Journal: Frontiers in Cell and Developmental Biology

Article Title: Plasma Exosomes Derived From Patients With End-Stage Renal Disease and Renal Transplant Recipients Have Different Effects on Vascular Calcification

doi: 10.3389/fcell.2020.618228

Figure Lengend Snippet: Fetuin-A and MGP were decreased in ESRD-Ex. (A) The protein concentration of exosomes in Nor-Ex, ESRD-Ex, and RTR-Ex. (B,C) The content of Fetuin-A and MGP in Nor-Ex, ESRD-Ex, and RTR-Ex were measured by ELISA. (D) Western blot analyses detected the levels of Fetuin-A and MGP protein. n = 3. (E) The CAC total score of Nor, ESRD, and RTR patients were calculated by a multilayer spiral CT. n = 8. The representative images were shown. The arrows indicated the calcified part of different branches of the coronary artery. (F,G) The spearman analysis showed the correlations between the content of plasma exosomes Fetuin-A and MGP with CAC total score, respectively. n = 24. *** p < 0.001, ** p < 0.01, * p < 0.05, compared with Nor. ## p < 0.01, # p < 0.05, compared with ESRD. ns, not significant; Nor-Ex, the plasma exosomes derived from normal health control; ESRD-Ex, the plasma exosomes derived from ESRD patients; RTR-Ex, the plasma exosomes derived from renal transplant recipients; CAC, coronary artery calcification.

Article Snippet: Primary antibodies including CD9 (ab92726, 1:1,000, Abcam), CD63 (ab68418, 1:1,000, Abcam), CD81 (ab79559, 1:1,000, Abcam), Runx2 (ab76956; 1:1,000, Abcam), Fetuin-A (16571-1-AP, 1:1,000, Proteintech), MGP (10734-1-AP, 1:1,000, Proteintech), Annexin-A2 (11256-1-AP, 1:1,000, Proteintech), BMP-2 (ab214821, 1:1,000, Abcam), Rankl (23408-1-AP, 1:500, Proteintech), and β-actin (ab6276, 1:3,000, Abcam).

Techniques: Protein Concentration, Enzyme-linked Immunosorbent Assay, Western Blot, Clinical Proteomics, Derivative Assay, Control

Journal: bioRxiv

Article Title: A Tough Biointerface in Human Knee Empowered by Dynamic Phase-transforming Minerals in Collagenous Matrix

doi: 10.1101/2024.08.03.606023

Figure Lengend Snippet: ( A ) Schematic illustration of the experimental procedure of proteomic analysis. ( B ) Classification of interface-enriched proteins based on functions described in Genecards Database. ( C ) Volcano plots of protein expression differences between interface and bone, and interface and root. Proteins with statistically significant differences in expression were colored in orange (significantly higher in interface) and blue (significantly lower in interface) ( p adjusted value < 0.05, fold change > 2). ( D ) Immunofluorescent staining of MGP at the S-H interface. ( E ) MGP protein expression profile across the S-H interface quantified using fluorescent signal intensity which increased in the mineralized tissue region and S-H interface (denoted by dashed line). ( F ) Functional verification of MGP’s role as a mineralization inhibitor through in vitro mineralization using nano-ACP. TEM images showed the increase in HAP crystals in the non-treated group was less than in the MGP-treated group on day 1 and day 3. Inlets revealed diffraction patterns of associated areas. ( G ) Quantification of HAP crystal density in TEM snapshots (n=5) of MGP-treated and non-treated groups. Statistical significance is illustrated as: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Abbreviations: LC-MS/MS, liquid chromatography−tandem mass spectrometry; MGP, matrix Gla protein.

Article Snippet: 200-mesh golden grids with a carbon support film were added with 3 μL recombinant matrix gla protein (MGP) (100 μg/mL, CUSABIO, China) diluted with deionized water and the control group was added with 3 μL deionized water, and both were incubated for 20 minutes at room temperature.

Techniques: Expressing, Staining, Functional Assay, In Vitro, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: A Tough Biointerface in Human Knee Empowered by Dynamic Phase-transforming Minerals in Collagenous Matrix

doi: 10.1101/2024.08.03.606023

Figure Lengend Snippet: The left panel illustrates the hierarchical structure of the meniscus root-bone interface under static condition. From macroscale to nanoscale: histological zones of the root-bone interface; transformation of mineral aggregates from ACP, immature HAP to HAP alongside mineralization of collagen fibrils; ACP, immature HAP and collagen form molecular bonds with collagen fibrils, while MGP protein and proteoglycans interact with the mineral aggregates and collagen to mediate mineralization. The right panel demonstrates the multiscale mechanical response upon mechanical loading. From macroscale to nanoscale: soft tissue responds to stress first via fiber stretching and straightening while the interface responds last, and cracks would be deflected or blunted; the ACP and immature aggregates dissipate stress (denoted by “E”) through sliding in the interfibrillar space, and crack would be arrested by stiff mineral aggregates; bonds among mineral aggregates and collagen could break for energy dissipation, and new bonds formation could occur simultaneously. Abbreviations: LR, ligamentous root; FC, fibrocartilage; MFC; mineralized fibrocartilage; ACP, amorphous calcium phosphate; HAP, hydroxyapatite; MGP, matrix gla protein.

Article Snippet: 200-mesh golden grids with a carbon support film were added with 3 μL recombinant matrix gla protein (MGP) (100 μg/mL, CUSABIO, China) diluted with deionized water and the control group was added with 3 μL deionized water, and both were incubated for 20 minutes at room temperature.

Techniques: Transformation Assay

Journal: PLoS ONE

Article Title: Calcification in dermal fibroblasts from a patient with GGCX syndrome accompanied by upregulation of osteogenic molecules

doi: 10.1371/journal.pone.0177375

Figure Lengend Snippet: (A) Immunofluorescence staining with an antibody specific for ucMGP. The level of ucMGP was higher in GGCX dermal fibroblasts (Pt) than in normal dermal fibroblasts (CON). The bar depicts 50 μm (original magnification, ×400). (B) RT-PCR analysis of MGP mRNA levels in normal (CON) and GGCX (Pt) dermal fibroblasts. The MGP mRNA levels in normal dermal fibroblasts are shown as mean ± SD (n = 4) and those in GGCX dermal fibroblasts as mean. (C) Quantification of ucMGP in normal (CON) and GGCX (Pt) dermal fibroblasts. Values in normal dermal fibroblasts are shown as mean ± SD (n = 3) and those in GGCX dermal fibroblasts as mean. These experiments were performed at least twice, and a representative data set is shown.

Article Snippet: ucMGP concentrations in GGCX dermal fibroblasts were quantified using a human ucMGP ELISA Kit according to the manufacturer’s protocol (#CSB-EC013789HU, Cusabio Biotech Co., Wuhan, China).

Techniques: Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction