Conversion of Contaminated Post-Consumer Polyethylene Terephthalate into a Thermoset Alkyd Coating Using Biosourced Monomers.

The synthesis of a high-performance oxidative cross-linked thermoset alkyd coating is described that utilizes a novel recycling strategy from contaminated postconsumer waste polyethylene terephthalate (wPET). A single-stage "depolymerization-repolymerization" process has been developed that allows the exploitation of a waste stream from a commercial PET recycling process with 95% efficiency, which, when copolymerized with glycerol and tall oil fatty acid, delivers a sustainable fatty acid-functional polyester suitable for use in thermoset alkyd coatings. Physical drying challenges have been tackled via the development of a convergent polymer formulation strategy from a single source of wPET and the formulation of the resulting fatty acid-functional polymers with commercial alkyd driers, delivering a thermoset alkyd coating suitable for industrial applications.

Rational design of a cyclohexanone dehydrogenase for enhanced α,ß-desaturation and substrate specificity.

The selective α,ß-desaturation of cyclic carbonyl compounds, which are found in the core of many steroid and bioactive molecules, using green chemistry is highly desirable. To achieve this task, we have for the first time described and solved the de novo structure of a member of the cyclohexanone dehydrogenase class of enzymes. The breadth of substrate specificity was investigated by assaying the cyclohexanone dehydrogenase, from Alicycliphilus denitrificans, against several cyclic ketones, lactones and lactams. To investigate substrate binding, a catalytic variant, Y195F, was generated and used to obtain a crystallographic complex with the natural substrate, cyclohexanone. This revealed substrate-active site interactions, as well as the proximity of the cofactor, flavin adenine dinucleotide, and enabled us to propose a mechanistic function to key amino acids. We then used molecular dynamic simulations to guide design to add functionality to the cyclohexanone dehydrogenase enzyme. The resulting W113A variant had overall improved enzyme activity and substrate scope, i.e., accepting the bulkier carbonyl compound, dihydrocoumarin. Structural analysis of the W113A variant revealed a broader, more open active site, which helped explain the modified substrate specificity. This work paves the way for future bespoke regioselective α,ß-desaturation in the synthesis of important bioactive molecules via rational enzyme engineering.

Kidney macrophages tap the stream.

Tissue-resident macrophages are essential for maintaining organismal homeostasis, but the precise mechanisms that macrophages use to perform this function are not fully understood. In this issue of Immunity, He et al. demonstrate that renal macrophages surveil and sample urine particles, ensuring optimal collecting duct flow and preventing kidney stone development.

Use of remote sensing to assess vegetative stress as a proxy for soil contamination.

We report, for the first time, a multimodal investigation of current crude oil reprocessing and storage sites to assess their impact on the environment after 50 years of continuous operation. We have adopted a dual approach to investigate potential soil contamination. The first approach uses conventional analytical techniques i.e. energy dispersive X-ray fluorescence (ED-XRF) for metal analysis, and a complementary metabolomic investigation using hydrophilic liquid interaction chromatography hi-resolution mass spectrometry (HILIC-MS) for organic contaminants. Secondly, the deployment of an unmanned aerial vehicle (UAV) with a multispectral image (MSI) camera, for the remote sensing of vegetation stress, as a proxy for sub-surface soil contamination. The results identified high concentrations of barium (mean 21 017 ± 5950 µg g-1, n = 36) as well as metabolites derived from crude oil (polycyclic aromatic hydrocarbons), cleaning processes (surfactants) and other organic pollutants (e.g. pesticides, plasticizers and pharmaceuticals) in the reprocessing site. This data has then been correlated, with post-flight data analysis derived vegetation indices (NDVI, GNDVI, SAVI and Cl green VI), to assess the potential to identify soil contamination because of vegetation stress. It was found that strong correlations exist (an average R2 of >0.68) between the level of soil contamination and the ground cover vegetation. The potential to deploy aerial remote sensing techniques to provide an initial survey, to inform decision-making, on suspected contaminated land sites can have global implications.

Use of an unmanned aerial vehicle for monitoring and prediction of oilseed rape crop performance.

