Laboratory of Nanomaterials, Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China.
PLoS One. 2010 Apr 26;5(4):e10343. doi: 10.1371/journal.pone.0010343.
The culture and demonstration of putative nanobacteria (NB) and calcifying nanoparticles (CNP) from human and animal tissues has relied primarily on the use of a culture supplement consisting of FBS that had been gamma-irradiated at a dose of 30 kGy (gamma-FBS). The use of gamma-FBS is based on the assumption that this sterilized fluid has been rid entirely of any residual NB/CNP, while it continues to promote the slow growth in culture of NB/CNP from human/animal tissues. We show here that gamma-irradiation (5-50 kGy) produces extensive dose-dependent serum protein breakdown as demonstrated through UV and visible light spectrophotometry, fluorometry, Fourier-transformed infrared spectroscopy, and gel electrophoresis. Yet, both gamma-FBS and gamma-irradiated human serum (gamma-HS) produce NB/CNP in cell culture conditions that are morphologically and chemically indistinguishable from their normal serum counterparts. Contrary to earlier claims, gamma-FBS does not enhance the formation of NB/CNP from several human body fluids (saliva, urine, ascites, and synovial fluid) tested. In the presence of additional precipitating ions, both gamma-irradiated serum (FBS and HS) and gamma-irradiated proteins (albumin and fetuin-A) retain the inherent dual NB inhibitory and seeding capabilities seen also with their untreated counterparts. By gel electrophoresis, the particles formed from both gamma-FBS and gamma-HS are seen to have assimilated into their scaffold the same smeared protein profiles found in the gamma-irradiated sera. However, their protein compositions as identified by proteomics are virtually identical to those seen with particles formed from untreated serum. Moreover, particles derived from human fluids and cultured in the presence of gamma-FBS contain proteins derived from both gamma-FBS and the human fluid under investigation-a confusing and unprecedented scenario indicating that these particles harbor proteins from both the host tissue and the FBS used as feeder. Thus, the NB/CNP described in the literature clearly bear hybrid protein compositions belonging to different species. We conclude that there is no basis to justify the use of gamma-FBS as a feeder for the growth and demonstration of NB/CNP or any NB-like particles in culture. Moreover, our results call into question the validity of the entire body of literature accumulated to date on NB and CNP.
从人体和动物组织中培养和展示假定的纳米细菌（NB）和钙化纳米颗粒（CNP）主要依赖于使用含有胎牛血清（FBS）的培养基，该血清已用 30 kGy（γ-FBS）进行伽马射线照射。使用γ-FBS 的依据是，这种无菌液体已经完全去除了任何残留的 NB/CNP，同时它继续促进来自人体/动物组织的 NB/CNP 的缓慢生长。我们在这里表明，伽马射线照射（5-50 kGy）会导致广泛的剂量依赖性血清蛋白分解，这可以通过紫外和可见光分光光度法、荧光法、傅里叶变换红外光谱法和凝胶电泳法来证明。然而，γ-FBS 和γ-照射的人血清（γ-HS）都在细胞培养条件下产生 NB/CNP，其形态和化学性质与正常血清对照物无法区分。与早期的说法相反，γ-FBS 并没有增强从几种人体体液（唾液、尿液、腹水和滑液）中测试的 NB/CNP 的形成。在存在额外沉淀离子的情况下，γ-照射的血清（FBS 和 HS）和γ-照射的蛋白质（白蛋白和胎球蛋白-A）保留了与未处理的对应物相同的内在双重 NB 抑制和接种能力。通过凝胶电泳，可以看到从γ-FBS 和γ-HS 形成的颗粒已经将相同的弥散蛋白图谱同化到它们的支架中，这些蛋白图谱也存在于γ-照射的血清中。然而，通过蛋白质组学鉴定的它们的蛋白质组成与从未处理的血清形成的颗粒几乎相同。此外，在γ-FBS 存在的情况下从人体液体中培养的颗粒包含源自γ-FBS 和正在研究的人类液体的蛋白质-这是一个令人困惑和前所未有的情况，表明这些颗粒含有来自宿主组织和用作饲料的 FBS 的蛋白质。因此，文献中描述的 NB/CNP 显然具有属于不同物种的混合蛋白质组成。我们得出的结论是，没有理由将γ-FBS 用作生长和展示 NB/CNP 或任何 NB 样颗粒的培养基。此外，我们的结果质疑迄今为止积累的关于 NB 和 CNP 的整个文献的有效性。
