Investigating the physiological roles of low-efficiency D-mannonate and D-gluconate dehydratases in the enolase superfamily: pathways for the catabolism of L-gulonate and L-idonate.

The sequence/function space in the D-mannonate dehydratase subgroup (ManD) of the enolase superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10(3)-10(4) M(-1) s(-1)) for dehydration of D-mannonate, low-efficiency (kcat/KM = 10-10(2) M(-1) s(-1)) for dehydration of D-mannonate and/or D-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the D-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087-4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for L-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for L-gulonate catabolism in Escherichia coli CFT073 and L-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential.

Assessing asthma symptoms in children: qualitative research supporting the development of the Pediatric Asthma Diary-Child (PAD-C) and Pediatric Asthma Diary-Observer (PAD-O).

BACKGROUND: Pediatric asthma has been identified by regulators, clinicians, clinical trial sponsors, and caregivers as an area in need of novel fit-for-purpose clinical outcome assessments (COAs) developed in accordance with the U.S. Food and Drug Administration's (FDA's) regulatory guidance for evaluating clinical benefit in treatment trials. To address this gap, the Patient-Reported Outcome (PRO) Consortium's Pediatric Asthma Working Group has continued development of 2 COAs to assess asthma signs and symptoms in pediatric asthma clinical trials to support efficacy endpoints: a PRO measure, the Pediatric Asthma Diary-Child (PAD-C) for children 8-11 years old (y.o.) and an observer-reported outcome measure, the Pediatric Asthma Diary-Observer (PAD-O) for caregivers of children 4-11 y.o. This qualitative research aimed to generate evidence regarding the content validity of the PAD-C and PAD-O. METHODS: Semi-structured combined concept elicitation and cognitive interviews were conducted with a diverse sample of U.S. participants (15 children 8-11 y.o. and 30 caregivers of children 4-11 y.o.). All children had clinician-diagnosed mild to severe asthma. Interviews explored the experience of pediatric asthma and assessed the understanding and relevance of both measures. Interviews were conducted across 3 iterative rounds to allow for modifications. RESULTS: Concept elicitation findings demonstrated that the core sign/symptom and impact concepts assessed in the PAD-C (cough, hard to breathe, out of breath, wheezing, chest tightness, and nighttime awakenings/symptoms) and PAD-O (cough, difficulty breathing, short of breath, wheezing, and nighttime awakenings/signs) correspond to those most frequently reported by participants; concept saturation was achieved. All PAD-C and PAD-O instructions and core items were well understood and considered relevant by most participants. Feedback from participants, the Pediatric Asthma Working Group, advisory panel, and FDA supported modifications to the measures, including addition of 1 new item to both measures and removal of 1 caregiver item. CONCLUSIONS: Findings provide strong support for the content validity of both measures. The cross-sectional measurement properties of both measures and their user experience and feasibility in electronic format will be assessed in a future quantitative pilot study with qualitative exit interviews, intended to support the reliability, construct validity, final content, and, ultimately, FDA qualification of the measures.

Pediatric asthma is one of the most common chronic diseases in children. However, there are problems of underdiagnosis, poor disease management, and undertreatment for many pediatric asthma patients, pressuring healthcare systems worldwide. Evaluating asthma symptoms is an important part of the development of treatments for pediatric asthma. However, there are few clinical outcome assessments (COAs) developed in line with regulatory guidance to directly assess symptom severity and evaluate the benefit of new treatments in children with asthma. In this study, we continued the development of the Pediatric Asthma Diary­Child (PAD-C) and the Pediatric Asthma Diary­Observer (PAD-O), according to regulatory guidance, to assess asthma signs and symptoms in children 4 through 11 years old and address this unmet need. The study aimed to explore the experience of pediatric asthma and assess how well-understood and relevant the measures are. Three rounds of qualitative interviews were conducted with 15 children 8 through 11 years old and 30 caregivers of children 4 through 11 years old with asthma. Results show that both measures are well-understood and assess the relevant and important aspects of pediatric asthma reported by children and caregivers. Findings provide evidence supporting the PAD-C and PAD-O as measures of symptom severity and their future use in pediatric asthma treatment trials. Further research is underway to evaluate their measurement properties and assess the user experience and feasibility of electronic completion, to ultimately support the PAD-C and PAD-O in an ongoing COA qualification process by the United States Food and Drug Administration.

Measuring dengue illness intensity: Development and content validity of the dengue virus daily diary (DENV-DD).

