Most Recent Activity

The BSRC Biophysics Suite

Part of the ISSF money was used to assemble a state-of-the-art Biophysics Suite at the BSRC, which opened officially in January 2016. This departmental facility is available to all students and staff within BSRC and provides a set of equipment to interrogate macromolecular structure and interaction.

The suite contains a stopped-flow spectrofluorometer, which allows the investigation of millisecond-timescale kinetics; an isothermal calorimeter (ITC), ideal for thermodynamic dissection of molecular interaction; a surface-plasmon resonance (SPR) spectrometer, where kinetic and equilibrium binding to small amounts of immobilised molecules are probed; and a circular dichroism (CD) spectrometer, suitable for investigating protein folding and unfolding based on secondary structure. Experimental highlights of the biophysics suite are reported below.

Peptide macrocyclases are enzymes that catalyse amide bond formation on a linear peptide to produce a circularised one. As many of these peptides possess therapeutic effects, elucidation of the enzymatic properties and mechanisms of macrocyclases are essential to help speed up and diversify macrocycle production. Using the stopped-flow, the complete kinetic sequence of the fungal macrocyclase PopB from Galerina marginata was determined. This identified product release as the rate-liming step of the reaction, while chemistry proceeds faster. Among other insights, these results point to which step should be targeted in optimising catalysis.

PopB and another macrocyclase, the PatGmac from the cyanobacterium Prochloron didemmi, have had their thermal stability investigated by CD spectroscopy thermal melting. Both enzymes have Tm’s around 44 °C. This is high enough for PopB, since it has significant catalytic rate at temperatures as low as 20 °C. However, PatGmac has negligible catalytic rate below 37 °C, a temperature too close to the Tm to ensure stability for long reaction times. Therefore, mutations on PatGmac are being made to try to increase the enzyme’s Tm, which will be again assessed via CD spectroscopy.

Sp2CBMTD is an engineered protein biologic that protects mice against a lethal challenge with H1N1 influenza virus. The biologic, which is based on a carbohydrate-binding module (CBM) from Streptococcus pneumoniae sialidase, binds to sialic acid on the host cell surface and also has an immuno-stimulatory effect. In order to understand the anti-viral mechanism we require CBM variants with reduced sialic acid binding. SPR is being used to measure the affinity between a biotinylated form of 2,3-sialyllactose immobilized to a streptavidin chip and the CBM variants.

Another important macrocyclase is the PCY1 from the plant S. vaccaria. Using ITC, dissociation constants and thermodynamic signatures have been determined for the interaction between PCY1 and several substrate variants. Those findings have improved the understanding of the enzyme’s substrate binding mechanism and are integral to the diversification of the substrate specificity of the enzyme.

 

David Hughes

Summary of Outcomes: The principal motivation for applying for WT ISSF funding, was to provide me with the opportunity to, “develop my independence as a career scientist”. In my proposal, I stated that, “I fully expect that by the end of the first year I will be in a competitive position to apply for independent fellowships…” Using WT ISSF funding, I was able to generate preliminary data that has supported an MRC New Investigator Research Grant application, submitted in January 2016:  ‘Investigating the impact of herpesvirus infection on the viral and host ‘acetylomes’.

This proposal was reviewed by four experts, who, unanimously scored the proposal as ‘Excellent (5/6)’, and all reviews were overwhelmingly positive. The proposal has been shortlisted with the outcome expected at the end of June 2016.

In addition, WT ISSF support has allowed me to develop a strong collaboration with Dr Michael Nevels and Dr Christina Paulus (MMN group). We recently worked in collaboration on a project that we have just submitted for publication (open access and acknowledging WT ISSF support):

  • Wood JJ, Boyne JR, Paulus C, Jackson BR, Nevels MM, Whitehouse A, Hughes DJ*. ARID3B: A Novel Regulator of the Kaposi’s sarcoma-associated Herpesvirus Lytic Cycle. Journal of Virology. Under review. *Corresponding author.

This report focusses on two projects that were initiated at St. Andrews using WT ISSF funding, and a third that was started at Leeds University, and completed at St. Andrews.

  1. Exploring the relationship between RNA virus infection and host SUMOylation. The Mononegalovirales (negative-sense ssRNA viruses) encompasses a variety of viruses that significantly impact human and animal health, including the measles virus, Ebola virus, Newcastle disease virus and respiratory syncytial virus (RSV). Human RSV (HRSV) is the leading cause of paediatric lower respiratory tract infections with >90% of infants infected by the age of two and, worldwide causes around 200,000 deaths per year. Critically, there is no vaccine. In preliminary data leading to my application for WT ISSF support, I had shown that HRSV infection led to increases in SUMOylated proteins, particularly the promyelocytic leukaemia protein (PML) – a factor with known antiviral activity. Furthermore, I had shown that the RSV nucleocapsid protein physically interacted with SUMO. This led to the hypothesis that:

SUMOylation is an integral part of the RSV lifecycle, that RNA virus infection leads to a dramatic induction of the host SUMOylation machinery and that understanding this process will inform the development of vaccines or targeted therapies.

Subsequent studies found that PML and SUMOylation do not play a role in negative-sense RNA virus biology: Several functional experiments demonstrated that neither SUMOylation, nor PML protein function played a significant role in RSV biology.

RNA virus infection and PML. Upon starting WT ISSF funding, I confirmed my earlier observations showing that PML protein expression was induced following HRSV infection (Fig. 1A-B). Additionally, by comparing infection of A549 cells (lung epithelial cells) with that of A549s engineered to not respond to the antiviral interferon (IFN) system (due to the stable expression of the viral IFN antagonist NPro), quantitative immunofluorescence analyses showed that the majority of the PML response was due to IFN signalling (Fig. 1C).

To ascertain the role PML plays during virus infection, I established a collaboration with Dr Alejandro Rojas-Fernandez (in the laboratory of Prof. Ron Hay, Dundee), who, using CRISPR-Cas9 technology, had generated PML-/- cells on a U2OS background. These cells were infected with a recombinant HRSV that expresses a luciferase reporter gene which, has been inserted into the viral genome (RSV-Luc). Following infection at low multiplicity of infection (MOI of 0.1), we used both luciferase activity and HRSV protein expression as a readout for virus infection. Surprisingly, I showed that a complete lack of PML had no significant effect on HRSV biology (Fig. 2). There are 6 splicoforms of PML, and, individual replacement of their expression in PML-/- cells also played no role in HRSV infection (Fig. 2).

PML-nuclear body components do not play cooperative antiviral roles against HRSV infection. The PML protein is the main constituent in discrete nuclear bodies (known as PML nuclear bodies or PML-NB). There are several (at least 70 reported) additional proteins that associate with PML-NBs, whose interaction is SUMOylation-dependent. Some of the best characterised PML-NB-associating proteins are sp100 and Daxx, and together, these proteins have been shown to possess antiviral activity. Therefore, it was possible that these proteins played cooperative roles during the antiviral response, and that PML alone was not sufficient during the antiviral response, as has been shown for herpes simplex virus type 1 (HSV-1) infection. Therefore, we knocked down sp100 and Daxx using siRNA (using 4 individual siRNAs per gene), and infected these cells with RSV-Luc at low MOI (0.1). We initially did this in HEp2 cells, a commonly used cell line in HRSV research due to its ability to robustly support infection. We clearly show that we achieved excellent knockdown of sp100 (2/4 siRNAs) and Daxx (4/4 siRNAs) (Fig. 3A); however, this had no appreciable effect on virus biology, as shown by RSV protein expression (Fig. 3B) or luciferase activity (Fig. 3C). This confirmed that individual ablation of PML-NB components had no effect on HRSV infection.

To determine if removal of these proteins together had any HRSV infection, we transfected our PML-/- cells (PMLKO, Fig. 3D) with either sp100 siRNA alone, Daxx siRNA alone or both, and infected them with RSV-Luc. Again, concomitant knockdown of these PML-NB components had no effect on virus biology (Fig. 3D). We noticed that RSV-Luc did not appear to propagate well in the PML-/- cells; therefore, we repeated the experiment in the parental cell line, U2OS cells (Fig. 3E), and the model cell line HEp2 (Fig. 3F). RSV-Luc did grow slightly better in U2OS cells with wtPML, yet double knockdown of sp100 and Daxx had no effect on virus biology. Similarly, HEp2 cells demonstrated a robust RSV-Luc infection which, was not affected by knockdown of PML-NB components. Additional data (not shown) suggests that inferior virus growth in PML-/- cells was due to cell fitness, rather than any effect on virus biology.

PML is not important during the antiviral response against negative-sense RNA viruses. At least for the PML studies, we extended this to numerous other negative-sense RNA viruses (parainfluenza virus (PIV)2, PIV3, PIV5, Bunyamwera virus, influenza A), and similarly found no significant effect on virus biology.  It might be possible that an apparent lack of any phenotype was due to the cell systems employed (although this was tested in both HEp2 and U2OS cells). We therefore attempted to generate PML-/- primary cultures, as these often provide more robust models for studying antiviral responses. However, this was not successful. Reluctantly, it was decided that it was not good value for money to pursue this any further. Attention was switched to other projects.

  1. Investigating Kaposi’s sarcoma associated herpesvirus (KSHV) and protein acetylation. The following work has led to a grant application (MRC NIRG award).

Human herpesviruses cause a multitude of diseases that place a significant burden on healthcare resources both in the developed and developing world. As a family, they are associated with a wide spectrum of diseases including usually benign cold sores (herpes simplex virus), debilitating diseases such as glandular fever (Epstein-Barr virus, EBV), serious congenital conditions (human cytomegalovirus, HCMV) and cancer (EBV and Kaposi’s sarcoma-associated herpesvirus, KSHV). Indeed, EBV causes a staggering 1% of all cancers worldwide, and KSHV causes Kaposi’s sarcoma (KS), the most prevalent cancer in regions with endemic HIV. Unfortunately, there are few treatment options, and those that are available have limited efficacy. Current anti-herpesvirus drugs (e.g. ganciclovir, foscarnet and cidofovir) block viral genome replication; however, these drugs are often associated with adverse side effects and drug-resistant strains have emerged, reminiscent of the current problems of antimicrobial resistance (AMR). Without an improved understanding of their biology it will be impossible to develop urgently needed new therapeutics.

A hallmark of all herpesviruses is their biphasic life cycle: the lytic cycle (production of new viral particles) and the latent phase (a persistent and silent infection with highly restricted viral gene expression). The lytic cycle is necessary in the majority of herpesvirus diseases. For example, the KSHV lytic cycle is indispensable for the pathogenesis of KS; indeed active virus replication and increased viral loads are associated with poorer clinical outcomes. Additionally, lytic cycle-associated proteins contribute to the development of KS and sustained reinfection from released virus maintains infection of the malignant tissue.

Herpesviruses express immediate early (IE) proteins that are responsible for initiating the lytic cycle. The KSHV IE protein, replication and transcriptional activator (RTA, a product of ORF50), is both necessary and sufficient for initiating the KSHV lytic cycle. RTA activates transcription of lytic genes by directly interacting with RTA-responsive elements (RREs) found in some lytic gene promoters, or indirectly via interactions with cellular transcription factors. It is also responsible for docking the viral pre-replication complex at the cis-acting origins of lytic replication (oriLyt) to initiate viral DNA replication. Therefore, IE proteins, or the mechanisms that regulate their function represent worthy therapeutic targets.

Posttranslational modifications (PTMs) as antiviral targets? As with all other viruses, herpesviruses rely critically on cellular processes, including PTMs, to promote infection. Disruption of these cellular processes are seen as an attractive therapeutic option, particularly as they are less likely to mutate (compared to viral targets) thus reducing the risk of antiviral resistance. Indeed, my own research has shown that inhibition of NEDDylation – a ubiquitin-like PTM – proved cytotoxic to KSHV-infected lymphoma cells by modulating latency-associated gene expression, in addition to blocking the KSHV lytic cycle by preventing the viral pre-replication complex from docking at oriLyt (Hughes et al. 2015. PLoS Path), thus establishing PTMs, and their control, as promising antiviral targets.

My preliminary work, funded by the WT ISSF, has demonstrated a clear link between KSHV RTA and the PTM acetylation. For example, the expression of RTA was able to induce the expression of the entire nucleosome remodelling and deacetylase (NuRD) complex, as well as several acetyltransferase proteins, including GCN5, CBP and MYST. This led us to ask, and then show for the first time that RTA was itself acetylated (Fig. 4). Further work has also shown that the C-terminal portion of RTA, that contains its transcription activation domain, is the target of acetylation (Fig. 5). Given the clear interplay between KSHV infection and acetylation (as shown by others and this work), we hypothesise that:

RTA expression, and the KSHV lytic cycle have profound effects on the cellular ‘acetylome’; moreover, acetylation functions as a molecular switch that regulates RTA’s lytic cycle activities.

We have proposed to provide an in-depth analysis of how KSHV infection remodels the cellular and KSHV acetylome, and how acetylation of RTA regulates its lytic cycle activities. Also, we will identify critical components of the cellular acetylation system that modulate KSHV infection. Together, these will inform the development of novel anti-KSHV (or pan-herpesviral) therapeutics. Furthermore, a global and comprehensive investigation of the effect of virus infection on the cellular acetylome has never been performed before. This is despite the gaining popularity of drugs designed to modulate acetylation, particularly for the treatment of cancer, but also as potential antivirals.

These studies will define the first acetylome study involving a virus infection, providing a catalogue of acetylated sites at individual residue resolution. We will also identify acetylation-specific pathways that are effected by KSHV infection. Importantly, it will provide a framework for future studies aimed at identifying novel acetylation therapeutics and to better understand the specificities of already available, or novel, histone deacetylase inhibitors.

  1. Identification of a novel regulator of the KSHV lytic cycle. This project was started at the University of Leeds, but completed at the University of St. Andrews (with WT ISSF support), in collaboration with Dr Michael Nevels and Dr Christina Paulus. This work is currently under review (Journal of Virology).

KSHV is the causative agent of fatal malignancies of immunocompromised individuals, including Kaposi’s sarcoma (KS). Herpesviruses are able to establish a latent infection, where they escape immune detection by restricting viral gene expression. Importantly however, reactivation of productive viral replication (the lytic cycle) is necessary for the pathogenesis of KS. Therefore, it is important that we comprehensively understand the mechanisms that govern lytic reactivation, to better understand disease progression. In this study, we identified a novel cellular protein (AT-rich interacting domain protein 3B, ARID3B) that we show is able to moderate lytic reactivation. We showed that the master lytic switch protein, RTA, enhanced ARID3B levels, which then interacted with viral DNA in a lytic cycle dependent manner. Therefore, we have added a new factor to the list of cellular proteins that regulate the KSHV lytic cycle, which has implications for our understanding of KSHV biology.

While work at the University of Leeds had clearly demonstrated the link between ARID3B and the lytic cycle (e.g. ARID3B relocalised to replication compartments upon lytic reactivation and siRNA knockdown of ARID3B enhances the lytic cycle; Fig. 6), we did not have any mechanistic information that could explain how ARID3B achieved this.

It has been predicted that ARID3B is a DNA binding protein with preference for A/T-rich sequences, and the preponderance of these sequences in regulatory elements of the KSHV genome (e.g. RTA responsive elements in the promoters of some lytic genes and in the origins of lytic DNA replication – oriLyt) led us to ask if ARID3B influenced KSHV lytic reactivation via interactions with the viral genome. To investigate this, we established a collaboration with MMN group who are experts in investigating viral chromatin interactions. To that end, we developed a chromatin immunoprecipitation assay (ChIP) that showed us that ARID3B did indeed interact with the viral genome. Furthermore, we demonstrated that ARID3B specifically interacted specifically with A/T-rich elements in the KSHV oriLyt, and this was dependent of lytic cycle reactivation (Fig. 7).

