Investigating the function of the hereditary spastic paraplegia protein spastin in the endomembrane system

Hereditary spastic paraplegias (HSPs) are genetically inherited neurological diseases characterised by the distal axonal degeneration of corticospinal neurons. Of the 80 genes currently associated with HSP, mutations in SPAST, encoding the protein spastin, are by far the most common cause of pathology. Spastin functions as a microtubule remodelling enzyme by using energy derived from ATP hydrolysis by its ATPase domain. The location of this activity is governed by spastin’s localisation domains which mediate recruitment to membrane sites including endosomes and the ER. In this thesis I aimed to elucidate the function of spastin at these sites, as well as to analyse the resulting effects on the cell surface proteome. Through this work, I have shown that spastin functions to mediate the fission of endosomal recycling tubule and have confirmed spastin’s localisation to ER exit sites, but show using synchronised secretion assays that spastin is dispensable for generalised cargo secretion of at least 2 classes of secretory cargo. Finally, through quantitative cell surface proteomics, I show that mutation of spastin’s ATPase domain induces substantial remodelling of the cell surface proteome, and through this have generated a list of pathological candidates whose change in surface abundance could drive the pathogenicity of spastin-HSP.Medical Research Council PhD Studentship Cambridge Institute of Medical Researc

Internal Robustness: systematic search for systematic bias in SN Ia data

A great deal of effort is currently being devoted to understanding, estimating and removing systematic errors in cosmological data. In the particular case of type Ia supernovae, systematics are starting to dominate the error budget. Here we propose a Bayesian tool for carrying out a systematic search for systematic contamination. This serves as an extension to the standard goodness-of-fit tests and allows not only to cross-check raw or processed data for the presence of systematics but also to pin-point the data that are most likely contaminated. We successfully test our tool with mock catalogues and conclude that the Union2.1 data do not possess a significant amount of systematics. Finally, we show that if one includes in Union2.1 the supernovae that originally failed the quality cuts, our tool signals the presence of systematics at over 3.8-sigma confidence level.Comment: 14 pages, 15 figures; matches version accepted for publication in MNRA

ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication

The β-coronavirus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the global Covid-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release and pathogenesis. We developed a non-disruptive tagging strategy for SARS-CoV-2 E and find that at steady-state, it localises to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for Endoplasmic Reticulum (ER)-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP Ribosylation Factor-1/Adaptor Protein-1 (ARFRP1/AP-1) dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and by developing a trans-complementation assay for SARS-CoV-2 structural proteins, we show that lysosomal delivery of E and its viroporin activity are necessary for efficient viral replication and release

ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic

Funder: National Institute for Health Research; doi: http://dx.doi.org/10.13039/501100000272Funder: Gates Cambridge Trust; doi: http://dx.doi.org/10.13039/501100005370Abstract: Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER–endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic

Trends in chlamydia and gonorrhoea testing and positivity in Western Australian Aboriginal and non-Aboriginal women 2001-2013: a population based cohort study

Aims: To examine trends in chlamydia and gonorrhoea testing and positivity in Aboriginal and non-Aboriginal women of reproductive age. Methods: A cohort of 318002 women, born between 1974-1995, residing in Western Australia (WA) was determined from birth registrations and the 2014 electoral roll. This cohort was then probabilistically linked to all records of chlamydia and gonorrhoea nucleic acid amplification tests (NAAT) conducted between 1st January 2001 and 31st December 2013 by two large WA pathology laboratories. Trends in chlamydia and gonorrhoea testing and positivity were investigated over time and stratified by Aboriginality and age group. Results: The proportion of women tested annually for chlamydia increased significantly between 2001 and 2013 from 24% to 37% in Aboriginal and 4.0% to 8.5% in non-Aboriginal women (both p-values80%) and so patterns of gonorrhoea testing were similar. Chlamydia and gonorrhoea positivity were substantially higher in Aboriginal compared to non-Aboriginal women; age-, region- and year-adjusted Incidence Rate Ratio’s 1.52(95%CI 1.50-1.69, p Conclusion: Between 2001 and 2013 in WA chlamydia and gonorrhoea positivity remained highest in young Aboriginal women despite chlamydia positivity increasing among young non-Aboriginal women. More effective prevention strategies, particularly in young Aboriginal women are needed to addres these disparitie

Risk of pelvic inflammatory disease in relation to chlamydia and gonorrhea testing, repeat testing, and positivity: A population-based cohort study

Background: There is uncertainty around whether the risks of pelvic inflammatory disease (PID) differ following Chlamydia trachomatis (chlamydia) and Neisseria gonorrhoeae (gonorrhea) infection. We quantified the risk of PID associated with chlamydia and gonorrhea infection and subsequent repeat infections in a whole-population cohort. Methods: A cohort of 315123 Western Australian women, born during 1974–1995, was probabilistically linked to chlamydia and gonorrhea testing records and to hospitalizations and emergency department presentations for PID from 2002 to 2013. Time-updated survival analysis was used to investigate the association between chlamydia and gonorrhea testing, and positivity, and risk of PID. Results: Over 3199135 person-years, 120748 women had pathology test records for both chlamydia and gonorrhea, 10745 chlamydia only, and 653 gonorrhea only. Among those tested, 16778 (12.8%) had ≥1 positive chlamydia test, 3195 (2.6%) ≥1 positive gonorrhea test, and 1874 (1.6%) were positive for both. There were 4819 PID presentations (2222 hospitalizations, 2597 emergency presentations). Adjusting for age, Aboriginality, year of follow-up, health area, and socioeconomic status, compared to women negative for chlamydia and gonorrhea, the relative risk (adjusted incidence rate ratio) of PID was 4.29 (95% confidence interval [CI], 3.66–5.03) in women who were both chlamydia and gonorrhea positive; 4.54 (95% CI, 3.87–5.33) in those only gonorrhea positive; and 1.77 (95% CI, 1.61–1.94) in those only chlamydia positive. Conclusions: Gonorrhea infection conferred a substantially higher risk than chlamydia of hospitalization or emergency department presentation for PID. The emergence of gonorrhea antimicrobial resistance may have a serious impact on rates of PID and its associated reproductive health sequelae

