Replication of damaged DNA

DNA is under constant attack from numerous damaging agents and our cells deal with thousands of lesions every day. With such constant damage it is inevitable that the template will not be completely cleared of lesions before the replication complex arrives. The consequences of the replisome meeting an obstacle will depend upon the nature of the obstacle. I have focussed upon replication in Escherichia coli and the effect of UV-induced lesions, which would block synthesis by the replicative polymerases. It is accepted that a UV lesion in the lagging strand template can be bypassed by the replisome complex, but the consequences of meeting a lesion in the leading strand template remain unclear. A lesion in the leading strand template could block replisome progression and the fork might require extensive processing in order to restart replication. However, it has also been proposed that the replisome could progress past these lesions by re‐priming replication downstream and leaving a gap opposite the lesion. The results of my studies revealed that all modes of synthesis are delayed after UV. I have demonstrated that when synthesis resumed, the majority reflected the combined effects of oriC firing and the initiation of inducible stable DNA replication. These modes of synthesis mask the true extent of the delay in synthesis at existing replication forks. The results also revealed that all synthesis after UV is dependent upon DnaC, suggesting that the replicative helicase and possibly the entire replisome, needs to be reloaded. A functional RecFOR system is required for efficient replication restart, without these proteins replication is capable of resuming but only after a long delay. My data support models proposing that replication forks require extensive processing after meeting a lesion in the leading strand template. Whilst I cannot exclude the possibility that replication forks can progress past some such lesions, my data indicate that they cannot progress past many before stalling. Overall, my results demonstrate the importance of measuring all modes of DNA synthesis when assessing the contribution of any particular protein to recovery after UV irradiation. Thus, although net synthesis in cells lacking RecG appears similar to wild type after UV, the mode of replication is in fact quite different. A dramatic increase in the level of stable DNA replication appears to account for much of the overall synthesis detected and coincides with a major chromosome segregation defect. The importance of stable DNA replication in irradiated recG cells has not previously been considered because the different modes of synthesis were ignored. The significance of this pathology and of the other findings reported in this thesis is discussed in relation to current models of DNA repair and replication restart

Replication of damaged DNA

DNA is under constant attack from numerous damaging agents and our cells deal with thousands of lesions every day. With such constant damage it is inevitable that the template will not be completely cleared of lesions before the replication complex arrives. The consequences of the replisome meeting an obstacle will depend upon the nature of the obstacle. I have focussed upon replication in Escherichia coli and the effect of UV-induced lesions, which would block synthesis by the replicative polymerases. It is accepted that a UV lesion in the lagging strand template can be bypassed by the replisome complex, but the consequences of meeting a lesion in the leading strand template remain unclear. A lesion in the leading strand template could block replisome progression and the fork might require extensive processing in order to restart replication. However, it has also been proposed that the replisome could progress past these lesions by re‐priming replication downstream and leaving a gap opposite the lesion. The results of my studies revealed that all modes of synthesis are delayed after UV. I have demonstrated that when synthesis resumed, the majority reflected the combined effects of oriC firing and the initiation of inducible stable DNA replication. These modes of synthesis mask the true extent of the delay in synthesis at existing replication forks. The results also revealed that all synthesis after UV is dependent upon DnaC, suggesting that the replicative helicase and possibly the entire replisome, needs to be reloaded. A functional RecFOR system is required for efficient replication restart, without these proteins replication is capable of resuming but only after a long delay. My data support models proposing that replication forks require extensive processing after meeting a lesion in the leading strand template. Whilst I cannot exclude the possibility that replication forks can progress past some such lesions, my data indicate that they cannot progress past many before stalling. Overall, my results demonstrate the importance of measuring all modes of DNA synthesis when assessing the contribution of any particular protein to recovery after UV irradiation. Thus, although net synthesis in cells lacking RecG appears similar to wild type after UV, the mode of replication is in fact quite different. A dramatic increase in the level of stable DNA replication appears to account for much of the overall synthesis detected and coincides with a major chromosome segregation defect. The importance of stable DNA replication in irradiated recG cells has not previously been considered because the different modes of synthesis were ignored. The significance of this pathology and of the other findings reported in this thesis is discussed in relation to current models of DNA repair and replication restart

Determining the source and eruption dynamics of a stealth CME using NLFFF modelling and MHD simulations

