Journal: Nature Communications

Article Title: Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

doi: 10.1038/s41467-022-33247-3

Figure Lengend Snippet: a ThT fluorescence kinetics of β2m solutions at various conditions ( n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge$$\end{document} ≥ 4). Serum was collected from a non-dialysis control #9 (DT(−)). b CD spectra of the initial monomers at 37, 60, and 95 °C, and amyloid fibrils formed by ultrasonication. c Thermal denaturation curve of β2m monomer monitored by the CD ellipticity at 230 nm. T m is the denaturation midpoint obtained from the fitted curve. d High performance reversed-phase chromatograms for intact monomers (upper) and amyloid fibrils formed by ultrasonication (lower). TEM images of ( e ) ThT-positive aggregates formed by ultrasonication, ( f ) aggregates seeded and elongated from the amyloid fibrils formed by ultrasonication, and ( g ) ThT-negative aggregates formed in the presence of 15% (v/v) serum. In TEM observations, five images were acquired for each sample. The images in panel ( e , f , and g ) are the representative image, and remaining four images are shown in Supplementary Fig. . a. u., arbitrary units. Source data are provided as a Source Data file.

Article Snippet: The equilibrated solution was ultracentrifugated at 100,000 × g for 1 h, and then, the concentration of β2m monomers in the supernatant was determined by the ELISA method (KGE019, Human beta 2-Microglobulin Parameter Assay Kit, R&D Systems).

Techniques: Fluorescence, Circular Dichroism

Journal: Nature Communications

Article Title: Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

doi: 10.1038/s41467-022-33247-3

Figure Lengend Snippet: a Clinical characteristics of non-dialysis control (DT(−), N = 30) and dialysis patient (DT(+, Pre), N = 30) groups. For the non-dialysis group, their glomerular filtration rates (GFR) are used as an index of kidney function, because they have never received dialysis treatments. b Representative ThT fluorescence kinetics using sera collected from non-dialysis controls and dialysis patients. For each sample, measurement was performed multiple times for independent solutions ( n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\,\ge$$\end{document} ≥ 4). Comparison of the effect of sera with or without dialysis treatment on β2m fibril formation in terms of the ( c ) lag time and ( d ) ThT fluorescence intensity. The p -values were caluculated by the unpaired one-sided t -test. For all 60 samples, row data are shown in Supplementary Fig. . Error bars in panels ( c and d ) denote the standard deviation among independent serum samples ( N = 30). Dialysis-vintage dependence of ( e ) the lag time and ( f ) ThT fluorescence intensity ( N = 30). The r -values indicate the Pearson correlation coefficient. a. u., arbitrary units. Source data are provided as a Source Data file.

Article Snippet: The equilibrated solution was ultracentrifugated at 100,000 × g for 1 h, and then, the concentration of β2m monomers in the supernatant was determined by the ELISA method (KGE019, Human beta 2-Microglobulin Parameter Assay Kit, R&D Systems).

Techniques: Filtration, Fluorescence, Comparison, Standard Deviation

Journal: Nature Communications

Article Title: Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

doi: 10.1038/s41467-022-33247-3

Figure Lengend Snippet: a Effects of maintenance dialysis treatment on the serum β2m concentration and body weight of dialysis patients ( N = 28), who donated their serum immediately before (DT(+, Pre)) and after (DT(+, Post)) maintenance dialysis treatment. The relative changes in the two parameters were shown with respect to their values before dialysis treatment. b ThT fluorescence kinetics of β2m fibril formation with 5% (v/v) addition of sera collected from an identical dialysis patient immediately before (DT(+, Pre), #15) and after (DT(+, Post), #15) a single maintenance dialysis treatment. Significance examination for the effects of serum before and after dialysis treatment in terms of the ( c ) lag time and ( d ) ThT fluorescence intensity ( N = 28). The p -values were calculated by the paired one-sided t -test. For all dialysis patients, row data are shown in Supplementary Fig. . Error bars in panels ( c and d ) denote the standard deviation among independent serum samples ( N = 28). Correlation between change in relative weight and ( e ) lag time and ( f ) ThT fluorescence intensity. a. u., arbitrary units. Source data are provided as a Source Data file.