Summary. While it was disappointing that I showed SUMOylation and PML played no role during the antiviral response to RSV infection, as originally hypothesised, I was able to quickly refocus my research. The aims of my current research programme is to investigate the disease-associated reactivation of the herpesvirus lytic cycle. Specifically, I aim to develop a deeper understanding of virus-host interactions that govern this process. In doing so, we may uncover novel therapeutic targets, with the primary rationale that ‘drugable cellular processes that hinder virus biology are harder to develop resistance to’. I recently showed that targeting PTMs, such as NEDDylation, are viable therapeutic targets for KSHV-associated malignancies. In new work, supported by WT ISSF funding, I have shown that the KSHV lytic switch protein RTA is associated with various acetylation-associated pathways which led us to ask, and then show for the first time, that RTA is itself acetylated. This preliminary work has led to an MRC NIRG award application, which has been shortlisted (decision expected late June). If successful, WT ISSF support will have placed in a strong position to establish a permanent position within the School of Biology, which is the ultimate goal.

 

Michael Nevels

ISSF Report (Christina Paulus, May 2016)

Project summary

Human Cytomegalovirus (CMV) is a leading cause of birth defects and severe disease in immunocompromised patients. There is no preventive vaccine for CMV, and antiviral therapy is limited to only a few drugs with major downsides including toxic side effects and emergence of resistant virus strains. All systemic drugs approved for CMV target the viral DNA polymerase and subsequent late events in infection. Drugs directed at earlier steps, including viral immediate-early (IE) or host innate immune functions, have not been developed. Dr Paulus has previously shown that the CMV IE1 protein targets the human signal transducer and activator of transcription (STAT) 2 protein to block type I interferon (IFN) signaling (http://www.ncbi.nlm.nih.gov/pubmed/16497831), arguably our most efficient innate immune response. She also discovered that IE1 forms a complex with STAT3 (http://www.ncbi.nlm.nih.gov/pubmed/23903834). Through mutual binding to STAT2 and STAT3 IE1 confers type I IFN resistance to CMV and rewires gene expression linked to viral pathogenesis, respectively.

Since March 2016, Dr Paulus has been working as an ISSF-funded Research Fellow at the BSRC. In line with her proposed aims, she set out to identify small molecule compounds targeting the CMV IE1 protein. These compounds are expected to render CMV sensitive to the IFN response and may therefore be developed into new antiviral drugs. Dr Paulus utilised an innovative screening approach developed by Dr Adamson and Prof Randall at the BSRC, which is based on a reporter cell line (A549/pr(ISRE).GFP) carrying a green fluorescent protein (GFP) coding sequence under the control of an IFN-responsive promoter. She transduced these cells to express IE1 in an inducible fashion, since long-term expression of the viral protein proved to be incompatible with normal cell division and survival. Following induction of IE1 expression, IFN-activated GFP expression was completely blocked in theses cells. Subsequently, Dr Paulus screened the 16,000 compound Maybridge library for drug-like molecules restoring GFP expression by interfering with IE1-STAT2 interaction and/or enhancing cell viability by antagonising IE1-related cytotoxicity. A total of four hit compounds were identified from this screening, three of which proved to inhibit CMV replication in infected primary human fibroblasts. Dr Paulus is currently conducting experiments to confirm the specificity of the antiviral mechanism. She is also looking into patent applications and ways of involving commercial partners to develop the compounds into antiviral drug candidates.

Importance of ISSF funding

In 2015, Dr Paulus applied for a Marie-Skłodowska-Curie Individual Fellowship from the EU. The application received excellent scores, but has not been funded. However, she will take the chance to re-apply by the next submission deadline (14 September 2016). If funded, the fellowship would cover her salary and material expenses for two years. The ISSF funding has been vital to bridge salary support for Dr Paulus until funds from the EU fellowship or another funding source are available.

Collaborators inside the BSRC

–   Dr Catherine S Adamson

–   Dr David J Hughes (ISSF-funded)

–   Dr C Paulus (ISSF-funded)

–   Prof Richard E Randall

Collaborators outside the BSRC

–   Prof Minhua Luo (Wuhan Institute of Virology: http://english.whiov.cas.cn)

Publications

(all in revision and not yet published; ISSF funding acknowledged in each)

–   Wood JJ, Boyne JR, Paulus C, Jackson BR, Nevels M, Whitehouse A, Hughes DJ. ARID3B: a novel regulator of the Kaposi’s sarcoma-associated herpesvirus lytic cycle. J. Virol.

–   Wu CC, Liu XJ, Li XJ, Yang B, Ye HQ, Harwardt T, Gan L, Zhao F, Cheng S, Jiang X, Xia HM, Hu F, Ming YZ, Britt WJ, Paulus C, Nevels M , Luo MH. Human cytomegalovirus IE1 mediates SOX2 depletion from neural progenitor cells by inhibiting STAT3: implications for congenital brain development disorders. PLoS Pathog.

–   Harwardt T, Lukas S, Zenger M, Reitberger T, Danzer D, Übner T, Munday D, Nevels M, Paulus C. Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNg-like response. PLoS Pathog.

 

Maciej Antokowtak (Medicine) in collaboration with Gareth Miles (Psychology & Neuroscience)

Understanding complex neural networks using novel optical electrophysiology.

Summary of aims:   This interdisciplinary project aims to explore and demonstrate the applicability of the novel technique of purely optical electrophysiology (OEP) to studies on the dynamics and connectivity in normal and diseased neural networks. This recently developed unique non-invasive method to control and interrogate large neural networks, both in vivo and in vitro, enables unprecedented insight into how nerve cells function and interact in complex networks. This project aims at applying this technique to neural networks formed by primary mammalian neurons and human neural cells obtianed from patient-derived iPS cells providing the most accurate human model of synaptic plasticity and neurodegeneration.

In more detail, the intended aims of the project are to:

  • Use patch-clamp electrophysiology as a benchmark to establish OEP as a robust and quantitative technique and to optimize the optical methods used in the experimental system.
  • Develop optical protocols for long term multicellular studies on dynamics in large networks formed by patient specific iPS-derived neural cells (in particular dementia and motor neuron disease).
  • Identify the main opportunities created by OEP as a breakthrough tool in studies on connectivity and dynamics in neural networks formed by diseased human neural cells in vitro and provide pilot data for future grant applications.

Progress in the first 10 months.

Following the original plan for this project we have, with the assistance of a research assistant supported by ISSF, focused on the first aim and progressed well in learning the traditional and optical techniques of whole-cell electrophysiology. Benefitting from her strong background in experimental neuroscience she has already developed a dedicated experimental setup and custom-made software to perform novel modalities of optical electrophysiology and is currently performing experiments that verify the quantitative equivalence of the two techniques. She will then focus on applying her experimental approach to stem cell-derived human neural cells and networks (Aim 2).

Marjet Dirks is an exceptionally talented and dedicated research assistant and is progressing very well with in this challenging interdisciplinary project.

Outcome in the first 10 months

It is too early to report any publications resulting directly from this ISSF project although I am convinced that a peer reviewed publication should be ready in the 2nd year of the project. Some of the initial data will be presented at the SLAS High-Content Screening Symposium in June 2016.

A further research award from Northwood Trust (30k) closely related to this project has been secured in April 2016. The existing support from ISSF in the form of the already active research assistant has significantly contributed to the success of this application.

Importantly, this project has already delivered on the promise of accelerating and strengthening the new interdisciplinary collaboration between the two groups involved, leading to development of new ideas for future grant applications.

