A body bag can save your life: a novel method of cold water immersion for heat stroke treatment.

Non-exertional heat stroke is a life-threatening condition characterized by passive exposure to high ambient heat, a core body temperature of 40°C (104°F) or greater, and central nervous system dysfunction. Rapid cooling is imperative to minimize mortality and morbidity. Although evaporative and convective measures are often used for cooling heat stroke patients, cold water immersion produces the fastest cooling. However, logistical difficulties make cold water immersion challenging to implement in the emergency department. To our knowledge, there is no documented case utilizing a body bag (ie, human remains pouch) as a cold water immersion tank for rapid resuscitation of heat stroke. During a regional heat wave an elderly woman was found unconscious in a parking lot with an oral temperature of 40°C (104°F) and altered mental status. She was cooled to 38.4°C (101.1°F) in 10 minutes by immersion in an ice- and water-filled body bag. The patient rapidly regained normal mentation and was discharged home from the ED. This case highlights a novel method for efficient and convenient cold water immersion for heat stroke treatment in the emergency department.

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes.

Dengue virus (DENV), an arthropod-borne ("arbovirus") virus, causes a range of human maladies ranging from self-limiting dengue fever to the life-threatening dengue shock syndrome and proliferates well in two different taxa of the Animal Kingdom, mosquitoes and primates. Mosquitoes and primates show taxonomic group-specific intolerance to certain codon pairs when expressing their genes by translation. This is called "codon pair bias". By necessity, dengue viruses evolved to delicately balance this fundamental difference in their open reading frames (ORFs). We have undone the evolutionarily conserved genomic balance in the DENV2 ORF sequence and specifically shifted the encoding preference away from primates. However, this recoding of DENV2 raised concerns of 'gain-of-function,' namely whether recoding could inadvertently increase fitness for replication in the arthropod vector. Using mosquito cell lines and two strains of Aedes aegypti we did not observe any increase in fitness in DENV2 variants codon pair deoptimized for humans. This ability to disrupt and control DENV2's host preference has great promise towards developing the next generation of synthetic vaccines not only for DENV but for other emerging arboviral pathogens such as chikungunya virus and Zika virus.

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes.

Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2Min, a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2Min, 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either (a) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or (b) by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation.

Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference.

The protein synthesis machineries of two distinct phyla of the Animal kingdom, insects of Arthropoda and mammals of Chordata, have different preferences for how to best encode proteins. Nevertheless, arboviruses (arthropod-borne viruses) are capable of infecting both mammals and insects just like arboviruses that use insect vectors to infect plants. These organisms have evolved carefully balanced genomes that can efficiently use the translational machineries of different phyla, even if the phyla belong to different kingdoms. Using dengue virus as an example, we have undone the genome encoding balance and specifically shifted the encoding preference away from mammals. These mammalian-attenuated viruses grow to high titers in insect cells but low titers in mammalian cells, have dramatically increased LD50s in newborn mice, and induce high levels of protective antibodies. Recoded arboviruses with a bias toward phylum-specific expression could form the basis of a new generation of live attenuated vaccine candidates.

Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies.

AOC3 is highly expressed in adipocytes and smooth muscle cells, but its function in these cells is currently unknown. The in vivo substrate(s) of AOC3 is/are also unknown, but could provide an invaluable clue to the enzyme's function. Expression of untagged, soluble human AOC3 in insect cells provides a relatively simple means of obtaining pure enzyme. Characterization of enzyme indicates a 6% titer for the active site 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor and corrected k(cat) values as high as 7 s(-1). Substrate kinetic profiling shows that the enzyme accepts a variety of primary amines with different chemical features, including nonphysiological branched-chain and aliphatic amines, with measured k(cat)/K(m) values between 10(2) and 10(4) M(-1) s(-1). K(m)(O(2)) approximates the partial pressure of oxygen found in the interstitial space. Comparison of the properties of purified murine to human enzyme indicates k(cat)/K(m) values that are within 3 to 4-fold, with the exception of methylamine and aminoacetone that are ca. 10-fold more active with human AOC3. With drug development efforts investigating AOC3 as an anti-inflammatory target, these studies suggest that caution is called for when screening the efficacy of inhibitors designed against human enzymes in non-transgenic mouse models. Differentiated murine 3T3-L1 adipocytes show a uniform distribution of AOC3 on the cell surface and whole cell K(m) values that are reasonably close to values measured using purified enzymes. The latter studies support a relevance of the kinetic parameters measured with isolated AOC3 variants to adipocyte function. From our studies, a number of possible substrates with relatively high k(cat)/K(m) have been discovered, including dopamine and cysteamine, which may implicate a role for adipocyte AOC3 in insulin-signaling and fatty acid metabolism, respectively. Finally, the demonstrated AOC3 turnover of primary amines that are non-native to human tissue suggests possible roles for the adipocyte enzyme in subcutaneous bacterial infiltration and obesity.