The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen from a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the () gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes,, also known as or, and Interestingly, our mutations were complemented by both and, leading us to delete, clone, and overexpress ,, , and in both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Infection by the opportunistic pathogen is the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

The energetics and hydrogen bonding profiles of the helix-to-coil transition were found to be an additive property and to increase linearly with chain length, respectively, in alanine-rich a-helical peptides. A model system of polyalanine repeats was used to establish this hypothesis for the energetic trends and hydrogen bonding profiles. Numerical measurements of a synthesized polypeptide Ac-Y(AEAAKA) k F-NH 2 and a natural a-helical peptide a2N (1-17) provide evidence of the hypothesis's generality. Adaptive steered molecular dynamics was employed to investigate the mechanical unfolding of all of these alaninerich polypeptides. We found that the helix-to-coil transition is primarily dependent on the breaking of the intramolecular backbone hydrogen bonds and independent of specific side-chain interactions and chain length. The mechanical unfolding of the a-helical peptides results in a turnover mechanism in which a 3 10 -helical structure forms during the unfolding, remaining at a near constant population and thereby maintaining additivity in the free energy. The intermediate partially unfolded structures exhibited polyproline II helical structure as previously seen by others. In summary, we found that the average force required to pull alanine-rich a-helical peptides in between the endpoints-namely the native structure and free coil-is nearly independent of the length or the specific primary structure.

Introduction CDC estimated more than 87,000 fire/burn injuries in 2017 among children 0–15 years old. Scald burns are the most common form of burn injury for children in the United States and are estimated to cost $44 million annually. Understanding the circumstances surrounding burn incidents and caretakers’ responses to them are needed to better guide prevention and education offerings. Methods We used a mixed methods approach to better understand pediatric scald burn mechanisms along with caregivers’ general knowledge, first aid responses and ability to accurately report characteristics of their child’s injury. Caregivers of children < 15 year old attending a burn follow up clinic with their child were recruited. Participants completed a 39-item, iPad-based survey that captured information about the circumstances, location and cause of the scald burn, whether first aid was provided, and caregiver/child demographics. With permission of the caregiver, the child’s medical record was accessed to collect burn descriptors (e.g., burn severity, total body surface area (TBSA), etc) to compare with parent self-report. Data collection occurred between May 2018 - May 2019. The study was approved by our institutional review board. Results Surveys were completed by 55 parents; 75% agreed to medical record review. The typical respondent was the female caregiver (80%) with more than a high school education (67%). The typical burn incident occurred in the kitchen (60%) of the family’s home (80%) and in the presence of the caregiver (71%). Seventy percent of respondents reported applying first aid; among those, 50% administered the proper first aid (using cool water on the burn) despite most (44%) reporting feeling “not confident” in the ability to do so. More than half (53%) didnot know their child’s TBSA; of those who did, self-reported TBSA ranged from 1 to 50%. Medical record review revealed that only 5 participants accurately reported their child’s TBSA. Conclusions Our data reveal that most scald burns occurred in the kitchen, of the child’s home, and in the presence of a caretaker. Half received incorrect first aid and few parents could accurately report their child’s TBSA. These results support the need to strengthen primary prevention offerings and improve parents’ understanding of their child’s burn injury. Applicability of Research to Practice The results of this research can help guide education efforts for primary prevention programs related to pediatric scald burn injuries. Furthermore, results can guide education practices for caregivers of children being treated for a scald burn injury.

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