Defining Protein Synthesis: New Technologies to Elucidate Translational Control

Protein translation has emerged as an important mediator of cellular activity. Here, we discuss efforts to develop and apply new technologies designed to gain insights into translational control. We begin with the application of ribosome profiling to a RiboTag Glioma mouse model which enables translational profiling of transformed cellular populations. This approach demonstrates a number of abnormalities of translation in transformed cells. We go on to report the development of an inexpensive and rapid library preparation methodology which enables high-throughput sequencing of ribosome-protected footprints from small amounts of input material. We apply this technique to a CAMKII RiboTag mouse model to make new insights into cell-type specific translation. Finally, we describe efforts to investigate translation regulatory networks through the development of a technique which couples large-scale perturbation with a genome-wide readout of translation. Molecular dissection of tissues through the ectopic expression of modified ribosomal proteins commonly relies on tissue-specific genes which act as drivers. In the case of glioma, a gene specific to transformed tissue, but not expressed in normal brain tissue, has not been identified. Chapter 2 focuses on efforts to bypass this through the development of a RiboTag Glioma mouse model which allows for concurrent transformation and the expression of an epitope-tagged ribosomal protein in virally infected cells. This model made possible the isolation of translating mRNA from transformed cellular populations and was used to demonstrate the existence of a number of translational abnormalities in transformed cells. Conventional ribosome profiling is a powerful tool which allows for the identification of ribosome-protected mRNA footprints. However, it is time-consuming, expensive, and difficult to implement. Based on our experiences with conventional ribosome profiling, we sought to develop a method which could decrease the overall number of enzymatic reactions and purification steps, thereby reducing the time and cost associated with the procedure; these efforts are discussed in Chapter 3. Utilizing a ligation-free library preparation process, which incorporates poly(A)-polymerase, template switching and bead-based purification, we reduced the time, costs and input requirements required to generate a ribosome profiling library while maintaining high library complexity. We applied our ligation-free ribosome profiling technique to a CAMKII RiboTag mouse model which enabled us to identify patterns of cell-type specific translation and the effects of mTOR inhibition in CAMKII-expressing excitatory neurons. Regulation of protein expression is an essential and highly complex cellular activity. Aberrations of translational control are central to a host of pathologies and have direct clinical relevance. However, our knowledge of the networks which control translation is limited. Chapter 4 details our efforts to develop a highly-scalable technology which enables the identification of gene-specific translational alteration in response to perturbation. Coupled with a large-scale perturbation screen, this technique could lead to the generation of a network for translational control, similar to efforts previously undertaken to understand transcriptional control. By combining the recently developed PLATE-Seq method, which utilizes unique barcode identifiers and pooled library construction, with a technique for the identification and isolation of ribosome associated mRNA, we are able to rapidly and inexpensively determine genome-wide translational states in a highly scalabl

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Has the growth of real GDP in the UK been overstated because of mis-measurement of banking output?

If official figures overstated the growth of banking output in the UK in the recent boom, does this mean that GDP growth was overstated too? The answer is no. It is truer to say that if banking output was overstated then the output of some other industry or industries must have been understated, leaving GDP relatively unaffected. The reason is that the Office for National Statistics measures the real growth of GDP primarily from the expenditure side. And from the expenditure side most of the problematic part of banking output drops out since it constitutes intermediate consumption not final expenditure. Consequently, the effect of any mis-measurement of banking output on GDP growth in the boom of 2000-2007 is likely to have been small: GDP growth might have been overstated by about 0.1% p.a

The Natural History of a Patient With COVID-19 Pneumonia and Silent Hypoxemia.

A patient who declined all interventions, including oxygen, and recovered highlights the importance of treating the individual instead of clinical markers and provides a time course for recovery from pneumonia and severe hypoxemia

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Maura Paterson (Birkbeck, University of London) visited our Department to present her seminar on “Applications of Disjoint Difference Families”. She also kindly took time out with Julia Böttcher (LSE) to answer a few questions on her research interests and how she takes a break from mathematics

HRV and Treatment Outcomes

A substantial body of literature indicates that autonomic functioning, and heart rate dynamics specifically, is related to a range of psychiatric disorders (Alvares, Quintana, Hickie, & Guastella, 2015; Clamor et al., 2014; Kandola, Ashdown-Franks, Stubbs, Osborn, & Hayes, 2019; Kemp, Brunoni, et al., 2014; Kemp, Quintana, Quintana, Quinn, Hopkinson, & Harris, 2014; Latvala et al., 2016; Paulus, Argo, & Egge, 2013). Furthermore, recent research points toward associations between cardiac functioning with psychotherapy outcomes, which might be driven by bidirectional causal pathways (Angelovski et al., 2016; Blanck et al., 2019; Chalmers et al., 2014; Kemp et al., 2014). Resting heart rate (HR) and heart rate variability (HRV) or the changes in the time intervals between consecutive heart beats, have been conceptualized as transdiagnostic biomarkers for health and disease (Beauchaine & Thayer, 2015; Kemp & Quintana, 2013). Specifically, lower resting HR and higher resting HRV, reflected in greater variability in beat-to-beat heart rate dynamics, are cross-sectionally and prospectively associated with lower depressive and anxiety symptom severity (Chalmers et al., 2014; Latvala et al., 2016; Nelson et al., 2020). Moreover, higher resting HR and lower resting HRV are associated with unfavorable psychological and physical health outcomes as reflected in higher subsequent morbidity and mortality (Kemp & Quintana, 2013). HR and HRV are regulated by prefrontal and subcortical brain regions associated with cognition, affect, and emotion regulation (Lemogne et al., 2011; Mather & Thayer, 2018; Shaffer et al., 2014; Shaffer & Ginsberg, 2017; Thayer et al., 2009), indicating that they may be modifiable transdiagnostic biomarkers amenable to clinical interventions aimed at reducing depressive and anxiety symptoms. HRV has been classically assessed through several different sets of metrics, spanning the time, frequency, and non-linear domains, each of which may offer a unique lens through which to improve our understanding of the neurovascular networks underlying transdiagnostic symptom changes over time during treatment. Thus, the current study will examine whether wearable assessed resting HR and HRV are associated with exposure to treatment, whether they can predict depressive and anxiety trajectories as well as treatment outcomes during an evidence-based therapist-supported digital mental health intervention