Proteomic evaluation of biological nanoparticles isolated from human kidney stones and calcified arteries

Abstract

Calcifying biologic nanoparticles (NPs) develop under cell culture conditions from homogenates of diverse tissue samples displaying extraosseous mineralization, including kidney stones and calcified aneurysms. Probes to definitively identify NPs in biologic systems are lacking. Therefore, the aim of this study was to begin to establish a proteomic biosignature of NPs in order to facilitate more definitive investigation of their contribution to disease. Biologic NPs derived from human kidney stones and calcified aneurysms were completely decalcified by overnight treatment with EDTA or brief incubation in HCl, as evidenced by lack of a calcium shell and of Alizarin Red S staining, by transmission electron microscopy and confocal microscopy, respectively. Decalcified NPs contained numerous proteins including some from bovine serum and others of prokaryotic origin. Most prominent of the latter group was EF-Tu, which appeared identical to EF-Tu from S. epidermidis. A monoclonal antibody against human EF-Tu recognized a protein in Western blots of total NP lysate, as well as in intact NPs by immunofluorescence and immunogold EM. Approximately 8% of NPs were quantitatively recognized by the antibody by flow cytometry. Therefore, we have defined methods to reproducibly decalcify biologic NPs, and identified key components of their proteome. These elements, including EF-Tu, can be used as biomarkers to further define processes which mediate propagation of biologic NPs and their contribution to disease.

The role of calcifying nanoparticles in biology and medicine

“…Bacterioscopic (DNA-specific dyes Hoechst 33258, propidium iodide, PicoGreen, staining after a demineralization is optimal) using scanning and transmission electron microscopy, [16][17][18][19][20][21][22][23][24] von Kossa staining, which is specific to calcium compounds, 16 staining by 2% uranyl acetate (possibly with lead citrate) to detect specific mucus on the hydroxyapatite shell, 1 staining by alizarin red S in the mineralized state, 26 staining by phosphotungstic acid, 16 after the long-term cultivation light microscopy with von Kossa staining is possible to be used for detection. 1,16 Bacteriological (cultivation in DMEM or RPMI-1640 without serum under 37°C for 4-6 weeks after filtration through 0.10-0.22 μm pores), 1,16 replication can be assessed by spectrophotometry (650 nm wavelength).…”

“…Therefore, CNPs being life forms cannot be ruled out based on their extremely small size alone. Also, it is not possible to rule out the uniqueness of the replicative and metabolic mechanisms Susceptibility to a number of antimicrobial chemotherapeutics possessing antimetabolic activity and not possessing the chelating activity, 30 (nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxasole), to 5-fluorouracil, cytosine arabinoside, 6-aminocaproic acid, 30 antimycin A, sodium azide, potassium cyanide 22 Morphological similarity to various mineral and mineralo-protein complexes containing inorganic calcium, phosphorus, and apatite compounds, and also proteins and phospholipids 2,6,[8][9][10] Acceleration of replication and reduction of formation of biofilms after photobiostimulation 29 Resistance to DNase and RNase activity 5 Presence of proteins, including bacterial ones, after a demineralization 16,20,21 (EF-Tu, 22,26 EF-G, 26 of CNPs requiring a smaller size for proper functioning, and these mechanisms can feasibly be characterized by a greater compactness compared with their analogs in other organisms.…”

Clinical implications of calcifying nanoparticles in dental diseases: a critical review

“…39,35,36 Many findings and data oppose the hypothesis that CNPs are mineral-protein complexes. Although the formation of complexes of minerals and protein serum and other biological liquid under homeostasis was proposed by Martel and Young, 9 Wu et al, 6 Young et al, 7,8 and Raoult et al, 10 Kutikhin et al 2 believed that the presence of CNPs in an organism is clearly a pathological process.…”

Do calcifying nanoparticles really contain 16S rDNA?

“…Second, after this report, multiple evidence-based studies were conducted in order to better understand the actual biological composition and self-propagation of nanobacteria. [5][6][7] None of these findings are conclusive; however, biological insights of this mystery are now emerging.…”