Genetic characterisation of Escherichia coli RecN protein as a member of SMC family of proteins

YesThe proteins of SMC family are characterised by having Walker A and B sites. The Escherichia coli RecN protein is a prokaryotic member of SMC family that involved in the induced excision of Tn10 and the repair of the DNA double strand breaks. In this work, the Walker A nucleotide binding site of the E. coli RecN protein was mutated by changing the highly conserved lysine residue 35 to the aspartic acid (D), designated as recN(K35D). Reverse genetics was utilized to delete the entire recN gene (Delta recN108) or introduce the recN(K35D) gene into the E. coli chromosomal DNA. The recN(K35D) cells showed decreasing in the frequency of excision of Tn10 from gal7

Phenomenology of Intrusive Trauma Memory in Psychosis and its Relationship with Hallucinations and Persecutory Beliefs

This thesis is presented in three parts, and is focused on developing the theoretical understanding of the role of trauma memory in psychosis. The systematic literature review investigates the relationship between psychosis symptom severity and re-experiencing of traumatic memories. 13 studies published since 1980 were identified as meeting the review criteria. Overall, findings suggest that people with more severe hallucinations and paranoia experiences report more re-experiencing of traumatic memories. However, this relationship was not seen when looking at more global symptoms of psychosis. The role of trauma memory in the development and maintenance of psychosis therefore warrants further investigation. The empirical paper (a joint project with Carr (2016), “Developing a brief trauma screening tool for use in psychosis”) explores the phenomenology of intrusive trauma memory in psychosis and investigates its relationship to hallucinations and persecutory beliefs. In line with theoretical accounts (Steel et al, 2005), it was hypothesised that increased memory fragmentation would be associated with more severe hallucinations. Twenty participants described an intrusive trauma memory and its phenomenological characteristics. Findings indicated that subjective fragmentation of intrusive memories was associated with more severe hallucinations but not persecutory beliefs, although the relationship between the two ratings of objective memory fragmentation and hallucinations were equivocal, with a negative correlation for one rating and no relationship for the other. Participants with psychosis also reported more frequent and vivid intrusions, with an increased sense of reliving, compared to non-clinical sample. The study suggests a potential role for memory fragmentation in hallucinatory experience, although the complexities of assessing memory characteristics are highlighted. The critical appraisal focuses on the experience of the research process, which includes reflections on methodological issues in memory assessment, challenges to recruitment in psychosis services and the role of the research process in the author’s professional development

Optimisation of Cleaning Detergent use in Brewery Fermenter Cleaning

This paper investigates improvement possibilities in the cleaning operations undertaken at an industrial brewery. Experiments were performed on a bench scale cleaning rig which was designed to simulate ‘real life’ cleaning conditions of a clean-in-place (CIP) set in the brewery. The rig was used to clean consistently fouled coupons using difficult soils from the brewery. The objective of the experiments was to determine the reduction in effective cleaning performance with varied levels of Na2CO3 in the detergent from NaOH degradation and the maximum level that may be present before cleaning quality is impacted. The shear force of the cleaning fluid across the surface of the coupon was also varied to determine the impact on cleaning performance. Data collected from these offline measurements has been used to predict the end point of the detergent usage based on cost optimisation within the empirically determined limits. The results show that the NaOH detergent usage can be extended while achieving the same time to clean without impacting the cleaning quality and preventing premature disposal. This will provide an increased confidence level when cleaning fermenters with NaOH. It will also reduce cleaning costs and benefit the environment by reducing chemical effluent and minimising water consumption

Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA

AbstractBiochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53–DNA complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53–DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53–mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA

The role of p53 in the chemotherapeutic responses to cisplatin, doxorubicin and 5-fluorouracil treatment

A panel of tumour models used extensively for in vivo evaluation of new drugs was characterised for their p53 status. Basal p53 protein levels were measured by immunodetection on both formalin-fixed tumour tissue and from protein extracts of fresh tumours. High levels of nuclear-specific staining, indicative of p53 mutation, was seen in 15/25 tumours, with the remainder showing intermittent or no staining. The functional status of p53 cDNA from these tumours was assayed within the functional analysis of separated alleles in yeast (F.A.S.A.Y.) reporter system. The cDNA from those tumours with high levels of p53 protein showed 14/15 failing to activate the reporter gene. The cDNA from tumours with low or non-detectable p53 levels showed 8/10 with wild-type p53. Tumours were grown subcutaneously in mice (n=10). Each mouse was given maximum tolerated doses for either doxorubicin, 5-fluorouracil or cisplatin. Tumour volumes were measured daily, alongside untreated controls. The specific growth delay values for each tumour were separated into two groups, those with functional p53 (wild-type) and those without (mutant and null status). The Mann-Whitney U test was performed on the groups of data, to evaluate differences in their response on the basis of p53 status. Cisplatin was moderately active against tumours with wild-type and mutant p53 genes with no significant difference seen between both groups. However, a significant difference in specific growth delay was seen between the two groups when treated with doxorubicin or 5-fluorouracil (P=0.05), indicating a role for p53 protein in modulating the in vivo efficacy of these agents

Measurement of the decay of laser-driven linear plasma wakefields.

