COVID-19 Symptom Reporting Compliance Rates Among University Performing Arts Majors.

AIMS: The COVID-19 pandemic negatively affected the performing arts sector by temporarily closing venues. As venues reopened, COVID-19 symptom self-reporting and monitoring were one tool to identify potentially symptomatic cast and crew, who would then undergo contact tracing, testing, or isolation to prevent spreading COVID-19. However, the compliance rates for submitting a COVID-19 self-monitoring checklist among undergraduate performing art majors remain unknown. METHODS: This retrospective medical chart review investigated 282 cast and crew (68 males and 214 females) regarding their daily COVID-19 symptom report documents across the genres of dance, musical theater, and vocal performance throughout the production runs of 11 performances at a midwestern United States university's integrated performing arts campus. Compliance regarding the completion of the COVID-19 symptom checklist was compared between gender identity, performance semester, and performance type throughout the 2020-2021 academic year. RESULTS: There was no statistically significant difference on masking compliance when comparing males (mean 73.6%) and females (73.8%). The completion of the symptom self-reporting during the fall 2020 semester was statistically significantly higher than in the spring 2021 semester (F = 6.065, t = 4.485, df = 229.661, p = 0.014, d = 0.52). Additionally, those participating in musical theater were more compliant than those in vocal performance (F(2,280) = 4.410, p = 0.013, d = 0.031). There was no statistically significant difference between dance and musical theater or vocal performance and dance genres regarding overall compliance. CONCLUSIONS: These findings can help understand the groups of performing artists who would comply with the public health measure of completing a daily symptom checklist for COVID-19 or similar communicable diseases.

Genetic Influences of the Microbiota on the Life Span of Drosophila melanogaster.

To better understand how associated microorganisms ("microbiota") influence organismal aging, we focused on the model organism Drosophila melanogaster We conducted a metagenome-wide association (MGWA) as a screen to identify bacterial genes associated with variation in the D. melanogaster life span. The results of the MGWA predicted that bacterial cysteine and methionine metabolism genes influence fruit fly longevity. A mutant analysis, in which flies were inoculated with Escherichia coli strains bearing mutations in various methionine cycle genes, confirmed a role for some methionine cycle genes in extending or shortening fruit fly life span. Initially, we predicted these genes might influence longevity by mimicking or opposing methionine restriction, an established mechanism for life span extension in fruit flies. However, follow-up transcriptome sequencing (RNA-seq) and metabolomic experiments were generally inconsistent with this conclusion and instead implicated glucose and vitamin B6 metabolism in these influences. We then tested if bacteria could influence life span through methionine restriction using a different set of bacterial strains. Flies reared with a bacterial strain that ectopically expressed bacterial transsulfuration genes and lowered the methionine content of the fly diet also extended female D. melanogaster life span. Taken together, the microbial influences shown here overlap with established host genetic mechanisms for aging and therefore suggest overlapping roles for host and microbial metabolism genes in organismal aging.IMPORTANCE Associated microorganisms ("microbiota") are intimately connected to the behavior and physiology of their animal hosts, and defining the mechanisms of these interactions is an urgent imperative. This study focuses on how microorganisms influence the life span of a model host, the fruit fly Drosophila melanogaster First, we performed a screen that suggested a strong influence of bacterial methionine metabolism on host life span. Follow-up analyses of gene expression and metabolite abundance identified stronger roles for vitamin B6 and glucose than methionine metabolism among the tested mutants, possibly suggesting a more limited role for bacterial methionine metabolism genes in host life span effects. In a parallel set of experiments, we created a distinct bacterial strain that expressed life span-extending methionine metabolism genes and showed that this strain can extend fly life span. Therefore, this work identifies specific bacterial genes that influence host life span, including in ways that are consistent with the expectations of methionine restriction.

Health care in high school athletics in West Virginia.

