The Planemo toolkit for developing, deploying, and executing scientific data analyses in Galaxy and beyond.

There are thousands of well-maintained high-quality open-source software utilities for all aspects of scientific data analysis. For more than a decade, the Galaxy Project has been providing computational infrastructure and a unified user interface for these tools to make them accessible to a wide range of researchers. To streamline the process of integrating tools and constructing workflows as much as possible, we have developed Planemo, a software development kit for tool and workflow developers and Galaxy power users. Here we outline Planemo's implementation and describe its broad range of functionality for designing, testing, and executing Galaxy tools, workflows, and training material. In addition, we discuss the philosophy underlying Galaxy tool and workflow development, and how Planemo encourages the use of development best practices, such as test-driven development, by its users, including those who are not professional software developers.

A blended future? A cross-sectional study demonstrating the impacts of the COVID-19 pandemic on student experiences of well-being, teaching and learning.

INTRODUCTION: The COVID-19 pandemic necessitated emergency changes to teaching, learning and assessment across higher education. Healthcare courses were particularly affected because of their interdependence with overstretched health services. We used this unprecedented situation to provide insight into how students react to unexpected crises and how institutions can most effectively support them. MATERIALS AND METHODS: This cohort study explored students' experiences of the pandemic across programmes and stages from five schools (medicine, dentistry, biomedical sciences, psychology and health professions) in a health faculty in a UK university. We carried out an inductive thematic analysis on the data collected. RESULTS: Many students reported fluctuating emotions and struggled to adapt to home working. Students' changes in motivation and coping strategies varied, many found structure, recreation and social interaction important. Opinions on how well online learning worked relative to face-to-face diverged across programmes. CONCLUSION: A one-size-fits-all blended learning response is unlikely to be appropriate. Our study shows students across one faculty, within one institution, responded diversely to an emergency affecting them all. Educators need to be flexible and dynamic in delivering curricula and supporting students responding to an unexpected crisis during their higher education.

GalaxyCloudRunner: enhancing scalable computing for Galaxy.

SUMMARY: The existence of more than 100 public Galaxy servers with service quotas is indicative of the need for an increased availability of compute resources for Galaxy to use. The GalaxyCloudRunner enables a Galaxy server to easily expand its available compute capacity by sending user jobs to cloud resources. User jobs are routed to the acquired resources based on a set of configurable rules and the resources can be dynamically acquired from any of four popular cloud providers (AWS, Azure, GCP or OpenStack) in an automated fashion. AVAILABILITY AND IMPLEMENTATION: GalaxyCloudRunner is implemented in Python and leverages Docker containers. The source code is MIT licensed and available at https://github.com/cloudve/galaxycloudrunner. The documentation is available at http://gcr.cloudve.org/.

No more business as usual: Agile and effective responses to emerging pathogen threats require open data and open analytics.

The current state of much of the Wuhan pneumonia virus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) research shows a regrettable lack of data sharing and considerable analytical obfuscation. This impedes global research cooperation, which is essential for tackling public health emergencies and requires unimpeded access to data, analysis tools, and computational infrastructure. Here, we show that community efforts in developing open analytical software tools over the past 10 years, combined with national investments into scientific computational infrastructure, can overcome these deficiencies and provide an accessible platform for tackling global health emergencies in an open and transparent manner. Specifically, we use all SARS-CoV-2 genomic data available in the public domain so far to (1) underscore the importance of access to raw data and (2) demonstrate that existing community efforts in curation and deployment of biomedical software can reliably support rapid, reproducible research during global health crises. All our analyses are fully documented at https://github.com/galaxyproject/SARS-CoV-2.

Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa.

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/- KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

The metabolic response to inflammation in astrocytes is regulated by nuclear factor-kappa B signaling.