 

St Andrews Bioinformatics Unit (StABU)

Publications – with substantial contribution by the Bioinformatics Unit

Strobl MAR, Barker D. On simulated annealing phase transitions in phylogeny reconstruction. Molec. Phylogenet. Evol. Volume 101, August 2016, doi:10.1016/j.ympev.2016.05.001

Warne B, Harkins CP, Harris SR, Vatsiou A, Stanley-Wall N, Parkhill J, Peacock SJ, Palmer T, Holden MT. The Ess/Type VII secretion system of Staphylococcus aureus shows unexpected genetic diversity. BMC Genomics. 2016 Mar 11;17(1):222. doi: 10.1186/s12864-016-2426-7.

Publications – facilitated by the Bioinformatics Unit (e.g. analyses run on the bioinformatics server)

Parker, D. J., Cunningham, C. B., Walling, C. A., Stamper, C. E., Head, M. L., Roy-Zokan, E. M., McKinney, E. C., Ritchie, M. G., Moore, A. J. 2015. Transcriptomes of parents identify parenting strategies and sexual conflict in a subsocial beetle. Nature Communications. 6:8449.

Shore R, Covill L, Pettigrew KA, Brandler WM, Diaz R, Xu Y, Tello JA, Talcott JB, Newbury DF, Stein J, Monaco AP, Paracchini S. 2016. The handedness-associated PCSK6 locus spans an intronic promoter regulating novel transcripts. Human Molecular Genetics. 2016 Feb 21. pii: ddw047.

Pettigrew K.A., E. Reeves, R. Leavett, E. Hayiou-Thomas, A. Sharma, N.H. Simpson, P. Thompson, M. Snowling, D.F. Newbury, S. Paracchini 2015 A de novo deletion at chromosome 15q13.1-13.3 in a child with reading and language impairment. PLoS One 10 (8), e0134997

Vatsiou AI, Bazin E, Gaggiotti OE. Changes in selective pressures associated with human population expansion may explain metabolic and immune related pathways enriched for signatures of positive selection. BMC Genomics (conditionally accepted pending revisions)

Vatsiou AI, Bazin E, Gaggiotti OE. 2016. Detection of selective sweeps in structured populations: a comparison of recent methods. Molecular Ecology 25:89-103.

de Villemereuil P, Gaggiotti OE. 2015. A new FST-based method to uncover local adaptation using environmental variables. Methods in Ecology and Evolution 6:1248-1258.

Frichot E, Schoville SD, de Villemereuil P, Gaggiotti OE, Francois O. 2015. Detecting adaptive evolution based on association with ecological gradients: orientation matters! Heredity 115:22-28.

Cunningham, C. B., Ji, L., Wiberg, R. A. W., Shelton, J., McKinney, E. C., Parker, D. J., Meagher, R. B., Benowitz, K. M., Roy-Zokan, E. M., Ritchie, M. G., Brown, S. J., Schmitz, R. J. & Moore, A. J. (2015). The genome and methylome of a beetle with complex social behavior, Nicrophorus vespilloides (Coleoptera: Silphidae). Genome Biology and Evolution 7: 3383-3396.

Highlights/Success/Achievements

  • We have improved our computing capacity. StABU continues to maintain an 11 node compute cluster for NGS projects with a total 70 TB storage system for datasets, with a groups system so that datasets maybe shared for efficiency. The addition of new machine brings the total processing cores to 208. This is a high grade webserver for web-based projects which provide higher visibility to certain projects.
  • StABU continues to support a wide variety of projects across different school contributing to the research of more than 15 research groups across the Schools of Medicine, Biology and Chemistry.
  • Following Miguel Pinheiro’s resignation, we have re-advertised the position over the summer. We received an impressive number of high quality applications. Ramon Fallon has accepted the position and started in November 2015. Since starting, Ramon has contributed to improving our service:

-Installation of over 50 software packages (either requested by users or to add extra functionality)

-Improvement of machine functionality (storage, upgrades, remote management) and handling of support request (introduction of time tracking system).

-Consolidating links with HPC facilities in St. Andrews (Cheminformatics and IT Services).

-Attended training courses

  • Through funding raised on over-heads we have employed for 3 months Alexandra Vatsiou, a newly graduated St Andrews PhD student willing to gain NGS training. Alexandra has now been appointed as Post-doctoral fellow at The Institute of Cancer Research
  • StABU continues to provide a platform to facilitate larger collaborations. As specific example, it supports Axel Wiberg and Mike Ritchie in the DrosEU consortium for computations for Comparitive Genomics of D. Melanogaster.

Events

  • In January 2016 StABU organised a workshop to bring together the St Andrews bioinformatics community. The workshop was attended by about 50 participants from different Schools and the feedback was extremely positive.
  • Ramon application for a CAPOD Training Award was successful. Through this initiative Ramon will organise a training session “Unix for Biological Data Processing” aimed at PG students to be held in September 2016. This course follows the increasing number of PhD students requiring access to the cluster. We are planning a separate course aimed at handling RNA-seq and transcriptomics data.
  • Bimonthly engagement in NextGenBug, a community of thought leaders in Next Generation Sequencing based at University of Edinburgh. The June 2016 meeting will be held in St Andrews and is supported by StABU member Matt Holden

Successful grant awards

Currently three awards directly contribute towards time of a StABU Bioinformatician

PI                                 % Time    Sponsor Start Date                  End Date

Matt Holden              20%         CSO          01-Apr-15                 31/03/2020

Helder Fererer        10%         BBSRC     01-Apr-15                 31/03/2018

Lesley Torrance     5%            BBSRC     01-Sep-16                30/11/2017

Grant application submissions that included Bioinformatics Unit resources and support

  • An increasing number of grant applications have included StABU support in their costing. Most recent application:
PI Start Date End Date Sponsor More details
Tom Meagher 10/15 09/18 NERC Biodiversity species distribution in Caatinga regions.
Mike Ritchie 04/16 03/19 NERC Inositol Pathways and cold tolerance in Drosophila
David Ferrier 04/16 10/19 Leverhulme Trust Operaculum plate generation In Spirobranchus lamarki
Janet Cox-Singh 02/16 08/19 Newton Fund UK Sequencing of macaque genome to identify potential reservoir of pathogen emergence
Gilbert Smith 2016 2021 NERC Role of gene expression regulation in evolution of phenotypic plasticity
Andy Gardner 11/15 10/16 NERC Simulations of genomic Imprinting
LesleyTorrance 04/16 09/17 BBSRC Processing NGS data for multiple viral infections in crops
David Shuker 04/16 03/20 NERC genomic ecology of an adaptation: sex allocation in parasitoid wasps
Silvia Paracchini 01/17 01/21 ERC Genome Wide Association for handedness in different cohorts, N= 10000
Janet Cox-Singh 02/16 08/19 Wellcome Trust Characterising putative virulent genes in two malaria parasites
Janet Cox-Singh 07/16 12/17 Wellcome Trust Seed Characterising virulent loci in malaria parasite
Anne Magurran 06/16 06/18 Carnegie Trust for the Universities of Scotland Structure in Trinidad bird populations
Anne Magurran 08/16 01/18 ERC BioCHANGE, biodiversity assemblage data
V. Anne Smith 05/17 04/21 James S. McDonnell Foundation Maps of Network-Dynamic Space
Maria Dornelas 06/17 05/20 NERC IOF Biodiversity Global Detection Attribution in the Anthropocene
Janet Cox-Singh 01/17 12/17 School of Medicine Synthetic Long Reads of Plasmodium knowlesi
Gillian Brown 01/17 12/19 Medical Research Council RNAseq of rat brain tissue for studies in Autism
Matthew Holden 05/16 05/19 NERC AMR theme 3, Evolution and transmission of AMR pathogens in the host
Stephen Gillespie 8/16 8/21 MRC AMR Theme 2, MOAT: Mining the Oceans for novel Antibiotics with new Targets

 

Michael Nevels – ISSF Report (May 2016)

Summary:   CMV is one of eight human herpesviruses and causes lifelong persistent infections in the majority of people worldwide. CMV-related symptoms are generally mild or absent in most people. However, CMV remains the leading cause of neurological damage in unborn children and a major threat to immunocompromised individuals including transplant recipients. Despite its clinical importance, awareness for CMV in the general population is low and the options for prevention or treatment are very limited. Moreover, CMV is one of the most complex known viruses, and its replication and persistence depends on numerous host cell interactions many of which remain to be elucidated.

