Discovery of Latent Cannabichromene Cyclase Activity in Marine Bacterial Flavoenzymes.

Production of phytocannabinoids remains an area of active scientific interest due to the growing use of cannabis by the public and the underexplored therapeutic potential of the over 100 minor cannabinoids. While phytocannabinoids are biosynthesized by Cannabis sativa and other select plants and fungi, structural analogs and stereoisomers can only be accessed synthetically or through heterologous expression. To date, the bioproduction of cannabinoids has required eukaryotic hosts like yeast since key, native oxidative cyclization enzymes do not express well in bacterial hosts. Here, we report that two marine bacterial flavoenzymes, Clz9 and Tcz9, perform oxidative cyclization reactions on phytocannabinoid precursors to efficiently generate cannabichromene scaffolds. Furthermore, Clz9 and Tcz9 express robustly in bacteria and display significant tolerance to organic solvent and high substrate loading, thereby enabling fermentative production of cannabichromenic acid in Escherichia coli and indicating their potential for biocatalyst development.

Cross-sectional and longitudinal associations between higher weight and eating disorder risk among collegiate athletes.

OBJECTIVES: To examine whether higher weight status is associated with increased eating disorder risk in athletes. DESIGN: Study 1 used cross-sectional (N = 942) and longitudinal (N = 483) data collected over a five-year period from Division I athletes at a Midwestern university. Study 2 used cross-sectional data from athletes competing at various levels at universities across the United States (N = 825). METHODS: Weight and height were measured in Study 1 and self-reported in Study 2; higher weight was defined as body mass index ≥ 25 kg/m2. Eating disorder risk was assessed with preliminary (Study 1) and final (Study 2) versions of the Eating Disorders Screen for Athletes. Study 1 examined weight status in relation to both concurrent and one-year changes in Eating Disorder Screen for Athletes score. Study 2 examined weight status in relation to both the Eating Disorders Screen for Athletes score and elevated eating disorder risk using the established Eating Disorders Screen for Athletes cut-off. All models adjusted for gender and sport type; Study 2 additionally adjusted for race/ethnicity, age, and competition level. RESULTS: Across studies, higher weight status was significantly associated with increased eating disorder risk among athletes. This association was observed both cross-sectionally and longitudinally, and the prevelence of elevated eating disorder risk was 43 % greater among athletes with higher weight status than among those without higher weight status. CONCLUSIONS: Despite existing stereotypes that eating disorders tend to occur with low weight status, findings highlight the importance of screening all athletes for eating disorders, regardless of their weight status.

Caged luciferins enable rapid multicomponent bioluminescence imaging.

Bioluminescence is a sensitive technique for imaging biological features over time. Historically, though, the modality has been challenging to employ for multiplexed tracking due to a lack of resolvable luciferase-luciferin pairs. Recent years have seen the development of numerous orthogonal probes for multi-parameter imaging. While successful, generating such tools often requires complex syntheses and lengthy enzyme evolution campaigns. This work showcases an alternative strategy for multiplexed bioluminescence that takes advantage of already-orthogonal caged luciferins and established uncaging enzymes. These probes generate unique bioluminescent signals that can be distinguished via a linear unmixing algorithm. Caged luciferins enabled two- and three-component imaging on the minutes time scale. We further showed that the tools can be used in conjunction with endogenous enzymes for multiplexed studies. Collectively, this approach lowers the barrier to multicomponent bioluminescence imaging and can be readily adopted by the broader community.

Expedient Synthesis and Characterization of π-Extended Luciferins.

Bioluminescence imaging enables the sensitive tracking of cell populations and the visualization of biological processes in living systems. Bioluminescent luciferase/luciferin pairs with far-red and near-infrared emission benefit from the reduced competitive absorption by blood and tissue while also facilitating multiplexing strategies. Luciferins with extended π-systems, such as AkaLumine and recently reported CouLuc-1 and -3, can be used for bioluminescence imaging in this long wavelength regime. Existing synthetic routes to AkaLumine and similar π-extended compounds require a multistep sequence to install the thiazoline heterocycle. Here we detail the development of a two-step strategy for accessing these molecules via a Horner-Wadsworth-Emmons reaction and cysteine condensation sequence from readily available aldehyde starting materials. We detail an improved synthesis of AkaLumine, as well as the corresponding two-carbon homologues, Tri- and Tetra-AkaLumine. We then extended this approach to prepare coumarin- and naphthalene-derived luciferins. These putative luciferins were tested against a panel of luciferases to identify capable emitters. Of these, an easily prepared naphthalene derivative exhibits photon emission on par with that of the broadly used Akaluc/AkaLumine pair with similar emission maxima. Overall, this chemistry provides efficient access to several bioluminescent probes for a variety of imaging applications.

