Dysferlin Forms a Dimer Mediated by the C2 Domains and the Transmembrane Domain In Vitro and in Living Cells

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes

Assessment of Validity of Children’s Movement Skill Quotient (CMSQ) Based on the Physical Education Classroom Environment

The development of movement skills in children is a critical element in promoting physical activity and other positive health trajectories over their lifetime. A reliable and valid assessment tool is essential for evaluating children’s movement skills in daily physical education environments. The purpose of this study was to examine the validity of Children’s Motor Skills Quotient (CMSQ) used in the classroom setting. Six raters conducted evaluation to participants, and a total of 734 children completed all the test items and were included in the study. Descriptive statistics and Rasch analysis were used in this study. The descriptive statistics were mainly used for calculating the mean, standard deviation, percentage, and internal consistency coefficient. Rasch analysis was used to verify the fitting statistics, project difficulty, and functional differences of the items of the CMSQ. The findings showed that the CMSQ met the assumption of the Rasch model, including the unidimensionality, local independence, person measure, and item difficulty hierarchy. The CMSQ also demonstrated adequate interrater reliability and internal consistency. The differential item functioning (DIF) demonstrated a few items showing different probabilities across sex and age. To maintain the item difficulty hierarchy of the CMSQ, no items were deleted. Overall, the CMSQ seems to have appropriate test items with an appropriate rating scale structure for measuring 6-9-year-old children’s movement skills in the physical education classroom environment

Analysis on coercivity enhancement mechanism of grain-boundary-diffused and Dual-alloyed Nd-Fe-B Magnets

With the rapid development of application fields, such as the green energy industry, has resulted in increased demand for magnetic characteristics, especially the coercivity (Hcj). In this paper, two types of (Nd, Dy)-Fe-B magnets with comparable Hcj values were prepared using by grain boundary diffusion (GBD) method and dual alloy (DA) method. The composition, microstructure, domain structure, and magnetization reversal process of GBD and DA magnets were subjected to comparison. Additionally, an analysis of the specific coercivity mechanism was also conducted by fitting the Brown's equation. The results of composition and microstructure analysis revealed the formation of core-shell structural grains were formed in both GBD and DA methods. Specifically, the GBD method exhibited a higher concentration of Dy in the shell, constituting 26.8 % of the total rare earth (RE) content. In contrast the DA magnets had Dy accounting for 15.8 % of the total RE content in the shell. This discrepancy was identified as the primary factor contributing to the superior efficiency in enhancing the coercivity (Hcj) observed in the GBD method. The fitting results showed that GBD magnets were more inclined to nucleate in the core region in grain, whereas DA magnets nucleate at the edge of the grain shell layer. Both the observation of magnetic domains and magnetization reversal processes analysis revealed that the demagnetization process in GBD magnets is more uniform, which leads to better squareness. Our finding can provide theoretical and experimental basis for the preparation of high-performance Nd-Fe-B magnets

The reinforcement strategy of electrophoretic deposition coating assisted by PVP for grain boundary diffusion of Nd-Fe-B magnet

In this study, we aimed to enhance the quality of electrophoretic deposition (EPD) coatings for Nd-Fe-B grain boundary diffusion (GBD) by addressing issues related to the adhesion and densification of coating. We introduced a novel approach by incorporating polyvinyl pyrrolidone (PVP) into an alcohol suspension containing TbH3 nano-powders. The addition of PVP resulted in the formation of a dense coating with superior adhesion and a smooth surface. To evaluate the effectiveness of the EPD coating with PVP, we compared the EPD rate of the TbH3 nano-powders alcohol suspension with and without PVP. The EPD rate of the suspension with PVP exhibited a significant 30-fold increase compared to the suspension without PVP, indicating a substantial improvement in the electrophoretic efficiency. Based on our experimental findings, we recommend depositing the EPD coating at a voltage of 120 V and adjusting the deposition time accordingly to achieve a desired coating weight percentage of 1.0 wt% with a thickness of approximately 30 μm. Following the GBD process, the coercivity of the magnet with PVP increased from 1131 kA/m to 1896 kA/m, which improved the high-temperature stability of the magnet. Overall, our research offers a promising approach to overcome the limitations associated with EPD coatings for GBD applications, providing a potential solution for enhancing the performance and industrial viability of these coatings