The ISSF funds have been instrumental in setting up my new laboratory at the BSRC. The funds have been used to provide the salary for a Research Fellow (Dr C Paulus) and to purchase essential equipment including a CO2 incubator, a liquid nitrogen tank, a tabletop centrifuge, a vacuum aspiration system, a water bath and a specialized instrument for chromatin shearing (Bioruptor Pico: https://www.diagenode.com/p/bioruptor-pico-sonication-device). In addition, a range of basic consumables to start up experiments were purchased using these funds.

As proposed in the initial application, we have continued to study the molecular processes underlying CMV-host cell interactions in two areas: epigenetic regulation and innate immunity. Our worked has centred on the function of the viral immediate-early 1 (IE1) protein, which is both a nucleosome-associated epigenetic modifier and a major antagonist of innate immune responses. Following up on our initial finding that IE1 targets the cellular signal transducer and activator of transcription (STAT) 2 protein to block type I interferon signaling, we discovered that the viral protein rewires upstream STAT3 to downstream STAT1 signaling. This finding not only provides a mechanism so far unique for a viral protein, but may also have implications for CMV pathogenesis. In fact, collaborating with Prof M Luo (Wuhan Institute of Virology, Chinese Academy of Sciences) we found that inhibition of STAT3 signaling by IE1 is linked to depletion of the key stem cell factor SOX2 from CMV-infected neural progenitor cells and disruption of neural cell fate. This finding may link IE1 to neurological damage caused by congenital CMV infection. In collaboration with Dr C Adamson and Prof R Randall, we also successfully screened for drug-like compounds that disrupt IE1 function and thereby inhibit viral replication, providing a new way to target CMV. We are currently looking into filing a patent and involving commercial partners to develop these compounds into antiviral drug candidates. Finally, we started a collaboration with Dr D Hughes investigating chromatin interactions in a virus distantly related to CMV, the Kaposi’s sarcoma-associated herpesvirus.

Collaborators inside the BSRC

–   Dr Catherine S Adamson

–   Dr David J Hughes (ISSF-funded)

–   Dr C Paulus (ISSF-funded)

–   Prof Richard E Randall

Collaborators outside the BSRC

–   Prof Minhua Luo (Wuhan Institute of Virology: http://english.whiov.cas.cn)

Publications

(all in revision and not yet published; ISSF funding acknowledged in each)

–   Wood JJ, Boyne JR, Paulus C, Jackson BR, Nevels M, Whitehouse A, Hughes DJ. ARID3B: a novel regulator of the Kaposi’s sarcoma-associated herpesvirus lytic cycle. J. Virol.

–   Wu CC, Liu XJ, Li XJ, Yang B, Ye HQ, Harwardt T, Gan L, Zhao F, Cheng S, Jiang X, Xia HM, Hu F, Ming YZ, Britt WJ, Paulus C, Nevels M , Luo MH. Human cytomegalovirus IE1 mediates SOX2 depletion from neural progenitor cells by inhibiting STAT3: implications for congenital brain development disorders. PLoS Pathog.

–   Harwardt T, Lukas S, Zenger M, Reitberger T, Danzer D, Übner T, Munday D, Nevels M, Paulus C. Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNg-like response. PLoS Pathog.

Further funding

–   Tenovus Scotland (£15,000)

 

Tracey Gloster

ISSF seed-corn funding for Lei Yang

 Seed-corn funding was obtained to support the salary and consumables for a post-doctoral fellow, Dr Lei Yang, to work in the Gloster group for one year. Lei worked on a number of projects focussing on mechanistic, structural, and functional studies of carbohydrate degrading enzymes.

Lei’s primary focus was a human hexosaminidase, HexD, for which the cellular function and physiological substrate are unknown. Increased levels of HexD have been implicated in rheumatoid arthritis. The group had previously investigated the details of HexD mechanism (for which a publication in Biochemistry has recently been published) and structural studies are underway. The aim of the on-going studies were (1) to use the mechanistic information to inform the design and development of potent and specific inhibitors; (2) to develop a method for knocking out the gene encoding HexD and assess the effect on cell phenotype; (3) to investigate the localisation of HexD.

In collaboration with Prof. David Vocadlo (Simon Fraser University, Canada), we made and tested five novel inhibitors that were predicted to inhibit HexD, which displayed an unusual slow-onset inhibition. We performed localisation studies, and also did pull down experiments, followed by mass spectrometry, to identify potential interacting partners. We are currently analysing these data and will perform follow up studies to validate potential binding partners. We have also developed a method, using RNA interference, of knocking out the gene encoding HexD in cells.

This work will form the basis of a grant application to either the Wellcome Trust for a Senior Research Fellowship (in 2017/18) or to a research council. There has been one publication on the HexD mechanism work, and the subsequent work is currently being consolidated prior to publication.

Lei was also involved with work on a human alpha-hexosaminidase (NAGLU), which is localized to the lysosome and degrades heparan sulfate. We aim to gain molecular insights into the mechanism and structure of NAGLU, which will aid efforts in the development of therapeutic ‘molecular chaperones’. We are still trying to optimise recombinant expression of NAGLU in mammalian cells as we are unable to obtain sufficient levels of protein secreted into the media. We have tried purification of the protein from cells, but this is proving difficult to solubilise and efforts are on-going for this project.

Lei has also been involved with expression, crystallisation and structure solution of a novel bacterial mannosyltransferase. In addition to the apo structure, we have obtained four complexes between the enzyme and enzyme substrates or product. This work is currently being written up for publication.

 

 

Upgrade of BSRC cell culture facilities, microscopy, and image analysis

 The virus-free tissue culture room (BMS 2.40) is now operational and in daily use for projects in the Sleeman and Smith (Terry) groups. The bench-top centrifuge, in particular, has been instrumental in refining protocols for sub-cellular fractionation, data from which have been included in two recent grant proposals (Sleeman PI, Smith co-I) currently under consideration by Muscular Dystrophy UK and the Leverhulme Trust. This work will also be incorporated into a joint publication currently in development. The image analysis work-station has been used for data processing and analysis by the Sleeman, Gunn-Moore and Adamson groups. Quantitative microscopy and western blot data analysed on this system forms a large part of a manuscript currently under preparation (Jordan et al, Sleeman group).

Cell culture facilities for the groups based in the BMS annex have been upgraded. Two rooms have now been commandeered, one for insect cell culture (BMS 2.38, used by the Gloster, Naismith, and White groups) and mammalian cell culture (BMS 2.34, used by the Gloster, Naismith, Taylor and Schwarz-Linek groups). A sink, freezer, and other pieces of small equipment have been installed in the mammalian cell culture room to make it fit for purpose, and a hood installed in the insect cell culture room. A Forma CO2 incubator with a two-tier shaker support and shakers was purchased to enable recombinant protein expression in mammalian cell suspension. This has enabled the production of protein in higher yield for mechanistic and structural studies in the Gloster group. In addition an EVOS FL digital inverted fluorescence microscope, with fluorescence capability of GFP, Texas Red/CherryFP and DAPI, and 10x, 20x, 40x LWD lenses for phase contrast and fluorite applications, was purchased. The microscope was supplemented with a Countess II module that is used to identify and count cells.