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging.

Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.

Longitudinal monitoring of individual infection progression in Drosophila melanogaster.

The innate immune system is critical for infection survival. Drosophila melanogaster is a key model for understanding the evolution and dynamics of innate immunity. Current toolsets for fly infection studies are limited in throughput and, because of their destructive nature, cannot generate longitudinal measurements in individual animals. We report a bioluminescent imaging strategy enabling non-invasive characterization of pathogen load. By using Escherichia coli expressing the ilux operon, we demonstrate that photon flux from autobioluminescent bacteria can be used to monitor pathogen loads in individual, living flies. Because animal sacrifice is not necessary to estimate pathogen load, stochastic responses to infection can be characterized in individuals over time. The high temporal resolution of bioluminescence imaging enables visualization of the dynamics of microbial clearance on the hours time-scale. This non-invasive imaging strategy provides a simple and scalable platform to observe changes in pathogen load in vivo over time.

Fluorogenic Cyclopropenones for Multicomponent, Real-Time Imaging.

Fluorogenic bioorthogonal reactions enable biomolecule visualization in real time. These reactions comprise reporters that "light up" upon reaction with complementary partners. While the spectrum of fluorogenic chemistries is expanding, few transformations are compatible with live cells due to cross-reactivities or insufficient signal turn-on. To address the need for more suitable chemistries for cellular imaging, we developed a fluorogenic reaction featuring cyclopropenone reporters and phosphines. The transformation involves regioselective activation and cyclization of cyclopropenones to form coumarin products. With optimal probes, the reaction provides >1600-fold signal turn-on, one of the highest fluorescence enhancements reported to date. The bioorthogonal motifs were evaluated in vitro and in cells. The reaction was also found to be compatible with other common fluorogenic transformations, enabling multicomponent, real-time imaging. Collectively, these data suggest that the cyclopropenone-phosphine reaction will bolster efforts to track biomolecule targets in their native settings.

Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging.

Multi-component bioluminescence imaging requires an expanded collection of luciferase-luciferin pairs that emit far-red or near-infrared light. Toward this end, we prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multi-component imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional fluorophore-derived probes for multiplexed imaging.

Caged Cumate Enables Proximity-Dependent Control Over Gene Expression.

Cell-cell interactions underlie diverse physiological processes yet remain challenging to examine with conventional imaging tools. Here we report a novel strategy to illuminate cell proximity using transcriptional activators. We repurposed cumate, a small molecule inducer of gene expression, by caging its key carboxylate group with a nitrile. Nitrilase-expressing activator cells released the cage, liberating cumate for consumption by reporter cells. Reporter cells comprising a cumate-responsive switch expressed a target gene when in close proximity to the activator cells. Overall, this strategy provides a versatile platform to image and potentially manipulate cellular interactions over time.

Seeing (and Using) the Light: Recent Developments in Bioluminescence Technology.

Bioluminescence has long been used to image biological processes in vivo. This technology features luciferase enzymes and luciferin small molecules that produce visible light. Bioluminescent photons can be detected in tissues and live organisms, enabling sensitive and noninvasive readouts on physiological function. Traditional applications have focused on tracking cells and gene expression patterns, but new probes are pushing the frontiers of what can be visualized. The past few years have also seen the merger of bioluminescence with optogenetic platforms. Luciferase-luciferin reactions can drive light-activatable proteins, ultimately triggering signal transduction and other downstream events. This review highlights these and other recent advances in bioluminescence technology, with an emphasis on tool development. We showcase how new luciferins and engineered luciferases are expanding the scope of optical imaging. We also highlight how bioluminescent systems are being leveraged not just for sensing-but also controlling-biological processes.

Vangl2-dependent regulation of membrane protrusions and directed migration requires a fibronectin extracellular matrix.

During zebrafish gastrulation the planar cell polarity (PCP) protein Vang-like 2 (Vangl2) regulates the polarization of cells that are engaged in directed migration. However, it is unclear whether Vangl2 influences membrane-protrusive activities in migrating gastrula cells and whether these processes require the fibronectin extracellular matrix. Here, we report that Vangl2 modulates the formation and polarization of actin-rich filopodia-like and large lamellipodia-like protrusions in ectodermal cells. By contrast, disrupted Glypican4/PCP signaling affects protrusion polarity but not protrusion number or directed migration. Analysis of fluorescent fusion protein expression suggests that there is widespread Vangl2 symmetry in migrating cells, but there is enrichment at membrane domains that are developing large protrusions compared with non-protrusive domains. We show that the fibronectin extracellular matrix is essential for cell-surface Vangl2 expression, membrane-protrusive activity and directed migration. Manipulation of fibronectin protein levels rescues protrusion and directed migration phenotypes in vangl2 mutant embryos, but it is not sufficient to restore either PCP, or convergence and extension movements. Together, our findings identify distinct roles for Vangl2 and Glypican4/PCP signaling during membrane protrusion formation and demonstrate that cell-matrix interactions underlie Vangl2-dependent regulation of protrusive activities in migrating gastrula cells.

Communications Technology and Motor Neuron Disease: An Australian Survey of People With Motor Neuron Disease.

BACKGROUND: People with Motor Neuron Disease (MND), of which amyotrophic lateral sclerosis (ALS) is the most common form in adults, typically experience difficulties with communication and disabilities associated with movement. Assistive technology is essential to facilitate everyday activities, promote social support and enhance quality of life. OBJECTIVE: This study aimed to explore the types of mainstream and commonly available communication technology used by people with MND including software and hardware, to identify the levels of confidence and skill that people with MND reported in using technology, to determine perceived barriers to the use of technology for communication, and to investigate the willingness of people with MND to adopt alternative modes of communication. METHODS: An on-line survey was distributed to members of the New South Wales Motor Neuron Disease Association (MND NSW). Descriptive techniques were used to summarize frequencies of responses and cross tabulate data. Free-text responses to survey items and verbal comments from participants who chose to undertake the survey by telephone were analyzed using thematic analysis. RESULTS: Responses from 79 MND NSW members indicated that 15-21% had difficulty with speaking, writing and/or using a keyboard. Commonly used devices were desktop computers, laptops, tablets and mobile phones. Most participants (84%) were connected to the Internet and used it for email (91%), to find out more about MND (59%), to follow the news (50%) or for on-line shopping (46%). A third of respondents used Skype or its equivalent, but few used this to interact with health professionals. CONCLUSIONS: People with MND need greater awareness of technology options to access the most appropriate solutions. The timing for people with MND to make decisions about technology is critical. Health professionals need skills and knowledge about the application of technology to be able to work with people with MND to select the best communication technology options as early as possible after diagnosis. If people with MND are willing to trial telehealth technology, there is potential for tele-consultations via Skype or its equivalent, with health professionals. People with MND can benefit from health professional involvement to match technology to their functional limitations and personal preferences. However, health professionals need a comprehensive understanding of the application of available technology to achieve this.

Spatiotemporal ablation of CXCR2 on oligodendrocyte lineage cells: Role in myelin repair.

BACKGROUND: Residual CXCR2 expression on CNS cells in Cxcr2 (+/-) →Cxcr2 (-/-) chimeric animals slowed remyelination after both experimental autoimmune encephalomyelitis and cuprizone-induced demyelination. METHODS: We generated Cxcr2 (fl/-) :PLPCre-ER(T) mice enabling an inducible, conditional deletion of Cxcr2 on oligodendrocyte lineage cells of the CNS. Cxcr2 (fl/-) :PLPCre-ER(T) mice were evaluated in 2 demyelination/remyelination models: cuprizone-feeding and in vitro lysophosphatidylcholine (LPC) treatment of cerebellar slice cultures. RESULTS: Cxcr2 (fl/-) :PLPCre-ER(T)(+) (termed Cxcr2-cKO) mice showed better myelin repair 4 days after LPC-induced demyelination of cerebellar slice cultures. Cxcr2-cKOs also displayed enhanced hippocampal remyelination after a 2-week recovery from 6-week cuprizone feeding. CONCLUSION: Using 2 independent demyelination/remyelination models, our data document enhanced myelin repair in Cxcr2-cKO mice, consistent with the data obtained from radiation chimerism studies of germline CXCR2. Further experiments are appropriate to explore how CXCR2 function in the oligodendrocyte lineage accelerates myelin repair.

Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

The ability of the Blood Brain Barrier (BBB) to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS) activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS): fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF) that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

Implications of multiple-choice testing in nursing education.

The evaluation of knowledge/competence is understood as an essential component of nursing education and practice. As such, nurse educators have a plethora of existing evaluation strategies from which to choose. A common written evaluative format used across all higher education settings is multiple-choice testing. This evaluation approach is accepted as a 'user-friendly' strategy to assess knowledge. Researchers from the disciplines of psychology and education have long been concerned with the consequences of multiple-choice testing on learning outcomes, a discussion that is essentially absent from the nursing literature. The purpose of this paper is to address the professional implications of multiple-choice testing in nursing. The potential knowledge consequences for nurse-learners, and by extension the provision of care to healthcare recipients, resultant from use of this testing modality are addressed within the context of the implementation of best practice guidelines in a long-term care home in a mid-sized rural and northern Canadian community with both regulated and non-regulated care providers.