title: Search for microbial signatures within human and microbial calcifications using soft x-ray spectromicroscopy.
abstract: BACKGROUND
  The origin of advanced arterial and renal calcification remains poorly understood. Self-replicating, calcifying entities have been detected and isolated from calcified human tissues, including blood vessels and kidney stones, and are referred to as nanobacteria. However, the microbiologic nature of putative nanobacteria continues to be debated, in part because of the difficulty in discriminating biomineralized microbes from minerals nucleated on anything else (eg, macromolecules, cell membranes). To address this controversy, the use of techniques capable of characterizing the organic and mineral content of these self-replicated structures at the submicrometer scale would be beneficial.
  
  
  METHODS
  Calcifying gram-negative bacteria (Caulobacter crescentus, Ramlibacter tataouinensis) used as references and self-replicating calcified nanoparticles cultured from human samples of calcified aneurysms were examined using a scanning transmission x-ray microscope (STXM) at the Advanced Light Source at Lawrence Berkeley National Laboratory. This microscope uses a monochromated and focused synchrotron x-ray beam (80-2,200 eV) to yield microscopic and spectroscopic information on both organic compounds and minerals at the 25 nm scale.
  
  
  RESULTS
  High-spatial and energy resolution near-edge x-ray absorption fine structure (NEXAFS) spectra indicative of elemental speciation acquired at the C K-edge, N K-edge, and Ca L(2,3)-edge on a single-cell scale from calcified C. crescentus and R. tataouinensis displayed unique spectral signatures different from that of nonbiologic hydroxyapatite (Ca(10)(PO(4))(6)(OH)(2)). Further, preliminary NEXAFS measurements of calcium, carbon, and nitrogen functional groups of cultured calcified nanoparticles from humans revealed evidence of organics, likely peptides or proteins, specifically associated with hydroxyapatite minerals.
  
  
  CONCLUSIONS
  Using NEXAFS at the 25 nm spatial scale, it is possible to define a biochemical signature for cultured calcified bacteria, including proteins, polysaccharides, nucleic acids, and hydroxyapatite. These preliminary studies suggest that nanoparticles isolated from human samples share spectroscopic characteristics with calcified proteins.
authors.affiliation: Institut de Minèralogie et de Physique des Milieux Condensés, UMR 7590 and Institut de Physique du Glove de Paris, Paris Cedex, France. karim.benzerara@impmc.jussieu.fr
authors.forename: Karim
authors.initials: K
authors.lastname: Benzerara
authors.forename: Virginia M
authors.initials: VM
authors.lastname: Miller
authors.forename: Gerard
authors.initials: G
authors.lastname: Barell
authors.forename: Vivek
authors.initials: V
authors.lastname: Kumar
authors.forename: Jennyfer
authors.initials: J
authors.lastname: Miot
authors.forename: Gordon E
authors.initials: GE
authors.lastname: Brown
authors.forename: John C
authors.initials: JC
authors.lastname: Lieske
mesh_terms: D000783:Aneurysm / Q000473:pathology; D002114:Calcinosis / Q000473:pathology*; D016935:Caulobacter crescentus / Q000648:ultrastructure; D006090:Gram-Negative Bacteria / Q000648:ultrastructure*; D006801:Humans; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D053758:Nanoparticles / Q000633:toxicity*; D013057:Spectrum Analysis / Q000379:methods*; D014965:X-Rays