Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase

\ua9 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA−A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly

Cation complexation by mucoid Pseudomonas aeruginosa extracellular polysaccharide

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung colonizer, producing an extracellular matrix (ECM) composed predominantly of the extracellular polysaccharide (EPS) alginate. The ECM limits antimicrobial penetration and, consequently, CF sufferers are prone to chronic mucoid P. aeruginosa lung infections. Interactions between cations with elevated concentrations in the CF lung and the anionic EPS, enhance the structural rigidity of the biofilm and exacerbates virulence. In this work, two large mucoid P. aeruginosa EPS models, based on β-D-mannuronate (M) and β-D-mannuronate-α-L-guluronate systems (M-G), and encompassing thermodynamically stable acetylation configurations–a structural motif unique to mucoid P. aeruginosa–were created. Using highly accurate first principles calculations, stable coordination environments adopted by the cations have been identified and thermodynamic stability quantified. These models show the weak cross-linking capability of Na+ and Mg2+ ions relative to Ca2+ ions and indicate a preference for cation binding within M-G blocks due to the smaller torsional rearrangements needed to reveal stable binding sites. The geometry of the chelation site influences the stability of the resulting complexes more than electrostatic interactions, and the results show nuanced chemical insight into previous experimental observations

Evaluation of the impact of a school gardening intervention on children's fruit and vegetable intake: a randomised controlled trial.

Background: Current academic literature suggests that school gardening programmes can provide an interactive environment with the potential to change children’s fruit and vegetable intake. This is the first cluster randomised controlled trial (RCT) designed to evaluate whether a school gardening programme can have an effect on children’s fruit and vegetable intake. Methods: The trial included children from 23 schools; these schools were randomised into two groups, one to receive the Royal Horticultural Society (RHS)-led intervention and the other to receive the less involved Teacher-led intervention. A 24-hour food diary (CADET) was used to collect baseline and follow-up dietary intake 18 months apart. Questionnaires were also administered to evaluate the intervention implementation. Results: A total of 641 children completed the trial with a mean age of 8.1 years (95% CI: 8.0, 8.4). The unadjusted results from multilevel regression analysis revealed that for combined daily fruit and vegetable intake the Teacher-led group had a higher daily mean change of 8 g (95% CI: −19, 36) compared to the RHS-led group -32 g (95% CI: −60, −3). However, after adjusting for possible confounders this difference was not significant (intervention effect: −40 g, 95% CI: −88, 1; p = 0.06). The adjusted analysis of process measures identified that if schools improved their gardening score by 3 levels (a measure of school gardening involvement - the scale has 6 levels from 0 ‘no garden’ to 5 ‘community involvement’), irrespective of group allocation, children had, on average, a daily increase of 81 g of fruit and vegetable intake (95% CI: 0, 163; p = 0.05) compared to schools that had no change in gardening score. Conclusions: This study is the first cluster randomised controlled trial designed to evaluate a school gardening intervention. The results have found very little evidence to support the claims that school gardening alone can improve children’s daily fruit and vegetable intake. However, when a gardening intervention is implemented at a high level within the school it may improve children’s daily fruit and vegetable intake by a portion. Improving children’s fruit and vegetable intake remains a challenging task

Safety Profile of Good Manufacturing Practice Manufactured Interferon \u3b3-Primed Mesenchymal Stem/Stromal Cells for Clinical Trials

Mesenchymal stem/stromal cells (MSCs) are widely studied by both academia and industry for a broad array of clinical indications. The collective body of data provides compelling evidence of the clinical safety of MSC therapy. However, generally accepted proof of therapeutic efficacy has not yet been reported. In an effort to generate a more effective therapeutic cell product, investigators are focused on modifying MSC processing protocols to enhance the intrinsic biologic activity. Here, we report a Good Manufacturing Practice-compliant two-step MSC manufacturing protocol to generate MSCs or interferon \u3b3 (IFN\u3b3) primed MSCs which allows freshly expanded cells to be infused in patients on a predetermined schedule. This protocol eliminates the need to infuse cryopreserved, just thawed cells which may reduce the immune modulatory activity. Moreover, using (IFN\u3b3) as a prototypic cytokine, we demonstrate the feasibility of priming the cells with any biologic agent. We then characterized MSCs and IFN\u3b3 primed MSCs prepared with our protocol, by karyotype, in vitro potential for malignant transformation, biodistribution, effect on engraftment of transplanted hematopoietic cells, and in vivo toxicity in immune deficient mice including a complete post-mortem examination. We found no evidence of toxicity attributable to the MSC or IFN\u3b3 primed MSCs. Our data suggest that the clinical risk of infusing MSCs or IFN\u3b3 primed MSCs produced by our two-step protocol is not greater than MSCs currently in practice. While actual proof of safety requires phase I clinical trials, our data support the use of either cell product in new clinical studies

Proposed therapeutic range of treosulfan in reduced toxicity pediatric allogeneic hematopoietic stem cell transplant conditioning: results from a prospective trial

