specicalpain 10 and hepatocyte nuclear factor 4 alpha, have been identified as genes related to T2D  and non-modifiable risk factors  . Non-modd to T2D . While gd to T2D –22. Furtd to T2D .Type 2 diabetes family history health education interventions have positive effects on lifestyle behaviors among adults. For example, people who know that a family member has been diagnosed with T2D are more likely to lose weight, increase PA, and practice healthy eating –26 as wevia administrative academic advisors. A total of 7,600 students were contacted across the four institutions, 905 of which responded (12% response rate). All study procedures were vetted by the Institutional Review Boards for each respective institution. $40 major-retailer gift card incentives were given to 13 participants via random drawing.Sample consisted of undergraduate students (18 or older) enrolled in four colleges/universities across a southern state. Institutional size ranged from 18,413 to 58,577 students. Solicitations were sent to student’s school emails via the item: “Have any of your family members  ever been diagnosed with T2D?” Possible responses were “No,” “Yes,” “I don’t know” and “Not applicable.” The first level of analysis replicated prior literature by utilizing the binary level of T2D family history measurement (yes/no) on health behaviors; thus, responses were coded to create a dichotomous outcome variable for knowledge of T2D family history status, such that “Yes” (coded 1) indicated a positive family history while “No,” “I don’t know,” and “Not applicable” (coded 0) indicated no family history of T2D. The second level of analyses included only respondents who had a family history of T2D and then stratified the risk by number of relatives with T2D, closeness of relationship with a relative with T2D, and severity of relatives with T2D.Family history was assessed Participants were asked to “select all that apply” to the following question: “which of the following relatives currently has T2D: mother, father, brother or sister, any grandparents on mothers side, any grandparents on fathers side, aunts or uncles on mothers side, aunts or uncles on fathers side, any cousins on mothers side, any cousins on fathers side.” Responses were coded to create a dichotomous outcome variable per the degree of relationship with relative with T2D. Those who reported to have a first or second degree relative living with T2D was (coded 1), while those who indicated they had only third degree relatives with T2D were (coded 0). We coded the relationship this way because one of the hypotheses that guide this paper is that having a closer genetic and most likely social/cultural/environmental relationship with a relative with T2D will heighten motivation to engage in T2D-related protective behaviors.Participants were asked to write down the number of living family members with T2D.Respondents were asked to “Please select if your family member or members with T2D has experienced or is experiencing any of the following: kidney disease, heart disease, blindness, limb amputation, or an emergency room visit for a T2D-related issue.” Possible responses were “No,” “Yes,” and “I don’t know.” Responses were coded to create a dichotomous outcome variable for T2D relative severity status, such that “Yes” (coded 1) indicated that their family member had severe T2D “No,” and “I don’t know” (coded 0) indicated that their family member did not have severe T2D.Based on the Centers for Disease Control recommendations , body maVegetable consumption behaviors were measured by asking participants: “about how many cups of vegetables (including 100% pure vegetable juice) do you eat or drink each day? .” Possible responses for this question were “Never” (coded as 0), “1/2 cup or less” (coded as 1), “1/2 cup to 1 cup” (coded as 2), “1–2 cups” (coded as 3), “2–3 cups” (coded as 4), “3–4 cups” (coded as 5), and “4 or more cups” (coded as 6). This question was adapted from the Health Information National Trends Survey .via two items: “during the last 7 days, on how many days did you do moderate physical activities like carrying light loads, bicycling at a regular pace, or doubles tennis? Do not include walking.” 0 days (coded as 0), 1 day (coded as 1), 2 days (coded as 2), 3 days (coded as 3), 4 days (coded as 4), 5 days (coded as 5), 6 days (coded as 6), and 7 days (coded as 7); and “how much time in total did you usually spend on one of those days doing moderate physical activities?” Possible responses were open ended and participants were prompted to write the length of time in a box provided. All responses were in minutes. Responses to each of these items were multiplied to create a single, composite indicator of PA. For instance, if a respondent indicated they were at least moderately active 3 days a week, and they typically engaged in such activity for 40 min, then their weekly PA would be 120 min. “Higher scores indicated higher levels of PA each week.” This question was adapted from the Health Information National Trends Survey ; however, all other analyses of T2D family history, such as the number and type of relative with T2D, required that we extract only those with a T2D family history for the analyses (n = 441).Means and SDs for continuous variables and frequencies for binary and categorical variables were calculated. Multiple linear regression was