Inflammation and metabolism are intrinsically linked with inflammatory stimuli inducing metabolic changes in cells and, in turn, metabolic capacity determining cellular inflammatory responses. Although well characterized in peripheral immune cells there is comparatively less known about these "immunometabolic" responses in astrocytes. In this study, we tested the hypothesis that the astrocytic inflammatory response driven by nuclear factor-kappa B (NF-κB) signaling is dependent on glycolytic metabolism. Using mouse primary cortical astrocyte cultures, we assessed changes in cellular metabolism after exposure to lipopolysaccharide (LPS), with cytokine ELISAs and immunoblotting being used to measure inflammatory responses. Results indicate temporally distinct metabolic adaptations to pro-inflammatory stimulation in astrocytes: 3 hr LPS treatment increased glycolysis but did not alter mitochondrial metabolism, while following 24 hr of LPS treatment we observed increased oxidative phosphorylation, and decreased glycolytic capacity and glucose uptake, partly due to reduced glucose transporter 1 expression. Inhibition of NF-κB signaling with the IKK-beta inhibitor TPCA-1 prevented the LPS induced changes to glycolysis and oxidative phosphorylation. Furthermore, TPCA-1 treatment altered both glycolysis and oxidative phosphorylation independently from inflammatory stimulation, indicating a role for NF-κB signaling in regulation of basal metabolism in astrocytes. Inhibition of glycolysis with 2-deoxyglucose significantly attenuated LPS-induced cytokine release and NF-κB phosphorylation, indicating that intact glycolysis is required for the full inflammatory response to LPS. Together our data indicate that astrocytes display immunometabolic responses to acute LPS stimulation which may represent a potential therapeutic target for neuroinflammatory disorders.

The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update.

Galaxy (homepage: https://galaxyproject.org, main public server: https://usegalaxy.org) is a web-based scientific analysis platform used by tens of thousands of scientists across the world to analyze large biomedical datasets such as those found in genomics, proteomics, metabolomics and imaging. Started in 2005, Galaxy continues to focus on three key challenges of data-driven biomedical science: making analyses accessible to all researchers, ensuring analyses are completely reproducible, and making it simple to communicate analyses so that they can be reused and extended. During the last two years, the Galaxy team and the open-source community around Galaxy have made substantial improvements to Galaxy's core framework, user interface, tools, and training materials. Framework and user interface improvements now enable Galaxy to be used for analyzing tens of thousands of datasets, and >5500 tools are now available from the Galaxy ToolShed. The Galaxy community has led an effort to create numerous high-quality tutorials focused on common types of genomic analyses. The Galaxy developer and user communities continue to grow and be integral to Galaxy's development. The number of Galaxy public servers, developers contributing to the Galaxy framework and its tools, and users of the main Galaxy server have all increased substantially.

Basal fatty acid oxidation increases after recurrent low glucose in human primary astrocytes.

AIMS/HYPOTHESIS: Hypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG). METHODS: To test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios. RESULTS: AMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure. CONCLUSIONS/INTERPRETATION: Taken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes.

Jupyter and Galaxy: Easing entry barriers into complex data analyses for biomedical researchers.

What does it take to convert a heap of sequencing data into a publishable result? First, common tools are employed to reduce primary data (sequencing reads) to a form suitable for further analyses (i.e., the list of variable sites). The subsequent exploratory stage is much more ad hoc and requires the development of custom scripts and pipelines, making it problematic for biomedical researchers. Here, we describe a hybrid platform combining common analysis pathways with the ability to explore data interactively. It aims to fully encompass and simplify the "raw data-to-publication" pathway and make it reproducible.

Choline transporter mutations in severe congenital myasthenic syndrome disrupt transporter localization.

The presynaptic, high-affinity choline transporter is a critical determinant of signalling by the neurotransmitter acetylcholine at both central and peripheral cholinergic synapses, including the neuromuscular junction. Here we describe an autosomal recessive presynaptic congenital myasthenic syndrome presenting with a broad clinical phenotype due to homozygous choline transporter missense mutations. The clinical phenotype ranges from the classical presentation of a congenital myasthenic syndrome in one patient (p.Pro210Leu), to severe neurodevelopmental delay with brain atrophy (p.Ser94Arg) and extend the clinical outcomes to a more severe spectrum with infantile lethality (p.Val112Glu). Cells transfected with mutant transporter construct revealed a virtually complete loss of transport activity that was paralleled by a reduction in transporter cell surface expression. Consistent with these findings, studies to determine the impact of gene mutations on the trafficking of the Caenorhabditis elegans choline transporter orthologue revealed deficits in transporter export to axons and nerve terminals. These findings contrast with our previous findings in autosomal dominant distal hereditary motor neuropathy of a dominant-negative frameshift mutation at the C-terminus of choline transporter that was associated with significantly reduced, but not completely abrogated choline transporter function. Together our findings define divergent neuropathological outcomes arising from different classes of choline transporter mutation with distinct disease processes and modes of inheritance. These findings underscore the essential role played by the choline transporter in sustaining acetylcholine neurotransmission at both central and neuromuscular synapses, with important implications for treatment and drug selection.