We present measurements of the temporal decay rate of one-dimensional (1D), linear Langmuir waves excited by an ultrashort laser pulse. Langmuir waves with relative amplitudes of approximately 6% were driven by 1.7J, 50fs laser pulses in hydrogen and deuterium plasmas of density n_{e0}=8.4×10^{17}cm^{-3}. The wakefield lifetimes were measured to be τ_{wf}^{H_{2}}=(9±2) ps and τ_{wf}^{D_{2}}=(16±8) ps, respectively, for hydrogen and deuterium. The experimental results were found to be in good agreement with 2D particle-in-cell simulations. In addition to being of fundamental interest, these results are particularly relevant to the development of laser wakefield accelerators and wakefield acceleration schemes using multiple pulses, such as multipulse laser wakefield accelerators

Low-density hydrodynamic optical-field-ionized plasma channels generated with an axicon lens

We demonstrate optical guiding of high-intensity laser pulses in long, low density hydrodynamic optical-field-ionized (HOFI) plasma channels. An axicon lens is used to generate HOFI plasma channels with on-axis electron densities as low as $n_e(0) = 1.5\times 10^{17}\, \mathrm{cm}^{-3}$ and matched spot sizes in the range $20 \mu \mathrm{m} \lesssim W_M \lesssim 40 \mu \mathrm{m}$. Control of these channel parameters via adjustment of the initial cell pressure and the delay after the arrival of the channel-forming pulse is demonstrated. For laser pulses with a peak axial intensity of $4 \times 10^{17}\, \mathrm{W\,cm}^{-2}$, highly reproducible, high-quality guiding over more than 14 Rayleigh ranges is achieved at a pulse repetition rate of 5 Hz, limited by the available channel-forming laser and vacuum pumping system. Plasma channels of this type would seem to be well suited to multi-GeV laser wakefield accelerators operating in the quasi-linear regime

Meter-Scale, Conditioned Hydrodynamic Optical-Field-Ionized Plasma Channels

We demonstrate through experiments and numerical simulations that low-density, low-loss, meter-scale plasma channels can be generated by employing a conditioning laser pulse to ionize the neutral gas collar surrounding a hydrodynamic optical-field-ionized (HOFI) plasma channel. We use particle-in-cell simulations to show that the leading edge of the conditioning pulse ionizes the neutral gas collar to generate a deep, low-loss plasma channel which guides the bulk of the conditioning pulse itself as well as any subsequently injected pulses. In proof-of-principle experiments we generate conditioned HOFI (CHOFI) waveguides with axial electron densities of $n_\mathrm{e0} \approx 1 \times 10^{17} \; \mathrm{cm^{-3}}$, and a matched spot size of $26 \; \mathrm{\mu m}$. The power attenuation length of these CHOFI channels is $L_\mathrm{att} = (21 \pm 3) \; \mathrm{m}$, more than two orders of magnitude longer than achieved by HOFI channels. Hydrodynamic and particle-in-cell simulations demonstrate that meter-scale CHOFI waveguides with attenuation lengths exceeding 1 m could be generated with a total laser pulse energy of only $1.2$ J per meter of channel. The properties of CHOFI channels are ideally suited to many applications in high-intensity light-matter interactions, including multi-GeV plasma accelerator stages operating at high pulse repetition rates

A novel SMC-like protein, SbcE (YhaN), is involved in DNA double-strand break repair and competence in Bacillus subtilis

Bacillus subtilis and most Gram positive bacteria possess four SMC like proteins: SMC, SbcC, RecN and the product of the yhaN gene, termed SbcE. SbcE is most similar to SbcC but contains a unique central domain. We show that SbcE plays a role during transformation in competent cells and in DNA double-strand break (DSB) repair. The phenotypes were strongly exacerbated by the additional deletion of recN or of sbcC, suggesting that all three proteins act upstream of RecA and provide distinct avenues for presynapsis. SbcE accumulated at the cell poles in competent cells, and localized as a discrete focus on the nucleoids in 10% of growing cells. This number moderately increased after treatment with DNA damaging agents and in the absence of RecN or of SbcC. Damage-induced foci of SbcE arose early after induction of DNA damage and rarely colocalized with the replication machinery. Our work shows that SMC-like proteins in B. subtilis play roles at different subcellular sites during DNA repair. SbcC operates at breaks occurring at the replication machinery, whereas RecN and SbcE function mainly, but not exclusively, at DSBs arising elsewhere on the chromosome. In agreement with this idea, we found that RecN-YFP damage-induced assemblies also arise in the absence of ongoing replication

Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins

Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3′ → 5′ polarity in the presence of Mn2+ and low inorganic phosphate (Pi) concentration, or to extend a 3′-OH end in the presence dNDP·Mn2+. Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3′-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