conducted to examine the impact of T2D familial risk profiles on related protective behaviors. Variables that have been established by the scientific literature as influencing health behaviors, such as age, sex, and race/ethnicity were controlled for –45. To an = 736), White , and the average age was 21 (SD = 1). Almost half  had a family history of T2D. Majority of the sample consumed less than two cups of vegetables   or PA behaviors  after controlling for demographic variables , approximately 441 reported a family member with T2D. The sample was primarily female . Most respondents were white . The average age of n = 207), and majority had a first or second degree relative diagnosed with T2D . Number of relatives with T2D ranged from 1 to 9 . Mean number of cups of vegetables eaten in a week was 2.23 (SD = 1.3), and average number of minutes PA per week was 75.7 (SD = 113.5). Mean BMI was 25.3 (SD = 5.8)  and with vegetable consumption  after controlling for demographic variables ; and with vegetable consumption  after controlling for demographic variables , and with vegetable consumption  after controlling for demographic variables  relatives with severe T2D, (b) greater number of relatives with T2D, and (c) a close genetic relationship with a relative with T2D is associated with increased PA levels and consumption of healthy food options. It is noteworthy that these associations would not have been detectable unless a more nuanced assessment of T2D risk was employed. In other words, a dichotomous family history measure, which simply indicates whether or not a family member has been diagnosed with T2D, did not identify a statistically significant relationship between T2D family history and health behaviors. Consequently, we recommend practitioners and researchers cease using simple, dichotomous measures of family history, which ignore important factors, such as familial risk profiles and “closeness” of relatives. If dichotomous measures are employed, it is imperative that follow-up assessment examine contextual factors  for those indicated presence of a family T2D history. While the results herein document associations between T2D risk and PA and dietary behaviors, it is beyond the scope of this investigation to outline the underlying mechanisms of these associations. Previous research, however, contend that representations of illness can create feelings of vulnerability, which can in turn motivate an individual to action . For exaEach of the individual components of the familial risk profile  was an indicator of risk for T2D and demonstrated a statistically significant relationship with preventive health behaviors. It is possible that this relationship is because family members encourage healthy behaviors among the youth and adolescents in the family to protect them from developing T2D, particularly when the family is burdened with disability and financial stress. Although friends are the most influential on adolescent PA behaviors , 30, it R-squared values in the results are low; this could be because some confounding variables were not included in the analyses. For example, the amount of time an individual has known about their family member’s T2D may influence when participation in T2D-related behaviors begin. Suppose most of our respondents knew about their family members T2D for only 1–3 weeks before participating in this study, their responses may have influenced the outcomes and strength of the R-squared values. Finally, the survey questions examining PA specifically excluded walking, which may have resulted in an underestimate of PA behaviors.Despite the novel nature of this study, there are several limitations, which need to be noted. First, the data are cross-sectional, which limits inferring causality. Second, it is possible that cases of T2D are still undiagnosed; thus, participants may have a family history of T2D but were unaware . AdditioSeveral notable implications stem from this study. Although it has become increasingly popular to collect information of T2D family history, most of the information collected is in a binary form . As the results herein highlight, more nuanced family history measures represent better measurement tools. Thus, collecting information on various familial risk profiles such as severity, type, and number of relatives with T2D can provide greater insights into an individual’s level of risk, particularly among overweight/obese populations. Stratifying familial risk information can also provide avenues to systematically organize screening procedures. Early screening among those ranking high in the strata is essential because the time lag between the onset of T2D and diagnosis is approximately 7 years . These hAll study procedures were vetted and permitted by the Institutional Review Boards of Texas A&M University, Prairie View A&M University, and University of the Incarnate Word. Since it was an online and completely anonymous survey, participants were given informed consent to read via email solicitations sent out, all participants were advised that clicking on the survey link indicated consent.AA came up with the idea for the paper, ran all the analyses, and drafted the methods and results. WJ drafted the introduction. RM drafted the discussion. AB contributed throughout the paper and edited the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.