The flowering stage of oilseed rape (Brassica napus L.) is of vital interest in precision agriculture. It has been shown that data describing the flower production of oilseed rape (OSR), at stage 3, in spring can be used to predict seed yield at harvest. Traditional field-based techniques for assessing OSR flowers are based on a visual assessment which is subjective and time consuming. However, a high throughput phenotyping technique, using an unmanned aerial vehicle (UAV) with multispectral image (MSI) camera, was used to investigate the growth stages of OSR (in terms of crop height) and to quantify its flower production. A simplified approach using a normalised difference yellowness index (NDYI) was coupled with an iso-cluster classification method to quantify the number of OSR flower pixels and incorporate the data into an OSR seed yield estimation. The estimated OSR seed yield showed strong correlation with the actual OSR seed yield (R2 = 0.86), as determined using in-situ sensors mounted on the combine harvester. Also, using our approach allowed the variation in crop height to be assessed across all growing stages; the maximum crop height of 1.35 m OSR was observed at the flowering stage. This methodology is proposed for effectively predicting seed yield 3 months prior to harvesting.

Plasma membrane abundance dictates phagocytic capacity and functional crosstalk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they eat, how much they eat, and when they move after eating. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G-protein subunit Gb4 exhibit profound plasma membrane expansion due to enhanced production of sphingolipids. This increased membrane allocation dramatically enhances phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. Gb4 deficient neutrophils are also defective in the normal inhibition of migration following cargo uptake. In Gb4 knockout mice, myeloid cells exhibit enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. These results reveal an unexpected, biophysical control mechanism lying at the heart of myeloid functional decision-making.

Maternal diet during early gestation influences postnatal taste activity-dependent pruning by microglia.

A key process in central sensory circuit development involves activity-dependent pruning of exuberant terminals. Here, we studied gustatory terminal field maturation in the postnatal mouse nucleus of the solitary tract (NST) during normal development and in mice where their mothers were fed a low NaCl diet for a limited period soon after conception. Pruning of terminal fields of gustatory nerves in controls involved the complement system and is likely driven by NaCl-elicited taste activity. In contrast, offspring of mothers with an early dietary manipulation failed to prune gustatory terminal fields even though peripheral taste activity developed normally. The ability to prune in these mice was rescued by activating myeloid cells postnatally, and conversely, pruning was arrested in controls with the loss of myeloid cell function. The altered pruning and myeloid cell function appear to be programmed before the peripheral gustatory system is assembled and corresponds to the embryonic period when microglia progenitors derived from the yolk sac migrate to and colonize the brain.

Early life high fructose exposure disrupts microglia function and impedes neurodevelopment.

Despite the success of fructose as a low-cost food additive, recent epidemiological evidence suggests that high fructose consumption by pregnant mothers or during adolescence is associated with disrupted neurodevelopment 1-7 . An essential step in appropriate mammalian neurodevelopment is the synaptic pruning and elimination of newly-formed neurons by microglia, the central nervous system's (CNS) resident professional phagocyte 8-10 . Whether early life high fructose consumption affects microglia function and if this directly impacts neurodevelopment remains unknown. Here, we show that both offspring born to dams fed a high fructose diet and neonates exposed to high fructose exhibit decreased microglial density, increased uncleared apoptotic cells, and decreased synaptic pruning in vivo . Importantly, deletion of the high affinity fructose transporter SLC2A5 (GLUT5) in neonates completely reversed microglia dysfunction, suggesting that high fructose directly affects neonatal development. Mechanistically, we found that high fructose treatment of both mouse and human microglia suppresses synaptic pruning and phagocytosis capacity which is fully reversed in GLUT5-deficient microglia. Using a combination of in vivo and in vitro nuclear magnetic resonance- and mass spectrometry-based fructose tracing, we found that high fructose drives significant GLUT5-dependent fructose uptake and catabolism, rewiring microglia metabolism towards a hypo-phagocytic state. Importantly, mice exposed to high fructose as neonates exhibited cognitive defects and developed anxiety-like behavior which were rescued in GLUT5-deficient animals. Our findings provide a mechanistic explanation for the epidemiological observation that early life high fructose exposure is associated with increased prevalence of adolescent anxiety disorders.

WNK1 enforces macrophage lineage fidelity.

