Development of an Acute Method to Deliver Transgenes Into the Brains of Adult Xenopus laevis.

The central vocal pathway of the African clawed frog, Xenopus laevis, is a powerful vertebrate model to understand mechanisms underlying central pattern generation. However, fast and efficient methods of introducing exogenous genes into the neurons of adult X. laevis are currently not available. Here, we systematically tested methods of transgene delivery into adult X. laevis neurons. Although successfully used for tadpole neurons for over a decade, electroporation was not efficient in transfecting adult neurons. Similarly, adeno-associated virus (AAV) was not reliable, and lentivirus (LV) failed to function as viral vector in adult Xenopus neurons. In contrast, vesicular stomatitis virus (VSV) was a fast and robust vector for adult X. laevis neurons. Although toxic to the host cells, VSV appears to be less virulent to frog neurons than they are to mice neurons. At a single cell level, infected neurons showed normal physiological properties up to 7 days post infection and vocal circuits that included infected neurons generated normal fictive vocalizations up to 9 days post infection. The relatively long time window during which the physiology of VSV-infected neurons can be studied presents an ideal condition for the use of optogenetic tools. We showed that VSV does not gain entry into myelinated axons, but is taken up by both the soma and axon terminal; this is an attractive feature that drives transgene expression in projection neurons. Previous studies showed that VSVs can spread across synapses in anterograde or retrograde directions depending on the types of glycoprotein that are encoded. However, rVSV did not spread across synapses in the Xenopus central nervous system. The successful use of VSV as a transgene vector in amphibian brains not only allows us to exploit the full potential of the genetic tools to answer questions central to understanding central pattern generation, but also opens the door to other research programs that focus on non-genetic model organisms to address unique questions.

Effects of fuel components and combustion particle physicochemical properties on toxicological responses of lung cells.

The physicochemical properties of combustion particles that promote lung toxicity are not fully understood, hindered by the fact that combustion particles vary based on the fuel and combustion conditions. Real-world combustion-particle properties also continually change as new fuels are implemented, engines age, and engine technologies evolve. This work used laboratory-generated particles produced under controlled combustion conditions in an effort to understand the relationship between different particle properties and the activation of established toxicological outcomes in human lung cells (H441 and THP-1). Particles were generated from controlled combustion of two simple biofuel/diesel surrogates (methyl decanoate and dodecane/biofuel-blended diesel (BD), and butanol and dodecane/alcohol-blended diesel (AD)) and compared to a widely studied reference diesel (RD) particle (NIST SRM2975/RD). BD, AD, and RD particles exhibited differences in size, surface area, extractable chemical mass, and the content of individual polycyclic aromatic hydrocarbons (PAHs). Some of these differences were directly associated with different effects on biological responses. BD particles had the greatest surface area, amount of extractable material, and oxidizing potential. These particles and extracts induced cytochrome P450 1A1 and 1B1 enzyme mRNA in lung cells. AD particles and extracts had the greatest total PAH content and also caused CYP1A1 and 1B1 mRNA induction. The RD extract contained the highest relative concentration of 2-ring PAHs and stimulated the greatest level of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFα) cytokine secretion. Finally, AD and RD were more potent activators of TRPA1 than BD, and while neither the TRPA1 antagonist HC-030031 nor the antioxidant N-acetylcysteine (NAC) affected CYP1A1 or 1B1 mRNA induction, both inhibitors reduced IL-8 secretion and mRNA induction. These results highlight that differences in fuel and combustion conditions affect the physicochemical properties of particles, and these differences, in turn, affect commonly studied biological/toxicological responses.