Metabolomics Reveals a "Trimeric" γ-Actinorhodin from Streptomyces coelicolor M145.

Streptomyces coelicolor is a prolific producer of natural products and serves as a model organism for their study. It produces several pigmented antibiotics, the best-studied of which are the actinorhodins. We used a combination of liquid chromatography-mass spectrometry (LC-MS) and computational tools used for annotating the detected species (e. g., spectral matching, in-silico predictors, molecular networking) to identify putative new actinorhodin analogs. These studies led to the discovery of the first trimeric benzoisochromanequinone, θ-actinorhodin (1). Further metabolomics analysis revealed that the relative amounts of shunt products produced were similar between the two growth conditions explored. This suggests that, while substantially different products were being produced, the biosynthetic gene clusters were similarly active. Overall, this work describes the discovery of the first trimeric benzoisochromanequinone and explores the biosynthetic processes that might lead to its production by metabolomics analysis of relevant intermediates.

Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins.

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

Comparison of Bioorthogonal ß-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of ß-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal ß-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Ion Mobility Mass Spectrometry as an Efficient Tool for Identification of Streptorubin B in Streptomyces coelicolor M145.

Ion mobility spectrometry was utilized to corroborate the identity of streptorubin B (2) as the natural product produced by Streptomyces coelicolor. Natural product 2 was initially assigned as butylcycloheptylprodigiosin (3), and only relatively recently was this assignment clarified. We present additional evidence of this assignment by comparing collisional cross sections (Ω) of synthetic standards of 2, 3, and metacycloprodigiosin (4) to the cyclic prodiginine produced by S. coelicolor. Calculated theoretical Ω values demonstrate that cyclic prodiginines could be identified without standards. This work highlights ion mobility as an efficient tool for the dereplication of natural products.

Automatic Torso Detection in Images of Preterm Infants.

Imaging systems have applications in patient respiratory monitoring but with limited application in neonatal intensive care units (NICU). In this paper we propose an algorithm to automatically detect the torso in an image of a preterm infant during non-invasive respiratory monitoring. The algorithm uses normalised cut to segment each image into clusters, followed by two fuzzy inference systems to detect the nappy and torso. Our dataset comprised overhead images of 16 preterm infants in a NICU, with uncontrolled illumination, and encompassing variations in poses, presence of medical equipment and clutter in the background. The algorithm successfully identified the torso region for 15 of the 16 images, with a high agreement between the detected torso and the torso identified by clinical experts.

Preliminary study of automated oxygen titration at birth for preterm infants.

OBJECTIVE: To study the feasibility of automated titration of oxygen therapy in the delivery room for preterm infants. DESIGN: Prospective non-randomised study of oxygenation in sequential preterm cohorts in which FiO2 was adjusted manually or by an automated control algorithm during the first 10 min of life. SETTING: Delivery rooms of a tertiary level hospital. PARTICIPANTS: Preterm infants <32 weeks gestation (n=20 per group). INTERVENTION: Automated oxygen control using a purpose-built device, with SpO2 readings input to a proportional-integral-derivative algorithm, and FiO2 alterations actuated by a motorised blender. The algorithm was developed via in silico simulation using abstracted oxygenation data from the manual control group. For both groups, the SpO2 target was the 25th-75th centile of the Dawson nomogram. MAIN OUTCOME MEASURES: Proportion of time in the SpO2 target range (25th-75th centile, or above if in room air) and other SpO2 ranges; FiO2 adjustment frequency; oxygen exposure. RESULTS: Time in the SpO2 target range was similar between groups (manual control: median 60% (IQR 48%-72%); automated control: 70 (60-84)%; p=0.31), whereas time with SpO2 >75th centile when receiving oxygen differed (manual: 17 (7.6-26)%; automated: 10 (4.4-13)%; p=0.048). Algorithm-directed FiO2 adjustments were frequent during automated control, but no manual adjustments were required in any infant once valid SpO2 values were available. Oxygen exposure was greater during automated control, but final FiO2 was equivalent. CONCLUSION: Automated oxygen titration using a purpose-built algorithm is feasible for delivery room management of preterm infants, and warrants further evaluation.

Automated control of oxygen titration in preterm infants on non-invasive respiratory support.

OBJECTIVE: To evaluate the performance of a rapidly responsive adaptive algorithm (VDL1.1) for automated oxygen control in preterm infants with respiratory insufficiency. DESIGN: Interventional cross-over study of a 24-hour period of automated oxygen control compared with aggregated data from two flanking periods of manual control (12 hours each). SETTING: Neonatal intensive care unit. PARTICIPANTS: Preterm infants receiving non-invasive respiratory support and supplemental oxygen; median birth gestation 27 weeks (IQR 26-28) and postnatal age 17 (12-23) days. INTERVENTION: Automated oxygen titration with the VDL1.1 algorithm, with the incoming SpO2 signal derived from a standard oximetry probe, and the computed inspired oxygen concentration (FiO2) adjustments actuated by a motorised blender. The desired SpO2 range was 90%-94%, with bedside clinicians able to make corrective manual FiO2 adjustments at all times. MAIN OUTCOME MEASURES: Target range (TR) time (SpO2 90%-94% or 90%-100% if in air), periods of SpO2 deviation, number of manual FiO2 adjustments and oxygen requirement were compared between automated and manual control periods. RESULTS: In 60 cross-over studies in 35 infants, automated oxygen titration resulted in greater TR time (manual 58 (51-64)% vs automated 81 (72-85)%, p<0.001), less time at both extremes of oxygenation and considerably fewer prolonged hypoxaemic and hyperoxaemic episodes. The algorithm functioned effectively in every infant. Manual FiO2 adjustments were infrequent during automated control (0.11 adjustments/hour), and oxygen requirements were similar (manual 28 (25-32)% and automated 26 (24-32)%, p=0.13). CONCLUSION: The VDL1.1 algorithm was safe and effective in SpO2 targeting in preterm infants on non-invasive respiratory support. TRIAL REGISTRATION NUMBER: ACTRN12616000300471.