CPAP pressure and flow data at 2 positive pressure levels and multiple controlled breathing rates from a trial of 30 adults

Objectives: A unique dataset of airway flow/pressure from healthy subjects on Continuous Positive Airway Pressure (CPAP) ventilation was collected. This data can be used to develop or validate models of pulmonary mechanics, and/or to develop methods to identify patient-specific parameters which cannot be measured non-invasively, during CPAP therapy. These models and values, particularly if available breath-to-breath in real-time, could assist clinicians in the prescription or optimisation of CPAP therapy, including optimising PEEP settings. Data description: Data was obtained from 30 subjects for model-based identification of patient-specific lung mechanics using a specially designed venturi sensor system comprising an array of differential and gauge pressure sensors. Relevant medical information was collected using a questionnaire, including: sex; age; weight; height; smoking history; and history of asthma. Subjects were tasked with breathing at five different rates (including passive), matched to an online pacing sound and video, at two different levels of PEEP (4 and 7 cmH2O) for between 50 and 180 s. Each data set comprises ~ 17 breaths of data, including rest periods between breathing rates and CPAP levels

Is there a standard measuring rod in the Universe?

The Caltech-Jodrell Bank very long baseline interferometry (VLBI) Surveys give detailed 5 GHz VLBI images of several hundred milliarcsecond (mas) radio sources, and the full width at half-maximum angular sizes of the corresponding compact cores. Using the latter, I have constructed an angular-diameter/redshift diagram comprising 271 objects, which shows clearly the expected features of such a diagram, without redshift binning. Cosmological parameters are derived which are compatible with existing concensus values, particularly when the VLBI data are combined with recent Baryon Accoustic Oscillations observations; the figures are presented as indications of what might be expected of larger samples of similar data. The importance of beaming and relativistic motion towards the observer is stressed; a model of the latter indicates that the emitting material is close to the observer's line of sight and moving with a velocity which brings it close to the observer's rest frame. With respect to linear size, these objects compare well in variance with type Ia supernovae; the efficacy of the latter is improved by the brighter-slower and brighter-bluer correlations, and by the inverse-square law.Comment: 5 pages, 3 figure

CMB observations from the CBI and VSA: A comparison of coincident maps and parameter estimation methods

We present coincident observations of the Cosmic Microwave Background (CMB) from the Very Small Array (VSA) and Cosmic Background Imager (CBI) telescopes. The consistency of the full datasets is tested in the map plane and the Fourier plane, prior to the usual compression of CMB data into flat bandpowers. Of the three mosaics observed by each group, two are found to be in excellent agreement. In the third mosaic, there is a 2 sigma discrepancy between the correlation of the data and the level expected from Monte Carlo simulations. This is shown to be consistent with increased phase calibration errors on VSA data during summer observations. We also consider the parameter estimation method of each group. The key difference is the use of the variance window function in place of the bandpower window function, an approximation used by the VSA group. A re-evaluation of the VSA parameter estimates, using bandpower windows, shows that the two methods yield consistent results.Comment: 10 pages, 6 figures. Final version. Accepted for publication in MNRA

Cosmological parameter estimation using Very Small Array data out to l=1500

We estimate cosmological parameters using data obtained by the Very Small Array (VSA) in its extended configuration, in conjunction with a variety of other CMB data and external priors. Within the flat $\Lambda$CDM model, we find that the inclusion of high resolution data from the VSA modifies the limits on the cosmological parameters as compared to those suggested by WMAP alone, while still remaining compatible with their estimates. We find that $\Omega_{\rm b}h^2=0.0234^{+0.0012}_{-0.0014}$, $\Omega_{\rm dm}h^2=0.111^{+0.014}_{-0.016}$, $h=0.73^{+0.09}_{-0.05}$, $n_{\rm S}=0.97^{+0.06}_{-0.03}$, $10^{10}A_{\rm S}=23^{+7}_{-3}$ and $\tau=0.14^{+0.14}_{-0.07}$ for WMAP and VSA when no external prior is included.On extending the model to include a running spectral index of density fluctuations, we find that the inclusion of VSA data leads to a negative running at a level of more than 95% confidence ($n_{\rm run}=-0.069\pm 0.032$), something which is not significantly changed by the inclusion of a stringent prior on the Hubble constant. Inclusion of prior information from the 2dF galaxy redshift survey reduces the significance of the result by constraining the value of $\Omega_{\rm m}$. We discuss the veracity of this result in the context of various systematic effects and also a broken spectral index model. We also constrain the fraction of neutrinos and find that $f_{\nu}< 0.087$ at 95% confidence which corresponds to $m_\nu<0.32{\rm eV}$ when all neutrino masses are the equal. Finally, we consider the global best fit within a general cosmological model with 12 parameters and find consistency with other analyses available in the literature. The evidence for $n_{\rm run}<0$ is only marginal within this model