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Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways. P450 oxidoreductase (POR) selectively activates numerous cytochromes P450 (CYP), crucial for metabolism of drugs, steroids and xenobiotics and natural product biosynthesis. Here, the authors identify ligands that bind POR and bias its specificity towards CYP redox partners, activating distinct metabolic cascades in cells.

Extracellular vesicle (EV) secretion rate is stimulated by hypoxia that causes increased reactive oxygen species (ROS) production by the mitochondrial electron transport chain (ETC) and hypoxia‐induced factor (HIF)‐1 signaling; however, their contribution to the increased EV secretion rate is unknown. We found that the EV marker secretion rate in our EV reporter cell line CD9truc‐EGFP was unaffected by the HIF‐1α stabilizer roxadustat; yet, ETC stimulation by dichloroacetic acid (DCA) significantly increased EV secretion. The DCA‐induced EV secretion was blocked by the antioxidant TEMPO and rotenone, an inhibitor of the ETC's Complex I. Under hypoxic conditions, the limited oxygen reduction impedes the ETC's Complex III. To mimic this, we inhibited Complex III with antimycin A, which increased ROS‐dependent EV secretion. The electron transport between Complex I and III is accomplished by coenzyme Q created by the mevalonate pathway and tyrosine metabolites. Blocking an early step in the mevalonate pathway using pitavastatin augmented the DCA‐induced EV secretion, and 4‐nitrobenzoate—an inhibitor of the condensation of the mevalonate pathway with tyrosine metabolites—increased ROS‐dependent EV secretion. Our findings indicate that hypoxia‐mimetics targeting the ETC modify EV secretion and that ROS produced by the ETC is a potent stimulus for EV secretion. The mitochondrial electron transport chain (ETC) creates reactive oxygen species (ROS), and mitochondrial ROS production is affected by cellular metabolism. Here, we used pharmacological interventions on cultured cells to show that high ETC activity increases EV secretion through a ROS‐dependent mechanism. Together, our data indicate that cellular metabolism is linked to EV secretion.

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. As a result, there is an incentive to understand the mechanisms underlying lipase activity at the molecular level, so as to be able to design new lipase variants with tailor-made functionalities. Our understanding of lipase function primarily relies on bulk assay averaging the behavior of a high number of enzymes masking structural dynamics and functional heterogeneities. Recent advances in single molecule techniques based on fluorogenic substrate analogues revealed the existence of lipase functional states, and furthermore so how they are remodeled by regulatory cues. Single particle studies of lipases on the other hand directly observed diffusional heterogeneities and suggested lipases to operate in two different modes. Here to decipher how mutations in the lid region controls Thermomyces lanuginosus lipase (TLL) diffusion and function we employed a Single Particle Tracking (SPT) assay to directly observe the spatiotemporal localization of TLL and rationally designed mutants on native substrate surfaces. Parallel imaging of thousands of individual TLL enzymes and HMM analysis allowed us to observe and quantify the diffusion, abundance and microscopic transition rates between three linearly interconverting diffusional states for each lipase. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states.

Abstract Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identified ligands that dock on POR and bias its specificity towards CYP redox partners. Single molecule FRET studies revealed ligand docking to alter POR conformational sampling, which resulted in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand docking on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease related, metabolic pathways. Competing Interest Statement The authors have declared no competing interest.

Three series of ionic self-assembled materials based on anionic azo-dyes and cationic benzalkonium surfactants were synthesized and thin films were prepared by spin-casting. These thin films appear isotropic when investigated with polarized optical microscopy, although they are highly anisotropic. Here, three series of homologous materials were studied to rationalize this observation. Investigating thin films of ordered molecular materials relies to a large extent on advanced experimental methods and large research infrastructure. A statement that in particular is true for thin films with nanoscopic order, where X-ray reflectometry, X-ray and neutron scattering, electron microscopy and atom force microscopy (AFM) has to be used to elucidate film morphology and the underlying molecular structure. Here, the thin films were investigated using AFM, optical microscopy and polarized absorption spectroscopy. It was shown that by using numerical method for treating the polarized absorption spectroscopy data, the molecular structure can be elucidated. Further, it was shown that polarized optical spectroscopy is a general tool that allows determination of the molecular order in thin films. Finally, it was found that full control of thermal history and rigorous control of the ionic self-assembly conditions are required to reproducibly make these materials of high nanoscopic order. Similarly, the conditions for spin-casting are shown to be determining for the overall thin film morphology, while molecular order is maintained.

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it’s membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies. - Lipidating the therapeutic peptide salmon calcitonin alters its biophysical characteristics, including oligomer size, hydrophobicity and membrane activity. - Fluorescent microscopy of single GUVs enables the determination of peptide mediated reporter dye influx behavior as either graded or All-or-None, which is coupled to either smaller membrane perturbations or peptide pore formation. - Modulating the number of hydrocarbons constituting the lipidation moieties determines the membrane permeation mechanism. - By increasing the lipid chain length lipidated of salmon calcitonin goes from displaying smaller membrane perturbations to a peptide pore formation mechanism. - Effective membrane translocation of lipidated salmon calcitonin requires a peptide mediated pore forming mechanism.

