Control of Unsaturation in De Novo Fatty Acid Biosynthesis by FabA.

Carrier protein-dependent biosynthesis provides a thiotemplated format for the production of natural products. Within these pathways, many reactions display exquisite substrate selectivity, a regulatory framework proposed to be controlled by protein-protein interactions (PPIs). In Escherichia coli, unsaturated fatty acids are generated within the de novo fatty acid synthase by a chain length-specific interaction between the acyl carrier protein AcpP and the isomerizing dehydratase FabA. To evaluate PPI-based control of reactivity, interactions of FabA with AcpP bearing multiple sequestered substrates were analyzed through NMR titration and guided high-resolution docking. Through a combination of quantitative binding constants, residue-specific perturbation analysis, and high-resolution docking, a model for substrate control via PPIs has been developed. The in silico results illuminate the mechanism of FabA substrate selectivity and provide a structural rationale with atomic detail. Helix III positioning in AcpP communicates sequestered chain length identity recognized by FabA, demonstrating a powerful strategy to regulate activity by allosteric control. These studies broadly illuminate carrier protein-dependent pathways and offer an important consideration for future inhibitor design and pathway engineering.

Pediatric Chronic Pain, Resilience and Psychiatric Comorbidity in Canada: A Retrospective, Comparative Analysis.

Objective: Chronic pain compromises child and adolescent well-being and development. This study aimed to identify risk factors for chronic pain and exploration of how young people negotiate such risks and express resilience. We hypothesized children and youth with chronic pain would report greater prevalence of mental health disorders than the general population; and those demonstrating greater resilience would demonstrate less psychiatric comorbidity. Method: A retrospective chart review was conducted for all patients (ages 7-17) attending the sole pediatric chronic pain clinic in Manitoba, from 2015 to 2019 (N = 116). Patients' demographic information and psychiatric illness burden were compared to provincial epidemiological data using Chi-Square tests. Pain sites, family history, psychiatric illness, psychosocial functioning, treatment history and treatment recommendations were explored. Results: The sample was predominantly female (74%; N = 114). Sixty-eight percent of patients reported a family history of chronic pain. Thirty-seven percent of the patients (vs. 14.0% anticipated; N = 326 260) reported comorbid psychiatric disorder, X2 (1, N = 114) = 53.00, p < 0.001. Thirty-two percent reported diagnosis of mood and/or anxiety disorder (vs. 7.3%), X2 (1, N = 114) = 99.34, p < 0.001. Children and youth demonstrating resilience through engagement in more prosocial behaviors reported fewer psychiatric symptoms (rs = -0.292, N = 114, p = 0.002, Spearman's correlation). Conclusions: Female sex, family history, and lower socioeconomic status were associated with chronic pain. Psychiatric conditions were more prevalent in chronic pain patients than in the general population. Approaching chronic pain from a mind-body perspective, while building on patients' strengths, is central to informing treatment.

Protein-protein interaction based substrate control in the E. coli octanoic acid transferase, LipB.

Lipoic acid is an essential cofactor produced in all organisms by diverting octanoic acid derived as an intermediate of type II fatty acid biosynthesis. In bacteria, octanoic acid is transferred from the acyl carrier protein (ACP) to the lipoylated target protein by the octanoyltransferase LipB. LipB has a well-documented substrate selectivity, indicating a mechanism of octanoic acid recognition. The present study reveals the precise protein-protein interactions (PPIs) responsible for this selectivity in Escherichia coli through a combination of solution-state protein NMR titration with high-resolution docking of the experimentally examined substrates. We examine the structural changes of substrate-bound ACP and determine the precise geometry of the LipB interface. Thermodynamic effects from varying substrates were observed by NMR, and steric occlusion of docked models indicates how LipB interprets proper substrate identity via allosteric binding. This study provides a model for elucidating how substrate identity is transferred through the ACP structure to regulate activity in octanoyl transferases.

Decoding allosteric regulation by the acyl carrier protein.

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522-542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Ploskon et al., Chem. Biol. 17, 776-785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein-protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528-547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein-protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis.

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

Optimizing Numeric Pain Rating Scale administration for children: The effects of verbal anchor phrases.

Background: The 0-10 Verbal Numeric Rating Scale (VNRS) is commonly used to obtain self-reports of pain intensity in school-age children, but there is no standard verbal descriptor to define the most severe pain. Aims: The aim of this study was to determine how verbal anchor phrases defining 10/10 on the VNRS are associated with children's reports of pain. Methods and Results: Study 1. Children (N = 131, age 6-11) rated hypothetical pain vignettes using six anchor phrases; scores were compared with criterion ratings. Though expected effects of age and vignette were found, no effects were found for variations in anchors. Study 2. Pediatric nurses (N = 102) were asked how they would instruct a child to use the VNRS. Common themes of "the worst hurt you could ever imagine" and "the worst hurt you have ever had" to define 10/10 were identified. Study 3. Children's hospital patients (N = 27, age 8-14) rated pain from a routine injection using four versions of the VNRS. Differences in ratings ranging from one to seven points on the scale occurred in the scores of 70% of children when the top anchor phrase was changed. Common themes in children's descriptions of 10/10 pain intensity were "hurts really bad" and "hurts very much." Discussion: This research supports attention to the details of instructions that health care professionals use when administering the VNRS. Use of the anchor phrase "the worst hurt you could ever imagine" is recommended for English-speaking, school-age children. Details of administration of the VNRS should be standardized and documented in research reports and in clinical use.

Contexte: L'échelle numérique verbale (ENV) de 0 à 10 est fréquemment utilisée pour l'auto-évaluation de l'intensité de la douleur chez les enfants d'âge scolaire, mais il n'existe pas de descripteur verbal pour définir la douleur la plus aiguë.But: Déterminer de quelle manière les énoncés définissant 10/10 sur l'ENV sont associés à la douleur rapportée par les enfants.Méthodes et résultats: Étude 1. Des enfants (N = 131, âgés de 6 à 11 ans) ont évalué des vignettes représentant une douleur hypothétique à l'aide de six énoncés; les scores obtenus ont été comparés aux évaluations de référence. Bien que les effets attendus en ce qui concerne l'âge et la vignette aient été observés, aucun effet n'a été observé pour les variations dans les énoncés. Étude 2. On a demandé à des infirmières pédiatriques (N = 102) de quelle manière elles enseigneraient à un enfant à utiliser l'ENV. Les thèmes communs de « la pire douleur que tu puisses imaginer ¼ et « la pire douleur que tu aies ressentie ¼ pour définir 10/10 ont été identifiés. Étude 3. Les patients d'un hôpital pour enfants (N = 27, âgés de 8 à 14 ans) ont évalué la douleur d'une injection de routine à l'aide de quatre versions de l'ENV. Des différences dans l'évaluation allant d'un à sept points sur l'échelle sont apparues dans les scores de 70 % des enfants lorsque l'énoncé relatif à la douleur la plus élevée a été modifié. Les thèmes communs dans les descriptions des enfants en ce qui concerne l'intensité de douleur 10/10 étaient « hurts really bad ¼ (fait vraiment mal) and « hurts very much ¼ (fait très mal).Discussion: Cette étude démontre l'importance de porter attention aux détails dans les instructions que les professionnels de la santé utilisent lorsqu'ils ont recours à l'ENV. L'utilisation de l'énoncé « the worst hurt you could ever imagine ¼ (la pire douleur que tu puisses imaginer) est recommandée pour les enfants d'âge scolaire anglophones. Les détails concernant l'utilisation de l'ENV devraient être uniformisés et documentés dans les rapports de recherche ainsi que dans le cadre de leur usage clinique.