The appropriate development of macrophages, the body's professional phagocyte, is essential for organismal development, especially in mammals. This dependence is exemplified by the observation that loss-of-function mutations in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities owing to an absence of macrophages. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for development of tissue-resident macrophages (TRMs). Myeloid-specific deletion of Wnk1 resulted in a dramatic loss of TRMs, disrupted organ development, systemic neutrophilia, and mortality between 3 and 4 weeks of age. Strikingly, we found that myeloid progenitors or precursors lacking WNK1 not only failed to differentiate into macrophages, but instead differentiated into neutrophils. Mechanistically, the cognate CSF1R cytokine macrophage-colony stimulating factor (M-CSF) stimulates macropinocytosis by both mouse and human myeloid progenitors and precursor cells. Macropinocytosis, in turn, induces chloride flux and WNK1 phosphorylation. Importantly, blocking macropinocytosis, perturbing chloride flux during macropinocytosis, and inhibiting WNK1 chloride-sensing activity each skewed myeloid progenitor differentiation from macrophages into neutrophils. Thus, we have elucidated a role for WNK1 during macropinocytosis and discovered a novel function of macropinocytosis in myeloid progenitors and precursor cells to ensure macrophage lineage fidelity. Highlights: Myeloid-specific WNK1 loss causes failed macrophage development and premature deathM-CSF-stimulated myeloid progenitors and precursors become neutrophils instead of macrophagesM-CSF induces macropinocytosis by myeloid progenitors, which depends on WNK1Macropinocytosis enforces macrophage lineage commitment.

Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

Metabolic adaptation supports enhanced macrophage efferocytosis in limited-oxygen environments.

Apoptotic cell (AC) clearance (efferocytosis) is performed by phagocytes, such as macrophages, that inhabit harsh physiological environments. Here, we find that macrophages display enhanced efferocytosis under prolonged (chronic) physiological hypoxia, characterized by increased internalization and accelerated degradation of ACs. Transcriptional and translational analyses revealed that chronic physiological hypoxia induces two distinct but complimentary states. The first, "primed" state, consists of concomitant transcription and translation of metabolic programs in AC-naive macrophages that persist during efferocytosis. The second, "poised" state, consists of transcription, but not translation, of phagocyte function programs in AC-naive macrophages that are translated during efferocytosis. Mechanistically, macrophages efficiently flux glucose into a noncanonical pentose phosphate pathway (PPP) loop to enhance NADPH production. PPP-derived NADPH directly supports enhanced efferocytosis under physiological hypoxia by ensuring phagolysosomal maturation and redox homeostasis. Thus, macrophages residing under physiological hypoxia adopt states that support cell fitness and ensure performance of essential homeostatic functions rapidly and safely.

Engineered living photosynthetic biocomposites for intensified biological carbon capture.

Carbon capture and storage is required to meet Paris Agreement targets. Photosynthesis is nature's carbon capture technology. Drawing inspiration from lichen, we engineered 3D photosynthetic cyanobacterial biocomposites (i.e., lichen mimics) using acrylic latex polymers applied to loofah sponge. Biocomposites had CO2 uptake rates of 1.57 ± 0.08 g CO2 g-1biomass d-1. Uptake rates were based on the dry biomass at the start of the trial and incorporate the CO2 used to grow new biomass as well as that contained in storage compounds such as carbohydrates. These uptake rates represent 14-20-fold improvements over suspension controls, potentially scaling to capture 570 tCO2 t-1biomass yr-1, with an equivalent land consumption of 5.5-8.17 × 106 ha, delivering annualized CO2 removal of 8-12 GtCO2, compared with 0.4-1.2 × 109 ha for forestry-based bioenergy with carbon capture and storage. The biocomposites remained functional for 12 weeks without additional nutrient or water supplementation, whereupon experiments were terminated. Engineered and optimized cyanobacteria biocomposites have potential for sustainable scalable deployment as part of humanity's multifaceted technological stand against climate change, offering enhanced CO2 removal with low water, nutrient, and land use penalties.

Self-Assembled, Hierarchical Structured Surfaces for Applications in (Super)hydrophobic Antiviral Coatings.