Solid-liquid phase separation diffused Nd-Fe-B sintered magnets by using DyH3 nanopowder realize high-efficiency coercivity improvement and overcome thickness limit

In this study, new grain boundary diffusion (GBD) technology is applied to Nd-Fe-B sintered magnets with different thicknesses utilizing DyH3 nanopowder. The weight ratio is 0.25 wt.%. For comparison, the GBD processes include solid-liquid phase separation diffusion (SepD) and solid-liquid phase simultaneous diffusion (SimD). The magnetic properties and microstructure of GBD magnets with different thicknesses are thoroughly investigated. The comprehensive magnetic properties of SepD magnets are higher than SimD magnets. When the magnets are 8 mm thick, the SepD magnet obtains a coercivity of 18.10 kOe, which is higher than the SimD magnets (17.00 kOe). It was determined that SepD can manufacture thick magnets with excellent coercivity. In SepD magnets, the Dy element diffusion distance is deeper than in SimD magnets. For SepD magnets, there are core-shell structures formed in the surface region, while there are anti-core-shell structures that are unfavorable to magnetic properties in the surface region of the SimD magnets. The enhanced coercivity and temperature stability of the SepD magnets is primarily due to the more continuous core-shell structures produced by the deeper diffusion depth of the Dy element. This study will provide more theoretical guidance for the application of SepD in magnets with different thicknesses

Summary of FCS experiments.

<p>Parameters shown are observed diffusion correlation time (τ_D), diffusion coefficient (D), and apparent hydrodynamic radius (r_h).</p

Mapping the determinants of dysferlin dimerization.

<p>(A) Dependence of FRET efficiencies on the protein expression levels of C2A to C2D domains of dysferlin. Hyperbolic fitting showed that the C2B (red), C2C (green) and C2D (purple) domains of dysferlin all mediate the self-interaction. There was no FRET for C2A construct (black). (B) Dependence of FRET efficiencies on the protein expression levels of C2E to C2G domains and the transmembrane (TM) domain of dysferlin. Hyperbolic fitting showed that the C2E (black), C2F (red), C2G (green) and TM (purple) domains of dysferlin also mediate the self-interaction. In A and B, the data were pooled for easy comparison. (C) Independence of dysferlin dimer FRET efficiencies on acceptor concentrations within the range examined. (D) Summary of FRET_max values obtained by fitting (expressed as mean ± S.E.M.). (E) Summary of <i>K_D</i> values obtained by fitting (expressed as mean ± S.E.M.). ND: not determined.</p

Fluorescence correlation spectroscopy and photon counting histogram analyses of dysferlin dimerization.

<p>(A) FCS data obtained from YFP control (black) showed a significantly faster diffusion compared to full length YFP dysferlin in detergent solution. The boxed region is enlarged and reproduced in the next panel. (B) Diffusion of YFP-dysferlin in 1% CHAPS (red) and 0.5% DDM (blue) were well described by a single species diffusion model (<b>Equation 1</b>), with a correlation time of 2.7 ms. This the apparent diffusion time constant was decreased in SDS (green) to 2.1 ms, suggesting a smaller species half the size of YFP-dysferlin in CHAPS or DDM. Triton X-100 solubilization yielded a YFP-dysferlin correlation (gray) that was intermediate between SDS and DDM/CHAPS. These data were best described by a fit to 2 species differing in molecular weight by 2-fold (<b>Equation 3</b>). The data suggest that the dysferlin complex is partially destabilized by Triton X-100, but some dimers remain. (C) PCH analysis yielded the molecular brightness (ε) of diffusing species, normalized to the brightness of control monomeric YFP. The molecular brightness of monomers and dimers are highlighted (dotted red lines). YFP-dysferlin in CHAPS and DDM had a molecular brightness exactly twice the measured brightness of monomeric YFP, suggesting stable dimers in these detergents. YFP-dysferlin in Triton X-100 and individual C2-domains all yielded intermediate molecular brightness, which is compatible with a mixture of monomers and dimers. *** indicates p<0.001, ** p<0.01 and * p<0.05 when compared to the molecular brightness of YFP.</p