Microplate Subtractive Hybridization To Enrich for Bacteroidales Genetic Markers for Fecal Source Identification

The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog)

Systematic Assessment of Exposure Variations on Observed Bioactivity in Zebrafish Chemical Screening

The embryonic zebrafish is a powerful tool for high-throughput screening of chemicals. While this model has significant potential for use in safety assessments and chemical prioritization, a lack of exposure protocol harmonized across laboratories has limited full model adoption. To assess the potential that exposure protocols alter chemical bioactivity, we screened a set of eight chemicals and one 2D nanomaterial across four different regimens: (1) the current Tanguay laboratory’s standard protocol of dechorionated embryos and static exposure in darkness; (2) exposure with chorion intact; (3) exposure under a 14 h light: 10 h dark cycle; and (4) exposure with daily chemical renewal. The latter three regimens altered the concentrations, resulting in bioactivity of the test agents compared to that observed with the Tanguay laboratory’s standard regimen, though not directionally the same for each chemical. The results of this study indicate that with the exception for the 2D nanomaterial, the screening design did not change the conclusion regarding chemical bioactivity, just the nominal concentrations producing the observed activity. Since the goal of tier one chemical screening often is to differentiate active from non-active chemicals, researchers could consider the trade-offs regarding cost, labor, and sensitivity in their study design without altering hit rates. Taken further, these results suggest that it is reasonably feasible to reach agreement on a standardized exposure regiment, which will promote data sharing without sacrificing data content

Impacts of high dose 3.5 GHz cellphone radiofrequency on zebrafish embryonic development.

The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. In this study, we used the embryonic zebrafish model to assess the impacts of a 3.5 GHz RFR on biology- a frequency typically used by 5G-enabled cell phones and lies within the 4G and 5G bandwidth. We established a plate-based exposure setup for RFRs, exposed developing zebrafish to 3.5 GHz RFR, specific absorption rate (SAR) ≈ 8.27 W/Kg from 6 h post fertilization (hpf) to 48 hpf, and measured a battery of morphological and behavioral endpoints at 120 hpf. Our results revealed no significant impacts on mortality, morphology or photomotor response and a modest inhibition of startle response suggesting some levels of sensorimotor disruptions. This suggests that the cell phone radiations at low GHz-level frequencies are likely benign, with subtle sensorimotor effects. Through this assessment, we have established a robust setup for zebrafish RFR exposures readily amenable to testing various powers and frequencies. Future developmental exposure studies in zebrafish will evaluate a wider portion of the radio frequency spectrum to discover the bioactive regions, the potential molecular targets of RFR and the potential long-term effects on adult behavior