The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update.

High-throughput data production technologies, particularly 'next-generation' DNA sequencing, have ushered in widespread and disruptive changes to biomedical research. Making sense of the large datasets produced by these technologies requires sophisticated statistical and computational methods, as well as substantial computational power. This has led to an acute crisis in life sciences, as researchers without informatics training attempt to perform computation-dependent analyses. Since 2005, the Galaxy project has worked to address this problem by providing a framework that makes advanced computational tools usable by non experts. Galaxy seeks to make data-intensive research more accessible, transparent and reproducible by providing a Web-based environment in which users can perform computational analyses and have all of the details automatically tracked for later inspection, publication, or reuse. In this report we highlight recently added features enabling biomedical analyses on a large scale.

How does calcium interact with the cytoskeleton to regulate growth cone motility during axon pathfinding?

The precision with which neurons form connections is crucial for the normal development and function of the nervous system. The development of neuronal circuitry in the nervous system is accomplished by axon pathfinding: a process where growth cones guide axons through the embryonic environment to connect with their appropriate synaptic partners to form functional circuits. Despite intense efforts over many years to understand how this process is regulated, the complete repertoire of molecular mechanisms that govern the growth cone cytoskeleton and hence motility, remain unresolved. A central tenet in the axon guidance field is that calcium signals regulate growth cone behaviours such as extension, turning and pausing by regulating rearrangements of the growth cone cytoskeleton. Here, we provide evidence that not only the amplitude of a calcium signal is critical for growth cone motility but also the source of calcium mobilisation. We provide an example of this idea by demonstrating that manipulation of calcium signalling via L-type voltage gated calcium channels can perturb sensory neuron motility towards a source of netrin-1. Understanding how calcium signals can be transduced to initiate cytoskeletal changes represents a significant gap in our current knowledge of the mechanisms that govern axon guidance, and consequently the formation of functional neural circuits in the developing nervous system.

Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin.

Eye movements depend on correct patterns of connectivity between cranial motor axons and the extraocular muscles. Despite the clinical importance of the ocular motor system, little is known of the molecular mechanisms underlying its development. We have recently shown that mutations in the Chimaerin-1 gene encoding the signaling protein α2-chimaerin (α2-chn) perturb axon guidance in the ocular motor system and lead to the human eye movement disorder, Duane retraction syndrome (DRS). The axon guidance cues that lie upstream of α2-chn are unknown; here we identify candidates to be the Semaphorins (Sema) 3A and 3C, acting via the PlexinA receptors. Sema3A/C are expressed in and around the developing extraocular muscles and cause growth cone collapse of oculomotor neurons in vitro. Furthermore, RNAi knockdown of α2-chn or PlexinAs in oculomotor neurons abrogates Sema3A/C-dependent growth cone collapse. In vivo knockdown of endogenous PlexinAs or α2-chn function results in stereotypical oculomotor axon guidance defects, which are reminiscent of DRS, whereas expression of α2-chn gain-of-function constructs can rescue PlexinA loss of function. These data suggest that α2-chn mediates Sema3-PlexinA repellent signaling. We further show that α2-chn is required for oculomotor neurons to respond to CXCL12 and hepatocyte growth factor (HGF), which are growth promoting and chemoattractant during oculomotor axon guidance. α2-chn is therefore a potential integrator of different types of guidance information to orchestrate ocular motor pathfinding. DRS phenotypes can result from incorrect regulation of this signaling pathway.

Community-driven development for computational biology at Sprints, Hackathons and Codefests.