BACKGROUND: Dengue is the most prevalent arboviral infection causing an estimated 50-60 million cases of febrile illness globally per year, exacting considerable disease burden. Few instruments exist to assess the patient illness experience, with most based on healthcare provider assessment, lacking standardization in timepoints and symptom assessment. This study aimed to evaluate the content validity of the novel 'Dengue Virus Daily Diary (DENV-DD)', designed to measure symptom intensity and disease burden within outpatient infant to adult populations. METHODS: The Dengue Illness Index Report Card was used as a foundation to create the DENV-DD, consisting of patient- and observer-reported outcome (PRO/ObsRO) instruments. In two South American dengue-endemic communities, qualitative combined concept elicitation and cognitive debriefing interviews were conducted among individuals and caregivers of children with symptomatic laboratory-confirmed dengue. Interviews were conducted across two rounds allowing DENV-DD modifications. A small-scale quantitative assessment of the DENV-DD was also conducted with data from an independent Dengue Human Infection Model (DHIM) to generate early evidence of feasibility of DENV-DD completion, instrument performance and insight into the sign/symptom trajectory over the course of illness. RESULTS: Forty-eight participants were interviewed (20 adults, 20 older children/adolescents with their caregivers, 8 caregivers of younger children). A wide spectrum of signs/symptoms lasting 3-15 days were reported with fever, headache, body ache/pain, loss of appetite, and body weakness each reported by > 70% participants. DENV-DD instructions, items and response scales were understood, and items were considered relevant across ages. DHIM data supported feasibility of DENV-DD completion. CONCLUSIONS: Findings demonstrate content validity of the DENV-DD (PRO/ObsRO instruments) in dengue-endemic populations. Psychometric and cultural validity studies are ongoing to support use of the DENV-DD in clinical studies.

Dengue is the most common viral infection transmitted to humans by mosquitos, and affects an estimated 50­60 million individuals globally per year. However, there are few resources for understanding and capturing the patient experience of dengue throughout illness. Most research studies are based on healthcare provider assessment, which lack consistency in terms of assessment time points and the signs/symptoms assessed. The 'Dengue Illness Index Report Card (DII-RC)' was used as a foundation to create the new 'Dengue Virus Daily Diary (DENV-DD)' to better capture the patient experience of symptom intensity and dengue disease burden for the duration of illness. Forty-eight individuals and caregivers of younger children from Peru and Ecuador who recently had symptomatic dengue were interviewed to understand the patient experience over the time of illness and to test whether the DENV-DD is understood by patients and caregivers and includes all relevant and important signs/symptoms and health-related quality of life impacts. Nine individuals with active dengue infection also completed the DENV-DD daily for 28-days as part of a clinical study. We found that > 70% of patients experienced fever, headache, body ache/pain, loss of appetite and body weakness. The DENV-DD instructions, questions and response option(s) were well understood, feasible to complete and the concepts assessed by the DENV-DD were relevant to the dengue experience. Our study adds to the understanding of the dengue illness experience and supports the DENV-DD for use in future dengue studies as an assessment of signs/symptoms throughout the duration of illness.

A purified mutant HemA protein from Salmonella enterica serovar Typhimurium lacks bound heme and is defective for heme-mediated regulation in vivo.

Archaea, plants, and most bacteria synthesize heme using the C5 pathway, in which the first committed step is catalyzed by the enzyme glutamyl-tRNA reductase (GluTR or HemA). In some cases, an overproduced and purified HemA enzyme contains noncovalently bound heme. The enteric bacteria Salmonella enterica and Escherichia coli also synthesize heme by the C5 pathway, and the HemA protein in these bacteria is regulated by proteolysis. The enzyme is unstable during normal growth due to the action of Lon and ClpAP, but becomes stable when heme is limiting for growth. We describe a method for the overproduction of S. enterica HemA that yields a purified enzyme containing bound heme, identified as a b-type heme by spectroscopy. A mutant of HemA (C170A) does not contain heme when similarly purified. The mutant was used to test whether heme is directly involved in HemA regulation. When expressed from the S. enterica chromosome in a wild-type background, the C170A mutant allele of hemA is shown to confer an unregulated phenotype, with high levels of HemA regardless of the heme status. These results strongly suggest that the presence of bound heme targets the HemA enzyme for degradation and is required for normal regulation.

Limited role for the DsrA and RprA regulatory RNAs in rpoS regulation in Salmonella enterica.

RpoS, the sigma factor of enteric bacteria that responds to stress and stationary phase, is subject to complex regulation acting at multiple levels, including transcription, translation, and proteolysis. Increased translation of rpoS mRNA during growth at low temperature, after osmotic challenge, or with a constitutively activated Rcs phosphorelay depends on two trans-acting small regulatory RNAs (sRNAs) in Escherichia coli. The DsrA and RprA sRNAs are both highly conserved in Salmonella enterica, as is their target, an inhibitory antisense element within the rpoS untranslated leader. Analysis of dsrA and rprA deletion mutants indicates that while the increased translation of RpoS in response to osmotic challenge is conserved in S. enterica, dependence on these two sRNA regulators is much reduced. Furthermore, low-temperature growth or constitutive RcsC activation had only modest effects on RpoS expression, and these increases were, respectively, independent of dsrA or rprA function. This lack of conservation of sRNA function suggests surprising flexibility in RpoS regulation.