 

 

David Ferrier

The aim of this bridge funding was to support Dr Myles Garstang between the end of his PhD and the potential start of a PDRA position on a BBSRC grant. Because the BBSRC application was ultimately unsuccessful only three months of bridge support was activated from the ISSF funds. The specific aims for these three months of support were for some final data to be gathered in order for submission of a manuscript. In particular the objectives were to (1) complete whole mount in situ hybridisations (WMISH) for two genes (SCP1 and CHIC) neighbouring the amphioxus ParaHox gene cluster; (2) clone the 5’ end of one of these genes (SCP1) to establish the exonic organisation of the 5’UTR; (3) attempt long-range PCR to independently confirm the genomic location of the SCP1 gene in the European species of amphioxus relative to the known genomic location in the American species of amphioxus; (4) begin drafting a manuscript based on this data alongside the bioinformatics and preliminary WMISH data from Myles’s PhD.

All of these aims were within the context of establishing that the SCP1 gene had evolved by a mechanism known as retrogene replacement and that following its insertion next to the ParaHox gene cluster the SCP1 retrogene had come under the control of a CHIC gene promoter, which was not under the influence of any pan-cluster regulatory mechanisms operating over the ParaHox gene cluster. The SCP1 expression data also adds to the accumulating evidence that this “meiosis” gene is highly likely to have further functions besides its established role in synaptonemal complex formation during meiosis. Aims (1) and (2) were entirely successful, with the RT-PCR used for aim (2) having the additional benefit that two different splice forms of SCP1 were discovered. The long-range PCR in aim (3) was not successful, but an alternative route to confirming the genomic location of SCP1 in Branchiostoma lanceolatum was via data from the genome project of this species, used in comparison to data from two further amphioxus genome projects. The drafting of the manuscript (aim 4) has begun and the paper should be submitted within the next couple of months, to an open access journal and acknowledging Wellcome ISSF support.

 

John Lucocq  

The Seahorse metabolic profiling machine funded in part from ISSF funds has been used in projects from two laboratories so far. The Lucocq lab (holding a Wellcome programme grant which runs until Dec 2016) has investigated the host parasite interactions of obligate intracellular parasitic organisms called microsporidia. Microsporidia are extremely genetically reduced and have adapted for an intracellular lifestyle by losing inherent metabolic capacity while reshaping metabolic processes in the host to their own advantage. Our recent studies suggest these organisms secrete regulators that modulate host cell metabolism to increase the supply of ATP and to divert carbon rich metabolites for growth. We have been testing the hypothesis that the metabolism mirrors that previously found in cancer cells (the Warburg effect).  The Seahorse machine has been a key tool in these studies. Our lab expertise in this machine has been used to support undergraduate practicals in the Biology division. The group of Frank Gunn Moore has used the Seahorse to establish the changes in cells expressing our Alzheimer’s disease related protein called ABAD when we lower or raise external glucose levels. This has uncovered a potential new mechanism of how ABAD activity is controlled by post-translation modification. We have also utilised the equipment in a new undergraduate practical for 3rd year biologists studying Bioenergetics.

 

Martin Ryan

Dr. Minskiya (a former MRC-funded post-doc in my laboratory) was employed on ISSF funds from 5/1/15 to 30/4/15 following the summary dismissal of Dr. Pathania and our subsequent discovery that virtually all of the FMDV replicon clones he had purportedly generated were incorrect. Dr. Minskiya rapidly generated new, correct, clones and verified their replication competence using our InCucyte Zoom fluorescence microscope. This work was invaluable since it allowed us to transfer these clones to the Pirbright Institute where corresponding infectious copies have been made and FMDV viruses rescued. The assistance provided by the ISSF scheme allowed us to retain our research ‘momentum’ at a critical time, and the data she generated directly led to further applications to the BBSRC (BB-P002633-1 – Vaccinology Initiative, currently under review; Global Challenges Initiative – to be submitted in June). Importantly, during this period Dr. Minskiya was also applying for posts and was successful in gaining a position at the Instituto de Medicina Molecular Faculdade de Medicina da Universidade de Lisboa. The flexibility provided by the ISSF scheme was critical for both our research project, and, to act as a vital ‘bridge’ for Dr. Minskiya in seeking a subsequent post.

 

Stephen Gillespie

Illumina MiSeq machine

This machine has played a central role in the activities of the CSO funded Scottish Healthcare Associated Infection Prevention Institute (SHAIPI) consortium, to which St Andrews has contributing to the genomics workstream. The University of St Andrews received £1.0 million in funding, and the MiSeq is at the heart of the of near-real time sequencing capacity that has been established within the School of Medicine. The SHAIPI funding has paid for a full time RA (Kerry Pettigrew) to run the machine, and for machine maintenance and consumables. On the SHAIPI project in the last year, a total 324 isolate’s genomes have been sequenced. These include: 69 E. coli, 25 VRE, 5 E. cloacae, 8 K. pneumonia, 144 S. aureus, 27 P. aeruginosa, 24 S. epidermidis, 22 S. pyogenes.

The genome sequencing has been conducted in collaboration with consortium partners to investigate a number of outbreaks and possible transmissions. These have ranged from transmission in healthcare settings as well as community settings.

Examples of these include:

NHS Scotland & Scottish Microbiology Reference LaboratoriesS aureus outbreak in the west of Scotland involving multiple healthcare facilities. An outbreak of skin and soft tissue infections (SSTI) caused by S. aureus and Group A streptococci (GAS, Streptococcus pyogenes) in IVDUs associated with the injection of a legal high (BURST). E coli O157 Shiga-ve isolates. Staphylococcus epidermidis hospital-associated infections and transmission associated with a healthcare worker.

NHS Fife:  S aureus bacteremias and SSTI in intravenous drug users (IVDUs). Healthcare-associated infections (HAIs): S. pyogenes, S. aureus, Enterobacter cloacae

NHS Grampian:  HAIs: E. coli, VRE, Kleibsella pneumonia, S. aureus, Pseudomonas aeruginosa

 NHS Tayside:  HAIs: P. aeruginosa & VRE

The data that these projects have yielded have been used to inform infection control and patient management at the local level (NHS board), and at the national level (Health protection Scotland and NHS National Services Scotland), and is being written up in several publications.

Collaborations:  The MiSeq has also been used to establish collaborative project across the university, and internationally.

Collaborative projects studying hospital-associated S. aureus infections have been established with the Oxford University Clinical Research Unit in Vietnam (OUCRU). To facilitate the collaboration with OUCRU and share skill sets using the MiSeq (OUCRU have just got their own MiSeq) we have hosted a visiting scientist (Mr Phat Voong Vinh; June 2015), and a member Dr Kerry Pettigrew has spent 3 weeks at OUCRU in January 2016 helping them with their sequencing.

Within the university we have been collaborating with Ulrich Schwarz-Linek and Ona Miller on a project looking at the vancomycin resistant enterococci (VRE) surface proteins. In this project we have sequenced clinical isolates of VRE from local hospitals and provided sequence data that has facilitated the identification of novel surface protein isolates.

The MiSeq has also been used to in a pilot project funded by NHS Fife to investigate the genome epidemiology of S. aureus infections in injecting drug users. This study is being written up and will form the basis of a grant application for a larger study.

Support for PhD students:  A number of PhD students within the School of Medicine make considerable use of the MiSeq as part of their experimental work.

 

Matt Holden

Public Engagement and Outreach  University of St Andrews Space School 2016

A series of visits by Fife primary schools pupils to different departments in the University to learn about aspects of space and space travel from the different disciplines. I participated in the Medical School Space School on the 7th of May, running a session teaching 70 P7 children them about the internal organs of the human body and space.

Capacity Building    Wellcome Genome Campus Advanced Course: Genomics and Epidemiological Surveillance of Bacterial Pathogens, Buenos Aires, Argentina, 17th Apr 2016 – 22nd Apr 2016.   Wellcome Trust Advanced Course: Genomics and Epidemiological Surveillance of Bacterial Pathogens, Costa Rica, 1st Feb 2015 – 6th Feb 2015 .   I helped design the course and demonstrated on these 5-day workshops, teaching clinical scientists and researchers from across Latin America how to use and apply bioinformatics and whole genome sequencing for public health.

