Effects of 700 and 3500 MHz 5G radiofrequency exposure on developing zebrafish embryos.

Telecommunications industries are rapidly deploying the fifth generation (5G) spectrum and there is public concern about the safety and health impacts of this type of Radio Frequency Radiation (RFR), in part because of the lack of comparable scientific evidence. In this study we have used a validated commercially available setting producing a uniform field to expose zebrafish embryos (ZFe) to unmodulated 700 and 3500 MHz frequencies. We have combined a battery of toxicity, developmental and behavioral assays to further explore potential RFR effects. Our neurobehavioral profiles include a tail coiling assay, a light/dark activity assay, two thigmotaxis anxiety assays (auditory and visual stimuli), and a startle response - habituation assay in response to auditory stimuli. ZFe were exposed for 1 and 4 h during the blastula period of development and endpoints evaluated up to 120 hours post fertilization (hpf). Our results show no effects on mortality, hatching or body length. However, we have demonstrated specific organ morphological effects, and behavioral effects in activity, anxiety-like behavior, and habituation that lasted in larvae exposed during the early embryonic period. A decrease in acetylcholinesterase activity was also observed and could explain some of the observed behavioral alterations. Interestingly, effects were more pronounced in ZFe exposed to the 700 MHz frequency, and especially for the 4 h exposure period. In addition, we have demonstrated that our exposure setup is robust, flexible with regard to frequency and power testing, and highly comparable. Future work will include exposure of ZFe to 5G modulated signals for different time periods to better understand the potential health effects of novel 5G RFR.

Spermcast mating with release of zygotes in the small dioecious bivalve Digitaria digitaria.

Digitaria digitaria, a small astartid usually less than 10 mm in length, has a non-brooding behaviour in spite of its limited space for gonad development. This species lives in highly unstable environments with strong currents, which represent a challenge for fertilization and larval settlement. The studied population of D. digitaria from the Strait of Gibraltar area was dioecious, with significant predominance of females and sexual dimorphism, where females are larger than males. The reproductive cycle is asynchronous throughout the year, without a resting period, but with successive partial spawning events. The presence of stored sperm in the suprabranchial chamber and inside the gonad of some females, together with the release of eggs along the dorsal axis of both gills, points to internal oocyte fertilization. Bacteriocytes were found in the female and male follicle walls, but no bacteria were observed inside any of the gametes. Digitaria digitaria could represent a "missing link" between spermcast mating bivalves with brooded offspring and bivalves with broadcast release of eggs and sperm. The small size, limiting the oocyte production, together with the unstable environment could represent evolutionary pressures towards sperm uptake in D. digitaria.

Constructing recombinant DNA molecules by PCR.

This unit describes the use of PCR to construct hybrid DNA molecules. The unit provides an overview of how PCR can be exploited to accomplish numerous cloning strategies. The Basic Protocol outlines the PCR amplification and cloning strategies. The Commentary includes a troubleshooting guide for problems most frequently encountered in PCR cloning, plus four specific examples of the application of this technique to create in-frame fusion proteins, to create recombinant DNA products, to generate deletions and inversions by inverse PCR, and to introduce mutagenized PCR products or specific mutations or fusions by gap repair in yeast.

Characterization of the aspartate kinase from Saccharomyces cerevisiae and of its interaction with threonine.

Aspartate kinase (AK) from Saccharomyces cerevisiae has been characterized to elucidate its quaternary structure and the effect of the allosteric inhibitor threonine on the enzyme conformation. The homogeneously purified enzyme was inhibited by threonine (K(i) 1.4 mM) and was found to bind this compound (K(d) 0.97 mM) in a hyperbolic manner. Gel filtration and native gel electrophoresis indicated that yeast AK is a homohexamer of 346 kDa composed by 58 kDa subunits. Threonine caused a decrease in the apparent molecular mass of AK as evidenced by size-exclusion chromatography (from 345 to 280 kDa) and blue native gel electrophoresis (from 346 to 297 kDa); no other molecular species were detected. This shift in the hydrodynamic size was threonine-specific and was reversed by rechromatography in the absence of threonine. No change in the apparent molecular mass was induced by threonine in an AK mutant insensitive to inhibition by this amino acid, which was observed to be unable to bind threonine. These results indicate that the allosteric transition elicited by binding of threonine to yeast AK involves a large conformational change of the protein that isomerizes from a relaxed active conformation to a more compact inactive one of smaller molecular dimensions.