BACKGROUND: Computational biology comprises a wide range of technologies and approaches. Multiple technologies can be combined to create more powerful workflows if the individuals contributing the data or providing tools for its interpretation can find mutual understanding and consensus. Much conversation and joint investigation are required in order to identify and implement the best approaches. Traditionally, scientific conferences feature talks presenting novel technologies or insights, followed up by informal discussions during coffee breaks. In multi-institution collaborations, in order to reach agreement on implementation details or to transfer deeper insights in a technology and practical skills, a representative of one group typically visits the other. However, this does not scale well when the number of technologies or research groups is large. Conferences have responded to this issue by introducing Birds-of-a-Feather (BoF) sessions, which offer an opportunity for individuals with common interests to intensify their interaction. However, parallel BoF sessions often make it hard for participants to join multiple BoFs and find common ground between the different technologies, and BoFs are generally too short to allow time for participants to program together. RESULTS: This report summarises our experience with computational biology Codefests, Hackathons and Sprints, which are interactive developer meetings. They are structured to reduce the limitations of traditional scientific meetings described above by strengthening the interaction among peers and letting the participants determine the schedule and topics. These meetings are commonly run as loosely scheduled "unconferences" (self-organized identification of participants and topics for meetings) over at least two days, with early introductory talks to welcome and organize contributors, followed by intensive collaborative coding sessions. We summarise some prominent achievements of those meetings and describe differences in how these are organised, how their audience is addressed, and their outreach to their respective communities. CONCLUSIONS: Hackathons, Codefests and Sprints share a stimulating atmosphere that encourages participants to jointly brainstorm and tackle problems of shared interest in a self-driven proactive environment, as well as providing an opportunity for new participants to get involved in collaborative projects.

Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM).

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins. In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart. To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.

Drebrin controls neuronal migration through the formation and alignment of the leading process.

Formation of a functional nervous system requires neurons to migrate to the correct place within the developing brain. Tangentially migrating neurons are guided by a leading process which extends towards the target and is followed by the cell body. How environmental cues are coupled to specific cytoskeletal changes to produce and guide leading process growth is unknown. One such cytoskeletal modulator is drebrin, an actin-binding protein known to induce protrusions in many cell types and be important for regulating neuronal morphology. Using the migration of oculomotor neurons as a model, we have shown that drebrin is necessary for the generation and guidance of the leading process. In the absence of drebrin, leading processes are not formed and cells fail to migrate although axon growth and pathfinding appear grossly unaffected. Conversely, when levels of drebrin are elevated the leading processes turn away from their target and as a result the motor neuron cell bodies move along abnormal paths within the brain. The aberrant trajectories were highly reproducible suggesting that drebrin is required to interpret specific guidance cues. The axons and growth cones of these neurons display morphological changes, particularly increased branching and filopodial number but despite this they extend along normal developmental pathways. Collectively these results show that drebrin is initially necessary for the formation of a leading process and subsequently for this to respond to navigational signals and grow in the correct direction. Furthermore, we have shown that the actions of drebrin can be segregated within individual motor neurons to direct their migration independently of axon guidance.

Exploring basic science knowledge retention within a cohort of undergraduate medical students in the United Kingdom: A longitudinal study.

BACKGROUND: Clinical reasoning is reliant on students having acquired a strong foundation in the basic sciences. However, there remains uncertainty regarding whether medical students are maintaining this knowledge over the span of their degrees. Therefore, this project aimed to assess long-term retention of basic science knowledge within a cohort of students from an undergraduate medical school in the United Kingdom (UK). METHODS: This longitudinal study followed a cohort of students, from their first to final year. In their final year, participants sat a bespoke formative basic science knowledge assessment that utilised 46 single-best-answer questions. To examine for long-term attainment differences, these scores were compared with those achieved in first-year assessments. RESULTS: Of the eligible students, 40% partook in the study (n = 22). Comparing assessment scores highlighted an enhancement in overall basic science knowledge between first and final year (p < 0.01). Although most basic science domains remained unchanged between both time points, anatomy and physiology scores increased (p = 0.03 and p = 0.02, respectively), whereas biochemistry scores were the only ones to decrease (p = 0.02). DISCUSSION: This project provides insight into how well students are retaining the basic sciences during their studies. Underperforming science domains were identified, alongside pedagogical explanations for their individual shortcomings; for instance, students' perceived relevance of a domain is seen as a driver for its retention. Subsequently, a group of recommendations were derived to reinforce the most affected domains. The inclusion of more questions on the underperforming sciences, in clinically focussed assessments, is one such suggestion.

Using iRT, a normalized retention time for more targeted measurement of peptides.

Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide-specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT-peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than four times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments.