Identification of important error fields in stellarators using Hessian matrix method

Error fields are predominantly attributed to inevitable coil imperfections. Controlling error fields during coil fabrication and assembly is crucial for stellarators. Excessively tight coil tolerance increases time and cost, and, in part, led to the cancellation of NCSX and delay of W7-X. In this paper, we improve the recently proposed Hessian matrix method to rapidly identify important coil deviations. Two of the most common figures of merit, magnetic island size and quasi-symmetry, are analytically differentiated over coil parameters. By extracting the eigenvectors of the Hessian matrix, we can directly identify sensitive coil deviations in the order of the eigenvalues. The new method is applied to the upcoming CFQS configuration. Important perturbations that enlarge n/m=4/11 islands and deteriorate quasi-axisymmetry of the magnetic field are successfully determined. The results suggest each modular coil should have separate tolerance and some certain perturbation combinations will produce significant error fields. By relaxing unnecessary coil tolerance, this method will hopefully lead to a substantial reduction in time and cost.Comment: Accepted by Nuclear Fusio

Representative images for detecting the localisation of wild-type and variants of full-length-DSG2-EYFP in HT1080 for three independent transfection experiments:

<p><b>DSC2b-HT1080 cells were transfected with full-length(fl)-DSG2-pEYFP; live cells were analysed with a fluorescence miscroscope one day after transfection.</b> R46Q, D154E, D187G, K294E and V392I indicate the sequence variant in fl-DSG2-EYFP, wt fl-DSG2-wt-pEYFP, and C the LFA mock transfected control. Chimeric DSG2-proteins localised preferentially to the cell borders. ARVC-associated variations had no detectable influence on the localisation of fl-DSG2-EYFP in DSC2b-HT1080. Images were acquired through YFP and phase-contrast filters. Scale (red bar) = 10 µm.</p

Comparison of rECD fragment ion peaks generated by MALDI-ISD using QuPE [<b>71</b>].

<p><b>A</b> The location of <i>ECD</i> and <i>Pro-ECD</i> and the positions of the corresponding c-ions are illustrated on the schematic view of the rECD. <b>B+C</b> Each row shows extracts of the MALDI-ISD spectra for the particular rECDs. Peaks in one column represent the ions of the m/z ratios indicated below the negative control. The corresponding c-ions are indicated for each peak. The calculated monoisotopic masses [M+H]⁺ are shown at the bottom of each column. <b>B</b> Fragment ion peaks representing the <i>ECD</i>-c-ions; only rECD-R46Q shows no fragment ions corresponding to <i>ECD </i><b>C</b> Fragment ion peaks representing the <i>Pro-ECD-</i>c-ions; only rECD-R46Q shows fragment ions corresponding to <i>Pro-ECD</i>.</p

Investigated DSG2-variants.

<p>All data were obtained from the ARVC database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanderZwaag1" target="_blank">[64]</a> and the corresponding references. TFC = task force criteria, EC = extracellular cadherin domain, DCM = dilatative cardiomyopathy.</p>a<p>The prevalence in controls for the DSG2-V392I was adapted to the results in our research group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Klauke1" target="_blank">[35]</a>.</p>b<p>Grantham, R. (1974). “Amino acid difference formula to help explain protein evolution.” Science 185(4154):862–864.; Li, W. H., C. I. Wu, et al. (1984). “Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications.” J Mol Evol 21(1): 58–71.</p>c<p>Kumar, P., S. Henikoff, et al. (2009). “Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.” Nat Protoc 4(7): 1073–1081.</p>d<p>Ramensky, V., P. Bork, et al. (2002). “Human non-synonymous SNPs: server and survey.” Nucleic Acids Res 30(17): 3894–3900.</p

Adhesion properties of rECDs.

<p><b>A+B</b> Flow cytometry-based assay for the binding of 0.8 µM rECD-wt or -variants to HT1080. <b>A</b> Representative histograms of FITC- fluorescence for binding of rECD-wt- and rECD-R46Q (as indicated). Bound rECD was detected with anti-HisFITC. As a negative control, HT1080 cells were incubated with only anti-HisFITC (grey filled area). <b>B</b> Column plots representing the ratio of rECD-binding related to the negative control (ratio_rECD-bound) as detected by flow cytometry. Ratios_rECD-bound are indicated as mean± SEM of 7 independent measurements for rECD-variants and 9 independent measurements for rECD-wt with rECDs from at least 3 different purifications. Statistical analysis was performed by one-way ANOVA with Dunnett’s posttest using rECD-wt as a control (GraphPad Prism 5.01). rECD-R46Q-binding to HT1080 is increased 1.8-fold as compared to rECD-wt. Other ARVC-associated variants have no influence on rECD-binding to HT1080. <b>C</b> Representative Western blot (with anti-DSG2-10G11) of rECDs crosslinked in a 5 mM CaCl₂ containing buffer with BS³ (+) or of controls (-) reveals that rECD wild-type and variants exist in solution as monomers (m), dimers (d), and oligomers (o).</p

Evaluation of CD data.