Funding: S.L.Y. would like to acknowledge STFC for support via the consolidated grant SMC1/YST037 and alsoNERC for funding via the SWIMMR Aviation Risk Modelling (SWARM) Project, grant number NE/V002899/1. P.P.would like to thank the ERC for support via grant No. 647214. D.H.M. would like to thank the STFC for support via consolidated grant ST/N000609/1 and, the Leverhulme trust, and the ERC under the Synergy Grant: The Whole Sun (grant agreement no. 810218) for financial support. P.P. and D.H.M. would like to thank STFC for IAA funding under grant number SMC1-XAS012. L.A.U. was supported by the NSF Atmospheric and Geospace Sciences Postdoctoral Research Fellowship Program (Award AGS-1624438).Context. Coronal mass ejections (CMEs) that exhibit weak or no eruption signatures in the low corona, known as stealth CMEs, are problematic as upon arrival at Earth they can lead to geomagnetic disturbances that were not predicted by space weather forecasters. Aims. We investigate the origin and eruption of a stealth event that occurred on 2015 January 3 that was responsible for a strong geomagnetic storm upon its arrival at Earth. Methods. To simulate the coronal magnetic field and plasma parameters of the eruption we use a coupled approach. This approach combines an evolutionary nonlinear force-free field model of the global corona with a MHD simulation. Results. The combined simulation approach accurately reproduces the stealth event and suggests that sympathetic eruptions occur. In the combined simulation we found that three flux ropes form and then erupt. The first two flux ropes, which are connected to a large AR complex behind the east limb, erupt first producing two near-simultaneous CMEs. These CMEs are closely followed by a third, weaker flux rope eruption in the simulation that originated between the periphery of AR 12252 and the southern polar coronal hole. The third eruption coincides with a faint coronal dimming, which appears in the SDO/AIA 211 Å observations, that is attributed as the source responsible for the stealth event and later the geomagnetic disturbance at 1 AU. The incorrect interpretation of the stealth event being linked to the occurrence of a single partial halo CME observed by LASCO/C2 is mainly due to the lack of STEREO observations being available at the time of the CMEs. The simulation also shows that the LASCO CME is not a single event but rather two near-simultaneous CMEs. Conclusions. These results show the significance of the coupled data-driven simulation approach in interpreting the eruption and that an operational L5 mission is crucial for space weather forecastingPostprintPeer reviewe

Epileptogenic effects of NMDAR antibodies in a passive transfer mouse model

Most patients with N-methyl D-aspartate-receptor antibody encephalitis develop seizures but the epileptogenicity of the antibodies has not been investigated in vivo. Wireless electroencephalogram transmitters were implanted into 23 C57BL/6 mice before left lateral ventricle injection of antibody-positive (test) or healthy (control) immunoglobulin G. Mice were challenged 48 h later with a subthreshold dose (40 mg/kg) of the chemo-convulsant pentylenetetrazol and events recorded over 1 h. Seizures were assessed by video observation of each animal and the electroencephalogram by an automated seizure detection programme. No spontaneous seizures were seen with the antibody injections. However, after the pro-convulsant, the test mice (n = 9) had increased numbers of observed convulsive seizures (P = 0.004), a higher total seizure score (P = 0.003), and a higher number of epileptic 'spike' events (P = 0.023) than the control mice (n = 6). At post-mortem, surprisingly, the total number of N-methyl D-aspartate receptors did not differ between test and control mice, but in test mice the levels of immunoglobulin G bound to the left hippocampus were higher (P < 0.0001) and the level of bound immunoglobulin G correlated with the seizure scores (R2 = 0.8, P = 0.04, n = 5). Our findings demonstrate the epileptogenicity of N-methyl D-aspartate receptor antibodies in vivo, and suggest that binding of immunoglobulin G either reduced synaptic localization of N-methyl D-aspartate receptors, or had a direct effect on receptor function, which could be responsible for seizure susceptibility in this acute short-term model