Article Snippet: The equilibrated solution was ultracentrifugated at 100,000 × g for 1 h, and then, the concentration of β2m monomers in the supernatant was determined by the ELISA method (KGE019, Human beta 2-Microglobulin Parameter Assay Kit, R&D Systems).

Techniques: Concentration Assay, Fluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

doi: 10.1038/s41467-022-33247-3

Figure Lengend Snippet: a – c The effects of serum albumin (ALB) and polyethylene glycol (PEG) on β2m amyloid fibril formation. a ThT fluorescence kinetics in the presence of 5 mg/mL serum albumin (ALB) or polyethylene glycol (PEG) ( n = 5), ( b ) their lag times, and ( c ) ThT fluorescence intensities. Centers for error bars and error bars denote the mean and standard deviation among independent sample solutions ( n = 5), respectively. d , e Interactions between β2m monomer and serum albumin monitored by QCM experiments ( n = 3): ( d ) Relative frequency change and ( e ) calculation of the dissociation constant, K D , between β2m monomer and serum albumin. The error bars denote the standard deviation among independent measurements ( n = 3). f Relative intensity changes in the NMR signal of the 15 N-labeled β2m monomer at different albumin concentrations. The error bars indicate errors in estimation of relative peak intensities. g Relative intensity changes in the NMR signals averaged over each strand. The error bars indicate errors in estimation of relative peak intensities. h Thermal denaturation curves of β2m monomers in the presence of serum albumin with various stoichiometries. T m value is a denaturation midpoint of β2m monomer at each serum albumin concentration. a. u., arbitrary units. Source data are provided as a Source Data file.

Article Snippet: The equilibrated solution was ultracentrifugated at 100,000 × g for 1 h, and then, the concentration of β2m monomers in the supernatant was determined by the ELISA method (KGE019, Human beta 2-Microglobulin Parameter Assay Kit, R&D Systems).

Techniques: Fluorescence, Standard Deviation, Labeling, Concentration Assay

Journal: Nature Communications

Article Title: Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

doi: 10.1038/s41467-022-33247-3

Figure Lengend Snippet: a Schemes of β2m amyloid fibril formation in the presence of serum albumin. The molecular illustrations were depicted using PyMOL Molecular Graphics (ver. 2.5.1). b – d Dependences on total β2m ([β2m] T ) and serum albumin ([ALB] T ) concentrations of ( b ) native β2m-serum albumin complex, ( c ) denatured monomer, and ( d ) amyloid fibrils, before (red mesh) and after (blue mesh) breakdown of supersaturation. In panel ( c) , the axis of the supersaturation ratio, σ = [D] S /[D] C , is also shown. e – g Dependences as shown in panels ( b – d ) are represented by fractions of ( e ) native β2m-serum albumin complex, ( f ) native monomer, and ( g ) amyloid fibrils. h The relative change in the lag time as a function of [β2m] T and [ALB] T . In panels ( b – h ), the blue, red, and green stars indicate the representative values for non-dialysis controls (DT(−), [β2m] T = 2 μg/mL (0.17 μM) and [ALB] T = 45 mg/mL (680 μM)) and dialysis patients before maintenance dialysis (DT(+, Pre), [β2m] T = 50 μg/mL (4.24 μM) and [ALB] T = 35 mg/mL (529 μM)) and dialysis patients after maintenance dialysis (DT(+, Post), [β2m] T = 20 μg/mL (1.69 μM) and [ALB] T = 38 mg/mL (574 μM)), respectively. The black arrows show the change in [β2m] T and [ALB] T by maintenance dialysis treatment. i Fluctuations of concentrations of the β2m monomer and serum albumin, temporary risk, TR , and accumulated risk, AR , as a function of the time. j Estimation of the onset of DRA by the accumulation of three risk factors at different conditions.

Article Snippet: The equilibrated solution was ultracentrifugated at 100,000 × g for 1 h, and then, the concentration of β2m monomers in the supernatant was determined by the ELISA method (KGE019, Human beta 2-Microglobulin Parameter Assay Kit, R&D Systems).

Techniques: