Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp.niger) endospores or Fusarium moniliformeconidia. The hot-detergent–bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells orB. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.

As a continuation of our effort to understand degradation mechanisms of a eutectic mixture of bis(2,2-dinitropropyl)acetal (BDNPA) and bis(2,2dinitropropyl)formal (BDNPF) (referred to as NP) under various environmental conditions, we investigated the thermal stability of NP under water and 74% relative humidity (RH) environments at temperatures below 70 °C. Based on a comprehensive characterization of samples aged over a period of two years, we conclude that in the presence of water the reaction pathways of the NP degradation are different from those observed in air or under nitrogen atmosphere. We found that the physical state of water molecules plays an important role as it determines the ability of oxygen to participate in the NP aging process. Based on the results obtained in Parts A and B of these studies, we conclude that the rate of NP degradation increases in the order: nitrogen < water < air < water vapor + air.

We investigated the chemical and thermal stability of a eutectic mixture of bis(2,2-dinitropropyl)acetal (BDNPA) and bis(2,2-dinitropropyl)formal (BDNPF) (referred to as NP) in various environments at temperatures below 70 °C. Changes in the chemical composition of aged samples were characterized using TGA, FTIR, GPC, ESI-MS, and 1 H NMR spectroscopies over a period of two years. The results show that the initial signs of NP degradation can be detected as early as 12 months at 70 °C in air. The initial step in the degradation is the elimination of HONO molecules, followed by the formation of nitroso-alcohol isomers. While the temperature plays a key role in determining the degradation kinetics of the initial stages, the absence or presence of oxygen determines the types and rates of formations of various isomers and intermediates during the thermal decomposition processes. In addition, oxygen accelerates the decomposition of the isomers and intermediates, whereas nitrogen has a stabilizing effect. BDNPA shows higher reactivity than BDNPF regardless of the aging conditions, which is attributed to the presence of an extra methyl group in its structure.

Quantum dots (QDs) are semiconductor nanocrystals exhibiting unique optical properties that can be exploited for many practical applications ranging from photovoltaics to biomedical imaging and drug delivery. A significant number of studies have alluded to the cytotoxic potential of these materials, implicating Cd-leaching as the causal factor. Here, we investigated the role of heavy metals in biological responses and the potential of CdSe-induced genotoxicity. Our results indicate that, while negatively charged QDs are relatively noncytotoxic compared to positively charged QDs, the same does not hold true for their genotoxic potential. Keeping QD core composition and size constant, 3 nm CdSe QD cores were functionalized with mercaptopropionic acid (MPA) or cysteamine (CYST), resulting in negatively or positively charged surfaces, respectively. CYST-QDs were found to induce significant cytotoxicity accompanied by DNA strand breakage. However, MPA-QDs, even in the absence of cytotoxicity and reactive oxygen species formation, also induced a high number of DNA strand breaks. QD-induced DNA damage was confirmed by identifying the presence of p53 binding protein 1 (53BP1) in the nuclei of exposed cells and subsequent diminishment of p53 from cytoplasmic cellular extracts. Further, high-throughput real-time PCR analyses revealed upregulation of DNA damage and response genes and several proinflammatory cytokine genes. Most importantly, transcriptome sequencing revealed upregulation of the metallothionein family of genes in cells exposed to MPA-QDs but not CYST-QDs. These data indicate that cytotoxic assays must be supplemented with genotoxic analyses to better understand cellular responses and the full impact of nanoparticle exposure when making recommendations with regard to risk assessment.

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