Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor.

In response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel-Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

Enhancing our understanding of sport practitioner perspectives on developing effective sporting environments.

Research has increasingly focused on the environmental features within talent and performance development settings. However, practitioner perspectives on their role in optimizing these environments are scarce. This study aimed to examine practitioner perspectives of the role of the environment, specifically, how they plan, deliver and review (p-D-R) to optimize environmental conditions for athletes. Ten sports practitioners (including managers, coaches and multidisciplinary support staff) took part in semi-structured interviews. Data was analysed using a reflexive thematic analysis and generated themes associated with Planning (Conceptualization, Planning and Meeting Athlete's Needs), Delivering (Explicit, Implicit, Support, Communication, Holistic Approach) and Reviewing (KPIs, Evaluation & Monitoring, Rolling Review, Review Process). Findings suggest that to offer the best possible experiences to participants, practitioners must have a clear view of their objectives and involve all stakeholders associated with delivery at the planning stage. Much of the delivery aspect aligned with notions of effective TDEs suggesting practitioners had a clear awareness of what works for them in their contexts. Reviewing the environment appeared to be the activity practitioners undertook the least, this may reflect the complex and dynamic nature of the environment in sports settings.

The effect of face mask wearing on language processing and emotion recognition in young children.

Face mask wearing was an important preventative strategy for the transmission of the COVID-19 virus. However, the effects that occluding the mouth and nose area with surgical masks could have on young children's language processing and emotion recognition skills have received little investigation. To evaluate the possible effects, the current study recruited a sample of 74 children from the North West of England (aged 4-8 years). They completed two computer-based tasks with adults wearing or not wearing surgical face masks to assess (a) language processing skills and (b) emotion recognition ability. To control for individual differences, age, sex, receptive vocabulary, early reading skills, and parent-reported social-emotional competence were included in analyses. The findings from the study highlighted that although younger children were less accurate than older children, face masks did not significantly impair basic language processing ability. However, they had a significant effect on the children's emotion recognition accuracy-with masked angry faces more easily recognized and masked happy and sad faces less easily recognized. Children's age and social-emotional skills also played a role. The findings suggest that the effects of face masks should continue to be evaluated.

Dynamic trafficking and turnover of JAM-C is essential for endothelial cell migration.

Junctional complexes between endothelial cells form a dynamic barrier that hinders passive diffusion of blood constituents into interstitial tissues. Remodelling of junctions is an essential process during leukocyte trafficking, vascular permeability, and angiogenesis. However, for many junctional proteins, the mechanisms of junctional remodelling have yet to be determined. Here, we used receptor mutagenesis, horseradish peroxidase (HRP), and ascorbate peroxidase 2 (APEX-2) proximity labelling, alongside light and electron microscopy (EM), to map the intracellular trafficking routes of junctional adhesion molecule-C (JAM-C). We found that JAM-C cotraffics with receptors associated with changes in permeability such as vascular endothelial cadherin (VE-Cadherin) and neuropilin (NRP)-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic JAM-C trafficking and degradation are necessary for junctional remodelling during cell migration and angiogenesis. By identifying new potential trafficking machinery, we show that a key point of regulation is the ubiquitylation of JAM-C by the E3 ligase Casitas B-lineage lymphoma (CBL), which controls the rate of trafficking versus lysosomal degradation.

The lexical semantics of adjective-noun phrases in the human brain.

As a person reads, the brain performs complex operations to create higher order semantic representations from individual words. While these steps are effortless for competent readers, we are only beginning to understand how the brain performs these actions. Here, we explore lexical semantics using magnetoencephalography (MEG) recordings of people reading adjective-noun phrases presented one word at a time. We track the neural representation of single word representations over time, through different brain regions. Our results reveal two novel findings: (a) a neural representation of the adjective is present during noun presentation, but this representation is different from that observed during adjective presentation and (b) the neural representation of adjective semantics observed during adjective reading is reactivated after phrase reading, with remarkable consistency. We also note that while the semantic representation of the adjective during the reading of the adjective is very distributed, the later representations are concentrated largely to temporal and frontal areas previously associated with composition. Taken together, these results paint a picture of information flow in the brain as phrases are read and understood.

North Carolina College of Emergency Physicians' Guidance Document on Emergency Medical Services.

The North Carolina College of Emergency Physicians (NCCEP) Emergency Medical Services (EMS) Committee uses an evidence-based approach in writing its protocols and procedures. The most recent revision of the NCCEP document, which was started in late 2010, lasted for more than 1 year and utilized committee members from across the state. Four meetings were held at locations across North Carolina. In addition, 2 surveys were sent to get input from EMS providers. Since 2010, the document has been updated on an ongoing basis, aligning it with the latest evidence-based medicine.

Multivariate analysis of correlation between electrophysiological and hemodynamic responses during cognitive processing.

Animal and human studies have frequently shown that in primary sensory and motor regions the BOLD signal correlates positively with high-frequency and negatively with low-frequency neuronal activity. However, recent evidence suggests that this relationship may also vary across cortical areas. Detailed knowledge of the possible spectral diversity between electrophysiological and hemodynamic responses across the human cortex would be essential for neural-level interpretation of fMRI data and for informative multimodal combination of electromagnetic and hemodynamic imaging data, especially in cognitive tasks. We applied multivariate partial least squares correlation analysis to MEG-fMRI data recorded in a reading paradigm to determine the correlation patterns between the data types, at once, across the cortex. Our results revealed heterogeneous patterns of high-frequency correlation between MEG and fMRI responses, with marked dissociation between lower and higher order cortical regions. The low-frequency range showed substantial variance, with negative and positive correlations manifesting at different frequencies across cortical regions. These findings demonstrate the complexity of the neurophysiological counterparts of hemodynamic fluctuations in cognitive processing.

Exploring athletic identity in elite-level English youth football: a cross-sectional approach.

This study is the first empirical investigation that has explored levels of athletic identity in elite-level English professional football. The importance of understanding athletes' psychological well-being within professional sport has been well documented. This is especially important within the professional football industry, given the high attrition rate (Anderson, G., & Miller, R. M. (2011). The academy system in English professional football: Business value or following the herd? University of Liverpool, Management School Research Paper Series. Retrieved from http://www.liv.ac.uk/managementschool/research/working%20papers/wp201143.pdf ) and distinct occupational practices (Roderick, M. (2006). The work of professional football. A labour of love? London: Routledge). A total of 168 elite youth footballers from the English professional football leagues completed the Athletic Identity Measurement Scale (AIMS). Multilevel modelling was used to examine the effect of playing level, living arrangements and year of apprentice on the total AIMS score and its subscales (i.e., social identity, exclusivity and negative affectivity). Football club explained 30% of the variance in exclusivity among players (P = .022). Mean social identity was significantly higher for those players in the first year of their apprenticeship compared to the second year (P = .025). All other effects were not statistically significant (P > .05). The novel and unique findings have practical implications in the design and implementation of career support strategies with respect to social identity. This may facilitate the maintenance of motivation over a 2-year apprenticeship and positively impact on performance levels within the professional football environment.

Myosin-1C augments endothelial secretion of von Willebrand factor by linking contractile actomyosin machinery to the plasma membrane.

ABSTRACT: Blood endothelial cells control the hemostatic and inflammatory response by secreting von Willebrand factor (VWF) and P-selectin from storage organelles called Weibel-Palade bodies (WPBs). Actin-associated motor proteins regulate this secretory pathway at multiple points. Before fusion, myosin Va forms a complex that anchors WPBs to peripheral actin structures, allowing for the maturation of content. After fusion, an actomyosin ring/coat is recruited and compresses the WPB to forcibly expel the largest VWF multimers. Here, we provide, to our knowledge, the first evidence for the involvement of class I myosins during regulated VWF secretion. We show that the unconventional myosin-1C (Myo1c) is recruited after fusion via its pleckstrin homology domain in an actin-independent process. This provides a link between the actin ring and phosphatidylinositol 4,5-bisphosphate (PIP2) at the membrane of the fused organelle and is necessary to ensure maximal VWF secretion. This is an active process requiring Myo1c ATPase activity because inhibition of class I myosins using the inhibitor pentachloropseudilin or expression of an ATPase-deficient Myo1c rigor mutant perturbs the expulsion of VWF and alters the kinetics of the exocytic actin ring. These data offer a novel insight into the control of an essential physiological process and provide a new way in which it can be regulated.

