A novel live cell assay to measure diacylglycerol lipase α activity

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays

Amorphous Solid Dispersions of Lidocaine and Lidocaine HCl Produced by Ball Milling with Well-Defined RAFT-Synthesised Methacrylic Acid Polymers

This study focuses on the use of methacrylic acid polymers synthesised via the Reversible Addition Fragmentation chain Transfer (RAFT) polymerisation method for the production of amorphous solid dispersions (ASDs) by ball milling, to kinetically solubilize a poorly water-soluble model drug. The solid-state characteristics and the physical stability of the formulations were investigated using X-ray diffraction, differential scanning calorimetry, and infrared spectroscopy. This was followed by dissolution studies in different media. It was discovered that the acidic polymers of methacrylic acid were capable of interacting with the weakly basic drug lidocaine and its hydrochloride salt form to produce ASDs when a polymer to drug ratio of 70:30 w/w was used. The ASDs remained amorphous following storage under accelerated aging conditions (40 °C and 75% relative humidity) over 8 months. Fast dissolution and increased lidocaine solubility in different media were obtained from the ASDs owing to the reduced microenvironment pH and enhanced solubilization of the drug caused by the presence of the acidic polymer in the formulation. Production of ASDs using well-defined RAFT-synthesised acidic polymers is a promising formulation strategy to enhance the pharmaceutical properties of basic poorly water-soluble drugs

Coral reef ecosystem services in the Anthropocene

Coral reefs underpin a range of ecosystem goods and services that contribute to the well‐being of millions of people. However, tropical coral reefs in the Anthropocene are likely to be functionally different from reefs in the past. In this perspective piece, we ask, what does the Anthropocene mean for the provision of ecosystem services from coral reefs? First, we provide examples of the provisioning, regulating, cultural and supporting services underpinned by coral reef ecosystems. We conclude that coral reef ecosystem service research has lagged behind multidisciplinary advances in broader ecosystem services science, such as an explicit recognition that interactions between social and ecological systems underpin ecosystem services. Second, drawing on tools from functional ecology, we outline how these social–ecological relationships can be incorporated into a mechanistic understanding of service provision and how this might be used to anticipate future changes in coral reef ecosystem services. Finally, we explore the emergence of novel reef ecosystem services, for example from tropicalized coastlines, or through changing technological connections to coral reefs. Indeed, when services are conceived as coming from social–ecological system dynamics, novelty in services can emerge from elements of the interactions between people and the ecosystem. This synthesis of the coral reef ecosystem services literature suggests the field is poorly prepared to understand the changing service provision anticipated in the Anthropocene. A new research agenda is needed that better connects reef functional ecology to ecosystem service provision. This research agenda should embrace more holistic approaches to ecosystem service research, recognizing them as co‐produced by ecosystems and society. Importantly, the likelihood of novel ecosystem service configurations requires further conceptualization and empirical assessment. As with current ecosystem services, the loss or gain of services will not affect all people equally and must be understood in the context in which they occur. With the uncertainty surrounding the future of coral reefs in the Anthropocene, research exploring how the benefits to people change will be of great importance

Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond

Lifetime imaging microscopy with sub-micron resolution provides essential understanding of living systems by allowing both the visualisation of their structure, and the sensing of bio-relevant analytes in vivo using external probes. Chemistry is pivotal for the development of the next generation of bio-tools, where contrast, sensitivity, and molecular specificity facilitate observation of processes fundamental to life. A fundamental limitation at present is the nanosecond lifetime of conventional fluorescent probes which typically confines the sensitivity to sub-nanosecond changes, whilst nanosecond background autofluorescence compromises the contrast. High-resolution visualization with complete background rejection and simultaneous mapping of bio-relevant analytes including oxygen – with sensitivity orders of magnitude higher than that currently attainable – can be achieved using time-resolved emission imaging microscopy (TREM) in conjunction with probes with microsecond (or longer) lifetimes. Yet the microsecond timescale has so far been incompatible with available multiphoton excitation/detection technologies. Here we realize for the first time microsecond-imaging with multiphoton excitation whilst maintaining the essential sub-micron spatial resolution. The new method is background-free and expands available imaging and sensing timescales 1000-fold. Exploiting the first engineered water-soluble member of a family of remarkably emissive platinum-based, microsecond-lived probes amongst others, we demonstrate (i) the first instance of background-free multiphoton-excited microsecond depth imaging of live cells and histological tissues, (ii) over an order-of-magnitude variation in the probe lifetime in vivo in response to the local microenvironment. The concept of two-photon TREM can be seen as “FLIM + PLIM” as it can be used on any timescale, from ultrafast fluorescence of organic molecules to slower emission of transition metal complexes or lanthanides/actinides, and combinations thereof. It brings together transition metal complexes as versatile emissive probes with the new multiphoton-excitation/microsecond-detection approach to create a transformative framework for multiphoton imaging and sensing across biological, medicinal and material sciences

Local human impacts disrupt relationships between benthic reef assemblages and environmental predictors

Human activities are changing ecosystems at an unprecedented rate, yet large-scale studies into how local human impacts alter natural systems and interact with other aspects of global change are still lacking. Here we provide empirical evidence that local human impacts fundamentally alter relationships between ecological communities and environmental drivers. Using tropical coral reefs as a study system, we investigated the influence of contrasting levels of local human impact using a spatially extensive dataset spanning 62 outer reefs around inhabited Pacific islands. We tested how local human impacts (low versus high determined using a threshold of 25 people km−2 reef) affected benthic community (i) structure, and (ii) relationships with environmental predictors using pre-defined models and model selection tools. Data on reef depth, benthic assemblages, and herbivorous fish communities were collected from field surveys. Additional data on thermal stress, storm exposure, and market gravity (a function of human population size and reef accessibility) were extracted from public repositories. Findings revealed that reefs subject to high local human impact were characterised by relatively more turf algae (>10% higher mean absolute coverage) and lower live coral cover (9% less mean absolute coverage) than reefs subject to low local human impact, but had similar macroalgal cover and coral morphological composition. Models based on spatio-physical predictors were significantly more accurate in explaining the variation of benthic assemblages at sites with low (mean adjusted-R2 = 0.35) rather than high local human impact, where relationships became much weaker (mean adjusted-R2 = 0.10). Model selection procedures also identified a distinct shift in the relative importance of different herbivorous fish functional groups in explaining benthic communities depending on the local human impact level. These results demonstrate that local human impacts alter natural systems and indicate that projecting climate change impacts may be particularly challenging at reefs close to higher human populations, where dependency and pressure on ecosystem services are highest

Recent Progress in Stimuli-Responsive Antimicrobial Electrospun Nanofibers

Electrospun nanofibrous membranes have garnered significant attention in antimicrobial applications, owing to their intricate three-dimensional network that confers an interconnected porous structure, high specific surface area, and tunable physicochemical properties, as well as their notable capacity for loading and sustained release of antimicrobial agents. Tailoring polymer or hybrid-based nanofibrous membranes with stimuli-responsive characteristics further enhances their versatility, enabling them to exhibit broad-spectrum or specific activity against diverse microorganisms. In this review, we elucidate the pivotal advancements achieved in the realm of stimuli-responsive antimicrobial electrospun nanofibers operating by light, temperature, pH, humidity, and electric field, among others. We provide a concise introduction to the strategies employed to design smart electrospun nanofibers with antimicrobial properties. The core section of our review spotlights recent progress in electrospun nanofiber-based systems triggered by single- and multi-stimuli. Within each stimulus category, we explore recent examples of nanofibers based on different polymers and antimicrobial agents. Finally, we delve into the constraints and future directions of stimuli-responsive nanofibrous materials, paving the way for their wider application spectrum and catalyzing progress toward industrial utilization

The fundamental links between climate change and marine plastic pollution

Plastic pollution and climate change have commonly been treated as two separate issues and sometimes are even seen as competing. Here we present an alternative view that these two issues are fundamentally linked. Primarily, we explore how plastic contributes to greenhouse gas (GHG) emissions from the beginning to the end of its life cycle. Secondly, we show that more extreme weather and floods associated with climate change, will exacerbate the spread of plastic in the natural environment. Finally, both issues occur throughout the marine environment, and we show that ecosystems and species can be particularly vulnerable to both, such as coral reefs that face disease spread through plastic pollution and climate-driven increased global bleaching events. A Web of Science search showed climate change and plastic pollution studies in the ocean are often siloed, with only 0.4% of the articles examining both stressors simultaneously. We also identified a lack of regional and industry-specific life cycle analysis data for comparisons in relative GHG contributions by materials and products. Overall, we suggest that rather than debate over the relative importance of climate change or marine plastic pollution, a more productive course would be to determine the linking factors between the two and identify solutions to combat both crises

Fundamental movement skills in relation to weekday and weekend physical activity in preschool children.

OBJECTIVES: To examine associations between fundamental movement skills and weekday and weekend physical activity among preschool children living in deprived communities. DESIGN: Cross-sectional observation study. METHODS: Six locomotor skills and 6 object-control skills were video-assessed using The Children's Activity and Movement in Preschool Study Motor Skills Protocol. Physical activity was measured via hip-mounted accelerometry. A total of 99 children (53% boys) aged 3-5 years (M 4.6, SD 0.5) completed all assessments. Multilevel mixed regression models were used to examine associations between fundamental movement skills and physical activity. Models were adjusted for clustering, age, sex, standardised body mass index and accelerometer wear time. RESULTS: Boys were more active than girls and had higher object-control skill competency. Total skill score was positively associated with weekend moderate-to-vigorous physical activity (p=0.034) but not weekday physical activity categories (p>0.05). When subdomains of skills were examined, object-control skills was positively associated with light physical activity on weekdays (p=0.008) and with light (p=0.033), moderate-to-vigorous (p=0.028) and light- and moderate-to-vigorous (p=0.008) physical activity at weekends. Locomotor skill competency was positively associated with moderate-to-vigorous physical activity on weekdays (p=0.016) and light physical activity during the weekend (p=0.035). CONCLUSIONS: The findings suggest that developing competence in both locomotor and object-control skills may be an important element in promoting an active lifestyle in young children during weekdays and at weekends