 

Peter Coote

Introduction –   ISSF support was provided to P. Coote for a School of Biology-funded research assistant  (Ms. Frances Entwistle). She commenced work on the 4 year project in October 2015.

Multi-drug resistant tuberculosis (MDRTB) is a rising global threat and new treatments for this are urgently required. One bottleneck in the discovery of new treatments for antibiotic-resistant bacteria is the lack of a reliable, cheap and ethical in vivo infection model that can be employed for screening novel compounds/treatments prior to efficacy testing in more traditional mammalian models. In recent years researchers have studied the possibility of employing invertebrate infection models to overcome some of these difficulties. Thus, the aims of this ISSF-supported project are:

  1. To evaluate the use of the Galleria mellonella (Greater wax-moth) larvae as an in vivo infection model for studying Mycobacterial infections.
  2. To employ this model to screen for antibiotic efficacy versus Mycobacterial infections
  3. To employ the model to screen and identify novel drug combination treatments with enhanced efficacy versus Mycobacteria in vivo.

Results   –   To date, inoculum-size dependent infections have been successfully induced in G. mellonella larvae using strains of fast-growing Mycobacteria including Mycobacterium fortuitum, Mycobacterium marinum and Mycobacterium aurum. These acute infections have been established at 37oC over a period of 6 days. Mycobacterium bovis BCG strain is yet to be tested. The infections were observed to be dose-dependent in terms of the size of the infecting inoculum and required the presence of live bacteria because infections with identical doses of heat-killed bacteria did not result in any larval mortality.   Acute infections of G. mellonella induced by M. fortuitum and M. marinum have been successfully treated using a range of anti-Mycobacterial antibiotics including amikacin, ciprofloxacin, ethambutol, isoniazid and rifampicin. These drugs were able to reduce the mortality of infected larvae in a dose-dependent manner. In addition, measurement of the bacterial burden within infected larvae during antibiotic treatment revealed a close correlation between antibiotic efficacy and a decline in bacterial burden.   Current experiments aim to further validate the G. mellonella model for use with Mycobacteria by studying the innate immune response to infection using fluorescence microscopy to measure haemocyte density and viability during infection.

 

Justin Ales

Recieved ISSF seedcorn funding to support research assistant Fraser Aitken. Fraser has been designing and implementing experimental protocols to test models of information processing in humans.  The  first experimental protocol is now up and running and he is collecting data. Early pilot results are promising.  These experiments are on course to provide pilot data to support a Wellcome Trust grant application.

 

 

Helder Ferreira

I received ISSF seedcorn funding for a research assistant to work on “Using C. elegans to understand the functions of ATRX”. I recruited a talented individual, Karim Hussain, who started in October 2015. Karim has optimised an in vitro rolling circle amplification assay showing that xnp-1 (worm ATRX homolog) mutants have elevated levels of telomeric C-circles. This is indicative of ALT activity and an important phenotype that helps to link mutation of human ATRX to ALT positive cancers. Karim will be going to the European Worm meeting in Berlin next month to present his work as a poster.

 

Rafael da Silva

Enzymology of sub-class II ATP phosphoribosyltransferases for antibiotic development

Histidine biosynthesis is essential in bacteria but absent in humans. This makes this pathway’s enzymes attractive targets for antibiotic development. The first enzyme of the pathway is ATP phosphoribosyltransferase (ATPPRT), which catalyses the nucleophilic substitution reaction between ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) to form N1-5ʹ-phosphoribosyl-ATP (PRATP) and inorganic pyrophosphate. ATPPRT is allosterically inhibited by histidine.

We are investigating the mechanism of this enzyme in Staphylococcus aureus and expect to use the information to design specific inhibitors of the reaction. S. aureus possesses a sub-class II ATPPRT, where the holoenzyme is a hetero-oligomer made up of two proteins, HisG and HisZ. The former contributes the catalytic site, while the latter contains the allosteric site for histidine binding.

We have expressed SaHisZ gene in E. coli and purified its product in large amounts. However, the SaHisG product is only expressed in insoluble form in E. coli. It is now being cloned into a different vector to allow co-expression with SaHisZ.

Meanwhile, we have expressed and purified HisG and HisZ from the cold-adapted bacterium Psychrobacter arcticus. The catalytic subunits (HisG) of the two species share 38% sequence identity and are expected to share strong structural similarity. Therefore, we are using PaATPPRT as a surrogate for the S. aureus counterpart until we obtain soluble SaHisG.

Our results indicate that PaATPPRT follows a sequential kinetic mechanism with ternary complex formation. In addition, temperature studies suggest that the enzyme follow Arrhenius behaviour only up to 30 °C, after which it starts to denature.

In collaboration with Prof James Naismith, we have crystallized the enzyme complex and collected useful X-ray diffraction data, which are currently being processed to solve the three-dimensional structure of the enzyme. These results will pave they for further mechanistic investigations and for inhibitor design.

 

Daniela Balslev

The aim of the project is to understand how attention and the eye are coupled.  Alexandra Mitchell,  joined the project in October 2015. She will be using functional magnetic resonance imaging (fMRI) to investigate the connectivity between attention and oculomotor networks in the human brain.  Preliminary tests show the suitability of our behavioural tasks for separating the oculosensory and the oculomotor components of the gaze signal. This is a necessary step before imaging the cortical representations for these signals. There are no publications from this work yet. I presented preliminary work last month, in an invited talk at the Workshop on Oculomotor Readiness and Covert Attention at the University of Durham.

 

Stefan Pulver

Project Overview

The stated aim of my ISSF project was to explore how modulation of sodium potassium pump activity regulates excitability within neural networks. I proposed using Drosophila as a model organism in this study because the fruitfly genetic toolkit allows us to do experiments that are difficult to do in other model organisms . The proposed project involved forging a new collaboration with a computational neuroscientist at Emory University.

Aims:    My goals in the initial phase of this project were to 1) Recruit a PhD student 2) Build computing and research infrastructure in Pulver lab 2) Begin generating preliminary electrophysiology and modeling data. Longer term, my stated objective was to apply for a Wellcome Trust New Investigator Award within 3 years.

Outcomes to date:  PhD student – I recruited a PhD student (B. Nathanson) who established computing and research foundations for the lab and generated preliminary data.

Grants – During these first 7 months of my ISSF award, I won a Research Grant from the Royal Society (£14,795) and a BBSRC CASE PhD Studentship (£92,883). Both funding successes grew directly out of groundwork laid by my ISSF funding.

Impact – ISSF funding allowed me to get my lab up and running to the point that I was able to run an outreach event for local secondary school children from Kircaldy in fall of 2015. This provided a platform for me to apply successfully for a MacKnight Early Career Educator Award from the American Physiological Society (£6500). These funds will be used in part to continue impact activities in Fife.

Honours – I was invited to join the editoral board of Nature: Scientific Reports and the review panel for Newton International Fellowships (Royal Society). I have accepted both invitations.

 

Olivier Penacchio

Report of ISSF funding, Olivier Penacchio, 1st January – 4th April 2016

In the period ranging from 1st January to 4th April 2016, I have mainly extended the work that was being carried out for the BBSRC grant to Prof. Julie Harris YBB039 (Nov 2012- Dec 2015). The purpose of this grant was to better understand the potential role of countershading in visual camouflage.

We had previously published theoretical modelling to predict how terrestrial animals should be countershaded to reduce their visibility to predators. To test these predictions, and therefore validate or no visual camouflage as one of the evolutionary driving force for the evolution of countershading, we also had to: 1. Measure the effect of countershading on animal visibility and 2. Measure the countershading of real animals to check whether real patterns agree with theoretical predictions. All the data (measurement using a 3D scanner, psychophysical experiments with birds and humans) had been collected before the grant finished.

