Monte Carlo simulation of DNA origami self-assembly

The optimal design of DNA origami systems that assemble rapidly and robustly is hampered by the lack of a model capable of simulating the self-assembly process that is sufficiently detailed yet computationally tractable. In this thesis, we propose a model for DNA origami that strikes a balance between these two criteria by representing DNA origami systems on a lattice at the level of binding domains. Particular attention is paid to the constraints imposed by the double-helical twist, as they determine where strand crossovers between adjacent helices can occur. Because of the highly specific types of interaction and the length of the scaffold, standard Monte Carlo simulation methods for polymeric systems are found to be ineffective at sampling the dense, near-assembled states considered here. In order to address the issue of sampling such states, we develop Monte Carlo methods that extend the configurational bias and recoil growth methods, and consider the sampling of scaffold conformations independently from the sampling of staple binding states. We demonstrate the validity of our model and the feasibility of our sampling methods with simulations of a small origami design previously studied with the oxDNA model, as well as with designs that include staples that span longer scaffold segments. In other self-assembling systems, it is often the case that nucleation barriers control the self-assembly behaviour. We investigate whether there is a nucleation barrier along the self-assembly pathway of DNA origami. Our simulations reveal that for simple systems, stacking interactions govern a nucleation barrier, albeit one that is never prohibitively large relative to thermal fluctuations. These findings may prove useful in the design of DNA origami structures capable of controllable reversible folding for functional purposes and in assisting the optimization of assembly pathways at the design stage

Investigations into Zika virus-host interactions: A neurological perspective

Zika virus (ZIKV) is a member of the Flavivirus genus of the family Flaviviridae. Members of this genus possess a single-stranded positive-sense RNA genome which are flanked by 5’ and 3’ untranslated regions. Previously, ZIKV infection was thought to cause symptomatic infection in 20% of patients, characterised by a non-purulent rash and a mild fever. However, recent ZIKV outbreaks have seen the emergence of novel neurological sequelae associated with ZIKV infection. These symptoms affected the central and peripheral nervous systems (CNS and PNS, respectively) of neonates and adults (congenital ZIKV syndrome and Guillain-Barré syndrome, respectively). Consequently, prior to commencement of this thesis, the knowledge of ZIKV interactions within the CNS and PNS was limited. Therefore, the primary aim of this thesis was to expand the current knowledgebase of ZIKV infection within cells of the CNS and PNS. The work presented herein assessed available tools to study ZIKV infection in vitro, including testing previously uncharacterised commercial antibodies targeting the ZIKV envelope (ZIKV E) and NS1 proteins. Subsequently, a suitable antibody raised against the ZIKV E was identified and used in downstream analyses. A model system of ZIKV infection of the CNS and PNS using mixed-cell co-cultures derived from mouse spinal cord (CNS) or dorsal root ganglion (PNS) was used to determined cell-type specific susceptibility to ZIKV infection. It was found that cells of the PNS are refractory to ZIKV infection, whereas cells of the CNS are permissive to ZIKV infection. In the CNS, oligodendrocytes and their precursors were the most susceptible cell type to ZIKV infection. Moreover, when infecting CNS co-cultures at a time point which reflects prenatal life through to post-natal life, white matter structures (myelin sheath and axons) are injured. However, neuronal cell bodies remained healthy. Using purified primary neuronal cultures derived from mouse spinal cord, it was determined that neurons were refractory to ZIKV infection in the absence of accompanying glial cells. This suggests axonal damage may not be a result of direct infection of neurons themselves and may be a consequence of oligodendrocyte infection and injury. Transcriptomic analyses of ZIKV-infected CNS co-cultures revealed that ZIKV infection induces the upregulation of genes involved in antiviral responses and inflammatory pathways, including TNF and ROS/NO pathway components; these pathways may be involved in exacerbating injury to white matter structures within ZIKV-infected CNS-co-cultures. In summary, the results described in this thesis show that ZIKV can infect the early post-natal CNS. Furthermore, ZIKV-infection of this murine model demonstrates that infection induces the injury and depletion of myelin and axons. This is likely to be due to a combination of effects such as direct viral infection, cell death of oligodendrocytes, and secreted inflammatory factors. Myelination occurs late in foetal development and carries on into early adulthood. These data, in combination with precedent pathological findings, suggest that ZIKV-infected children born absent of microcephaly may develop other, more subtle, neurological sequelae later in life

Inhibition of type I interferon induction and signalling by mosquito-borne flaviviruses

The Flavivirus genus (Flaviviridae family) contains a number of important human pathogens, including dengue and Zika viruses, which have the potential to cause severe disease. In order to efficiently establish a productive infection in mammalian cells, flaviviruses have developed key strategies to counteract host immune defences, including the type I interferon response. They employ different mechanisms to control interferon signal transduction and effector pathways, and key research generated over the past couple of decades has uncovered new insights into their abilities to actively decrease interferon antiviral activity. Given the lack of antivirals or prophylactic treatments for many flaviviral infections, it is important to fully understand how these viruses affect cellular processes to influence pathogenesis and disease outcome. This review will discuss the strategies mosquito-borne flaviviruses have evolved to antagonise type I interferon mediated immune responses

