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.

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.

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.

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.

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.

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.

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.

Highly-automated, high-throughput replication of yeast-based logic circuit design assessments.

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of Saccharomyces cerevisiae by Gander et al. Our experimental campaign used a novel high-throughput experimentation framework developed under Defense Advanced Research Projects Agency's Synergistic Discovery and Design program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report and make some remarks about lessons learned. Graphical Abstract.

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.

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.].

Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence.

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

Gender bias, social bias, and representation: 70 years of B H ollywood.

We use a suite of cutting-edge natural language processing methods to quantify and characterize societal and gender biases in popular movie content. Our data set consists of English subtitles of popular movies from Bollywood-the Mumbai film industry-spanning 7 decades (700 movies). In addition, we include movies from Hollywood and movies nominated for the Academy Awards for contrastive purposes. Our findings indicate that while the overall portrayal of women has improved over time in popular movie dialogues from both Bollywood and Hollywood, modern films still exhibit considerable gender bias and are yet to achieve equal representation among genders. We also observe a strong bias favoring fair skin color in Bollywood content that occurred consistently across all time periods we considered. While our geographic representation analysis indicates improved inclusion over time for several Indian states, it also reveals a long-standing under-representation of many northeastern Indian states.

Erratum: Gender bias, social bias, and representation: 70 years of B H ollywood.

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

Combining computational controls with natural text reveals aspects of meaning composition.

To study a core component of human intelligence-our ability to combine the meaning of words-neuroscientists have looked to linguistics. However, linguistic theories are insufficient to account for all brain responses reflecting linguistic composition. In contrast, we adopt a data-driven approach to study the composed meaning of words beyond their individual meaning, which we term 'supra-word meaning'. We construct a computational representation for supra-word meaning and study its brain basis through brain recordings from two complementary imaging modalities. Using functional magnetic resonance imaging, we reveal that hubs that are thought to process lexical meaning also maintain supra-word meaning, suggesting a common substrate for lexical and combinatorial semantics. Surprisingly, we cannot detect supra-word meaning in magnetoencephalography, which suggests that composed meaning might be maintained through a different neural mechanism than the synchronized firing of pyramidal cells. This sensitivity difference has implications for past neuroimaging results and future wearable neurotechnology.

Assessing the Role of Gaze Tracking in Optimizing Humans-In-The-Loop Telerobotic Operation Using Multimodal Feedback.

A key challenge in achieving effective robot teleoperation is minimizing teleoperators' cognitive workload and fatigue. We set out to investigate the extent to which gaze tracking data can reveal how teleoperators interact with a system. In this study, we present an analysis of gaze tracking, captured as participants completed a multi-stage task: grasping and emptying the contents of a jar into a container. The task was repeated with different combinations of visual, haptic, and verbal feedback. Our aim was to determine if teleoperation workload can be inferred by combining the gaze duration, fixation count, task completion time, and complexity of robot motion (measured as the sum of robot joint steps) at different stages of the task. Visual information of the robot workspace was captured using four cameras, positioned to capture the robot workspace from different angles. These camera views (aerial, right, eye-level, and left) were displayed through four quadrants (top-left, top-right, bottom-left, and bottom-right quadrants) of participants' video feedback computer screen, respectively. We found that the gaze duration and the fixation count were highly dependent on the stage of the task and the feedback scenario utilized. The results revealed that combining feedback modalities reduced the cognitive workload (inferred by investigating the correlation between gaze duration, fixation count, task completion time, success or failure of task completion, and robot gripper trajectories), particularly in the task stages that require more precision. There was a significant positive correlation between gaze duration and complexity of robot joint movements. Participants' gaze outside the areas of interest (distractions) was not influenced by feedback scenarios. A learning effect was observed in the use of the controller for all participants as they repeated the task with different feedback combination scenarios. To design a system for teleoperation, applicable in healthcare, we found that the analysis of teleoperators' gaze can help understand how teleoperators interact with the system, hence making it possible to develop the system from the teleoperators' stand point.

Engineered yeast genomes accurately assembled from pure and mixed samples.

Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow-it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering.

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.