During the period of the ISSF funding I analysed the measurements of both the shape and colour pattern of several species of caterpillar and designed sophisticated methods to assess the extent to which these measurements match theoretical predictions. This study allowed me to show that in some species colour and shape are indeed optimal to reduce visibility in natural environments by reducing shading cues on the body. A paper on this important result is in progress.

I also wrote with my collaborators (Prof. Julie Harris, Prof. Innes Cuthill, Dr George Lovell and Prof. Graeme Ruxton) two papers exploring the effect of the countershading pattern on visual camouflage. One of them defines behavioural limits on countershaded animal body orientation in space to preserve the beneficial effect of countershading on visibility; this paper will be submitted to Scientific report soon. The second analyses how a countershaded pattern should be matched to the illumination of the scene to be efficient; this paper will be submitted to Animal Behaviour soon.

During the period covered by the ISSF grant I also worked in parallel on a project on underwater camouflage as well as on a characterisation of visually uncomfortable images informed by computational neurosciences. The first of these two projects has been carried out in collaboration with Professor Sönke Johnsen (Department of Biology, Duke University) and Prof. Julie Harris. This period allowed me to build mathematical models to understand underwater camouflage and has led to the submission of an abstract accepted for oral communication at the conference Light and Color in Nature, June, 2016. The corresponding paper will be drafted within a few months.

In a different project, on a computational characterisation of visual discomfort, the period covered by the ISSF grant allowed me to further my understanding of the consequences of a reduced availability of inhibitory activity in the visual cortex on the strength of activity in response to uncomfortable visual stimuli. This work provides an account for the huge individual differences in susceptibility to visual discomfort. The corresponding work, in collaboration with Prof. Julie Harris, Prof. Arnold Wilkins, University of Essex, and Dr Xavier Otazu, UAB, Barcelona, has been submitted as an abstract to this year European Conference on Visual Perception (ECVP 2016) and accepted for an oral presentation.

From 1st April 2016, I started working on a new BBSRC grant, to Harris & Penacchio (YBB091).

 

Bela Bode

ISSF bridging funds (Scheme 2) 6 months funding requested for Dr Katrin Ackermann (PDRA), 3 months funded.

The sumbitted grant for which bridging funds were requested was funded (EPSRC  “Intra-monomer distances in multimeric systems” EP/M024660/1) and matched by the university extending Katrin’s contract until August 2017. Another grant which came into force the same time was a royal society research grant (RG140723). Recently an eastBIO studentship was awarded (starting 09/2016). An MRC grant by U. Schwarz-Linek directly benefitted from the preliminary data generated by Katrin and partially from her being bridged. An ERC Starting Grant Proposal had progressed to interview in Brussels, but was unsuccessful.

Two publications (open and acknowledging WT funding) have directly benefitted from bridging Katrin.

J. Morten, J. R. Peregrina, M. Figueira-Gonzalez, K. Ackermann, B. E. Bode, M. F. White, J. C. Penedo, Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach, Nucleic Acids Res. 2015, 43, 10907-10924.

Valera, K. Ackermann, C. Pliotas, H. Huang, J. H. Naismith, B. E. Bode, Accurate Extraction of Nanometer Distances in Multimers by Pulse EPR, Chem. Eur. J. 2016, 22, 4700-4703.

Several manuscripts with Katrin’s input (1 first authorship and 2 co-authorships) are in preparation.

Further proposals for two different follow-up projects are submitted:

BBSRC ‘ Taking a molecular motor for a spin: helicase mechanism studied by EPR’, PI M.F. White, Co-I B.E. Bode

ERC ‘mustEPR – membrane channel multimeric structure by pulse Electron Paramagnetic Resonance: assessing structural changes in mechanosensation’, PI B. E. Bode

Outcomes 2/2

ISSF Seedcorn £10k equipment requested (and granted)

Electron Paramagnetic Resonance Sample Preparation and Storage Laboratory and Liquid Nitrogen EPR

The sample preparation laboratory and storage facilities as well as the liquid nitrogen EPR operation are in full force.

EPR samples produced by a variety of BSRC groups (Naismith, Penedo, Pliotas, Schwarz-Linek, Stewart, White) are stored or produced and stored in the lab and long-term archived.

Operation of cw and pulse EPR using nitrogen as cost-efficient cryogen can be routinely performed, however it proves mostly feasible for cw EPR.

Manuscripts benefitting from the use of the lab are (as in outcome 1/2)

J. Morten, J. R. Peregrina, M. Figueira-Gonzalez, K. Ackermann, B. E. Bode, M. F. White, J. C. Penedo, Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach, Nucleic Acids Res. 2015, 43, 10907-10924.Valera, K. Ackermann, C. Pliotas, H. Huang, J. H. Naismith, B. E. Bode, Accurate Extraction of Nanometer Distances in Multimers by Pulse EPR, Chem. Eur. J. 2016, 22, 4700-4703.   Several others in preparation.

 

Judith Sleeman

Adopting a BioID approach to study the minor spliceosome.

The proposal was to compare the results of interactome analysis the splicing factor, SNRNP48, using a conventional mCherry affinity isolation vs the newer proximity-labelling approach BioID.

Progress:  All plasmids required are present in the lab and have now been fully validated. I have repeated some of the analysis the localisation of mCherrySNRNP48 in stable cell lines and transient transfections and confirmed correct protein expression by western blot. I have recruited an undergraduate summer student to the lab (Kimberley Morrison, funded by the Callan Memorial Scholarship). This will be her project and she will start in the lab at the beginning of June.

 

David O’Hagan

I received two months salary (Feb-March) for Dr Davide Bello, to bridge between two EPSRC grants.

The EPSRC application was successful and Davide moved off the ISSF account from the 1st April.   Davide has developed a new analogue fluorovinyl thioether  of acetyl-coenzyme-A which mimics the enolate of acetyl coenzyme-A.  This analogue is a good inhibitor of citrate synthese and has the potential to form the basis of inhibitors of a range of acyl-CoA processing enzymes.  We have yet to publish on this work, and the contribution made in Feb-March 2016, although there will be papers in due course. He now works in a collaboration with the Naishmith lab (co-crystallisation with citrate synthase) and theory and modelling of the interaction of the inhibitor with the enzyme in an interaction from the Buehl lab.

 

Alan Stewart

Dr Simon Powis and I received funding from ISSF which has been used to support a research assistant, Dagmara Wiatrek (under St Andrews ISSF seedcorn funding initiative). Dagmara is now examining the roles of zinc in the immune system.  The project has developed well – she has been looking at the effects of zinc and lipopolysaccharide-stimulation on zinc transporter expression in dendritic cells using molecular and proteomic techniques (SWATH). We hope to write a paper on this in the coming months. Furthermore, she is examining the modulation of histidine-rich glycoprotein-complement interactions by zinc, a process that has relevance to the progression of age-related macular degeneration. This latter work has led to a new collaboration with Dr Imre Lengyel (UCL Institute of Ophthalmology) and contributed to a recent grant award to Stewart group of £110k from Fight for Sight.

I received funding from ISSF for 50% of the cost of a new MicroCal iTC200 isothermal titration calorimeter (under St Andrews ISSF equipment funding initiative). This instrument complements the University’s existing VP-ITC system but has the important advantage of requiring 7-times less sample. The School of Medicine matched the ISSF funding and the machine was installed in February 2014. It has been used not only by my group but also by members of the Naismith, White and Westwood groups. In addition, this equipment has provided pilot data central to awarded project and studentship grants from British Heart Foundation (of £191K and £108K, respectively) for Stewart group.

I received 6 months funding for Dr Siavash Khazaipoul (under St Andrews ISSF bridging funding initiative) in December 2014. Dr Khazaipoul’s post was successfully bridged and only 2 months (of the 6 months funds) were used due to the award of a British Heart Foundation project grant (as mentioned above). He started a new 3-year contract in June 2015.