Presynaptic self-depression at developing neocortical synapses

A central tenet of most theories of synaptic modification during cortical development is that correlated activity drives plasticity in synaptically connected neurons. Unexpectedly, however, using sensory-evoked activity patterns recorded from the developing mouse cortex in vivo, the synaptic learning rule that we uncover here relies solely on the presynaptic neuron. A burst of three presynaptic spikes followed, within a restricted time window, by a single presynaptic spike induces robust long-term depression (LTD) at developing layer 4 to layer 2/3 synapses. This presynaptic spike pattern-dependent LTD (p-LTD) can be induced by individual presynaptic layer 4 cells, requires presynaptic NMDA receptors and calcineurin, and is expressed presynaptically. However, in contrast to spike timing-dependent LTD, p-LTD is independent of postsynaptic and astroglial signaling. This spike pattern-dependent learning rule complements timing-based rules and is likely to play a role in the pruning of synaptic input during cortical development

NMDA-receptor antibodies alter cortical microcircuit dynamics

NMDA-receptor antibodies (NMDAR-Abs) cause an autoimmune encephalitis with a diverse range of EEG abnormalities. NMDAR-Abs are believed to disrupt receptor function, but how blocking this excitatory synaptic receptor can lead to paroxysmal EEG abnormalities-or even seizures-is poorly understood. Here we show that NMDAR-Abs change intrinsic cortical connections and neuronal population dynamics to alter the spectral composition of spontaneous EEG activity and predispose brain dynamics to paroxysmal abnormalities. Based on local field potential recordings in a mouse model, we first validate a dynamic causal model of NMDAR-Ab effects on cortical microcircuitry. Using this model, we then identify the key synaptic parameters that best explain EEG paroxysms in pediatric patients with NMDAR-Ab encephalitis. Finally, we use the mouse model to show that NMDAR-Ab-related changes render microcircuitry critically susceptible to overt EEG paroxysms when these key parameters are changed, even though the same parameter fluctuations are tolerated in the in silico model of the control condition. These findings offer mechanistic insights into circuit-level dysfunction induced by NMDAR-Ab

Subset of cortical layer 6b neurons selectively innervates higher order thalamic nuclei in mice

The thalamus receives input from 3 distinct cortical layers, but input from only 2 of these has been well characterized. We therefore investigated whether the third input, derived from layer 6b, is more similar to the projections from layer 6a or layer 5. We studied the projections of a restricted population of deep layer 6 cells (“layer 6b cells”) taking advantage of the transgenic mouse Tg(Drd1a-cre)FK164Gsat/Mmucd (Drd1a-Cre), that selectively expresses Cre-recombinase in a subpopulation of layer 6b neurons across the entire cortical mantle. At P8, 18% of layer 6b neurons are labeled with Drd1a-Cre::tdTomato in somatosensory cortex (SS), and some co-express known layer 6b markers. Using Cre-dependent viral tracing, we identified topographical projections to higher order thalamic nuclei. VGluT1+ synapses formed by labeled layer 6b projections were found in posterior thalamic nucleus (Po) but not in the (pre)thalamic reticular nucleus (TRN). The lack of TRN collaterals was confirmed with single-cell tracing from SS. Transmission electron microscopy comparison of terminal varicosities from layer 5 and layer 6b axons in Po showed that L6b varicosities are markedly smaller and simpler than the majority from L5. Our results suggest that L6b projections to the thalamus are distinct from both L5 and L6a projectionsZ.M.’s laboratory is supported by Medical Research Council (G00900901), Biotechnology and Biological Sciences Research Council (BB/1021833) and The Wellcome Trust (092071/Z/10/Z). E.G. held an MRC Doctoral Studentship; S.H. is supported from Daiichi Sankyo Foundation of Life Science, Japan, L.U. is supported by OXION Wellcome Trust Initiative, Oxford. Y.K. is supported from the Pennsylvania Department of Health using Tobacco CURE Funds SAP#4100062216; P.K. from National Institutes of Health (NIH) R01DC009607 and a visiting Fellowship at St. Catherine’s College, Oxford. F.C.’s laboratory is supported by Human Brain Project (European Flagship, Ref. GA 604102 and Ministerio de Economia y Competitividad MINECO (Spain; Grant BFU2017-88549-P)

Shared decision making or paternalism in nursing consultations? A qualitative study of primary care asthma nurses’ views on sharing decisions with patients regarding inhaler device selection. Health Expect

Abstract Background Although patients with asthma would like more involvement in the decision-making process, and UK government policy concerning chronic conditions supports shared decision making, it is not widely used in practice