Treosulfan is given off‐label in pediatric allogeneic hematopoietic stem cell transplant. This study investigated treosulfan's pharmacokinetics (PKs), efficacy, and safety in a prospective trial. Pediatric patients (n = 87) receiving treosulfan‐fludarabine conditioning were followed for at least 1 year posttransplant. PKs were described with a two‐compartment model. During follow‐up, 11 of 87 patients died and 12 of 87 patients had low engraftment (≤ 20% myeloid chimerism). For each increase in treosulfan area under the curve from zero to infinity (AUC(0‐∞)) of 1,000 mg hour/L the hazard ratio (95% confidence interval) for mortality increase was 1.46 (1.23–1.74), and the hazard ratio for low engraftment was 0.61 (0.36–1.04). A cumulative AUC(0‐∞) of 4,800 mg hour/L maximized the probability of success (> 20% engraftment and no mortality) at 82%. Probability of success with AUC(0‐∞) between 80% and 125% of this target were 78% and 79%. Measuring PK at the first dose and individualizing the third dose may be required in nonmalignant disease

The Neurocognitive Architecture of Individual Differences in Math Anxiety in Typical Children

Math Anxiety (MA) is characterized by a negative emotional response when facing math-related situations. MA is distinct from general anxiety and can emerge during primary education. Prior studies typically comprise adults and comparisons between high- versus low-MA, where neuroimaging work has focused on differences in network activation between groups when completing numerical tasks. The present study used voxel-based morphometry (VBM) to identify the structural brain correlates of MA in a sample of 79 healthy children aged 7–12 years. Given that MA is thought to develop in later primary education, the study focused on the level of MA, rather than categorically defining its presence. Using a battery of cognitive- and numerical-function tasks, we identified that increased MA was associated with reduced attention, working memory and math achievement. VBM highlighted that increased MA was associated with reduced grey matter in the left anterior intraparietal sulcus. This region was also associated with attention, suggesting that baseline differences in morphology may underpin attentional differences. Future studies should clarify whether poorer attentional capacity due to reduced grey matter density results in the later emergence of MA. Further, our data highlight the role of working memory in propagating reduced math achievement in children with higher MA

Contribution of Chondroitin Sulfate A to the Binding of Complement Proteins to Activated Platelets

Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.Human blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.This study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases

Climate change, the Great Barrier Reef and the response of Australians

© 2016, Palgrave Macmillan Ltd. All rights reserved. Inspiration, aspirations, attitudes, and perception of threats play a pivotal role in the way that individuals associate themselves with natural environments. These sentiments affect how people connect to natural places, including their behaviours, perceived responsibility, and the management interventions they support. World Heritage Areas hold an important place in the lives of people who visit, aspire to visit, or derive a sense of security and well-being from their existence. Yet, the connection between people and special places is rarely quantified and policymakers find it difficult to incorporate these human dimensions into decision-making processes. Here we describe the personal concern and connection that Australians have with the Great Barrier Reef and discuss how the results may help with its management. We utilize a statistically representative sample of Australian residents (n = 2,002) and show empirically that climate change is perceived to be the biggest threat to the Great Barrier Reef, and that the Great Barrier Reef inspires Australians, promotes pride, and instills a sense of individual identity and collective responsibility to protect it. An increased understanding of the high levels of personal connection to iconic natural resources may help managers to enhance public support for protecting climate-sensitive systems within Australia and around the world

Reduction in downstream test utilization following introduction of coronary computed tomography in a cardiology practice

To compare utilization of non-invasive ischemic testing, invasive coronary angiography (ICA), and percutaneous coronary intervention (PCI) procedures before and after introduction of 64-slice multi-detector row coronary computed tomographic angiography (CCTA) in a large urban primary and consultative cardiology practice. We utilized a review of electronic medical records (NotesMD®) and the electronic practice management system (Megawest®) encompassing a 4-year period from 2004 to 2007 to determine the number of exercise treadmill (TME), supine bicycle exercise echocardiography (SBE), single photon emission computed tomography (SPECT) myocardial perfusion stress imaging (MPI), coronary calcium score (CCS), CCTA, ICA, and PCI procedures performed annually. Test utilization in the 2 years prior to and 2 years following availability of CCTA were compared. Over the 4-year period reviewed, the annual utilization of ICA decreased 45% (2,083 procedures in 2004 vs. 1,150 procedures in 2007, P < 0.01) and the percentage of ICA cases requiring PCI increased (19% in 2004 vs. 28% in 2007, P < 0.001). SPECT MPI decreased 19% (3,223 in 2004 vs. 2,614 in 2007 P < 0.02) and exercise stress treadmill testing decreased 49% (471 in 2004 vs. 241 in 2007 P < 0.02). Over the same period, there were no significant changes in measures of practice volume (office and hospital) or the annual incidence of PCI (405 cases in 2004 vs. 326 cases in 2007) but a higher percentage of patients with significant disease undergoing PCI 19% in 2004 vs. 29% in 2007 P < 0.01. Implementation of CCTA resulted in a significant decrease in ICA and a corresponding significant increase in the percentage of ICA cases requiring PCI, indicating that CCTA resulted in more accurate referral for ICA. The reduction in unnecessary ICA is associated with avoidance of potential morbidity and mortality associated with invasive diagnostic testing, reduction of downstream SPECT MPI and TME as well as substantial savings in health care dollars