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Ming-jun Zhang,1 Sheng-nan Liu,1 Ge Xu,2 Ya-nan Guo,1 Jian-nan Fu,3 De-chun Zhang11Molecular Medicine and Tumor Research Center, Chongqing Medical University, 2Electron Microscopy Group, Department of Life Science, Chongqing Medical University, Chongqing, People's Republic of China; 3First People's Hospital of Jiulongpo District, Chongqing, People's Republic of ChinaBackground: The existing evidence that nanobacteria (NB) are closely associated with human disease is overwhelming. However, their potential toxicity against cancer cells has not yet been reported. The objective of this study was to investigate the cytotoxic effects of NB and nanohydroxyapatites (nHAPs) against human breast cancer cells and to elucidate the mechanisms of action underlying their cytotoxicity.Methodology/principal findings: NB were isolated from calcified placental tissue, and nHAPs were artificially synthesized. The viability of the MDA-MB-231 human breast cancer cell line was tested by using the Kit-8 cell counting kit assay. Apoptosis was examined by transmission electron microscopy and flow cytometry. The endocytosis of NB and nHAPs by MDA-MB-231 cells was initially confirmed by microscopy. Although both NB and nHAPs significantly decreased MDA-MB-231 cell viability and increased the population of apoptotic cells, NB were more potent than nHAPs. After 72 hours, NB also caused ultrastructural changes typical of apoptosis, such as chromatin condensation, nuclear fragmentation, nuclear dissolution, mitochondrial swelling, and the formation of apoptotic bodies.Conclusion/significance: In MDA-MB-231 human breast cancer cells, NB and nHAPs exerted cytotoxic effects that were associated with the induction of apoptosis. The effects exerted by NB were more potent than those induced by nHAPs. NB cytotoxicity probably emerged from toxic metabolites or protein components, rather than merely the hydroxyapatite shells. NB divided during culturing, and similar to cells undergoing binary fission, many NB particles were observed in culture by transmission electron microscopy, suggesting they are live microorganisms.Keywords: nanobacteria, nanohydroxyapatites, human breast cancer MDA-MB-231 cells, cytotoxicity, apoptosis

The existence of positive solutions for boundary value problems of nonlinear functional difference equations with -Laplacian operator is investigated. Sufficient conditions are obtained for the existence of at least one positive solution and two positive solutions.