A versatile method for the creation of multitier hierarchical structured surfaces is reported, which optimizes both antiviral and hydrophobic (easy-clean) properties. The methodology exploits the availability of surface-active chemical groups while also manipulating both the surface micro- and nanostructure to control the way the surface coating interacts with virus particles within a liquid droplet. This methodology has significant advantages over single-tier structured surfaces, including the ability to overcome the droplet-pinning effect and in delivering surfaces with high static contact angles (>130°) and good antiviral efficacy (log kill >2). In addition, the methodology highlights a valuable approach for the creation of mechanically robust, nanostructured surfaces which can be prepared by spray application using nonspecialized equipment.

Risk of Dysphagia and Dysphonia in Patients With Prior Thyroidectomy Undergoing Anterior Cervical Discectomy and Fusion.

STUDY DESIGN: Retrospective cohort study using PearlDiver database. OBJECTIVES: To evaluate the effect of prior thyroidectomy on complications of Anterior Cervical Discectomy and Fusion (ACDF) surgery. METHODS: PearlDiver was used to identify patients without prior dysphagia or dysphonia undergoing ACDF between the years 2010-2020Q1. Patients with and without prior thyroidectomy were matched by levels of fusion, alcohol use, and gastroesophageal reflux disease in a 1:5 ratio. Postoperative outcomes were assessed for each cohort with multivariable logistic regression, controlling for age, sex, and Elixhauser Comorbidity Index. RESULTS: Between 2010 and 2019, matched cohorts of 792 ACDF patients with prior thyroidectomy and 3960 ACDF only patients were included in the study. Of patients with previous thyroidectomy undergoing ACDF, 16.3% experienced dysphagia at 1-year compared with 10.6% for patients undergoing ACDF only (aOR=1.39, P=.004). Patients with previous thyroidectomy also had higher odds of dysphonia at 1-year following ACDF, as compared to patients with ACDF alone (2.7% vs 1.2%, aOR=1.74, P= .048). Patients undergoing ACDF with prior thyroidectomy did not have increased risk of revision at 1 year (aOR=1.10, P=.698), 2 years (aOR=1.16, P=.457), or 5 years (aOR=1.20, P=.255) following surgery. There were no differences in postoperative opioid utilization rates at 1 month (aOR=2.07, P=.138), 3 months (aOR=2.45, P=.095), 6 months (aOR=1.34, P=.520), and 12 months (aOR=1.69, P=.202). Prior thyroidectomy was not associated with reintubation following ACDF (P=.995). CONCLUSIONS: Patients with prior thyroidectomy undergoing ACDF surgery experience increased odds of dysphagia and dysphonia at 1-year follow-up compared to those without prior thyroidectomy.

Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo.

Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. We developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, we uncovered multiple qualitative and quantitative features of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo.

Modelling of environmental impacts of printed self-healing products.

Products utilising self-healing materials have the potential to restore some of their function following damage, thereby extending the product lifespan and contributing to waste prevention and increased product safety. Despite the growing interest in these products, there a lack of comprehensive studies on the environmental implications of self-healing products and the parameters that influence impacts. The study presented in this paper combined life cycle assessment combined with a Taguchi experimental design and analysis of variance to investigate the effect of various parameters across the life stages of a self-healing composite product manufactured by 3D printing using poly-lactic acid (PLA) and self-healing polyurethane (PU). The results of this study suggests that impacts are primarily affected by avoided production due to the increased service of the product, followed by electricity requirements and material deposition rate (efficiency) of 3D printing. In the case of water consumption raw material manufacturing of PLA and PU are the highest and hence should be a target for research on reducing their water footprint. When comparing self-healing vs. regular products it is evident that most of the impacts are dominated by the electricity consumption of the manufacturing process. These results suggest that maximising avoided production can play a major role in reducing impacts of 3D printed products. The results are important for maximising the circularity of additive manufacturing products while minimising their life cycle impact.

Effects of OP2113 on Myocardial Infarct Size and No Reflow in a Rat Myocardial Ischemia/Reperfusion Model.