Clinical Implications of Basic Research: Exploring the Transformative Potential of Spatial 'Omics in Uro-oncology.

New spatial molecular technologies are poised to transform our understanding and treatment of urological cancers. By mapping the spatial molecular architecture of tumours, these platforms uncover the complex heterogeneity within and around individual malignancies, offering novel insights into disease development, progression, diagnosis, and treatment. They enable tracking of clonal phylogenetics in situ and immune-cell interactions in the tumour microenvironment. A whole transcriptome/genome/proteome-level spatial analysis is hypothesis generating, particularly in the areas of risk stratification and precision medicine. Current challenges include reagent costs, harmonisation of protocols, and computational demands. Nonetheless, the evolving landscape of the technology and evolving machine learning applications have the potential to overcome these barriers, pushing towards a future of personalised cancer therapy, leveraging detailed spatial cellular and molecular data. PATIENT SUMMARY: Tumours are complex and contain many different components. Although we have been able to observe some of these differences visually under the microscope, until recently, we have not been able to observe the genetic changes that underpin cancer development. Scientists are now able to explore molecular/genetic differences using approaches such as "spatial transcriptomics" and "spatial proteomics", which allow them to see genetic and cellular variation across a region of normal and cancerous tissue without destroying the tissue architecture. Currently, these technologies are limited by high associated costs, and a need for powerful and complex computational analysis workflows. Future advancements and results through these new technologies may assist patients and their doctors as they make decisions about treating their cancer.

Downregulation of androgen receptor transcription by promoter g-quadruplex stabilization as a potential alternative treatment for castrate-resistant prostate cancer.

Androgen receptor (AR) signaling remains an important regulatory pathway in castrate-resistant prostate cancer, and its transcriptional downregulation could provide a new line of therapy. A number of small-molecule ligands have previously demonstrated the ability to stabilize G-quadruplex structures and affect gene transcription for those genes whose promoters contain a quadruplex-forming sequence. Herein, we report the probable formation of new G-quadruplex structure present in the AR promoter in a transcriptionally important location. NMR spectroscopy, circular dichroism, UV spectroscopy, and UV thermal melting experiments for this sequence are consistent with G-quadruplex formation. Fluorescence resonance energy transfer (FRET) melting studies have identified a novel compound, MM45, which appears to stabilize this G-quadruplex at submicromolar concentrations. The effects of MM45 have been investigated in prostate cancer cell lines where it has been shown to inhibit cell growth. A reporter assay intended to isolate the effect of MM45 on the G-quadruplex sequence showed dose-dependent transcriptional repression only when the AR promoter G-quadruplex sequence is present. Dose-dependent transcriptional repression of the AR by MM45 has been demonstrated at both a protein and mRNA level. This proof of concept study paves the route toward a potential alternative treatment pathway in castrate-resistant prostate cancer.

Neuropilin-1 interacts with VE-cadherin and TGFBR2 to stabilize adherens junctions and prevent activation of endothelium under flow.

Linear and disturbed flow differentially regulate gene expression, with disturbed flow priming endothelial cells (ECs) for a proinflammatory, atheroprone expression profile and phenotype. Here, we investigated the role of the transmembrane protein neuropilin-1 (NRP1) in ECs exposed to flow using cultured ECs, mice with an endothelium-specific knockout of NRP1, and a mouse model of atherosclerosis. We demonstrated that NRP1 was a constituent of adherens junctions that interacted with VE-cadherin and promoted its association with p120 catenin, stabilizing adherens junctions and inducing cytoskeletal remodeling in alignment with the direction of flow. We also showed that NRP1 interacted with transforming growth factor-ß (TGF-ß) receptor II (TGFBR2) and reduced the plasma membrane localization of TGFBR2 and TGF-ß signaling. NRP1 knockdown increased the abundance of proinflammatory cytokines and adhesion molecules, resulting in increased leukocyte rolling and atherosclerotic plaque size. These findings describe a role for NRP1 in promoting endothelial function and reveal a mechanism by which NRP1 reduction in ECs may contribute to vascular disease by modulating adherens junction signaling and promoting TGF-ß signaling and inflammation.

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.

Computational tools addressing various components of design-build-test-learn (DBTL) loops for the construction of synthetic genetic networks exist but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called Design Assemble Round Trip (DART). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions. Graphical Abstract.

Teaching open and reproducible scholarship: a critical review of the evidence base for current pedagogical methods and their outcomes.

In recent years, the scientific community has called for improvements in the credibility, robustness and reproducibility of research, characterized by increased interest and promotion of open and transparent research practices. While progress has been positive, there is a lack of consideration about how this approach can be embedded into undergraduate and postgraduate research training. Specifically, a critical overview of the literature which investigates how integrating open and reproducible science may influence student outcomes is needed. In this paper, we provide the first critical review of literature surrounding the integration of open and reproducible scholarship into teaching and learning and its associated outcomes in students. Our review highlighted how embedding open and reproducible scholarship appears to be associated with (i) students' scientific literacies (i.e. students' understanding of open research, consumption of science and the development of transferable skills); (ii) student engagement (i.e. motivation and engagement with learning, collaboration and engagement in open research) and (iii) students' attitudes towards science (i.e. trust in science and confidence in research findings). However, our review also identified a need for more robust and rigorous methods within pedagogical research, including more interventional and experimental evaluations of teaching practice. We discuss implications for teaching and learning scholarship.

Tracking neural coding of perceptual and semantic features of concrete nouns.

We present a methodological approach employing magnetoencephalography (MEG) and machine learning techniques to investigate the flow of perceptual and semantic information decodable from neural activity in the half second during which the brain comprehends the meaning of a concrete noun. Important information about the cortical location of neural activity related to the representation of nouns in the human brain has been revealed by past studies using fMRI. However, the temporal sequence of processing from sensory input to concept comprehension remains unclear, in part because of the poor time resolution provided by fMRI. In this study, subjects answered 20 questions (e.g. is it alive?) about the properties of 60 different nouns prompted by simultaneous presentation of a pictured item and its written name. Our results show that the neural activity observed with MEG encodes a variety of perceptual and semantic features of stimuli at different times relative to stimulus onset, and in different cortical locations. By decoding these features, our MEG-based classifier was able to reliably distinguish between two different concrete nouns that it had never seen before. The results demonstrate that there are clear differences between the time course of the magnitude of MEG activity and that of decodable semantic information. Perceptual features were decoded from MEG activity earlier in time than semantic features, and features related to animacy, size, and manipulability were decoded consistently across subjects. We also observed that regions commonly associated with semantic processing in the fMRI literature may not show high decoding results in MEG. We believe that this type of approach and the accompanying machine learning methods can form the basis for further modeling of the flow of neural information during language processing and a variety of other cognitive processes.

Exploring commonalities across participants in the neural representation of objects.

The question of whether the neural encodings of objects are similar across different people is one of the key questions in cognitive neuroscience. This article examines the commonalities in the internal representation of objects, as measured with fMRI, across individuals in two complementary ways. First, we examine the commonalities in the internal representation of objects across people at the level of interobject distances, derived from whole brain fMRI data, and second, at the level of spatially localized anatomical brain regions that contain sufficient information for identification of object categories, without making the assumption that their voxel patterns are spatially matched in a common space. We examine the commonalities in internal representation of objects on 3T fMRI data collected while participants viewed line drawings depicting various tools and dwellings. This exploratory study revealed the extent to which the representation of individual concepts, and their mutual similarity, is shared across participants.

Erratum: Gender bias, social bias, and representation in Bollywood and Hollywood.

[This corrects the article DOI: 10.1016/j.patter.2022.100442.][This corrects the article DOI: 10.1016/j.patter.2021.100409.].

pySBOL3: SBOL3 for Python Programmers.

The Synthetic Biology Open Language version 3 (SBOL3) provides a data model for representation of synthetic biology information across multiple scales and throughout the design-build-test-learn workflow. To support practical use of this data model, we have developed pySBOL3, a Python library that allows programmers to create and edit SBOL3 documents. Here we describe this library and key engineering decisions in its design. The resulting implementation is a compact and maintainable core that provides both a familiar, pythonic interface for manipulating SBOL3 objects as well as mechanisms for building additional extensions and representations on this base.