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The objective of this study was to investigate whether exposure of human monocytes to a pulsed ultra-wideband electromagnetic field (EMF) of 1 kV/cm average peak power triggers a signaling pathway responsible for the transcriptional regulation of NFKB (NF-kappaB)-dependent gene expression. Human Mono Mac 6 (MM6) cells were exposed intermittently to EMF pulses for a total of 90 min. The pulse width was 0.79+/-0.01 ns and the pulse repetition rate was 250 pps. The temperature of the medium was maintained at 37 degrees C in both sham- and EMF-exposed flasks. Total NFKB DNA-binding activity was measured in the nuclear extracts by the electrophoretic mobility shift assay. Cells exposed to the EMFs and incubated for 24 h postexposure showed a 3.5+/-0.2-fold increase in the NFKB DNA-binding activity. Since activation of NFKB was observed, the possibility of kappaB-dependent gene expression in response to exposure to the EMFs was investigated using NFKB signal-specific gene arrays. The results revealed no difference in the NFKB-dependent gene expression profiles at 8 or 24 h postexposure, indicating that activated NFKB does not lead to the differential expression of kappaB-dependent target genes. To determine whether the absence of the kappaB-dependent gene expression was due to compromised transcriptional regulation of NFKB, the functional activity of NFKB was examined in cells transiently transfected with Mercury Pathway constructs containing 4x NFKB binding sites associated either with the luciferase reporter system or a control vector. Pulsed EMF exposure did not induce NFKB-driven luciferase activity in these cells, indicating that the activation of NFKB at 24 h after the 1 kV/cm EMF exposure is functionally inactive. From these results, it is clear that the EMF-induced NFKB activation is only a transient response, with minimal or no downstream effect.

Aims: This study evaluated the effects of DNA extraction method, DNA purification and pooling of PCR amplification products on the description of bacterial and archaeal diversity. Methods and Results: Soil DNA was extracted by the Power Soil DNA extraction kit and a customized Griffiths' protocol. Both methods are based on cell disruption by bead beating. In total, we used three soils and six independent extractions from each soil obtained by each of the two methods. Then, three of the six extracts of each treatment were further purified by spin columns filled with Sepharose 2B and polyvinylpolypyrrolidone (PVPP). The V4 hypervariable region of the 16S rRNA gene was amplified from each extract using the 515F/806R primer pair in four independent reactions. Three amplification products were combined and sequenced as a pooled sample, while the additional amplification product was sequenced individually. The resulting 72 amplification products were sequenced by Illumina MiSeq platform. DNA extraction method had a statistically significant effect on the estimation of the composition of microbial communities that might overwhelm differences in microbial communities from distinct soils. On the other hand, a further DNA purification step or pooling of PCR amplification products had a minor effect on the description of bacterial and archaeal communities. Conclusions: DNA extraction had the strongest effect on the description of bacterial and archaeal communities; low concentration of impurities, which allow PCR amplification, can still generate a minor additional bias, while PCR stochastic variability had the lowest effect. Significance and Impact of the Study: Although it is well known that methodological factors affect the description of microbial communities, the relative importance of each step is still unknown. The present study determined that of the factors tested, the DNA extraction method had the strongest effects on the description of bacterial and archaeal communities. [ABSTRACT FROM AUTHOR]