<p>Results of the deconvolution with DichroWeb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Whitmore1" target="_blank">[67]</a> using the CONTINLL-algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Provencher1" target="_blank">[68]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanStokkum1" target="_blank">[69]</a> and the CRYST175 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Evans1" target="_blank">[70]</a> reference data set. The results are presented as means±SEM [%] for three independent measurements. α-helical content (<b>A</b>) was 5.9±0.8 and 5.9±0.5 without Ca²⁺ (-Ca²⁺) and 3.2±1.4 and 3.2±0.2 with 5 mM CaCl₂ for rECD-wt-nat and rECD-wt-denat, respectively. β-strand content (<b>B</b>) was 38.2±0.3 and 36.6±1.3 without Ca²⁺ and 40.5±0.8 and 40.7±0.6 with 5 mM CaCl₂. Analysis of the secondary structure with two-way ANOVA showed that the α-helix content (<b>A</b>) was significantly decreased (p<0.05) while the β-strand content (<b>B</b>) was significantly increased (p<0.01) by the addition of 5 mM CaCl₂ (+Ca²⁺). However, as shown by two-way ANOVA, purification conditions had no significant effect on the rECD secondary structure.</p

Flow cytometry-based assay for rECD-binding.

<p><b>A</b> Representative histograms of FITC-fluorescence for rECD-wt binding with 5 mM CaCl₂ (1) or with 2 mM EGTA (2). HT1080 cells were incubated with (black line) or without (negative control, grey filled area) rECD-wt. Bound rECD-wt was detected with anti-HisFITC. <b>B</b> Column plots representing the ratios of rECD-wt-binding (ratio_rECD-bound; for calculation see Supporting Information) indicated as mean±SEM of 3 independent measurements as detected by flow cytometry. The ratio_rECD-bound was significantly (p<0.01) decreased from 1.21±0.03 for samples incubated in 5 mM CaCl₂ (1) to 1.05±0.03 for samples incubated with 2 mM EGTA (2) showing that rECD-wt has a Ca²⁺-dependent binding to HT1080 cells. Statistical analysis was performed with unpaired student’s t-test (GraphPad Prism 5.01).</p

In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations - A

<p><b>Schematic view of the rECD with analysed ARVC-associated variations.</b> The dotted line shows the predicted PC cleavage site. SS = signal sequence, Pro = prodomain, EC1-EC4 = DSG2 extracellular cadherin subdomains 1-4. <b>B</b> Recombinantly expressed proteins were identified as DSG2-ECD with anti-DSG2-10G11 by Western blot analysis. The calculated apparent molecular weights were 67.5±1.5, 72.5±3.5, 70.0±3.0, 70.0±3.0, 70.5±2.5, and 69.0±4.0 (mean±SEM; n = 2) for the proteins in the traces in 1, 2, 3, 4, 5 and 6, respectively. <b>C</b> Coomassie-R-250 staining revealed the purity of the proteins. 1 = rECD-wt, 2-6 = rECDs as labelled in <b>A</b>.</p

Datasheet1_Cardiomyopathy related desmocollin-2 prodomain variants affect the intracellular cadherin transport and processing.pdf

BackgroundArrhythmogenic cardiomyopathy can be caused by genetic variants in desmosomal cadherins. Since cardiac desmosomal cadherins are crucial for cell-cell-adhesion, their correct localization at the plasma membrane is essential.MethodsNine desmocollin-2 variants at five positions from various public genetic databases (p.D30N, p.V52A/I, p.G77V/D/S, p.V79G, p.I96V/T) and three additional conserved positions (p.C32, p.C57, p.F71) within the prodomain were investigated in vitro using confocal microscopy. Model variants (p.C32A/S, p.V52G/L, p.C57A/S, p.F71Y/A/S, p.V79A/I/L, p.I96l/A) were generated to investigate the impact of specific amino acids.ResultsWe revealed that all analyzed positions in the prodomain are critical for the intracellular transport. However, the variants p.D30N, p.V52A/I and p.I96V listed in genetic databases do not disturb the intracellular transport revealing that the loss of these canonical sequences may be compensated.ConclusionAs disease-related homozygous truncating desmocollin-2 variants lacking the transmembrane domain are not localized at the plasma membrane, we predict that some of the investigated prodomain variants may be relevant in the context of arrhythmogenic cardiomyopathy due to disturbed intracellular transport.</p

13) 早期十二指腸癌の1例(I.一般演題, 第44回新潟癌治療研究会）

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