PURPOSE: The present study was to determine whether OP2113 could limit myocardial infarction size and the no-reflow phenomenon in a rat myocardial ischemia/reperfusion model. METHODS: Rat heart-isolated mitochondria (RHM) were used to investigate mitochondrial respiration and mitochondrial reactive oxygen species (mtROS) generation both in normal conditions and in ischemia/reperfusion-mimicking conditions (using high concentrations of succinate). Human skeletal muscle myoblasts (HSMM) in culture were used to investigate the cellular intermittent deprivation in energy substrates and oxygen as reported in ischemia/reperfusion conditions. In vivo, rats were anesthetized and subjected to 30 min of left coronary artery occlusion followed by 3 h of reperfusion. Rats were randomized to receive OP2113 as an intravenous infusion starting either 5 min prior to coronary artery occlusion (preventive), or 5 min prior to reperfusion (curative), or to receive vehicle starting 5 min prior to coronary artery occlusion. Infusions continued until the end of the study (3 h of reperfusion). RESULTS: RHM treated with OP2113 showed a concentration-dependent reduction of succinate-induced mtROS generation. In HSMM cells, OP2113 treatment (5-10 µM) during 48H prevented the reduction in the steady-state level of ATP measured just after reperfusion injuries and decreased the mitochondrial affinity to oxygen. In vivo, myocardial infarct size, expressed as the percentage of the ischemic risk zone, was significantly lower in the OP2113-treated preventive group (44.5 ± 2.9%) versus that in the vehicle group (57.0 ± 3.6%; p < 0.05), with a non-significant trend toward a smaller infarct size in the curative group (50.8 ± 3.9%). The area of no reflow as a percentage of the risk zone was significantly smaller in both the OP2113-treated preventive (28.8 ± 2.4%; p = 0.026 vs vehicle) and curative groups (30.1 ± 2.3%; p = 0.04 vs vehicle) compared with the vehicle group (38.9 ± 3.1%). OP2113 was not associated with any hemodynamic changes. CONCLUSIONS: These results suggest that OP2113 is a promising mitochondrial ROS-modulating agent to reduce no-reflow as well as to reduce myocardial infarct size, especially if it is on board early in the course of the infarction. It appears to have benefit on no-reflow even when administered relatively late in the course of ischemia.

Erratum: Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.024.].

Applied aerial spectroscopy: A case study on remote sensing of an ancient and semi-natural woodland.

An area of ancient and semi-natural woodland (ASNW) has been investigated by applied aerial spectroscopy using an unmanned aerial vehicle (UAV) with multispectral image (MSI) camera. A novel normalised difference spectral index (NDSI) algorithm was developed using principal component analysis (PCA). This novel NDSI was then combined with a simple segmentation method of thresholding and applied for the identification of native tree species as well as the overall health of the woodland. Using this new approach allowed the identification of trees at canopy level, across 7.4 hectares (73,934 m2) of ASNW, as oak (53%), silver birch (37%), empty space (9%) and dead trees (1%). This UAV derived data was corroborated, for its accuracy, by a statistically valid ground-level field study that identified oak (47%), silver birch (46%) and dead trees (7.4%). This simple innovative approach, using a low-cost multirotor UAV with MSI camera, is both rapid to deploy, was flown around 100 m above ground level, provides useable high resolution (5.3 cm / pixel) data within 22 mins that can be interrogated using readily available PC-based software to identify tree species. In addition, it provides an overall oversight of woodland health and has the potential to inform a future woodland regeneration strategy.

First we eat, then we do everything else: The dynamic metabolic regulation of efferocytosis.

Clearance of apoptotic cells, or "efferocytosis," is essential for diverse processes including embryonic development, tissue turnover, organ regeneration, and immune cell development. The human body is estimated to remove approximately 1% of its body mass via apoptotic cell clearance daily. This poses several intriguing cell metabolism problems. For instance, phagocytes such as macrophages must induce or suppress metabolic pathways to find, engulf, and digest apoptotic cells. Then, phagocytes must manage the potentially burdensome biomass of the engulfed apoptotic cell. Finally, phagocytes reside in complex tissue architectures that vary in nutrient availability, the types of dying cells or debris that require clearance, and the neighboring cells they interact with. Here, we review advances in our understanding of these three key areas of phagocyte metabolism. We end by proposing a model of efferocytosis that integrates recent findings and establishes a new paradigm for testing how efferocytosis prevents chronic inflammatory disease and autoimmunity.