INTRODUCTION: The purpose of this study was to determine the level of implementation of emergency preparedness procedures and administrative procedures to provide appropriate medical coverage to high school athletics in the predominantly rural US state of West Virginia. Particular attention was given to determine the extent to which the schools provided the recommendations for best practice in the National Athletic Trainers Association consensus statement outlining appropriate medical coverage for high school athletics. METHODS: A listing of all public schools participating in the state high school athletic association with at least one team participating in interscholastic competition was obtained from the state Department of Education office. An electronic survey was sent to the principal at each high school with instructions that an administrator or sports medicine professional complete the survey. A total of 62 respondents completed the survey (49.6% response rate). Most respondents were principals (92%), followed by athletic administrators (8%). The majority of schools reported a rural zip code at the school level based on the Rural Urban Community Area Codes. Measures assessed the school demographics, including size and rurality. Additional measures assessed the development and implementation of a comprehensive athletic healthcare administrative system, and the development and implementation of a comprehensive emergency action plan. RESULTS: The majority of respondents reported that there was a consent form on file for student athletes (91%) and comprehensive insurance was required for participation (80%). A third of the respondents (33%) reported that all members of the coaching staff were certified in first aid and cardiac pulmonary resuscitation (CPR) and 31% reported 'never' when asked if all coaches were required to be certified in CPR and first aid. When asked if there was a written emergency action plan (EAP) that outlines procedures to follow in emergency situations during athletic participation, 36% responded 'never' and 38% responded 'always'. When asked about specific limitations for health care to athletes the three main themes identified in qualitative analysis were lack of funding, lack of certified medical personnel, and the inability to locate certified medical personnel in a rural area. CONCLUSIONS: This study confirmed expected barriers to health care for high school athletes in West Virginia, specifically the lack of funding and resources available to rural schools. In order to prevent a life threatening emergency or possibly sudden cardiac death, preparing and planning for emergencies should be an essential part of high school athletic programs. Rural areas face significant challenges in regards to funding and qualified personnel. Requiring first aid and CPR certification for coaches and requiring an EAP are two steps that could improve the health care provided to athletes. These are inexpensive and achievable steps that could be taken to improve the safety for athletes at high schools in both rural and non-rural areas.

The Structure of an NDR/LATS Kinase-Mob Complex Reveals a Novel Kinase-Coactivator System and Substrate Docking Mechanism.

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. "Hippo" pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with "Mob" coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1-Mob2, to our knowledge the first of an NDR/LATS kinase-Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1's regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.

Site-saturation mutagenesis of position V117 in OXA-1 ß-lactamase: effect of side chain polarity on enzyme carboxylation and substrate turnover.

Class D ß-lactamases pose an emerging threat to the efficacy of ß-lactam therapy for bacterial infections. Class D enzymes differ mechanistically from other ß-lactamases by the presence of an active-site N-carboxylated lysine that serves as a general base to activate the serine nucleophile for attack. We have used site-saturation mutagenesis at position V117 in the class D ß-lactamase OXA-1 to investigate how alterations in the environment around N-carboxylated K70 affect the ability of that modified residue to carry out its normal function. Minimum inhibitory concentration analysis of the 20 position 117 variants demonstrates a clear pattern of charge and polarity effects on the level of ampicillin resistance imparted on Escherichia coli (E. coli). Substitutions that introduce a negative charge (D, E) at position 117 reduce resistance to near background levels, while the positively charged K and R residues maintain the highest resistance levels of all mutants. Treatment of the acidic variants with the fluorescent penicillin BOCILLIN FL followed by SDS-PAGE shows that they are active for acylation by substrate but deacylation-deficient. We used a novel fluorescence anisotropy assay to show that the specific charge and hydrogen-bonding potential of the residue at position 117 affect CO(2) binding to K70, which in turn correlates to deacylation activity. These conclusions are discussed in light of the mechanisms proposed for both class D ß-lactamases and BlaR ß-lactam sensor proteins and suggest a reason for the preponderance of asparagine at the V117-homologous position in the sensors.

Structures of the class D carbapenemase OXA-24 from Acinetobacter baumannii in complex with doripenem.

The emergence of class D ß-lactamases with carbapenemase activity presents an enormous challenge to health practitioners, particularly with regard to the treatment of infections caused by Gram-negative pathogens such as Acinetobacter baumannii. Unfortunately, class D ß-lactamases with carbapenemase activity are resistant to ß-lactamase inhibitors. To better understand the details of the how these enzymes bind and hydrolyze carbapenems, we have determined the structures of two deacylation-deficient variants (K84D and V130D) of the class D carbapenemase OXA-24 with doripenem bound as a covalent acyl-enzyme intermediate. Doripenem adopts essentially the same configuration in both OXA-24 variant structures, but varies significantly when compared to the non-carbapenemase class D member OXA-1/doripenem complex. The alcohol of the 6α hydroxyethyl moiety is directed away from the general base carboxy-K84, with implications for activation of the deacylating water. The tunnel formed by the Y112/M223 bridge in the apo form of OXA-24 is largely unchanged by the binding of doripenem. The presence of this bridge, however, causes the distal pyrrolidine/sulfonamide group to bind in a drastically different conformation compared to doripenem bound to OXA-1. The resulting difference in the position of the side-chain bridge sulfur of doripenem is consistent with the hypothesis that the tautomeric state of the pyrroline ring contributes to the different carbapenem hydrolysis rates of OXA-1 and OXA-24. These findings represent a snapshot of a key step in the catalytic mechanism of an important class D enzyme, and might be useful for the design of novel inhibitors.