Reducing Poverty in California…Permanently

If California were to seriously commit to equalizing opportunity and reducing poverty, how might that commitment best be realized? This is of course a hypothetical question, as there is no evidence that California is poised to make such a serious commitment, nor have many other states gone much beyond the usual lip-service proclamations. There are many reasons for California’s complacency, but an important one is that most people think that poverty is intractable and that viable solutions to it simply don’t exist. When Californians know what needs to be done, they tend to go forward and get it done. When, for example, the state’s roads are in disrepair, there are rarely paralyzing debates about exactly how to go about fixing them; instead we proceed with the needed repairs as soon as the funds to do so are appropriated. The same type of sure and certain prescription might appear to be unavailable when it comes to reducing poverty. It is hard not to be overwhelmed by the cacophony of voices yielding a thick stream of narrow-gauge interventions, new evaluations, and piecemeal proposals.1 Although the research literature on poverty is indeed large and may seem confusing, recent advances have in fact been so fundamental that it is now possible to develop a science-based response to poverty. In the past, the causes of poverty were not well understood, and major interventions, such as the War on Poverty, had to be built more on hunch than science. It is an altogether different matter now. The causes of poverty are well established, and the effects of many possible policy responses to poverty are likewise well established. The simple purpose of this essay is to assemble these advances into a coherent plan that would, if implemented, reduce poverty in California substantially

Late periorbital haemorrhage following functional endoscopic sinus surgery: a caution for potential day case surgery

BACKGROUND: Orbital complications following functional endoscopic sinus surgery (FESS) are fortunately rare. They are usually easily and rapidly recognizable. CASE PRESENTATION: We present an unusual case of a forty-five year old woman who underwent routine FESS and was not packed nasally after the procedure. Six hours later she started bleeding and nasal packs were inserted. She soon developed unilateral periorbital bruising and within hours her condition had worsened so much that the viability of the eye was thrown into question. She underwent medial and lateral canthotomies and made an uneventful post-operative recovery. CONCLUSION: This rare case demonstrates that late, brisk post-operative bleeding can occur after FESS with potentially catastrophic consequences. Clinicians should be aware that discharging patients after FESS too early may lead to medico-legal problems

Zika virus tropism and interactions in myelinating neural cell cultures: CNS cells and myelin are preferentially affected

The recent global outbreak of Zika virus (ZIKV) infection has been linked to severe neurological disorders affecting the peripheral and central nervous systems (PNS and CNS, respectively). The pathobiology underlying these diverse clinical phenotypes are the subject of intense research; however, even the principal neural cell types vulnerable to productive Zika infection remain poorly characterised. Here we used CNS and PNS myelinating cultures from wild type and Ifnar1 knockout mice to examine neuronal and glial tropism and short-term consequences of direct infection with a Brazilian variant of ZIKV. Cell cultures were infected pre- or post-myelination for various intervals, then stained with cell-type and ZIKV-specific antibodies. In bypassing systemic immunity using ex vivo culture, and the type I interferon response in Ifnar1 deficient cells, we were able to evaluate the intrinsic infectivity of neural cells. Through systematic quantification of ZIKV infected cells in myelinating cultures, we found that ZIKV infection is enhanced in the absence of the type I interferon responses and that CNS cells are considerably more susceptible to infection than PNS cells. In particular, we demonstrate that CNS axons and myelinating oligodendrocytes are especially vulnerable to injury. These results have implications for understanding the pathobiology of neurological symptoms associated with ZIKV infection. Furthermore, we provide a quantifiable ex vivo infection model that can be used for fundamental and therapeutic studies on viral neuroinvasion and its consequences

Mortality and readmission rates of patients discharged in-hours and out-of-hours from a British ICU over a 3-year period

Excess in-hospital mortality following out-of-hours ICU discharge has been reported worldwide. From preliminary data, we observed that magnitude of difference may be reduced when patients discharged for end-of-life care or organ donation are excluded. We speculated that these patients may be disproportionately discharged out-of-hours, biasing results. We now compare in-hospital mortality and ICU readmission rates following discharge in-hours and out-of-hours over 3 years, excluding discharges for organ donation or end-of-life care. This single-centre retrospective study includes patients discharged alive following ICU admission between 01/07/2015-31/07/2018, excluding readmissions and discharges for end-of-life care/organ donation. A multiple logistic regression model was fitted to estimate adjusted odds ratio of death following out-of-hours versus in-hours discharge. Characteristics and outcomes for both groups were compared. 4678 patients were included. Patients discharged out-of-hours were older (62 vs 59 years, p < 0.001), with greater APACHE II scores (15.7 vs 14.4, p < 0.001), length of ICU stay (3.25 vs 3.00 days, p = 0.01) and delays to ICU discharge (736 vs 489 min, p < 0.001). No difference was observed in mortality (4.6% vs 3.7%, p = 0.25) or readmission rate (4.1% vs 4.2%, p = 0.85). In the multiple logistic regression model out-of-hours discharge was not associated with in-hospital mortality (OR = 1.017, 95% CI 0.682-1.518, p = 0.93). Our findings present a possible explanation for reported excess mortality following out-of-hours ICU discharge, related to inclusion of organ donation and end-of-life care patients in data sets rather than standards of care delivered out-of-hours. We are not aware of any other studies investigating the influence of this group on reported post-ICU mortality rates

Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil

Background: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. Methodology/Principal findings: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. Conclusions/Significance: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions