Central Alabama Community College

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro . In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo . We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

A recent computational analysis of the stabilizing intramolecular OH⋯O contact in 1,2-dialkyl-2,3-epoxycyclopentanol diastereomers has been extended to thiiriane, aziridine and phosphirane analogues. Density functional theory (DFT), second-order Møller-Plesset perturbation theory (MP2) and CCSD(T) coupled-cluster computations with simple methyl and ethyl substituents indicate that electronic energies of the c i s isomers are lowered by roughly 3 to 4 kcal mol−1 when the OH group of these cyclopentanol systems forms an intramolecular contact with the O, S, N or P atom on the adjacent carbon. The results also suggest that S and P can participate in these stabilizing intramolecular interactions as effectively as O and N in constrained molecular environments. The stabilizing intramolecular OH⋯O, OH⋯S, OH⋯N and OH⋯P contacts also increase the covalent OH bond length and significantly decrease the OH stretching vibrational frequency in every system with shifts typically on the order of −41 cm−1.

The amount of sodium chloride (NaCl) that escapes a nebulizer cup, intended for patient inhalation, during a 5-min hypertonic saline jet nebulization (HSJN) breathing treatment is apparently unknown in the pure and applied scientific literature. This study aimed to address this void by focusing on NaCl mass changes prior to and after a typical HSJN breathing treatment using an ordinary household, medical-grade air compressor.

Saline solutions of varying concentrations were nebulized to room air for 5 min. Pre- and post-nebulization NaCl concentrations were determined from measured conductivities via calibration curve. The resulting data were used to quantify NaCl mass changed from the beginning and end of a typical HSJN breathing treatment.

Conductivity was a reliable metric in NaCl concentrations ranging from 2.10 × 10^-1 to 8.16 × 10^-3 M. Pre- and post-nebulization NaCl mass differences of 19-114 mg linearly correlated with saline concentration (wt%). The resulting trendline data reasonably predict how much NaCl is available for patient inhalation during a typical HSJN breathing treatment. Linearity in the data suggests that factors such as colligative properties (e.g., osmolarity) have a minimal influence on the amount of NaCl that escapes the nebulizer cup.

These results are the first to quantify how much NaCl escapes a nebulizer cup during a typical HSJN breathing treatment. Furthermore, the results represent a key starting point upon which future studies can be built to explore additional airflow rates, kinetics, and temperature effects. Collectively, these findings will play a critical role in ascertaining the mechanism of action in hypertonic saline breathing treatments.