Heterohexameric ring arrangement of the eukaryotic proteasomal ATPases: implications for proteasome structure and assembly.

The proteasome has a paramount role in eukaryotic cell regulation. It consists of a proteolytic core particle (CP) bound to one or two regulatory particles (RPs). Each RP is believed to include six different AAA+ ATPases in a heterohexameric ring that binds the CP while unfolding and translocating substrates into the core. No atomic-resolution RP structures are available. Guided by crystal structures of related homohexameric prokaryotic ATPases, we use disulfide engineering to show that the eukaryotic ATPases form a ring with the arrangement Rpt1-Rpt2-Rpt6-Rpt3-Rpt4-Rpt5 in fully assembled proteasomes. The arrangement is consistent with known assembly intermediates. This quaternary organization clarifies the functional overlap of specific RP assembly chaperones and led us to identify a potential RP assembly intermediate that includes four ATPases (Rpt6-Rpt3-Rpt4-Rpt5) and their cognate chaperones (Rpn14, Nas6, and Nas2). Finally, the ATPase ring structure casts light on alternative RP structural models and the mechanism of RP action.

The 1.4 A crystal structure of the class D beta-lactamase OXA-1 complexed with doripenem.

The clinical efficacy of carbapenem antibiotics depends on their resistance to the hydrolytic action of beta-lactamase enzymes. The structure of the class D beta-lactamase OXA-1 as an acyl complex with the carbapenem doripenem was determined to 1.4 A resolution. Unlike most class A and class C carbapenem complexes, the acyl carbonyl oxygen in the OXA-1-doripenem complex is bound in the oxyanion hole. Interestingly, no water molecules were observed in the vicinity of the acyl linkage, providing an explanation for why carbapenems inhibit OXA-1. The side chain amine of K70 remains fully carboxylated in the acyl structure, and the resulting carbamate group forms a hydrogen bond to the alcohol of the 6alpha-hydroxyethyl moiety of doripenem. The carboxylate attached to the beta-lactam ring of doripenem is stabilized by a salt bridge to K212 and a hydrogen bond with T213, in lieu of the interaction with an arginine side chain found in most other beta-lactamase-beta-lactam complexes (e.g., R244 in the class A member TEM-1). This novel set of interactions with the carboxylate results in a major shift of the carbapenem's pyrroline ring compared to the structure of the same ring in meropenem bound to OXA-13. Additionally, bond angles of the pyrroline ring suggest that after acylation, doripenem adopts the Delta(1) tautomer. These findings provide important insights into the role that carbapenems may have in the inactivation process of class D beta-lactamases.

Mutation of the active site carboxy-lysine (K70) of OXA-1 beta-lactamase results in a deacylation-deficient enzyme.

Class D beta-lactamases hydrolyze beta-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate and subsequently employ water to deacylate the beta-lactam and release product. Class D beta-lactamases are carboxylated on the epsilon-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 beta-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate but is impaired with respect to deacylation. We employed the fluorescent penicillin BOCILLIN FL to show that three different substitutions for K70 (alanine, aspartate, and glutamate) lead to the accumulation of significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL deacylation rates (t(1/2)) vary depending on the identity of the substituting residue, from approximately 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e., nonfluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation.

The role of OXA-1 beta-lactamase Asp(66) in the stabilization of the active-site carbamate group and in substrate turnover.

The OXA-1 beta-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser(67). In class A beta-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp(66) in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp(66) mutants expressed in Escherichia coli DH10B ranged from < or =8 microg/ml for cysteine, proline and the basic amino acids to > or =256 microg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys(70), that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria-Bertani-broth agar resulted in a > or =16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp(66) forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys(70) and thereby profoundly affects substrate turnover.