Background As the cost of sequencing has declined, clinical diagnostics based on next generation sequencing (NGS) have become reality. Diagnostics based on sequencing will require rapid and precise mapping against redundant databases because some of the most important determinants, such as antimicrobial resistance and core genome multilocus sequence typing (MLST) alleles, are highly similar to one another. In order to facilitate this, a novel mapping method, KMA ( k -mer alignment), was designed. KMA is able to map raw reads directly against redundant databases, it also scales well for large redundant databases. KMA uses k -mer seeding to speed up mapping and the Needleman-Wunsch algorithm to accurately align extensions from k -mer seeds. Multi-mapping reads are resolved using a novel sorting scheme (ConClave scheme), ensuring an accurate selection of templates. Results The functionality of KMA was compared with SRST2, MGmapper, BWA-MEM, Bowtie2, Minimap2 and Salmon, using both simulated data and a dataset of Escherichia coli mapped against resistance genes and core genome MLST alleles. KMA outperforms current methods with respect to both accuracy and speed, while using a comparable amount of memory. Conclusion With KMA, it was possible map raw reads directly against redundant databases with high accuracy, speed and memory efficiency.

Whilst the last decades have seen a clear shift in emphasis from managing natural hazards to managing risk, the majority of natural-hazard risk research still focuses on single hazards. Internationally, there are calls for more attention for multi-hazards and multi-risks. Within the European Union (EU), the concepts of multi-hazard and multi-risk assessment and management have taken centre stage in recent years. In this perspective paper, we outline several key developments in multi-(hazard-)risk research in the last decade, with a particular focus on the EU. We present challenges for multi-(hazard-)risk management as outlined in several research projects and papers. We then present a research agenda for addressing these challenges. We argue for an approach that addresses multi-(hazard-)risk management through the lens of sustainability challenges that cut across sectors, regions, and hazards. In this approach, the starting point is a specific sustainability challenge, rather than an individual hazard or sector, and trade-offs and synergies are examined across sectors, regions, and hazards. We argue for in-depth case studies in which various approaches for multi-(hazard-)risk management are co-developed and tested in practice. Finally, we present a new pan-European research project in which our proposed research agenda will be implemented, with the goal of enabling stakeholders to develop forward-looking disaster risk management pathways that assess trade-offs and synergies of various strategies across sectors, hazards, and spatial scales.

The objective was to develop evidence -based recommendations and a research and educational agenda for the non-pharmacological management of hip and knee osteoarthritis (OA). The multidisciplinary task force comprised 21 experts: nurses, occupational therapists, physiotherapists, rheumatologists, orthopaedic surgeons, general practitioner, psychologist, dietician, clinical epidemiologist and patient representatives. After a preliminary literature review, a first task force meeting and five Delphi rounds, provisional recommendations were formulated in order to perform a systematic review. A literature search of Medline and eight other databases was performed up to February 2012. Evidence was graded in categories I–IV and agreement with the recommendations was determined through scores from 0 (total disagreement) to 10 (total agreement). Eleven evidence-based recommendations for the non-pharmacological core management of hip and knee OA were developed, concerning the following nine topics: assessment, general approach, patient information and education, lifestyle changes, exercise, weight loss, assistive technology and adaptations, footwear and work. The average level of agreement ranged between 8.0 and 9.1. The proposed research agenda included an overall need for more research into non-pharmacological interventions for hip OA, moderators to optimise individualised treatment, healthy lifestyle with economic evaluation and long-term follow-up, and the prevention and reduction of work disability. Proposed educational activities included the required skills to teach, initiate and establish lifestyle changes. The 11 recommendations provide guidance on the delivery of non-pharmacological interventions to people with hip or knee OA. More research and educational activities are needed, particularly in the area of lifestyle changes.

Aerosol-induced absorption of shortwave radiation can modify the climate through local atmospheric heating, which affects lapse rates, precipitation, and cloud formation. Presently, the total amount of aerosol absorption is poorly constrained, and the main absorbing aerosol species (black carbon (BC), organic aerosols (OA), and mineral dust) are diversely quantified in global climate models. As part of the third phase of the Aerosol Comparisons between Observations and Models (AeroCom) intercomparison initiative (AeroCom phase III), we here document the distribution and magnitude of aerosol absorption in current global aerosol models and quantify the sources of intermodel spread, highlighting the difficulties of attributing absorption to different species. In total, 15 models have provided total present-day absorption at 550 nm (using year 2010 emissions), 11 of which have provided absorption per absorbing species. The multi-model global annual mean total absorption aerosol optical depth (AAOD) is 0.0054 (0.0020 to 0.0098; 550 nm), with the range given as the minimum and maximum model values. This is 28 % higher compared to the 0.0042 (0.0021 to 0.0076) multi-model mean in AeroCom phase II (using year 2000 emissions), but the difference is within 1 standard deviation, which, in this study, is 0.0023 (0.0019 in Phase II). Of the summed component AAOD, 60 % (range 36 %–84 %) is estimated to be due to BC, 31 % (12 %–49 %) is due to dust, and 11 % (0 %–24 %) is due to OA; however, the components are not independent in terms of their absorbing efficiency. In models with internal mixtures of absorbing aerosols, a major challenge is the lack of a common and simple method to attribute absorption to the different absorbing species. Therefore, when possible, the models with internally mixed aerosols in the present study have performed simulations using the same method for estimating absorption due to BC, OA, and dust, namely by removing it and comparing runs with and without the absorbing species. We discuss the challenges of attributing absorption to different species; we compare burden, refractive indices, and density; and we contrast models with internal mixing to models with external mixing. The model mean BC mass absorption coefficient (MAC) value is 10.1 (3.1 to 17.7) m2 g−1 (550 nm), and the model mean BC AAOD is 0.0030 (0.0007 to 0.0077). The difference in lifetime (and burden) in the models explains as much of the BC AAOD spread as the difference in BC MAC values. The difference in the spectral dependency between the models is striking. Several models have an absorption Ångstrøm exponent (AAE) close to 1, which likely is too low given current knowledge of spectral aerosol optical properties. Most models do not account for brown carbon and underestimate the spectral dependency for OA.