Assessment of naloxone as a therapeutic for inhaled carfentanil in the ferret.

Carfentanil is a powerful synthetic opioid that is approximately 100 times more potent than fentanyl and 10,000 times more potent than morphine. Carfentanil was originally intended to be used as a sedative for big game animals in a veterinary setting, but it is becoming increasingly recognized as a public health concern. We set out to investigate the effectiveness of naloxone against a potentially lethal dose of inhaled carfentanil in male ferrets. Ferrets were implanted with telemetry devices to study cardiac parameters and exposed to aerosolized carfentanil in a whole-body plethysmography chamber to record respiratory parameters. We observed profound respiratory depression in exposed animals, which led to apneic periods constituting 24-31 % of the exposure period. Concomitant with these apneic periods, we also observed cardiac abnormalities in the form of premature junctional contractions (PJCs). At our acute exposure dose, lethal in 3 % of our animals, naïve ferrets were unresponsive and incapacitated for a total of 126.1 ± 24.6 min. When administered intramuscularly at human equivalent doses (HEDs) of either 5 mg or 10 mg, naloxone significantly reduced the time that ferrets were incapacitated following exposure, although we observed no significant difference in the reduction of time that the animals were incapacitated between the treatment groups. Naloxone was able to quickly resolve the respiratory depression, significantly reducing the frequency of apneic periods in carfentanil-exposed ferrets. Our results suggest that naloxone, when administered via intramuscular injection following incapacitation, is a viable treatment against the effects of a potentially lethal dose of inhaled carfentanil.

Methylene blue and monosodium glutamate improve neurologic signs after fluoroacetate poisoning.

Fluoroacetate (FA) is a tasteless, odorless, water-soluble metabolic poison with severe toxicological effects. Characterized in the mid-1900s, it has been used as a rodenticide but is comparably lethal to all mammals. Many countries have restricted its use, and modern-day accidental human exposures are rare, but recently, concerns have been raised about its application as a chemical weapon with no known antidote. A combined treatment of methylene blue (MB), an antioxidant, and monosodium glutamate (MSG), a precursor of the citric acid cycle substrate alpha-ketoglutarate, has been recommended as an effective countermeasure; however, no peer-reviewed articles documenting the efficacy of this therapy have been published. Using a rodent model, we assessed the effects of MB and MSG on the neurologic, cardiac, and pulmonary systems. Transcriptomic analysis was used to elucidate inflammatory pathway activation and guide bioassays, which revealed the advantages and disadvantages of these candidate countermeasures. Results show that MB and MSG can reduce neurologic signs observed in rats exposed to sodium FA and improve some effects of intoxication. However, while this strategy resolved some signs of intoxication, ultimately it was unable to significantly reduce lethality.

Incorporation of Modified Amino Acids by Engineered Elongation Factors with Expanded Substrate Capabilities.

Noncanonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, in vivo incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proofreading aa-tRNAs, which substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Here, we demonstrate an alternative strategy leveraging computational analysis to broaden EF-Tu's substrate specificity. Evolutionary analysis of EF-Tu and a naturally evolved specialized elongation factor, SelB, provide the opportunity to engineer EF-Tu by targeting amino acid residues that are associated with functional divergence between the two ancient paralogues. Employing amber codon suppression, in combination with mass spectrometry, we identified two EF-Tu variants with non-native substrate compatibility. Additionally, we present data showing these EF-Tu variants contribute to host organismal fitness, working cooperatively with components of native and engineered translation machinery. These results demonstrate the viability of our computational method and lend support to corresponding assumptions about molecular evolution. This work promotes enhanced polyspecific EF-Tu behavior as a viable strategy to expand ncAA scope and complements ongoing research emphasizing the importance of a comprehensive approach to further expand the genetic code.