Automation of oxygen titration in preterm infants: Current evidence and future challenges.

For the preterm infant with respiratory insufficiency requiring supplemental oxygen, tight control of oxygen saturation (SpO2) is advocated, but difficult to achieve in practice. Automated control of oxygen delivery has emerged as a potential solution, with six control algorithms currently embedded in commercially-available respiratory support devices. To date, most clinical evaluations of these algorithms have been short-lived crossover studies, in which a benefit of automated over manual control of oxygen titration has been uniformly noted, along with a reduction in severe SpO2 deviations and need for manual FiO2 adjustments. A single non-randomised study has examined the effect of implementation of automated oxygen control with the CLiO2 algorithm as standard care for preterm infants; no clear benefits in relation to clinical outcomes were noted, although duration of mechanical ventilation was lessened. The results of randomised controlled trials are awaited. Beyond the gathering of evidence regarding a treatment effect, we contend that there is a need for a better understanding of the function of contemporary control algorithms under a range of clinical conditions, further exploration of techniques of adaptation to individualise algorithm performance, and a concerted effort to apply this technology in low resource settings in which the majority of preterm infants receive care. Attainment of these goals will be paramount in optimisation of oxygen therapy for preterm infants globally.

Enzyme-targeted fluorescent small-molecule probes for bacterial imaging.

Molecular imaging methods to visualize myriad biochemical processes in bacteria have traditionally been dependent upon molecular biology techniques to incorporate fluorescent biomolecules (e.g., fusion proteins). Such methods have been instrumental in our understanding of how bacteria function but are not without drawbacks, including potential perturbation to native protein expression and function. To overcome these limitations, the use of fluorescent small-molecule probes has gained much attention. Here, we highlight examples from the recent literature that showcase the utility of small-molecule probes for the fluorescence imaging of bacterial cells, including electrophilic, metabolic, and enzyme-activated probes. Although the use of these types of compounds for bacterial imaging is still relatively new, the selected examples demonstrate the exciting potential of these critical tools in the exploration of bacterial physiology.

Predicting Apnoeic Events in Preterm Infants.

Apnoea, a pause in respiration, is almost ubiquitous in preterm infants born before completing 30 weeks gestation. Apnoea often begets hypoxemia and/or bradycardia, and has the potential to result in adverse neurodevelopmental consequences. Our current inability to predict apnoeic events in preterm infants requires apnoea to first be detected by monitoring device/s in order to trigger an intervention by bedside (medical or nursing) staff. Such a reactive management approach is laborious, and makes the consequences of apnoeic events inevitable. Recent technological advances and improved signal processing have allowed the possibility of developing prediction models for apnoeic events in preterm infants. However, the development of such models has numerous challenges and is only starting to show potential. This paper identifies requisite components and current gaps in developing prediction models for apnoeic events, and reviews previous studies on predicting apnoeic events in preterm infants.

Physiological instability after respiratory pauses in preterm infants.

BACKGROUND: The factors influencing the severity of apnea-related hypoxemia and bradycardia are incompletely characterized, especially in infants receiving noninvasive respiratory support. OBJECTIVES: To identify the frequency and predictors of physiological instability (hypoxemia-oxygen saturation (SpO2 ) <80%, or bradycardia-heart rate (HR) < 100 bpm) following respiratory pauses in infants receiving noninvasive respiratory support. METHODS: Respiratory pause duration, derived from capsule pneumography, was measured in 30 preterm infants of gestation 30 (24-32) weeks [median (interquartile range)] receiving noninvasive respiratory support and supplemental oxygen. For identified pauses of 5 to 29 seconds duration, we measured the magnitude and duration of SpO2 and HR reductions over a period starting at the pause onset and ending 60 seconds after resumption of breathing. Temporally clustered pauses (<60 seconds separation) were analyzed separately. The relative contribution of respiratory pauses to overall physiological instability was determined, and predictors of instability were sought in regression analysis, including demographic, clinical and situational variables as inputs. RESULTS: In total, 17 105 isolated and 9180 clustered pauses were identified. Hypoxemia and bradycardia were more likely after longer duration and temporally-clustered pauses. However, the majority of such episodes occurred after 5 to 9 second pauses given their numerical preponderance, and short-lived pauses made a substantial contribution to physiological instability overall. Birth gestation, hemoglobin concentration, form of respiratory support, caffeine treatment, respiratory pause duration and temporal clustering were identified as predictors of instability. CONCLUSIONS: Brief respiratory pauses, especially when clustered, contribute substantially to hypoxemia and bradycardia in preterm infants.